Frontiers in Stem Cell and Regenerative Medicine Research: Volume 2 [1 ed.] 9781681081830, 9781681081847

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Frontiers in Stem Cell and Regenerative Medicine Research (Volume 2) Editors

Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK &

Shazia Anjum Department of Chemistry Cholistan Institute of Desert Studies The Islamia University of Bahawalpur Pakistan

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CONTENTS Preface

i

Contributors

ii

CHAPTERS 1.

Contribution of Stem Cells to Dental Tissue Regeneration: Isolation, Function, and Application

3

Atsushi Tomokiyo, Naohisa Wad and Hidefumi Maeda

2.

Epidermal Stem Cells and their use in Regenerative Applications for Severe Cutaneous Injuries

39

Yella H. Martin and Anthony D. Metcalfe

3.

Biological and Therapeutic Implications of Cancer Stem Cells

63

Ruth M. Risueño, Amaia Etxabe and Josep Maria Cornet-Masana

4.

Role of Stem Cells in Heart Regeneration

102

Jianhua Xiong

5.

Differentiation Potential of Stem Cells into Ovarian Cells

116

Derek Toms, Paul W. Dyce and Julang Li

6.

Challenges and Opportunities in the Development of Induced Pluripotent Stem Cell Therapeutics

157

James A. Smith, Anna French, Hannah Hurley, Benjamin Davies, Sue Dopson, Paul Fairchild, Mackenna Roberts, Paul Riley, Brock Reeve, David Williams, Laurence Daheron, Kim Bure, Andrew Carr, Jeff Karp, Ivan Wall and David Brindley

7.

Cell Cycle and Cell Cycle Regulators in the Process of Development, Pluripotency, Differentiation, and Reprogramming

176

Xiao Qi Wang and Eric J. Stanbridge

8.

Effect of Microenvironment Modulation on Stem Cell Therapy for Peripheral Nerve Injury

190

Sufang Liu and Feng Tao

9.

Transplantation of Umbilical Cord Blood Cells for Patients with Neonatal Hypoxic-Ischemic Encephalopathy and Cerebral Palsy: From Preclinical Studies to Ongoing Clinical Trials

225

Pedro M. Pimentel-Coelho, Paulo H. Rosado-de-Castro, Fernanda Gubert and Rosalia MendezOtero

10. Stem Cells for Treatment of Articular Cartilage Defects and Osteoarthritis

255

Shipin Zhang, Jun Sheng Wong, Fulya Ustunkan, Wern Cui Chu, Hee Hon Tan, Eng Hin Lee and Wei Seong Toh

Subject Index

289

The designed cover image is created by Bentham Science and Bentham Science holds the copyrights for the image.

i

PREFACE The second volume of ‘Frontiers in Stem Cell and Regenerative Medicine Research’ presents comprehensive reviews contributed by leading exponents in the exciting field of regenerative medicine. Tomokiyo et al., the Japanese group of scientists have dealt with dental tissue regeneration and elaborated the potential of dental stem cells in clinical application in Chapter 1. Martin and Metcalfe in Chapter 2 have reviewed epidermal stem cells in the context of clinical application for patients with severe cutaneous injuries. This holds the potential to significantly improve wound healing in patients and positively influence clinical outcomes. Cancer stem cells (CSCs) are a subset of cells within a tumor having self-renewal and differentiation capacity. Risueño et al. in Chapter 3 present the biological and therapeutic implications of CSCs in preclinical and clinical studies. Recent developments on stem cells in heart regeneration have stimulated studies directed towards potential clinical applications of this field. In Chapter 4, Xiong present an overview of the progress made towards unravelling the mechanisms underlying stem cell development and heart regeneration. In Chapter 5 of this volume Li et al. present the current state of research on the differentiation potential of stem cells into ovarian cells, their limitations and future prospects within the context of regenerative medicine. Smith et al. in Chapter 6 present the challenges and opportunities in the development of induced pluripotent stem cell therapeutics with special emphasis on immunocompatibility and immune suppression issues. The cell cycle machinery and its associated signaling pathways play important roles in regulation of stem cell properties. Wang and Stanbridge have summarized the current understanding of the role of the cell cycle and cell cycle regulators in the process of development, pluripotency, differentiation, and reprogramming in Chapter 7. Neural stem cells (NSCs) derived from the spinal cord have been shown to be useful in peripheral nerve regeneration. However, the stem cell therapy still exibits low efficiency. In Chapter 8 Liu and Tao discuss the effects of microenvironment on neural stem cell therapy for peripheral nerve injury and recent progress in this field. A growing number of studies on the beneficial effects of umbilical cord blood cells (UCBCs) have improved our understanding regarding fundamental neuroprotective action of transplanted cells in animal models of HIE, intrauterine hypoxia and neonatal stroke. In Chapter 9, Pimentel-Coelho et al. discussed recent data from several clinical trials and case reports that have estimated the safety and feasibility of UCBCs therapy in newborns with hypoxic-ischemic encephalopathy (HIE) and in children with cerebral palsy. In the last chapter, Toh et al. have reviewed the stem cell-based strategies that include direct intra-articular injection of mesenchymal stem cells and implantation of tissue-engineered cartilage grafts for treatment of cartilage defects and osteoarthritis. We owe our special thanks to all the contributors for their valuable contributions to the second volume of this book. We are also grateful to the editorial staff of Bentham Science Publishers, particularly Dr. Faryal Sami, Mr. Shehzad Naqvi and Mr. Mahmood Alam for their constant help and support.

Atta-ur-Rahman, FRS Honorary Life Fellow Kings College University of Cambridge Cambridge UK

Shazia Anjum Department of Chemistry Cholistan Institute of Desert Studies The Islamia University of Bahawalpur Pakistan

ii

CONTRIBUTORS Amaia Etxabe

Josep Carreras Leukaemia Research Institute, Barcelona, Spain

Andrew Carr

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK and Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

Anna French

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK

Anthony D. Metcalfe

Blond McIndoe Research Foundation, East Grinstead, West Sussex, United Kingdom and The Brighton Centre for Regenerative Medicine, University of Brighton, Brighton, East Sussex, United Kingdom

Atsushi Tomokiyo

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Higashiku, Japan

Benjamin Davies

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK and Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

Brock Reeve

Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA

David Brindley

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK; Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK; Centre for Behavioural Medicine, University College London, London, UK; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Stanford-UCSF FDA Centre for Regulatory Science and Innovation and Saïd Business School, University of Oxford, Oxford, UK

David Williams

Centre for Biological Engineering, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK

Derek Toms

Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada and Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, Canada

Eng Hin Lee

Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore and Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore

Eric J. Stanbridge

Department of Microbiology and Molecular Genetics, University of California, Irvine, School of Medicine, California, USA

iii

Feng Tao

Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, Texas, USA

Fernanda Gubert

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio Janeiro, Rio de Janeiro, Brazil

Fulya Ustunkan

Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Hannah Hurley

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK and Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

Hee Hon Tan

Faculty of Dentistry, National University of Singapore, Singapore

Hidefumi Maeda

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Higashiku, Japan

Ivan Wall

Department of Biochemical Engineering, University College London, London, WC1H 0AH, UK; Department of Nanobiomedical Science and BK21 Plus NBM Global Research Center of Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea and Institute of Tissue Regeneration Engineering, Dankook University Graduate School, Cheonan 330-714, Republic of Korea

James A. Smith

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK and Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

Jeff Karp

Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Division of Biomedical Engineering, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

Jianhua Xiong

Center for Molecular Medicine, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Josep Maria CornetMasana

Josep Carreras Leukaemia Research Institute, Barcelona, Spain

Julang Li

Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada

Jun Sheng Wong

Faculty of Dentistry, National University of Singapore, Singapore

Kim Bure

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK and Sartorius Stedim, Göttingen, Germany

Laurence Daheron

Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA

iv

Mackenna Roberts

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK

Naohisa Wada

Division of General Oral Clinic, Kyushu University Hospital, Higashi-ku, Japan

Paul Fairchild

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

Paul Riley

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

Paul W. Dyce

Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada and Department of Animal Sciences, Auburn University, Auburn, Alabama, USA

Paulo H. Rosado-deCastro

Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Pedro M. PimentelCoelho

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio Janeiro, Rio de Janeiro, Brazil

Rosalia MendezOtero

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio Janeiro, Rio de Janeiro, Brazil

Ruth M. Risueño

Josep Carreras Leukaemia Research Institute, Barcelona, Spain

Shipin Zhang

Faculty of Dentistry, National University of Singapore, Singapore

Sue Dopson

Saïd Business School, University of Oxford, Oxford, UK

Sufang Liu

Department of Physiology, Zhengzhou University School of Medicine, Zhengzhou, Henan Province, China

Wei Seong Toh

Faculty of Dentistry, National University of Singapore, Singapore and Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore

Wern Cui Chu

Faculty of Dentistry, National University of Singapore, Singapore

Xiao Qi Wang

Department of Surgery, The University of Hong Kong, Hong Kong

Yella H. Martin

Blond McIndoe Research Foundation, East Grinstead, West Sussex, United Kingdom and The Brighton Centre for Regenerative Medicine, University of Brighton, Brighton, East Sussex, United Kingdom

Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 2, 2016, 3-38

3

CHAPTER 1

Contribution of Stem Cells to Dental Tissue Regeneration: Isolation, Function, and Application Atsushi Tomokiyo1,*, Naohisa Wada2 and Hidefumi Maeda1 1

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Higashi-ku, Japan and 2Division of General Oral Clinic, Kyushu University Hospital, Higashi-ku, Japan Abstract: Tooth loss due to periodontitis, traumatic injury, or deep caries can cause facial aesthetic problems and difficulties with mastication. The ultimate goal of dental prosthetic treatment is to generate fully functioning organs to replace dental tissue that has been lost or damaged due to disease, injury or aging. Tissue engineering is a rapidly expanding field of applied biology and biomedical sciences, which aims to replace defective tissues with newly-generated tissue by combining cells, scaffolds, and biologically active molecules. Stem cells hold great promise for tissue engineering owing to their multipotency and self-renewal ability. In this article, we will present the current progress in stem cell-based dental tissue regeneration and elaborate on the potential of dental stem cells for clinical application.

Keywords: Alveolar bone, apical papilla, biological signals, bone marrowderived mesenchymal stem cells, cementum, clinical application, dental follicle, dental stem cells, dentin, exfoliated deciduous teeth, gingiva, multipotency, neural crest, periodontal ligament, pulp, regeneration, repair, scaffolds, self-renewal, tooth. 1. INTRODUCTION After the age of approximately 55–65 years, the average person loses around 8–10 of their permanent teeth [1]. The loss of one or more teeth negatively affects an individual’s oral health and quality of life because it reduces their facial aesthetic and can hamper speech and mastication. Surprisingly, a large German survey demonstrated that missing more than 19 teeth had a worse influence on healthrelated quality of life than having cancer, hypertension, or allergy [2]. A variety of options exist to replace missing teeth, such as dentures, bridges, or dental *Corresponding author Atsushi Tomokiyo: Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Higashi-ku, Japan; Tel: +8192-642-6432; Fax: +81-92-642-6366; E-mail: [email protected]

Atta-ur-Rahman & Shazia Anjum (Eds.) All rights reserved-© 2016 Bentham Science Publishers

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implants, however, each of these approaches have disadvantages, with patients often complaining of discomfort, color disagreement, and allergic responses. Deep dental caries and severe periodontal disease are two of the main attributing factors that lead to the need for tooth extraction; deep dental caries destructs the components of the tooth and severe periodontal disease damages the supportive tissues around teeth. Therefore, the ideal therapy for destructed tooth components, damaged supportive tissue, and missing teeth would be to apply a dental tissue replacement that looks, feels and functions just like natural dental tissue. Stem cells have the capacity for self-renewal and the ability to differentiate into multiple cell types of different tissues or organs, and thereby hold great promise as a potential cell source for use in cell-based therapies. Somatic stem cells, also known as adult stem cells, possess the same basic characteristics of all stem cells and are found among differentiated cells in most tissues throughout the human body. Somatic stem cells were firstly studied more than 70 years ago [3]. In the 1970s, it was discovered that bone marrow contains at least two types of stem cells; hematopoietic stem cells and bone marrow-derived mesenchymal stem cells (BMMSCs). Hematopoietic stem cells which have the capacity to differentiate into all blood cell types [4], whereas BMMSCs, which is a quite rare population of stromal cells, have the capacity to give rise to bone, cartilage, muscle, and fat cells, which are involved in the formation of blood and connective tissue [5]. Following these findings, somatic stem cells were reported to be present in many organs and tissues, such as the brain, skeletal muscle, skin, heart, gut, liver, ovarian epithelium, and testis [6]. Furthermore, somatic stem cells are believed to reside in a specific compartment within each tissue, termed a stem cell niche, that provides a particular microenvironment where stem cells can survive in an undifferentiated and self-renewable state [7]. The principal roles of somatic stem cells are maintaining and healing the tissues in which they reside. For example, epidermal cells undergo daily turnover as a part of their normal homeostatic process, which requires the constant use of somatic stem cells [8]. These cells are very active, expending and consuming vast amounts of energy during their migratory and differentiative processes. Conversely, dormant tissues such as adult skeletal muscle and brain also contain stem cell populations. These dormant tissue-derived stem cells are quiescent or they undergo extremely low division during normal homeostasis, but can respond efficiently to stimulation caused by injury to induce tissue repair [9]. The involvement of these cells in tissue homeostasis and repair has offered the potential for new clinical treatments using somatic cell transplantation. In fact,

Dental Tissue Regeneration

Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 2 5

adult bone marrow-derived hematopoietic or blood-forming stem cells have been applied in transplantation therapies for more than 40 years [10]. If researchers and clinicians can find a mechanism to control the differentiation of somatic stem cells in the laboratory and clinic, these cells could be guided to generate specialized cells and become the basis of transplantation-based therapies. Dental tissues are easily accessible for dentists during a routine extraction procedure in the dental clinic. Recently, a lot of reports have demonstrated the presence of somatic stem cells in various dental tissues, such as dental follicle, apical papilla, exfoliated deciduous teeth, periodontal ligament, and pulp. Furthermore, numerous in vitro and in vivo studies have shown the unique characteristics of these dental stem cells. Therefore, the aim of this article is to summarize the current status of the dental stem cell biology, along with the potential benefits of using dental stem cells to treat damaged tissues, and future prospective of dental stem cell-based regenerative therapies. 2. DENTAL FOLLICLE 2.1. Definition of Dental Follicle The dental follicle is an ectomesenchyme-derived component that surrounds the enamel organ and the dental papilla of the developing tooth germ before tooth eruption [11]. Dental follicle cells (DFCs) have been known to play important roles in the tooth development. In addition, when a tooth erupts, DFCs differentiate into periodontal ligament (PDL) cells to form the PDL, which anchors the tooth in its socket to the surrounding alveolar bone [12]. Moreover, DFCs near the forming root differentiate into cementum-forming cementoblasts and the cells towards the alveolar bone differentiate into osteoblasts, which secrete bone matrix. [12]. Therefore, it is believed that the dental follicle contains the stem/progenitor cells for the periodontium. There have been a number of studies that have investigated the characteristics of DFCs. Luan et al. established three mouse DFC lines (DF1, DF2, and DF3) using pSV3 plasmid DNA containing the SV40 large T antigen [13]. Surprisingly, their phenotypes were considerably different; DF1 cells exhibited a high proliferative rate, but did not form any mineralization nodules, DF2 cells revealed remarkably high alkaline phosphatase activity, and DF3 cells matched the mineralization characteristics of similar stage osteoblasts in terms of bone-related gene expression and nodule formation. This result indicated that DFCs might contain cells that are in diverse stages of differentiation. Moreover, DFCs strongly expressed the MSC-related cell surface markers, CD29, CD44, CD73, CD90, CD105, CD146 and HLA-I

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(Table 1) [13, 14]. DFCs and BMMSCs exhibited similar gene expression profiles for COL1, COL3, COL18, FGF7, FGFR1-IIIC, vimentin, and nestin [15]. Table 1: Surface marker expression in dental stem cells Antigen

DFC

SCAP

SHED

PDLSC

CD9

DPSC ○

CD10

○

CD13

○

CD24

○

○

○

CD26

○

CD29

○

CD44

○

○

○

○

○

○

○

○

CD49a

○

CD49b

○

CD49c

○

CD49d

○

○

CD49e

○

CD51/61

○

CD54

○

CD62E

○

CD71

○

○

CD73

○

○

○

○

○

CD90

○

○

○

○

CD102 CD105

○ ○

○

CD106

○

○

○

○

○

○

○

CD117

○

CD119

○

CD120a

○

CD123

○

CD140b CD146

○ ○

○

○

○

CD166

○

○

○

○

○

CD271

○

○

○

CD318

○

CD349 STRO-1

○ ○

○

Dental Tissue Regeneration

Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 2 7

Table 1: contd…

STRO-3

○

TNAP/MSCA-1

○

NG2 HLA-I

○ ○

DFC, dental follicle cells; SCAP, apical papilla stem cells; SHED, stem cells from human exfoliated deciduous teeth, PDLSC, periodontal ligament stem cells; DPSC, dental pulp stem cells

2.2. Differentiation of Dental Follicle Cells When DFCs were implanted into immunodeficient mice with hydroxyapatite (HA) scaffolds, they formed PDL-like fibrous and cementum-like mineralized tissues that expressed of cementum attachment protein (CAP), bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and collagen type I (COLI), suggesting that DFCs contain stem/progenitor cells that possess multilineage differentiative potential (Table 2). Subsequently, Saito et al. established a cell line of cementoblast progenitors from bovine DFCs through the expression of Bmi-1 and telomerase reverse transcriptase (TERT) [16]. After transplantation of these cells into immunodeficient mice, they also formed cementum-like tissue and the surrounding matrix, and expressed high levels of BSP, OCN, OPN, and COLI. Yokoi et al. also developed a cell line of mouse DFCs that exhibited high alkaline phosphatase (ALPase) activity and the expression of bone-related and PDLrelated genes [17]. After 4 weeks of their transplantation into immunodeficient mice, they generated PDL-like fibrous tissues and scattered bone-like tissues. In addition, DFCs were capable of forming colonies from single cells and differentiating into not only mesenchymal lineage cells (osteoblasts, chondrocytes, and adipocytes), but also ectodermal lineage cells (neural cells) (Table 2) [13, 18]. Thus, these cells exhibit prominent characteristics of stem cells. Although various studies have demonstrated the osteoblastic/cementoblastic differentiation potential of DFCs, the mechanism is very complicated and not known in detail. Both DFCs and osteoblasts have been known to have the potential to form mineralized matrices; however, they revealed differences in bone-related marker expression following their culture in osteoblast differentiation medium. Osteoblasts showed strong ALPase activity and BSP, OCN, OPN, and COLI expression, whereas DFCs revealed low ALPase activity and expressed only OPN and COLI [19]. Morsczeck investigated the expression of bone-related genes (DLX-3, DLX-5, MSX-2, Osterix, and Runx2) in DFCs during osteoblastic/cementoblastic differentiation and compared them with BMMSCs [20]. The expression of DLX-5, MSX-2, Osterix, and Runx2 was increased in BMMSCs during osteoblastic/cementoblastic differentiation;

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however, Runx2, DLX-5, and MSX-2 expression did not change and Osterix expression was not detected in DFCs. 2.3. Tissue-Forming Potential of Dental Follicle Cells Honda et al. transplanted cell pellets of three different types of DFCs without scaffolds into surgically-created full-thickness critical size parietal defects in rats Table 2 Overview of multipotency and tissue forming potential in dental stem cells Properties

Location

Multipotency

Tissue formation

DFC

SCAP

Dental follicle of Apical papilla of developing tooth developing root

SHED

PDLSC

DPSC

Exfoliated deciduous tooth

Periodontal ligament

Dental pulp

Osteoblasts, cementoblasts, chondrocytes, adipocytes, neural cells

Osteoblasts, odontoblasts, chondrocytes, adipocytes, neural cells, glial cells

Osteoblasts, Osteoblasts, odontoblasts, chondrocytes, Osteoblasts, chondrocytes, adipocytes, odontoblasts, adipocytes, chondrocytes, endothelial cells, neural cells, neural cells, adipocytes, melanocytes, glial cell, endothelial cells, oligodendrocytes, hepatocytes, neural cells pancreatic β-cells pancreatic β-cells

Bone, periodontal ligament, cementum, blood vessel, dentin, pulp

Bone, dentin, periodontal ligament

Bone, dentin, blood vessel, pulp, neuron, skin

Bone, periodontal Bone, periodontal ligament, ligament, cementum, cementum, dentin, pulp, dentin, pulp, neuron, tendon neuron, muscle

DFC, dental follicle cells; SCAP, apical papilla stem cells; SHED, stem cells from human exfoliated deciduous teeth, PDLSC, periodontal ligament stem cells; DPSC, dental pulp stem cells

[21]. After 4 weeks post-surgery, controls without cell transplantation formed many fibrous tissues and some bone at the site of the defect; however, in all DFC transplantation groups, the defects were robustly filled with newly-formed bonelike tissues. Perk et al. compared the osteogenic potential of BMMSCs, skinderived MSCs, and DFCs using in vivo mice model [22]; each cell type was mixed with demineralized bone matrix and a fibrin gel scaffold and transplanted into the subcutaneous tissue. At 4 weeks after implantation, all groups produced new bone-like tissues exhibiting high OCN expression and radio-opacity.

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Furthermore, the DFC-grafted group exhibited higher OCN expression and calcium content compared with BMMSCs and skin-derived MSCs. Yang et al. generated dental follicle cell sheets (DFCSs) using DFCs [23]. Surprisingly, DFCSs showed considerably high expression of COLI, ALP, BSP, and OCN, and formed more small mineralized particles on their surface compared with uninduced DFCs. Guo et al. formed DFCSs and PDL cell sheets (PDLCSs) and compared their features [24]. DFCSs secreted higher amounts of laminin and fibronectin than PDLCSs in vitro. Moreover, in vivo studies demonstrated that both DFCSs and PDLCSs generated periodontium structures, including PDL, cementum, and alveolar bone; however, DFCSs exhibited stronger periodontium regenerative potential than PDLCSs. They also implanted DFCSs in combination with human calcinated dentin into the dorsum of mice [24]. After 8 weeks posttransplantation, the cementum-PDL complex, which consisted of cementum, PDL-like fibers, and blood vessels, was generated outside the scaffolds. In addition, the dentin-pulp complex, which contained newly formed pre-dentin, polarizing odontoblast-like structure, collagen fibers and blood vessels, developed inside the scaffolds. Interestingly, the dentin-pulp complex contained fibers that were positive for the neural cell marker III-tubulin, suggesting that the newlyformed pulp was innervated by peripheral nerve (Table 2). These results indicated that DFCs, especially DFCSs, may be an ideal cell source to generate a bioengineered tooth root. More recently, the possibility that DFCs can be used to improve the microenvironment for PDL regeneration was suggested in in vitro coculture experiments, where DFCs enhanced the proliferative activity of periodontal ligament stem cells (PDLSCs), and induced embryonic stem cell (ESC)-related gene expression, and osteoblastic and adipocytic differentiation of PDLSCs [25]. Moreover, PDLSCs cocultured in vivo with DFCs enhanced the formation of a root/periodontal ligament-like complex and a periodontal ligament/bone-like complex, compared with mono-cultured PDLSCs [25]. 3. APICAL PAPILLA 3.1. Definition of Apical Papilla Stem Cells The dental papilla located at the apex of developing permanent teeth is known as the apical papilla. The apical papilla is a soft tissue that is loosely attached to the apex of the developing tooth root, making it is easy to detach and isolate this tissue. Sonoyama et al. firstly isolated and characterized the stem cell population from the root apical papilla of human tooth [26]; these stem cells from the apical papilla (SCAP) expressed mesenchymal stem cell (MSC)-related cell surface markers, CD29, CD73, CD90, CD105, CD106, CD146, CD166, and STRO-1, but were

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negative for the hematopoietic stem cell-related markers, CD18, CD34, CD45, CD18 and CD150 (Table 1). This expression pattern was similar to that of BMMSCs and dental pulp stem cells (DPSCs); however, CD24 was only detected in SCAP. Moreover, CD24 expression was decreased when SCAP were cultured in osteoblast differentiation medium, suggesting that CD24 is a useful marker to identify SCAP. They also demonstrated that SCAP showed higher potential for proliferation and migration than DPSCs [26]. In addition, it was also reported that SCAP expressed the ESC-related markers, Nanog and Oct4 [27], as well as the neural crest cell markers, nestin, musashi-1, p75NTR, snail-1, snail-2, slug, and Sox9 [28]. 3.2. Differentiation of Apical Papilla Stem Cells Previous reports demonstrated the potential of SCAP to differentiate into osteoblasts/odontoblasts (Table 2). Interestingly, SCAP showed higher population doubling capacity and produced significantly greater mineralized matrices than DPSCs [26]. Recently, several factors were reported to regulate the osteoblastic/odontoblastic differentiation of SACP; BMP4 induced the expression of Dlx2, which promoted ALPase activity, mineralized nodule formation and the expression of bone-related genes [29]. In addition, overexpressing of nuclear factor I-C or BMP2, stimulation of Wnt signaling using a GSK3 inhibitor, or exogenous addition of BMP9 to SCAP induced an upregulation of ALPase activity, mineral nodule formation, and bone- and dentin-related marker expression [30-33]. Conversely, insulin-like growth factor 1 treatment increased ALPase activity, bone-related marker expression, and bone-like tissue formation of SACP; however, dentin-related marker expression was decreased [34]. Conversely, activation of Sonic hedgehog (Shh) by the exogenous addition of Shh and overexpression of active mutant M2-Smoothened in SCAP resulted in decreased ALPase activity, mineral nodule formation, calcium content, and ALP and BSP mRNA levels in vitro, and decreased bone/dentin-like mineralized tissue formation in vivo [35]. SCAP also showed potential to differentiate into adipocytes and chondrocytes (Table 2) [31]. Recently, it was reported that lysine (K)-specific demethylase 2A was a key regulator for the adipogenic and chondrogenic differentiation of SCAP by inducing changes in SOX2 and NANOG mRNA expression [36]. Moreover, SCAP was reported to differentiate into neural cells and, interestingly, SCAP exhibited high levels of expression of neural and glial cell markers, without the induction of neural cell differentiation (Table 2) [37]. The coculture of SCAP and rat trigeminal neurons demonstrated that SCAP promoted neurite outgrowth by secreting BDNF [38], indicating that intact SCAP may have a similar phenotype to neural and/or glial cells.

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3.3. Involvement of Apical Papilla Stem Cells in Apexification and Apexogenesis Progressing dental caries or traumatic injury of the permanent teeth of young patients can lead to pulp inflammation and/or necrosis and apical periodontitis. These pathological lesions sometimes induce the development of an incompletely formed tooth root with thin root dentin and a wide apex, which can cause root fracture in the clinical management of pulp and periapical disease. Apexification is the most traditional method to treat infection of an immature tooth, by placing a calcified barrier in a root with an open apex or an incompletely formed root with necrotic pulp [39]. Apexogenesis is a vital pulp therapy procedure that allows the root of the immature tooth to continue developing, increasing its strength and chance of long term survival [40]. The developing process of the immature tooth root after apexification and apexogenesis is still unclear, but recent reports have indicated the involvement of SCAP in this process. Immature permanent teeth are supplied by rich cellular and vascular provisions that may help SCAP to survive infection, as suggested by several clinical case reports showing apexogenesis in immature teeth with pulpal necrosis [41, 42]. SCAP showed the potential to form a typical dentin-pulp-like complex when they were injected into immunodeficient mice [43]. Moreover, the surgical removal of SCAP at an early stage of root development caused an interruption of the developmental process despite the pulp tissue being intact, but other roots of the tooth containing SCAP showed normal growth and development, suggesting that SCAP would be the cell source of primary odontoblasts that have the potential to generate root dentin [44]. Mineral trioxide aggregate (MTA), one of the materials of choice for apexification and apexogenesis procedures, was shown to promote migration and proliferation of SCAP [45]. MTA also increased ALP activity, calcium deposition, and the expression of bone- and dentin-related markers in SCAP via the activation of the nuclear factor (NF)-κB signaling pathway [46]. In addition, SCAP transplanted into empty human root canals containing MTA and poly-D,L-lactide and glycolide (PLGA) induced the formation of vascularized human pulp tissue and a continuous layer of dentin-like tissues covering the root end in vivo [47]. This indicates that MTA may be an ideal material for apexification and apexogenesis because of its potential to accelerate the dentinogenesis of SCAP. 3.4. Tissue Forming Potential of Apical Papilla Stem Cells Wang et al. transplanted rat tooth root segment scaffolds containing SCAP into renal capsules of adult rats. Two weeks later, the formation of new mineralized tissues inside the root canal space or covering the canal orifice was confirmed. In

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addition, the strong expression of bone- and dentin-related markers was detected in newly formed mineralized tissues (Table 2) [48]. Conversely, the control tooth root segment without SCAP did not induce the production of mineralized tissues inside the root canal space. Sonoyama et al. developed a bioengineered tooth root that was composed of a root-shaped hyaluronic acid (HA)/tricalcium phosphate (TCP) scaffold loaded with SCAP and a membrane-like Gelfoam scaffold containing PDLSCs (Table 2) [26]. At 3 months post-transplantation of the bioengineered tooth root into the socket of an extracted tooth in minipigs, a layer of dentin-like tissue formed on the bioengineered tooth root surface and PDL-like tissue was generated that exhibited a natural relationship with the surrounding bone. Furthermore, when a pre-fabricated porcelain crown was attached to the bioengineered tooth root, this crown/root complex revealed high compressive strength. These results indicated that SCAP might have the potential to develop bioengineered human tooth roots. However, root dentin is normally formed via interactions of Hertwig’s epithelial root sheath cells and dental papilla cells during the development of the tooth root. Therefore, further research is needed to clarify the relationship between SCAP and Hertwig’s epithelial root sheath cells and/or dental papilla cells and their involvement in the formation of a new bioengineered tooth root, so that the bioengineered tooth root can perform the same function as a natural tooth root. 4. EXFOLIATED DECIDUOUS TEETH 4.1. Definition of Stem Cells from Human Exfoliated Deciduous Teeth In 2003, Miura et al. were the first to report that exfoliated human deciduous teeth contained stem cell populations, which they termed stem cells from human exfoliated deciduous teeth (SHED) [49]. SHED are particularly attractive sources of stem cells because they can be isolated noninvasively from naturally exfoliated deciduous teeth. SHED have been shown to express the MSC-related cell surface markers, CD13, CD29, CD44, CD73, CD90, CD105, CD146, CD166, and STRO1, but not the hematopoietic stem cell- or leukocyte-related markers, CD14, CD34, and CD45 (Table 1) [43, 49, 50]. Interestingly, SHED showed higher expression levels of STRO-1, CD73, and CD146, whereas they exhibited lower expression levels of CD105 and CD166 compared with BMMSCs [51]. SHED showed a higher proliferative potential than BMMSCs and DPSCs [49, 52]. Nakamura et al. revealed that SHED expressed high levels of the growth factors, basic fibroblast growth factor (bFGF), connective tissue growth factor (CTGF), transforming growth factor (TGF)-β2, and TGF-β3, compared with DPSCs [53]. These growth factors and known to regulate the biological activities of many

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types of cells, and thus, may be secreted from SHED and function in an autocrine manner to stimulate their proliferation. 4.2. Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth The differentiation potential of SHED into osteoblasts/odontoblasts was confirmed in in vitro and in vivo studies (Table 2); SHED highly expressed osteogenic-associated growth factor receptors [54], and showed the formation of mineralized nodules and an increase in osteoblast- and odontoblast-related marker expression after culturing in osteoblast differentiation medium [49]. SHED exhibited similar properties in mineralized tissue formation and bone-related marker expression compared with BMMSCs [51]. Gosau et al. also demonstrated that the potential of SHED and SCAP to develop the mineralized nodules was similar after osteogenic induction [55]. However, the dentin-related marker DMP1 was highly expressed in SCAP but not in SHED, whereas the bone-related marker BSP was highly expressed [55]. The authors indicated that SCAP had the potential to differentiate into primary odontoblasts, while SHED mainly differentiated into odontoblast-like cells or replacement odontoblasts of reparative dentine. SHED had the capacity to give rise to adipocytes that formed Oil red Opositive lipid droplets and expressed adipocyte-related marker genes (Table 2) [49]. Li et al. demonstrated that bFGF treatment enhanced the formation of Oil red-O positive lipid droplets in SHED, compared to that in untreated cells [56]; however, other studies reported their lower potential to generate lipid droplets and to express adipocyte-related markers compared with BMMSCs [51, 57]. Moreover, neither of two clonal strains derived from SHED by single-cell cloning could show the capacity to differentiate into adipocytes [50]. The chondrocytic differentiation potential of SHED was also determined by the significant upregulation of chondrocyte-related marker genes [57], and the development of Safranin O-positive glycosaminoglycans after culturing in chondrogenic medium (Table 2) [58]. SHED constitutively expressed vascular endothelial growth factor (VEGF) receptor (VEGFR)-1 and its co-receptor neuropilin-1, and moreover, could differentiate into endothelial cells via exogenous VEGF addition, as identified by the upregulation of endothelium-related marker expression and the formation of capillary-like sprouts (Table 2) [59]. Extracellular signal-related kinase (ERK), AKT, and signal transducer and activator of transcription 3 (STAT3) signaling was reported to be involved induction of endothelial cell differentiation of SHED [60]. Surprisingly, a recent study demonstrated that SHED also secreted soluble proangiogenic factors that promoted the formation of

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capillary-like sprouts in endothelial cells [61]. These results suggested that SHED possessed the potential not only to differentiate into endothelial cells, but also to induce the differentiation of endothelial cells that were located around them. SHED constantly expressed the neural cell-related markers, III-tubulin, nestin, Sox2, and ATP-binding cassette sub-family G member 2 (ABCG2) without any cell induction [62]. These markers were also detected in DFCs; however, the expression of a representative neural stem cell marker gene, PAX6, was only confirmed in SHED. In addition, SHED formed neurospheres, which are known to form during an early stage of neural differentiation, when they were exposed to serum-free medium containing epidermal growth factor (EGF) and bFGF; however, DFCs did not generate neurospheres under the same culture conditions [63]. After 4 weeks of neurogenic induction, SHED generated neurite-like multicytoplasmic processes and expressed a variety of neural cell markers, including glutamic acid decarboxylase (GAD), NeuN, glial fibrillary acidic protein (GFAP), neurofilament medium polypeptide (NFM), 2',3'-cyclic nucleotide 3'phosphodiesterase (CNPase), polysialylated neuronal cell adhesion molecule (PSA-NCAM), Tau, microtubule-associated protein 2 (MAP2), and tyrosine hydroxylase (TH) (Table 2) [49, 64]. 4.3. Dental Tissue Forming Potential of Stem Cells from Human Exfoliated Deciduous Teeth When subcutaneously transplanted into immunocompromised mice, SHED showed the capacity to produce bone-like structures on the surface of ceramic bovine bone scaffolds (Table 2) [52]. Miura et al. demonstrated that SHED induced new bone formation by the recruitment of host osteogenic cells into the transplanted sites, instead of their direct differentiation into osteoblasts in vivo [49]. Conversely, SHED exhibited the potential to differentiate into odontoblasts and endothelial cells, and consequently, promoted the formation of dentin and microvessels. Rosa et al. demonstrated that SHED cultured in tooth root complexes that were composed of the roots of human premolars within nanofiber hydrogel or collagen scaffolds upregulated the expression of dentin-related genes in vitro (Table 2) [65]. After implantation of these complexes into the dorsum of immunocompromised mice, SHED-derived connective tissues occupied the full extension of the root canal. Moreover, the tooth root complexes containing SHED demonstrated new dentin and microvessel development throughout the length of the root. Surprisingly, the pulp tissue engineered with SHED in tooth root complexes revealed similar cellularity and vascularization compared with normal human dental pulp.

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4.4 Non-Dental Tissue-Forming Potential of Stem Cells from Human Exfoliated Deciduous Teeth Several studies have examined the application of SHED to the repair and/or regeneration of non-dental tissues, including neuron, skin, and cranial bone (Table 2). The implantation of SHED into rats suffering from spinal cord contusion injuries increased oligodendrocyte- and myelination-related marker expression and improved general locomotor activities [66]. The injection of SHED into the striatum of 6OHDA-treated parkinsonian rats exhibited their long-time survival at the injury site and the potential for recovery in rotational behavior [63]. Following removal of a 10 mm segment of the sciatic nerve in rats, SHED were transplanted into the defect using tubular shaped electrospun poly(ε-caprolactone)/gelatin nanofibrous scaffolds [67]. Within 16 weeks after transplantation, SHED definitely promoted functional recovery and axonal regeneration of the damaged sciatic nerve, as evidenced by walking track analysis, plantar testing, electrophysiology and immunohistochemistry. SHED injected into full-thickness excisional skin wounds in nude mice exhibited decreased skin wound areas and an increase of hyaluronic acid production in wounded tissues, suggesting that they promoted wound healing by inducing re-epithelialization and attachment to the extracellular matrix [68]. Interestingly, the wound healing effect of SHED was significantly higher than those of phosphate-buffered saline controls and human fibroblasts. Seo et al. transplanted SHED into critical-size calvarial defects with HA/TCP particle scaffolds in immunocompromised mice [54]. The formation of bone-like tissues and the expression of bone-related markers were significantly upregulated in the SHED transplantation group compared with the control scaffold only group. In addition, newly-formed bone-like tissues, including osteocytes that were positive for anti-human-specific mitochondria antibody staining, suggested that SHED were involved in the bone regeneration of calvarial defects. 5. PERIODONTAL LIGAMENT 5.1. Definition of Periodontal Ligament Stem Cells The PDL is a highly specialized connective tissue that anchors the tooth root to the tooth socket bone and plays important roles in tooth anchorage, sensation, and facilitating nutrient delivery to surrounding cells [69]. From many years, the PDL fibroblast population has been believed to contain stem/progenitor cells that migrate from endosteal spaces to the PDL, where they differentiate into the critical PDL cell populations, fibroblasts, osteoblasts, and cementoblasts, in response to their microenvironment [70, 71]. In 2004, Seo et al. were the first to isolate PDLSCs from the PDL tissue of extracted human third molar teeth [72].

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Following their study, many researchers identified the presence of PDLSCs, not only in human but also in various animals. PDLSCs were shown to express the MSC-related cell surface markers, CD10, CD13, CD26, CD29, CD44, CD71, CD73, CD90, CD105, CD106, CD146, CD166, CD349, STRO-1, STRO-3, and tissue non-specific alkaline phosphatase (TNAP)/mesenchymal stem cell antigen (MSCA)-1 (Table 1) [73-75]. Interestingly, a recent report also demonstrated the expression of pericyte-related cell surface markers NG2 and CD140b in PDLSCs [76]. Moreover, PDLSCs expressed the ESC-related markers, c-Myc, Klf4, Nanog, OCT-4, TRA-1-60, TRA-1-81, SOX2, REX1, SSEA-1, SSEA-3 and SSEA-4, as well as the neural crest cell-related markers, Snail, Slug, Twist, SOX9, SOX10, Nestin, p75NTR, CD49d, and Tuj1 [73, 74, 77, 78]. PDLSCs have been known to have a capacity for self-renewal, whereby they can develop single-cell derived colonies when seeded on a culture dish at an extremely low density [79]. In addition, the STRO-1-positive fraction of PDLSCs exhibited significantly higher colony-forming capacity than the STRO-1-negative fraction [72]. One characteristic of MSCs is their ability to exhibit immunomodulatory behavior. Indeed, PDLSCs were also reported to exhibit an inhibitory effect on the proliferation of allogeneic and xenogeneic peripheral blood mononuclear cells (PBMCs) by suppressing the cell cycle [80]. Moreover, PDLSCs suppressed the secretion of interferon (IFN)-γ in PBMCs by indirect soluble mediators and direct cell-to-cell contact [81]. This inhibitory effect of PDLSCs on PBMCs was mediated by soluble factors, including transforming growth factor (TGF)-β, hepatocyte growth factor (HGF), and indoleamine-2,3 dioxygenase (IDO) [82]. 5.2. Differentiation of Periodontal Ligament Stem Cells Seo et al. cultured PDLSCs in osteogenic medium, which induced the formation of mineralized nodules and an upregulation of bone-related gene expression, suggesting that PDLSCs can differentiate into osteoblasts (Table 2) [72]. Additionally, several factors were demonstrated to regulate the osteoblastic differentiation of PDLSCs; whereby a cyclic tension force enhanced collagen synthesis, mineral deposit formation, and bone-related marker expression [83]. Furthermore, estrogen-related receptor (ERR)α, which was expressed throughout osteoblastic differentiation of PDLSCs, regulated ALP activity, mineralized nodule formation, and bone-related marker expression [84]. The differentiation capacity of PDLSCs into adipocytes and chondrocytes was also determined by the development of Oil red O-positive lipid droplets and Safranin O-positive glycosaminoglycans, respectively (Table 2) [72, 85]. However, a recent study demonstrated that the osteoblastic and adipocytic differentiation potential of PDLSCs was lower than that of BMMSCs [86]. This outcome may be related to

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the effect of Wnt/β-catenin signals, because the canonical Wnt pathway enhanced osteoblastic differentiation of BMMSCs, but suppressed it in PDLSCs [87]. PDLSCs were also differentiated into endothelial cells that expressed endothelial cell- and smooth muscle cell-related markers and constructed capillary-like sprouts with lumens [88]. Moreover, a phosphoinositide 3-kinse (PI3K) inhibitor suppressed the proliferation and endothelial cell-related marker expression in PDLSCs, suggesting that PI3K activation played crucial roles in the endothelial cell differentiation of PDLSCs. Previous reports suggested that PDLSCs can be induced to differentiate into cells with neural phenotypes relatively easily because they are derived from the neural crest and express various neural crest-related markers. Indeed, PDLSCs cultured in serum-free neural induction media were shown to generate free-floating neurospheres [89]. In addition, PDLSCs that migrated from adherent neurospheres gave rise cells with one, two, or more neurite-like processes, and distinctly expressed specific markers for neurons, glia, and oligodendrocytes (Table 2). Cells with one, two or three or more neurites are classed as unipolar, bipolar or multipolar, respectively. ERK1/2 signaling was suggested to be involved in the process of neural cell differentiation in PDLSCs [90]. Interestingly, Bueno et al. injected PDLSCderived neural cells into the hippocampus of immunosuppressed mice [78]. Three weeks after injection, these cells survived, migrated, and integrated in the hippocampus of the adult mouse brain. A recent study also generated retinal progenitors from PDLSCs via the formation of neurospheres, which was shown by the expression of eye field transcription factors and photoreceptor markers, and by a calcium transient in response to glutamate insult [91]. PDLSCs exposed to a three-dimensional culture in pancreatic differentiation medium formed tight cellular aggregations that resembled pancreatic islets (Table 2) [92]. RT-PCR and flow cytometry analyses revealed an increase of pancreatic marker expression in PDLSC-derived pancreatic islet-like cellular aggregations. Additionally, these aggregations were positive for insulin-producing β-cells-specific antibodies and secreted insulin in response to glucose in a manner similar to pancreatic β-cells. Our recent work revealed that semaphorin 3A (Sema3A) plays a dominant role in preserving stem cell properties of PDLSCs; the expression of Sema3A was detected at high levels in dental follicle, the origin of PDL tissue, only during the cap stage; however, its expression was significantly decreased in mesenchymal tissue surrounding the dental organ at the bud stage and in dental follicle tissue at the late bell stage [93]. The expression level of Sema3A was stronger in multipotent human PDL cell lines compared with low-differentiation potential

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lines. In addition, Sema3A-overexpressing low-differentiation potential PDL clones showed an upregulation of ESC- and MSC-related marker expression and an increased capacity to differentiate into osteoblasts and adipocytes. 5.3. Establishment of Human Periodontal Ligament Stem/Progenitor Cell Lines The stem cells in the PDL are a quite rare population; thus it is very difficult to acquire enough numbers of cells that can be used for repeated experiment to ensure consistency of results. Therefore, studies have focused on the development of immortalized PDL stem cell lines using the SV40 large T-antigen, human telomerase reverse transcriptase, human papillomavirus 16-related E6E7, Bmi-1, and BMP4. Initially, immortalized PDL cell lines were generated from mice, swine, and human PDL cells [94, 95]. More recently, Shirai et al. established clonal swine PDL cell lines that exhibited the potential to form mineralized nodules and vascular tube-like structures [96]. Recently, our group reported the establishment of an immortalized PDL cell line using the SV40 large T-antigen and human telomerase reverse transcriptase [97]. Following limiting dilution, two clonal human PDL cell lines with multipotency were isolated; cell line 1-11 showed the ability to differentiate into osteoblasts and adipocytes [98], whereas cell line 1-17 could differentiate into osteoblasts, chondrocytes, adipocytes and neural cells [99]. These cell lines strongly expressed several MSC-related cell surface markers; however, it was suggested that their characteristic was partly different from BMMSCs because both cell lines expressed the PDL cell-related markers, periostin and scleraxis, whereas BMMSCs do not. Cell lines 1-11 and 1-17 also exhibited several different properties in addition to their multipotency; cell line 1-17 strongly expressed OCT4 and Nanog mRNA, whereas their expression level was very low in cell line 1-11 [100]. Additionally, cell line 1-17 had a higher number of p75NTR-positive cells (38.41%) than cell line 1-11 (6.26%) (Fig. 1A). bFGF has been known to suppress matrix mineralization in immature human calvarial osteoblastic cells and promote it in more mature cells [101]. When cell lines 1-11 and 1-17 were exposed to osteoblastic differentiation medium, they generated nearly the same amount of calcified deposits (Fig. 1C, F). Conversely, bFGF enhanced calcium deposition in cell line 1-11 (Fig. 1D), whereas it almost completely suppressed it in cell line 1-17 (Fig. 1G) as well in the control medium (Fig. 1B, E). Following subcutaneous transplantation of both cell lines into the dorsal side of immunodeficient mice, cell line 1-11 generated bone-like tissues containing Sharpy’s fiber-like tissues [98]. Cell line 1-17 also showed the

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capacity to form bone-like tissues; however, no fibers were observed (data not shown). Furthermore, when these cell lines were injected into artificiallyfabricated periodontal defects, cell line 1-11 attached to the surfaces of alveolar bone and tooth root, and within the PDL tissue [100]. In contrast, cell line 1-17 was identified only within the PDL tissue [100]. These our results suggested that both cell lines had the typical characteristics of stem cells, but differed in maturity; cell line 1-17 was thought to be at a much earlier stage of differentiation than cell line 1-11, according to their differences in multipotency, ESC- and neural crest-related marker expression, their response to bFGF in osteoblastic differentiation, and the results of transplantation assays (Fig. 2). A

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Fig. (1). Difference in CD271 expression and calcified deposit formation between cell lines 1-11 and 1-17 cultured in DM+bFGF. (A) Flow cytometry analysis of cell line 1-11 and 1-17 using antibodies reactive to the IgG isotype control and the neural crest cell-related surface molecule CD271. (B–G) von Kossa staining images of cell lines 1-11 (B–D) and 1-17 (E–G) cultured in CM (B, E), DM (C, F), and DM+bFGF (D, G) for 4 weeks. Scale bar = 200 m. CM, control medium; DM, osteoblastic differentiation medium; bFGF, basic fibroblast growth factor.

5.4. Differentiation-Inducing Capacity of Periodontal Ligament Stem Cells It has been reported that BMMSCs express and secrete significant amounts of various growth factors that have the potential to promote repair and regeneration of damaged tissues [102]. Recent studies have suggested that PDLSCs also have the capacity to induce differentiation of other cell populations; when dedifferentiated fat cells were exposed to indirect coculture with PDLSCs, their methylation status was upregulated in adipogenic gene promoters, but downregulated in bone-related gene promoters [103]. Additionally, indirect coculture with PDLSCs promoted bone-related gene expression and the

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Fig. (2). Schematic of the different characteristics of cell lines 1-11 and 1-17, regarding cell surface marker expression, ESC-related marker expression, and PDL-related marker expression, osteoblastic differentiation in the presence of bFGF, and tissue formation after in vivo transplantation. ESC; embryonic stem cell, PDL; periodontal ligament.

suppression of adipocyte-related gene expression in dedifferentiated fat cells compared with non-coculture conditions. Cell pellets of PDLSCs and chondrocytes derived from the temporomandibular joint (TMJ-CH) revealed increased proliferation and glycosaminoglycan formation than pellets containing PDLSCs only and TMJ-CH only [104]. Furthermore, PDLSC-conditioned medium also promoted proliferation, glycosaminoglycan formation, and cartilagerelated gene expression in TMJ-CH. We performed coculture of rat adrenal pheochromocytoma-derived PC12 cells with cell line 1-17, which resulted in the induction of neural differentiation of PC12 cells [74]. In addition, coculture of PC12 cells on devitalized cell line 1-17 induced neural differentiation in a small number of PC12 cells; however, cell line 1-17-conditioned medium distinctly induced it. Furthermore, NGF secretion from cell line 1-17 was suggested to play crucial roles in the promotion of neural differentiation of PC12 cells following the discovery that addition of a NGF neutralization antibody or the NGF receptor antagonist K252a to the conditioned medium inhibited this effect. We also

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performed a coculture of both cell lines 1-11 and 1-17 in the presence of bFGF, by placing the cell lines in two different chambers that were separated by a permeable barrier (Fig. 3A). Although the formation of calcified deposits was not identified in the culture of cell line 1-17 alone (Fig. 3B, D), it was induced when it as cocultured with cell line 1-11 (Fig. 3C, D). The expression of BMP4 mRNA was detected in cell line 1–11, whereas cell line 1–17 did not express it (Fig. 3E). 1-11

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Fig. (3). Osteoblastic differentiation of cell line 1–17 cocultured with cell line 1–11. (A) The schematic of the two chambers with the permeable barrier used for the indirect coculture. (B, C) von Kossa staining images of cell line 1-17 cultured in DM+bFGF (B) and cocultured with cell line 1-11 in DM+bFGF (C) for 4 weeks. (D) Net values of the size of von Kossa-positive areas for each culture condition. **p80% double-positive). Furthermore, the researchers also observed the conversion of testosterone into estradiol and the production of AMH. As was the case with Woods et al. (2013) these granulosa cells appeared to be at an early stage of development as no LH receptor was detected. We have recently differentiated skin-derived stem cells into a granulosa cell phenotype that was capable of restoring estrus cycling in ovariectomized mice [172]. Somatic stem cells were isolated from newborn mouse skin and

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differentiated using a follicular fluid containing media to generate PGC-like cells in the presence of extracellular matrix (Matrigel™) to facilitate cell-cell interactions. The resulting cultures showed high expression of both aromatase and FSH receptor, and were able to synthesize estradiol in vitro [172]. Extended in vitro culture resulted in the formation of large, OLCs, similar to what we have seen previously [139, 151]. When we instead transplanted early-stage differentiated cultures into ovariectomized mice, serum estradiol levels were restored to those comparable to control mice with intact ovaries, and uterine atrophy was prevented. Some of the mice receiving transplants additionally resumed estrus cycling, although not as regularly as control mice. Furthermore, these transplants were capable of reestablishing the pituitary-gonadal axis resulting in decreased serum FSH, which rises following the removal of the ovary (or menopause) because of disruption to the negative feedback loop involving estradiol. FSH receptor-positive cells were found in the transplants, further supporting the characterization of these cells as granulosa-like [172]. While we were able to observe granulosa cell markers, as well as the restoration of endocrine signaling suggesting a granulosa cell phenotype, the possibility that other cell types were generated exists, although the potential of skin derived stem cells for hormone replacement therapy is encouraging. Stem Cell Transplantation to Restore Ovarian Function In Vivo Another area of research in this field is the restoration of fertility following chemotherapy, which causes apoptosis of the pregranulosa cells of primordial follicles [173, 174]. Using a mouse model of chemotherapy, treated with a combination of cyclophosphamide and busulfan, several attempts have been made to restore fertility using pluripotent stem cells. Following tail vein injection of GFP-labelled human amniotic epithelial cells (hAECs), Wang and colleagues found ovaries containing GFP-positive follicles up to 60 days post-injection [175]. Isolated hAECs express the stem cell markers OCT4, NANOG, and C-KIT, (negative for DAZL, VASA) suggesting their pluripotent nature. Fourteen days after transplantation, hAECs mitigated the chemotherapy-induced loss of primordial follicles while preventing the loss of primary and secondary follicles [175]. At the end of the two month trial period, it seemed that the hAECs had successfully migrated through the ovarian stroma and differentiated into granulosa cells of the follicle. Recipient ovaries that showed renewed folliculogenesis following hAEC injections stained positive for human specific nuclear antigen, human FSHR and AMH, supporting the differentiation of hAECs into granulosa cells in vivo [175]. However, no follicles showed contribution to eggs by hAECs. The positive response seen early, between seven and 28 days and before

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integration of differentiated hAECs could suggest that these pluripotent cells may also have trophic effects on endogenous ovarian cells, and may reactivate host GSC. Importantly, several follicles examined did not appear to contain human cells, suggesting that trophic effects from the transplants may have also benefited ovarian regeneration. With the positive effects seen from this study, it is encouraging to think that a more accessible pluripotent stem cell, derived from term placentas, are capable of restoring fertility following chemotherapy. It would have been interesting if the authors had extended the study to determine if fertility was improved and the follicles observed supported competent oocytes. In a similar study, mouse amniotic fluid stem cells (mAFSCs) were able to prevent follicle loss following chemotherapy, although this was far less effective and by five weeks control and mAFSC-injected mice showed similar decreases in follicle numbers [176]. Transplanted cells did not contribute to either the germ or somatic compartments of the ovary, and as mentioned, only a transient improvement in fertility after transplantation was observed. However, increased estrous cycling over a two-week period, more successful matings and larger litter sizes were observed in trials beginning one week after transplantation [176]. As mAFSCs were not found to contribute to either somatic or germ cell compartments, it is likely that these cells were solely responsible for trophic support to endogenous ovarian cells. Using skin-derived mesenchymal stem cells (SMSCs), Lai and coworkers (2014) showed that transplantation of undifferentiated cells maintained body weight and prevented ovarian and uterine atrophy in chemotherapy-treated mice [177]. Similar to the work performed by Wang et al. (2013), the authors observed a migration of SMSCs through the ovarian stroma to the granulosa cell compartment where these pluripotent cells were shown to differentiate into granulosa cells and express AMH two months after transplantation [177]. Interestingly, only 20% of recipient ovaries were positive for the donor red fluorescent protein (RFP) marker, yet all chemotherapy-treated mice showed improved fertility. Analysis of inflammatory-associated markers in the ovary showed decreases at seven days post-transplantation suggesting that this could be a mechanism by which the stem cell transplants improved fertility [177]. The lack of entirely RFP-positive follicles further supports the idea that multiple mechanisms may be at work and transplanted cells could be providing important trophic signals to enhance endogenous stem cells present in the ovary.

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Lai et al. (2013) reported that human amniotic fluid stem cells (hAFSCs) could also restore fertility in a chemotherapy mouse model by differentiating into granulosa cells. Characterized hAFSC expressed the germ and stem cell markers NANOG, OCT4, C-KIT, BLIMP1, STELLA, DAZL, VASA, C-MOS, ZPA, ZPC, SCP1, SCP3, and GDF9 [178]. They used a mesenchymal-like clone to transplant into chemotherapy-damaged ovaries. These cells restored morphology of both follicles and oocytes, and as was found by Wang (2013), GFP-positive cells were present around the oocyte and in the ovarian stroma. Expression of FSH receptor colocalized with human nuclear antigen, suggesting that the hAFSCs had differentiated into granulosa, but not germ cells in vivo [178]. Not all restored follicles contained GFP-positive cells, suggesting that hAFSCs may have restored folliculogenesis “via indirect trophism” on endogenous ovarian stem cells. The identity of endogenous ovarian stem cells, however, still remains a mystery. CURRENT RESEARCH LIMITATIONS AND FUTURE DIRECTIONS While several reports have suggested the appearance of OLCs from differentiated pluripotent cells, few have been able to successfully recapitulate meiosis [147, 179]. The few successes have been in vivo, where it is likely that endogenous processes are necessary to drive successful meiosis. A recent report suggests that oocyte differentiation is a separable phenomenon from meiosis [180]. They showed that in spite of a Stra8 knockout, which prevents meiotic initiation, some oocytes were capable of differentiating. These mutant oocytes produced zona pellucida and polar bodies, which contained random, disorganized genetic material. Even more surprisingly, superovulating these oocytes resulted in fertilizable eggs that were capable of undergoing cleavage despite massive abnormalities in chromosome segregation [180]. In light of these findings, it will be crucial going forward that OLCs that are generated meet all the necessary criteria [2] to be considered oocytes. Another consideration is the coordination of in vitro-generated germ cells with their supporting somatic cells. These two cell types are mutually indispensable for their development in vivo, and it therefore only makes sense that attempts to culture either cell type alone is hampered by the lack of the other. Indeed, many investigations that were successful in generating OLCs required aggregation with ovarian somatic cells [134, 148] or used culture conditions that generated both germ cells and supporting somatic cells [151, 167]. Even our own differentiation of granulosa-like cells from skin-derived stem cells was based on a protocol to derive PGCLCs, and had DAZL-positive cells present in the recovered transplants

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[172]. Continued research should focus on compatibility between OLCs and somatic follicle-like cells, and ensure that differentiation protocols equally support the production of both of these cell types. A limitation of most current protocols is the use of undefined media to generate both germ and somatic cells in vitro. Serum and follicular fluid are frequently used for great benefit, but because of their largely undefined nature, it becomes impossible to understand the mechanisms driving this differentiation process. Recent studies by Lan (2013) and Irie (2015) using defined medias reflect our growing understanding of signals regulating development of the follicle. The ability to fine-tune differentiation pathways afforded by wholly defined media will allow for rational optimization of these processes in vitro and move forward to clinical applications. In light of the limitations of current OLCs, it is unlikely that these in vitro generated germ cells will be ready to generate offspring in human medicine anytime soon. The success of the Saitou group in generating live offspring from in vitro-derived germ cells is certainly promising [148, 149], although important differences exist between mice and human development. One interesting observation is the length of time it takes to generate PGCLCs or OLCs in culture. Human ESCs differentiated in vitro have been shown to express VASA at day 7 [125], although in vivo, normally developing human germ cells do not typically express VASA until seven weeks [181]. Similarly, when mouse ESCs are aggregated with trophoblast cells or Bmp4-expressing cells, Vasa expression has been detected as early as one day following differentiation [135]. This suggests that an accelerated development is seen in vitro. Understanding the ramifications of this increased rate of differentiation in non-rodent species with longer gestation times will be a necessary first step before considering human applications. Although therapies utilizing human OLCs may be a long way from being realized, regenerating the somatic compartment of the ovarian follicle avoids many ethical concerns and may be on the horizon. Young patients with premature ovarian failure typically develop the disease before a woman achieves peak adult bone mass. They therefore suffer from a sex steroid deficiency for many years longer than menopausal women whose depletion of these hormones occurs via the physiological aging process. As a result, these premature ovarian failure patients are at higher risk for osteoporosis and cardiovascular disease [182]. Current hormone-based therapies have been shown to have side effects including an increased risk of breast cancer, stroke, and associated heart disease [183]. Thus, a

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more comprehensive and safe source of ovarian sex hormones supply is desirable. To this end, our recent study demonstrated encouraging results of restoring estradiol production and estrus cycle in an ovariectomized animal model via transplantation of ovarian cell-like cells differentiated from skin-derived stem cells [172]. This would allow for autologous transplantation, using readily accessible skin tissue, and have the additional benefit of reestablishing the pituitary-gonadal axis such that hormones are produced and released according to the body’s requirements. However, before the feasibility of this application can be recognized, many questions still remain to be addressed. As mentioned, identifying and then using defined factors responsible for inducing granulosa cell differentiation would increase yield and ensure that production of undesirable cell types are minimized. These differentiated granulosa cells may need further purification prior to transplantation. It will also be critical to test for potential systemic side effects of transplantation, and demonstrate in vivo stem cell potential in humans. Despite all the potential obstacles for using stem cell-derived ovarian cell-like cells in regenerative medicine, they nevertheless remain valuable for studying germ cell formation and development. The generally inaccessible nature of female germ cells makes these models critical in modeling and understanding associated diseases. Further improvement to in vitro ovarian cell differentiation systems will allow investigators to pinpoint specific factors and pathways, as well as required somatic cell-germ cell interactions that are crucial for these developmental processes. In addition, though current OLCs do not meet the gold standard of a real, functional oocyte, the exciting investigations taking place to identify what transcripts or proteins are missing or altered may provide clues into the genetic pathways governing oocyte development and unravel the molecular basis of meiosis. It is anticipated that these identifications, facilitated by the use of nuclease-based gene editing technologies, will allow for manipulation of genes within these in vitro cell models to provide novel insights into female germ cell differentiation in the near future. CONFLICT OF INTEREST The author confirms that author has no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS Declared none.

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Challenges and Opportunities in the Development of Induced Pluripotent Stem Cell Therapeutics James A. Smith*,1,2, Anna French1, Hannah Hurley1,2, Benjamin Davies1,2, Sue Dopson11, Paul Fairchild4, Mackenna Roberts1, Paul Riley5, Brock Reeve6, David Williams15, Laurence Daheron6, Kim Bure1,7, Andrew Carr1,2, Jeff Karp6,8,9, Ivan Wall#, 12,13,14 and David Brindley#,1,2,3,6,10,11 1

The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK; 2Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK; 3Centre for Behavioural Medicine, University College London, London, UK; 4Sir William Dunn School of Pathology, University of Oxford, Oxford, UK; 5Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 6Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; 7Sartorius Stedim, Göttingen, Germany; 8Division of Biomedical Engineering, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; 9Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; 10 Stanford-UCSF FDA Centre for Regulatory Science and Innovation; 11Saïd Business School, University of Oxford, Oxford, UK; 12Department of Biochemical Engineering, University College London, London, WC1H 0AH, UK; 13Department of Nanobiomedical Science and BK21 Plus NBM Global Research Center of Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea; 14Institute of Tissue Regeneration Engineering, Dankook University Graduate School, Cheonan 330-714, Republic of Korea and 15Centre for Biological Engineering, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK Abstract: Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have generated much excitement as a potential source of therapeutic cells. Diverse applications, including for diabetes, neurological and ocular disorders, and heart failure are currently being investigated, and new therapies could enter into a growing regenerative medicine market. Despite this, there are significant challenges in the development of iPSC therapeutics and their ultimate translation to clinical use. Heterogeneity in iPSC products can be introduced throughout the complex process of iPSC generation and selection. Challenges with, and current approaches to, developing scalable, consistent methods for reprogramming of somatic cells, and selection, validation and characterization of iPSCs are, therefore, discussed in the context of good manufacturing guidelines and quality assurance. Further, we discuss issues and considerations with immuno-compatibility and the possible need for immune suppression. The final barrier to the development of iPSC therapeutics discussed is intellectual property. *Corresponding

author James A. Smith: The Oxford - UCL Centre for the Advancement of Sustainable Medical Innovation (CASMI), University of Oxford, Oxford, UK; Tel: +44 01865 227374; Fax: +44 01865 737640; E-mail: [email protected] #Joint Senior Author: Ivan Wall and David Brindley Atta-ur-Rahman & Shazia Anjum (Eds.) All rights reserved-© 2016 Bentham Science Publishers

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Keywords: iPSC translation, iPSC therapy, cell therapy, reprogramming method, iPSC selection, iPSC validation, immunology, intellectual property, translation, healthcare risk management, risk: benefit analysis, commercialization, bio manufacturing, bioprocessing, intellectual property. INTRODUCTION All cells in the human body - somatic and germ line - originate from pluripotent embryonic stem cells (ESCs) that have the capacity to differentiate into any cell type. Since they were firstly reported [1], ESCs have been anticipated as a potentially key treatment for a variety of challenging disorders, such as Parkinson’s disease and spinal cord injury [2]. Though promising, there are considerable ethical and practical concerns with the use of ESCs. In 2006, it was shown that adult cells can be reprogrammed into a pluripotent state similar to that of ESCs following expression of four transcription factors (or reprogramming factors): the cells generated were termed induced pluripotent stem cells (iPSCs) [3]. The importance of iPSCs is considered so great that the 2012 Nobel Prize in Physiology and Medicine was awarded for “the discovery that mature cells can be reprogrammed to become pluripotent.” Though iPSCs were discovered relatively recently, the first realisation that differentiation was not unidirectional came in 1962 when Sir John Gurdon generated cloned frogs by transferring a somatic Xenopus tadpole cell nucleus into an oocyte [4]. This critical experiment in nuclear reprogramming demonstrated that somatic cells retain all genetic information necessary for pluripotency and that they can be rejuvenated upon artificial manipulation. Another important historical step in the discovery of iPSCs was the realisation that transcription factors could induce transdifferentiation: i.e. lineage committed cells could change fate when appropriate transcription factors were introduced [5]. By combining these two conceptual advances, induced pluripotency emerged (Fig. 1) [2]. iPSCs are considered to have great potential, both directly as a therapeutic and also as a tool for disease modelling and drug discovery (Fig. 2). This chapter focuses on their application as a therapeutic. Firstly, current therapeutic applications that are under development are discussed and areas in which iPSCs are anticipated to be useful are outlined. Despite these potential areas of application, at the moment, clinical use of iPSC therapies is very limited; the second section of this chapter discusses the challenges that are associated with bringing iPSC therapies to the clinic.

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Figure 1: Important advances leading to the development of iPSCs (adapted from [2]). A) Nuclear transfer generates cloned frogs, B) Lineage conversion of committed cells with transcription factors (TF) C) combining these ideas leads to induced pluripotency

Figure 2: Potential clinical applications of induced pluripotent stem cells iPSCs. This chapter focuses on the use of therapeutic application of iPSCs. (Orange = process, blue = product, green = application; adapted from [6])

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PROMISE OF iPSCs Cell transplantation therapy is likely to be one of the most important therapeutic uses for iPSCs. Because of their pluripotent state, iPSCs can, theoretically, be directed to differentiate into any adult cell type. Many diseases are characterised by defects in specific cell types, such that non-defective cells grown in vitro could be transplanted into diseased individuals to replace defective cells and restore normal function. Diseases caused by defects in single genes represent a natural opportunity for the use of iPSCs. iPSCs can be generated from adult cells, defective genes corrected, and the resulting cells directed to differentiate into the cell type affected by the genetic defect. In fact, the first proof of concept study for the therapeutic use of iPSCs was conducted in this context. A mouse model of sickle-cell anaemia, a blood disorder caused by a mutation in the β-globin gene, was rescued by transplantation of hematopoietic progenitor cells differentiated from autologous iPSC lines corrected by homologous recombination [7]. Though successful in this model, the use of autologous iPSCs would be likely to incur high costs and, for some disorders, the timeframe required for reprograming, expansion and differentiation of autologous cells is too long, thus, allogeneic therapies are sometimes considered to be the most promising route forward, at least in the near future [2, 8, 9]. Disorders for which transplantation of donor tissue is known to be a successful treatment option are, therefore, excellent targets for developing iPSC therapeutics as it is already known that allogeneic autologous tissues are appropriate. iPSC technology could lead to, essentially, limitless supplies of desired cells for transplantation, in contrast to the ubiquitous shortage of donor tissue for transplantation [9]. Diabetes exemplifies this principle: type 1 diabetics can become insulin independent for > 5 years with transplantation of human cadaveric islets [10]. However, a lack of donor islets and variations in donor tissue quality - both viability of the tissue and immunological mismatch -limit the utility of this approach. iPSCs represent a promising treatment. In 2014, a significant breakthrough was made: functional human pancreatic β cells were generated from iPSCs and ESCs via a scalable method that can rapidly produce large cell numbers [10], possibly eventually addressing the lack of donor islets available (Fig. 3). Though not yet tested in humans, data from mice and in vitro studies are promising. Immune rejection would, however, still require management.

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Figure 3: Summary of generation and application of β-cells in a recent, important work by Doug Melton's group. A) Human pancreatic β-cells are generated by a scalable protocol from induced pluripotent stem cells (iPSCs) by addition of soluble signals that induce β-cell formation via intermediate cell types. B) Transplantation of iPSC-derived β-cells into diabetic mice leads to recovery of blood glucose control. Eventually, it is hoped that similar techniques will be used treat human diabetics. (Adapted from [10])

Heart failure represents another potential application for iPSCs. iPSCs can be used to generate human cardiomyocytes which could support heart tissue regeneration [11]. Various small animal models have demonstrated favourable responses with the use of ESC-derived cardiomyocytes following myocardial infarction [12-14], and human ESC-derived cardiomyocytes have been shown to regenerate non-human primate (macaque) hearts [15]. Tackling neurological disease may be another important application of iPSCs. Some have gone as far as to say that “the impact of [iPSC technology] has been

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most strongly felt in the neurosciences” [6]. This impact has largely revolved around the use of iPSCs as a tool for modelling neurological disease; however, more direct therapeutic applications are emerging. Fetal neural stem cell (fNSC) transplantation has been investigated for several central nervous system conditions including Parkinson’s disease [16], Huntington’s disease [17] and amyotrophic lateral sclerosis (Lou Gehrig's Disease) [18]. However, outcomes have been inconsistent, possibly due to difficulties in cell standardization and limited availability of starting cell material [6]. The use of iPSCs could help to address these issues. The most advanced iPSC treatment, and indeed the only iPSC therapeutic currently in clinical study, is a treatment for wet-type age related macular degeneration (AMD) using autologous iPSC-derived retinal pigment epithelial (RPE) cells. A group led by Takahashi at the RIKEN Centre for Developmental Biology in Kobe, Japan, are using autologous iPSCs to generate monolayer sheets of RPE cells that can then be directly transplanted to the patient’s eye. In September 2014, the first participant underwent transplantation without complications [19] as part of a pilot study limited to 6 patients. The London Project to Cure Blindness also aims to use stem cell technology, albeit largely embryonic stem cells, to improve vision in patients with AMD and other visual impairments. A Promising Market In 2012, cell therapy and regenerative medicine products were estimated to be generating $900 million per year [20]. At present, none of the leading products is iPSC-based; however, the general success of this young market indicates that there is considerable potential for the infiltration of new regenerative medicine products. Given the vast potential therapeutic applications of iPSCs and lack, in many cases, of alternative treatments, the market can be expected to grow considerably as more scientific breakthroughs are translated into therapeutics [21]. For companies simply supplying iPSC research products, global revenue in 2012 was $837 million and is anticipated to exceed $1billion by 2015 [22]. CHALLENGES FACING iPSC THERAPEUTICS Despite the widespread potential therapeutic applications of iPSC technology and the promising market into which new regenerative medicine therapies can enter, there are significant challenges facing the successful translation of iPSCs to patients. The remainder of this chapter discusses these challenges. There are

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certain challenges, such as mode of delivery, cell retention and cell survival, that are applicable to all cell-therapy approaches. However, here we focus on the specific issues associated with iPSCs, where possible. Though allogeneic therapy may be cheaper and more feasible, it comes with its own set of challenges. Issues typically associated with blood, tissue and organ transplantation will likely be applicable, including donor consent, selection, screening and immune rejection [8]. As described above, one of the greatest theoretical benefits of iPSCs is the potential to meet the demand that cannot be met by donor tissue - another is complete immune compatibility from autologous iPSCs. Establishing ‘iPSC banks’ that store and readily allow access to diverse cell lines to ensure the best possible tissue match with patients is therefore important; however, it represents a key translational barrier [23]. Critical to success here will be regulatory compliance, including development of current good manufacturing practice (cGMP) standards, quality assurance (QA), and ethical guidelines [8, 24, 25]. Firstly, the technical challenges associated with generating, characterizing and validating iPSCs are discussed. The process of iPSC generation is outlined in Figs. (4) and (5). This discussion is applicable to both autologous and allogeneic iPSCs. Issues with the final iPSC product are then covered. Next, challenges associated with immuno-compatibility and iPSC banks are investigated. Finally, issues of intellectual property (IP) that must be taken into account when commercializing an iPSC product are explored.

Figure 4: A simple representation of product stages and processes during iPSC production. (Blue = product, green = process; [adapted from [26]]).

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Episomes

Adenovirus

Keratinocytes

Alkaline phosphatase staining

Sendai Virus

Dermal Fibroblasts

Minicircle

FACS

Immunohistochemistry Embryoid body development

Confirmed iPSCs

Protein

Live staining

Candidate iPSCs

Adipose derived stem cells

Modified mRNA

Multiple cell colonies

Blood cells

Somatic cell sample

Tissue sample

Morphology

Teratoma formation

Lentivirus (inducible)

VEE RNA

Figure 5: Summary of starting cellular materials that can be used for iPSC generation (green), reprogramming methods (red), colony selection (blue) and pluripotency confirmation (purple). FACS = fluorescence-activated cell sorting, VEE RNA = Venezuelan equine encephalitis virus RNA.

Starting Cell Material An important consideration in generating iPSCs is the starting cellular material. The choice of cell type from which to derive iPSCs is informed by ease of accessibility, proliferative capacity, and tractability for reprogramming. Numerous cell types have been considered as starting material for generating iPSCs, including fibroblasts[27], adipose-derived stem cells [28], cord blood mononuclear cells [29], T cells [30] and erythroblasts [31]. At present, blood is considered a particularly attractive source because isolation is straightforward and less invasive than tissue biopsies. Multipotent cells, such as adipose-derived stem cells, usually retain functional plasticity and may therefore be more amenable to reprogramming than certain blood cells. Establishing consensus on the optimal cell source will be important in minimising variability and increasing comparability. Given that culture times needed to expand and isolate reprogrammed cells can extend to several weeks, even low initial heterogeneity can result in large

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variability in cellular output if not appropriately managed. Biopsy technique, storage, transportation and culture conditions all contribute to variability in the final product [26]. Enriching cell subpopulations, for example by antibody-based separation [32], could reduce heterogeneity, however, identification of markers that allow this is challenging and the separation itself could be costly. Ethical considerations in sourcing starting cell materials are also important. Donors will be required to give informed consent to meet regulatory requirements. However, if cells are isolated originally for reasons other than generating iPSCs, iPSC generation may not be acceptable as the final use of the cells may be different to that of the original consent. Initiatives such as The DISCUSS Project are attempting to address such issues and have issued guidance on how best to manage ethical issues appropriately [33]. Reprogramming Method Once cell material is sourced, a reprogramming method must be chosen to generate iPSCs [26]. A critical challenge is to utilize methods that have high reprogramming efficiencies and minimize risks of instability and, therefore, ease regulatory concerns. In particular, the genomic ‘footprint’ that a method leaves, its efficacy, and the cost and speed of using the method need to be considered. Most reprogramming methods fall into three broad classes: integrating viruses, non-integrating viruses and non-viral methods. Integrating viruses, including retrovirus and lentivirus, are used as vectors for genetic constructs encoding reprogramming factors that are inserted into host genomes, therefore, ensuring a prolonged expression in transduced target cells. This technique is probably the most established, having been used in the seminal works of pluripotency induction in mice and human cells [3, 27]. Lentiviral vectors, can reach relatively high reprogramming efficiencies (0.1 - 1.5% [34]), could be excisable and are, therefore, widely used in research settings. However, viral integration into the host genome carries a risk of insertional mutagenesis, tumour formation via transgene reactivation, and leaves a genomic footprint which may be acceptable for most therapeutic applications [35, 36]. Non-integrating viral delivery of reprogramming factors, for example by adenovirus or Sendai virus, obviates this problem because genetic material is not incorporated into the host genome. Adenovirus, though non-integrating, has very low reprogramming efficiency (~0.0002% in human cells [37]) and is thus limited in utility until further optimized. Sendai virus is a more promising candidate for

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translational iPSC products: it is an RNA virus that is effectively diluted from cells over time. Further, high reprogramming efficiencies of 1% have been reported for fibroblasts and 0.1% for blood cells, and high levels of protein expression can be achieved [36]. Non-viral reprogramming methods are also footprint free, and have emerged more recently as a promising alternative to viral delivery. In particular, synthetic mRNA transfection of reprogramming factors achieves reprogramming efficiencies of 1.4% and, when cultured with an additional factor (LIN28) in lowoxygen environment, up to 4.4% [38]. Though promising, this approach suffers from being highly labor intensive, requiring approximately 12 consecutive days of mRNA transfection, and from lack of validation in human cell types other than fibroblasts. Episomal plasmids are another means to offer transient expression of reprogramming factors. Other non-viral approaches, for example miRNA, minicircle vectors and transposons such as PiggyBac, exist but all suffer various shortcomings or have not yet been extensively tested in human cells [36]. At present, the most effective and broadly used method to generate iPSCs for research use is probably Sendai virus. However, this is not available as a clinical grade reagent. The episomal vector method offers a current good manufacturing practice compliant process, and other techniques like mRNA are promising as high reprogramming efficiencies can be achieved. Ultimately, the method used may vary depending on the original cell source. Safety is a paramount concern, and achieving high reprogramming efficiency with safe, footprint-free methods across several cell types will be important. iPSC Selection Following generation of iPSCs, a heterogeneous population of fully reprogrammed, partially reprogrammed and partially differentiated cells results [39]. Developing high-throughput, scalable methods that select only fully reprogrammed iPSCs is a significant challenge. Manual, morphology based selection is currently the most widely used method, however, it is labor intensive and can be influenced by bias, which is difficult to control and disadvantageous from a regulatory perspective. Use of reporters for positive selection of iPSCs (e.g. green fluorescent protein [40]) may reduce bias but introduces greater process complexity and may have downstream effects on the iPSC product. Immunoselection is the most accurate method of iPSC identification. Cell surface antigens which are associated with pluripotency allow fluorescence or magnetic-

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activated cell sorting and enable multiparametric selection and high purity output [41]. Meeting cGMP guidelines with this technique is challenging, as removal of antibodies from iPSCs is required and all equipment and materials used need to be cGMP compliant, however, it has been achieved with the CliniMACS® System (Miltenyi Biotec). Validation and Characterization Validating the quality of the iPSC product is another central challenge to development of iPSC therapeutics; specifically, identity, pluripotency, purity and safety require validation [42]. Establishing standardized validation protocols will allow development of acceptable iPSC standards and eventually allow development of, and adherence to, QA specifications. Validation assays for purity and identity include cell selection based on morphology or cell surface markers; however, as previously discussed, morphology based selection is bias-prone and use of antibodies introduces complications. Staining of intracellular markers for microscopy or flow cytometry is another possibility but results in cell death so can only be used on a sub-sample of the product. Combinatorial approaches may be appropriate but, importantly, these assays report population level characteristics and do not necessarily allow detection of cells that are not genuine iPSCs, which may be problematic for meeting QA expectations. Confirmation of pluripotency is determined by the ability of iPSCs to differentiate into cells from each of the three germ layers. Teratoma formation in a rodent is currently regarded as the best validation method; however, it is labor-intensive and time consuming, taking 2-3 months to complete. Creating equally robust in vitro assays will be an important step in developing iPSC therapies. Some in vitro methods, such as the embryoid body (EB) assay, have been developed [43]. In this assay, culture conditions promote differentiation, and the resulting cells can be tested for presence of cells from all three germ layers. Reference maps of ESC and iPSC variation in DNA methylation and gene expression have also been developed to allow more rapid characterization of PSC lines. Scorecards, such as the TaqMan® hPSC Scorecard™ (Life Technologies) have been developed based on these reference maps that provide a highly quantitative approach to estimating differentiation potential and bias. As the field progresses, it is likely that similar quantitative methods will emerge for other important iPSC parameters and may help to reduce high costs presently associated with QA.

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Testing differentiation towards a specific fate may be acceptable for some applications, though demonstration of full pluripotency is likely to be required for iPSC banks as uses of the cell line may not be known at the time of banking. Finally, genomic stability is another critical attribute that will be required for therapeutic iPSCs (Box 1). Tests to ensure that iPSCs are free of genetic abnormalities and footprint free will be required, which may need to be as detailed as genome-wide sequencing in clinical-grade products. Box 1. Genetic abnormalities observed in iPSCs [44, 45] 

Changes in chromosomal number or structure



Copy number variations, for example subkaryotypic or subchromosomal amplifications and deletions



Loss of heterozygosity



Random or site specific integration of alien DNA into the host genome

Due to the complexity and multitude of required assays, iPSC validation represents a large barrier to iPSC therapeutic translation. Multiparametric approaches to characterization and validation may provide a rapid and robust methodology, and are in fact recommended by the FDA for stem cell-based products [46]. Conducting a panel of tests in parallel, such as morphological evaluation, detection of specific cell surface antigens, assessment of unique biochemical markers and genomic and/or proteomic analysis, may streamline the process and alleviate some of the problems discussed. Final iPSC Therapeutic Product A subsequent challenge is the generation of a usable therapeutic through expansion of the iPSCs and directed differentiation to the desired cell type. The difficulties here are twofold: first, producing cell numbers great enough for patient use is problematic, particularly at a feasible cost; second, ensuring that the product is functionally mature and no longer pluripotent upon transplantation is essential from the perspective of safety and efficacy. The issue of scale-up for patient treatment with iPSC-derived products is significant. Depending on the desired cell type, the exact differentiation protocol will vary; however, the need for efficient and cost-effective protocols is ubiquitous. Many groups are attempting to address these needs, such as for cardiomyocytes [15, 47] and β-cells [10], and cost-effective procedures are emerging. However, as most therapeutic

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applications are confined to animal models and human dosage is not yet fully known, the true scalability of these methods in the context of cell numbers for human patients remains to be seen. In applications of autologous iPSC-derived products, cost-effectiveness will be particularly challenging as economies of scale are not applicable. Differentiation protocols have been developed for a variety of cell types, however, the differentiated product is not always terminally differentiated; it can be functionally immature compared to adult cells. Generally, iPSC-derived cells are described as ‘foetal-like’ and yield lower levels of function than their adult cell counterparts [48]. For example, HSC-derived cardiomyocytes can spontaneously contract and exhibit ion channel expression, electrophysiological signals, gene expression patterns and physical phenotypes of foetal cardiomyocytes. As such, there is a need to rescue iPSC-derived cells through maturation pre- or postengraftment. For cardiomyocytes, 3D culture and passaging for up to one year can enhance maturation but are not fully effective and incur significant expense. Finally, there is some concern over the tumorigenic potential of iPSC-derived therapeutics. However, in a mouse model of AMD, iPSC-derived RPE cells were shown to have “negligible” risk of tumour formation: for example, no tumours resulted from iPSC-derived RPE sheets transplanted to the sub retinal space from 6 -12 months in 26 rats [49]. Ensuring and demonstrating that this is the case across iPSC-derived products will be essential from a regulatory perspective. Immunological Compatibility and iPSC Banks As mentioned earlier, establishing a bank of iPSC lines from which to select cells on a patient-by-patient basis is largely regarded as the most realistic route forward for iPSC therapeutics at present. This could allow iPSCs to be used that are a close immunological match to the patient and, therefore, reduce the chance of rejection and the need for immunosuppression. Further, they may be used to produce off-the-shelf therapeutic cells that could be rapidly delivered to patients. Many large scale iPSC banks are currently under development, for example stemBANCC, European Bank for Induced Pluripotent Stem Cells (EBISC), New York Stem Cell Foundation and the California Institute for Regenerative Medicine [50]. However, the majority of these are being developed for research purposes, not with the aim of developing therapeutics. Cellular Dynamics International are among the first to attempt to build a therapeutic-grade iPSC bank and it is currently in its early stages [51]. Donor and recipient matching at three

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loci, HLA-A, HLA-B and HLA-DR, is known to be responsible for the majority of benefits of tissue typing in whole-organ transplantation; matching beyond that is of limited utility due to rapidly decreasing probabilities of matching at further MHC loci [9]. It has been calculated that 150 homozygous HLA types from a database of 17 million volunteers would provide a sufficient match at these three loci for 93.16% of the UK population [52], and is therefore an achievable and reasonable aim. Even though beneficial, this level of MHC matching may not be sufficient to obviate the need for immune suppression upon cell transplantation [9]. In wholeorgan transplantation, recovery of end-stage organ failure is typically the aim, and the benefits of immunosuppression therefore outweigh the risks. However, many diseases that may be appropriate for iPSC therapeutics, such as AMD, reduce quality of life without necessarily having high, immediate risks of mortality. In such circumstances, the benefits of cell transplantation may be countered by the risks of immunosuppression. For pluripotent cells therapies, risks are particularly high as the cells are inherently tumorigenic and the immune system is responsible for eliminating malignant cells. Different forms of immune control, for example induction of antigen specific immunological tolerance, are therefore being developed [9]. Achieving low risk immune intervention is a critical challenge for iPSC therapeutics, and may explain the lack of attempts to develop iPSC banks for therapeutic purposes. Intellectual Property A further consideration in bringing iPSC therapeutics to the clinic, and, therefore, market, is the IP landscape. IP incentivizes innovation but when mismanaged can obstruct overall progress in a field and hinder clinical translation. Application of iPSCs as therapeutics requires the use of a complex chain of interrelated technologies, thus, IP is particularly important and challenging. Indeed, there has been much concern that the emerging and growing iPSC patent ecosystem is becoming prohibitive as patents for interrelated and possibly interdependent technologies are not owned by any one entity [53]. A recent analysis identified the largest group of iPSC patents to be differentiation technologies (1300-1400 patents), followed by those for iPSC production (650 patents), which include culture techniques, growth factors and other technologies needed for generation [53] (Fig. 6). Of concern is the thin patent distribution, with many entities holding few patents; such a pattern is expected of emerging technologies, though can lead to formation of a ‘patent thicket’ in which there is

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congestion of patents over interrelated enabling technologies. Though lack of ownership of the majority of patents by one entity may fuel competition and increase rates of innovation, it can also lead to difficulties in licensing. The formation of patent thickets is a concern for the industry, and preventing deceleration of progress may require innovative licensing strategies [53]. Transparency in licensing practices will greatly aid technological progress in the field, in much the same way that sharing of scientific resources already does. Various models for such an approach exist, such as ‘clearinghouses’ [54] and the ‘Easy access IP’ initiative [55], which do not require unrealistic selflessness on the patent holder’s behalf.

Figure 5: the iPSC patent landscape 'Higher' areas are representative of high patent activity, with each dot representing a patent family [53].

CONCLUDING REMARKS There is great therapeutic potential for iPSCs, and the journey to the clinic has already begun, with a clinical study underway for age related macular degeneration. However, a number of significant barriers stand in the way of widespread clinical translation at the moment, and have been highlighted here. Establishing standardized, scalable methods for reprogramming, selection, validation and characterisation are critical to ensure consistent quality and quantity of iPSCs for therapy. Immunological issues need to be taken into

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account; developing diverse iPSC banks will be useful but will not completely eliminate the need for immune intervention. Finally, as with all technology, IP needs to be considered. At present, there is a risk of the iPSC IP distribution hindering advances in the field. Addressing all of these issues may allow the great potential of iPSC therapeutics to be realised and applied. DISCLOSURE AND CONFLICTS OF INTEREST The content outlined herein represents the individual opinions of the authors and may not necessarily represent the viewpoints of their employers. D.A.B. gratefully acknowledges support from the SENS Research Foundation (Mountain View, CA). D.A.B. is a stockholder in Translation Ventures Ltd. (Charlbury, Oxfordshire, UK) and IP Asset Ventures Ltd. (Oxford, UK) companies that amongst other services provide bio manufacturing, regulatory, and financial advice to clients in the cell therapy sector. J.A.S is a consultant of IP Asset Ventures Ltd. D.A.B. is subject to the CFA Institute’s Codes, Standards, and Guidelines, and as such, this author must stress that this piece is provided for academic interest only and must not be construed in any way as an investment recommendation. Additionally, at time of publication, D.A.B. and the organizations with which he is affiliated may or may not have agreed and/or pending funding commitments from the organizations named herein. ACKNOWLEDGEMENTS We wish to express our sincere thanks to the following organizations that have contributed to the CASMI Translational Stem Cell Consortium (CTSCC) as funding and events partners — without whom the consortium and the benefits it will bring to stem cell translation would be constrained: GE Healthcare, Center for Commercialization of Regenerative Medicine (CCRM), Sartorius Stedim Biotech (formerly TAP Biosystems), Lonza, California Institute for Regenerative Medicine (CIRM), SENS Research Foundation, UK Cell Therapy Catapult, NIH Centre for Regenerative Medicine, New York Stem Cell Foundation (NYSCF), ThermoFisher Scientific, Eisai, Medipost (US), Medipost (Korea), Celgene, Roche and Oxford Biomedica. P.J.F. is supported by the Regenerative Medicine Initiative of the Britain-Israel Research and Academic Exchange Partnership (BIRAX). P.R. is supported by the British Heart Foundation. J.A.S gratefully acknowledges support from the CTSCC. The authors wish to thank Marli Silva, Richard Barker, Hae-Won Kim, Pete J. Coffey, Justin J. Cooper-White, Mahendra Rao, Evan Y. Snyder, Kelvin S. Ng and Benjamin E. Mead for their work on a previous draft of a publication that informed some of the present work.

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McKernan R, Watt FM. What is the point of large-scale collections of human induced pluripotent stem cells?. Nat Biotechnol 2013; 31(10): 875-7. Banking on iPSCs | The Scientist Magazine®. http://www.the-scientist.com/?articles.view/ articleNo/40376/ title/ Banking-on-iPSCs/, [accessed January 30, 2015]; n.d. Taylor CJ, Peacock S, Chaudhry AN, Bradley JA, Bolton EM. Generating an iPSC bank for HLAmatched tissue transplantation based on known donor and recipient HLA types. Cell Stem Cell 2012; 11(2): 147-52. Roberts M, Wall IB, Bingham I, et al. The global intellectual property landscape of induced pluripotent stem cell technologies. Nat Biotechnol 2014; 32(8): 742-8. Bergman K, Graff GD. The global stem cell patent landscape: implications for efficient technology transfer and commercial development. Nat Biotechnol 2007; 25(4): 419-24. Moran N. No-fee university licenses spur biotech partnerships. Nat Biotechnol 2013; 31(5): 376-6.

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CHAPTER 7

Cell Cycle and Cell Cycle Regulators in the Process of Development, Pluripotency, Differentiation, and Reprogramming Xiao Qi Wang1* and Eric J. Stanbridge2 1

Department of Surgery, The University of Hong Kong, Hong Kong and 2Department of Microbiology and Molecular Genetics, University of California, Irvine, School of Medicine, California, USA Abstract: The core cell cycle machinery and its associated signaling pathways play critical roles in regulation of stem cell properties. Cell cycle length and rate is a determining factor for stem cells to maintain pluripotency or undergo differentiation. Manipulating the cell cycle has functional consequences for tissue differentiation as well as somatic cell reprogramming. In addition, accumulating evidence indicates that cell cycle regulators may control pluripotency and differentiation by direct interaction with the pluripotency network and other essential signal pathways without altering the cell cycle per se. In this chapter, we summarize the current understanding of the role of the cell cycle and cell cycle regulators in the process of development, pluripotency, differentiation, and reprogramming.

Keywords: Asymmetric cell division, cell cycle, cell cycle regulators, cell fate decision, cyclin dependent kinases (CDKs), cyclins, differentiation, embryonic development, epigenetic regulation, p53, pluripotent stem cells (PSCs), Rb, selfrenewal, somatic reprogramming. G1 RULES IN CELL FATE DECISION Pluripotent stem cells (PSCs) are defined by their capacity to undergo indefinite self-renewal and to differentiate into all somatic cell lineages and germ cells [1], which includes embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Based on their pluripotent states, stem cells can be divided into ground state pluripotent stem cells (2i/LIF dependent) such as mouse ESCs (mESCs) and primed pluripotent stem cells including epiblast stem cells (EpiSCs) and human ESCs (hESCs) [2]. Pluripotent stem cells have unique cell cycle characteristics; for example, cycling cells have a short G1 phase and a large percentage of *Corresponding author Xiao Qi Wang: Department of Surgery, The University of Hong Kong, 21 Sassoon Rd, Pokfulam, Hong Kong; Tel: 852-39179653; Fax: 852-39179634; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds.) All rights reserved-© 2016 Bentham Science Publishers

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S phase cells, whereas upon induction of differentiation the G1 phase lengthens and the fraction of cells in S phase is reduced [3]. Numerous studies have identified the critical link between G1 length and differentiation, which can be manipulated by activities of CDK2, cyclin Ds and E, Rb-E2F, and environmental signals [1, 4-7]. The evidence that differentiation initiates in the G1 phase is based on the finding that G1 cells are prone to differentiation signaling cues, such as retinoic acid, while S phase cells are refractory to this retinoic acid signal [8]. More importantly, the transcripts levels of developmental genes have been found to be cell cycle regulated, peaking in G1 and declining in S phase, respectively [9]. Thus, G1 is the cell fate decisionmaking point; when pluripotent cells enter into G1, they become “poised” and permissive to either continued self-renewal or commitment to lineage specification [9]. G1 “rules” also represent a mechanism whereby a simple manipulation of the cell cycle, using small molecules, might direct pluripotent stem cells towards differentiation without the need of exogenous growth factors [5] (Fig. 1).

Figure 1: hESCs sense differentiation signals in G1 and complete differentiation in G2/M. Cell cycle specific cell cycle regulators and factors that control the process of pluripotency or differentiation are indicated.

S AND G2 PHASES FOR PLURIPOTENT STATE MAINTENANCE The G1 phase has been the focus for stem cell fate regulation in view of the assumption that G1 is the phase of the cell cycle that stem cells respond to differentiation signals. The importance of S/G2 in stem cell control is much less explored until a recent report by Gonzales et al. [10] who, in the setting of induction of hESC differentiation, used RNA-interference or small-molecule inhibitors to block regulators of each cell phase. Their results showed that suppression of regulators of the S and G2 phases, such as cyclin B1 led to a rapid decrease in expression of pluripotency markers, whereas suppression of the G1 phase regulators (CDK4/6 or cyclin D) had little effect on pluripotency markers. In addition to the cyclin B1

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pathway, the activation of the ATM/ATR-CHEK2-p53 pathway by “incomplete differentiation” in S phase could reinforce the pluripotency network to avoid the generation of “abnormal” cells [10, 11]. Importantly, the S and G2 phases are intrinsically associated with maintaining pluripotency. At de novo transcription levels of the pluripotency factors, NANOG and PRDM14 are preferentially transcribed during the S and G2/M phases compared to the G1 phase [10]. Moreover, inheritance of DNA methylation and histone marks occurs during the S and G2 phases, indicating that epigenetic regulation of a shift from pluripotency to a differentiated phenotype is an important parameter during these phases of the cell cycle [11]. These recent findings have led to propose a novel mechanistic model where cell fate specification starts in the G1 phase where hESCs can sense differentiation signals; then cell fate commitment is completed in G2/M, where pluripotency is dissolved and differentiation completed via cell cycle-dependent mechanisms [10, 11] (Fig. 1). ASYMMETRIC CELL DIFFERENTIATION

DIVISION

FOR

PLURIPOTENCY

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Many adult stem cells undergo asymmetric division in their natural environment, including neural, muscle, and hematopoietic stem/progenitor cells. This asymmetric cell division is essential for generating diverse cell types [12, 13]. Asymmetric division of stem cells generates two daughter cells, each with a distinctive cell fate: one retains its stem cell identity while the other one loses stem cell properties and becomes specialized [14]. Asymmetric division can be achieved by asymmetric segregation of intrinsic fate determinants, so that two daughter cells unequally inherit components such as protein, RNA, and organelles. Alternatively, asymmetric placement ensures only one daughter cell maintains contact with the stem cell niche, that is necessary for retaining self-renewal capability [12, 13]. Niche mechanisms are common in adult stem cells, whereas intrinsically asymmetric divisions predominate during development [13]. For example, when human mammary stemlike cells divide the more aged subpopulation of mitochondria are separated asymmetrically to the daughter cells. The daughter cells that receive the “younger” mitochondria maintain their stem-like properties [15]. Furthermore, asymmetric inheritance of cyclin D2 in early G1 is a critical event during mouse brain development that is functionally associated with the balance between the neural stem/progenitor cell pool and differentiated neurons [16]. CELL CYCLE AND EPIGENETIC REGULATION Promoters of regulatory genes in PSCs are frequently marked by overlapping activating (H3K4me3) and repressive (H3K27me3) histone marks [17]. These

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bivalent domains are considered to balance expression of developmental genes, allowing timely activation while sustaining repression in the absence of differentiation signals [18]. The chromatin is further modified by the Trithorax group (trxG) activators and the Polycomb group (PcG) silencer proteins [18,19]. In PSCs, H3K4me3 is established via the activity of trxG complexes containing MLL or SET enzymes [20, 21]. Bivalently marked developmental genes are transcribed during the late G1 phase in hPSCs [22], via the mechanism of CDK2dependent phosphorylation of the MLL2 histone methyl-transferase, to establish the H3K4me3-marked bivalent domain in G1 [17]. Taken together, bivalent domains are established to control the cell-cycle dependent activation of developmental genes so that differentiation initiates from the G1 phase. CELL CYCLE REGULATORS IN SOMATIC REPROGRAMMING Pluripotency can be induced with a combination of four transcription factors, namely Oct4, Sox2, Klf4, and c-Myc (OSKM) [23-25]. Using this technology, human iPSCs have been generated from various types of somatic cells including fibroblasts, hepatocytes, gastric epithelial cells, blood cells, and neural cells. While iPSCs can be reproducibly generated, the process suffers from low efficiency with only a small percentage of somatic cells that receive the reprogramming factors becoming iPSCs [26]. More recently, studies have been carried out to identify the factors and critical pathways that significantly enhance direct reprogramming. Among these, several cell cycle regulators have been shown to be functionally involved. p53 pathway activation, after ectopic expression of iPS factors, acts as a major reprogramming barrier [27]; therefore, in addition to the four factors, the addition of p53 shRNA, in order to decrease p53 expression, increases iPSC formation [28]. Furthermore, overexpression of cyclin D1 enhances reprogramming efficiency more than 3 fold [29, 30]. Expression of cyclin D2, cyclin E2 and, particularly, co-expression of CDK4 and cyclin D1 increase reprogramming efficiency via promoting cells to more rapidly enter into S phase [30]. Conversely, the proteins that induce cell cycle arrest in somatic cells such as p15, p16, and p53/p21 restrict reprogramming [30, 28]. Thus, overcoming cell cycle arrest (G1 arrest) seems to be a necessary event for successful reprogramming. However, a further study has demonstrated that many initially formed iPSC colonies turn from initially TRA-1-60 (+), one of the best markers for human pluripotent stem cells [31], back into TRA-1-60 (−) iPSC colonies during the subsequent culture. [26]. Thus, most of the TRA-1-6 (+) cells fail to complete the reprogramming process because of reversion, cell death, and other mechanisms. Cyclin D1 and p53 shRNA increase the efficiency of induction

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of iPSC colonies, mainly through promoting cellular proliferation or suppressing cell death, but have no role in promoting iPSC maturation towards a completed reprogramming [26]. Inactivation of Rb significantly enhances reprogramming efficiency [32, 30]. The effect of Rb on reprogramming is independent of cell cycle regulation [32]. Loss of Rb relieves its suppression of pluripotency genes by affecting chromatin structure to increase the active histone marks, H3Ac and H3K4me3 [32]. Thus, Rb plays a critical role in chromatin-mediated regulation of pluripotency genes such as Oct4, Sox2, and Nanog and demonstrates a direct link between cell cycle regulators and pluripotency genes [32]. Interestingly, Aurora A, a kinase for centrosome activity and spindle assembly [33], displays a contradictory role on somatic reprogramming. Overexpression of Aurora shows its positive role in improving reprograming efficiency via Aurora-mediated inhibition of p53 activity [34]. Conversely, another study shows that Aurora A is one of the reprogramming barrier kinases because Aurora A maintains GSK3β in an active state, whereas inhibition of GSK3β enhances reprogramming efficiency. Thus, inhibition of Aurora A kinase activity by small molecule kinase inhibitors significantly improves iPSC generation [35]. Moreover, reduction of cyclin A1 protein levels not only improves the pluripotency states. Interestingly, this condition also reduces the tumorigenic potential of iPSCs, possibly by cyclin A1 downregulating differentiation genes and up-regulating tumor suppressor genes indirectly or directly [36]. All these studies demonstrate the importance of cell cycle regulators on the somatic reprogramming process via cell cycle regulationdependent or -independent mechanisms. CYCLINS AND CYCLIN DEPENDENT KINASES (CDKs) In the sections below we will discuss the functional roles of each cyclin and CDK during embryonic development and biogenesis of tissue stem cells. CDK1 CDK1 is the most pleiotropic cell cycle regulator; it not only interacts with cyclin B to drive the G2-M transition, but also binds to all other interphase cyclins (cyclin D1, E, and A) to regulate G1 progression and G1-S transition [37]. In the absence of Cdk1, embryos fail to develop to the blastocyst stage, whereas in knock-out mice lacking all other Cdks (Cdk2, Cdk3, Cdk4, and Cdk6) Cdk1 alone is sufficient to drive mammalian cell cycle progression and embryonic development [38]. Cdk1 is also essential for meiosis in mouse oocytes [39] and

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suppression of Cdk1 leads to the differentiation of mouse trophoblast stem cells into giant cells [40]. However, liver-specific deletion of Cdk1 is well tolerated and does not impair liver regeneration [41]. Beyond its role as a key cell cycle regulator, studies have demonstrated that CDK1 is required for self-renewal in both mESCs and hESCs (hESCs) [42, 43], which might be related to its interaction with Oct4 [44]. Cdk1 has been predicted to be involved in a protein interaction network with Oct4 [45] and CDK1/CDK2 potentially regulate a large number of substrates (1220), including protein kinases, during induced hESC differentiation [46]. Recently, an in vitro study identified multiple phosphorylation sites on human Nanog, with some sites being specifically phosphorylated by Erk2 and CDK1 kinases [47]. All of these studies point to a strong association of CDK1 with pluripotency, although the mechanistic role of how CDK1 regulates pluripotency is not well defined. Our recent study demonstrated that CDK1 specifically targets phosphorylation of PDK1 and, consequently, activity of PI3K/Akt and its effectors Erk and Gsk3β. Inactivation of CDK1‐mediated differentiation can be blocked by inhibition of Akt signaling effectors suggesting that the CDK1‐PDK1‐PI3K/Akt kinase cascade is a functional signaling pathway for pluripotency of hESCs. CDK2 CDK2 binds and activates cyclins E, D1, and A, respectively, to regulate G1 and S phases of the cell cycle. Cdk2 is not an essential gene as Cdk2-null mice are viable. But Cdk2 is indispensable for germ cell development and meiosis [48, 49]. Cdk2 is required for maintenance of the undifferentiated progenitor pool in the subventricular zone (SVZ) of the adult brain [50]. Loss of Cdk2 promotes differentiation and remyelination of the oligodendrocyte progenitor cell (OPC) population in the adult central nervous system [51]. Cdk2 and Cdk4 double knockout mice display an ablation of the intermediate zone and cortical plate in the embryonic brain [52]. Together, Cdk2, as a cell-intrinsic determinant, is an important regulator for neurogenesis. Important questions to be asked are whether and how Cdk2 is associated with the process of pluripotency and differentiation of ESCs? CDK1/CDK2 potentially regulate a large number of substrates during induced hESC differentiation [46]. Cdk2-mediated Sox2 phosphorylation regulates pluripotency establishment during somatic reprogramming but not ESC maintenance [53]. It has also been demonstrated that, during differentiation of pluripotent cells (mESCs), Cdk2 activity switches from constitutive to cell cycle-dependent, which coincides with

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changes in cyclin A1 and E1 protein levels and activation of Rb-E2F pathways [4]. Similarly, down-regulation of CDK2 activity prolongs G1 phase progression both in mESCs and hESCs, resulting in differentiation [54, 55]. However, manipulating PI3K/Akt activity and the Smad2/3-regulated switch also induces hESC differentiation, whereas CDK2 levels remain unchanged [56]. Thus, differentiation of pluripotent stem cells does not necessarily result from G1 lengthening. CDK4/6 G1 cell cycle progression is mediated by activation of the CDK4/6-cyclin D complex and phosphorylation of Rb, and subsequent E2F-dependent transcription [57]. CDK4 and CDK6 display 71% of amino acid homology, therefore indicating that they may have redundant functions. Only the deletion of both genes leads to embryonic lethality at E14.5-E18.5, as a result of multi-lineage hematopoietic failure [58-60]. CDK6 is a key regulator for activation of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs)[61]; HSCs and LSCs share functional features and gene expression profiles, and have unlimited self-renewal capacity [62]. Cdk6−/− HSCs fail to repopulate upon competitive transplantation and Cdk6−/− LSCs fail to produce leukemias [61] Elevated levels of the cyclin D1CDK4 complex are associated with increased self-renewal of HSCs [63]. Furthermore, the Cdk4-E2f1 pathway regulates early pancreas development by increasing the pool of endocrine precursors [64] and promoting replication and activation of beta-cell progenitors in the ductal epithelium [65]. The expression level of CDK6 also controls differentiation of several types of stem cells, such as osteoblasts, cortical and neural progenitors [66]. The regulatory effects of CDK4 on stem cells are largely dependent on its kinase activity and, therefore, most probably related to its regulation of the cell cycle. Increased expression of the cyclin D1-CDK4 complex promotes the transit from G0 to G1 (for HSCs) and prevents G1 phase lengthening, resulting in: (a) protection from differentiation-inducing signals for HSCs [63]; and (b) expansion of the pool of neural stem cells and delayed neurogenesis [67]. It is, however, possible that the regulatory effects of CDK6 on stem cells might be kinaseindependent, with it acting as a direct regulator of transcription. For example, CDK6 transcriptionally suppresses Egr1 for HSCs activation (Egr1 is expressed at high levels in quiescent HSCs and its downregulation is required for initiating cell proliferation) [61]. CDK6 also has a key role in hematopoietic tumors. One example is that CDK6 transcriptionally induces Vegf-A expression, a proangiogenic factor for lymphoma formation, [68]. Thus, understanding the

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biological roles of CDK4 and CDK6 in stem cell regulation may provide a rational scientific basis for targeting CDK4/6 in the treatment of certain leukemias and solid malignancies. CYCLIN D MEMBERS Cyclin D members (cyclin Ds) show tissue-specific expression during mouse embryo gastrulation. Cyclin D1/D2 is expressed in mesoderm and neuroectoderm, cyclin D2 in endoderm, and cyclin D3 in trophectoderm [69]. Deficiency of all three D type cyclins, but not each cyclin D alone, leads to lethality at embryonic day 16 [70]. Most organogenesis and tissue development seems unaffected but hematopoietic development is significantly impacted, which is likely the cause of embryonic death [60, 70]. Differential expression of cyclin Ds also displays a cell type-specific function in human tissues. All cyclin Ds (D1, D2, and D3) are induced during hESC differentiation toward neuroectoderm, whereas cyclin D1 is significantly decreased in endoderm differentiation. Mesoderm differentiation shows upregulation of cyclin D2 [5]. Cyclin Ds controlling pluripotency/differentiation of hESCs largely depend on regulating the length of the G1 phase, which also follows tissue-specific expression patterns of cyclin Ds [5]. Low levels of cyclin Ds in hESCs lengthen the early G1 phase and activate Smad2/3 to induce hESC differentiation towards the endoderm lineage. Conversely, high levels of cyclin Ds in hESCs block Smad2/3 function in late G1 phase but initiate neuroectoderm differentiation [5, 71]. Cyclin D activity-manipulated G1 length also regulates tissue stem cell differentiation. Loss of cyclin D/CDK leads to an increased G1 phase, which is associated with neuronal stem cell differentiation into neurons [67, 72] and premature differentiation of hematopoietic stem cells [71]. Thus, manipulation of cyclin D-CDK4/6 activity by chemical inhibitors should have the potential to direct endoderm differentiation of hESCs or hiPSCs for regenerative medicine. Moreover, in regenerating tissues different cyclin D members are transcriptionally induced in response to critical growth signaling pathways such as Wnt, Hedgehog (Hh), Notch, and Hox, for cell division and proliferation [60]. Therefore, cyclin D proteins act as essential regulators of a variety of signaling pathways for stem cell division, differentiation and regeneration. CYCLIN A Mammalian cells express two A-type cyclins. There is the testis-specific cyclin A1 [73, 74], and somatic cells that ubiquitously express cyclin A2, which is

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considered to be an S phase cyclin [75]. Male knockout mice lacking cyclin A1 are sterile because cyclin A1 is required for the first meiotic division [76]. Cyclin A2 is an essential gene for cell proliferation. Homozygous loss of cyclin A2 leads to early embryonic lethality (E5.5 after implantation) [77]. In cyclin A (both cyclin A1 and A2) conditional knockout mice, fibroblasts proliferate normally, whereas hematopoietic stem cells (HSCs), as well as lineage-committed HSCs, disappear rapidly, resulting in death of the majority of mice [78]. Proliferation of ESCs derived from the blastocyst of cyclin A conditional knockout mice is also impaired [78]. More importantly, expression of cyclin A is particularly high in both ESCs and HSCs [78], indicating that these stem cells are dependent on cyclin A for self-renewal and proliferation during early development. Moreover, cyclin A2, normally silenced in the mammalian postnatal heart [79], can induce cardiac regeneration when constitutively expressed in myocardial infarction cellular and animal models, providing a potential therapeutic impact upon clinical treatment of damaged myocardium. [79, 80]. CYCLIN E MEMBERS Cyclin E1 and E2 constitute the cyclin E subfamily, which activate CDK2 to regulate the G1 to S transition of the cell cycle. In genetic knock out mice, deletion of cyclin E1 or cyclin E2 alone does not result in dramatic phenotypes, and their cell cycle dynamics are comparable to wild type cells [81, 82]. Double knockout embryos die at midgestation due to placental defects [82]. Interestingly, placental defects result from the trophoblast giant cells lacking endoreplicative capability, consequently failing to undergo multiple rounds of DNA synthesis [82]. The requirement for E-type cyclins can be abrogated in normal mitotic cycles but not in endoreplicating cells during embryonic development [82]. In addition, cyclin E1 plays a substantial role in organ-specific regeneration. Cyclin E1 is non-redundantly required for hematopoietic stem cell (HSC) cycling by mediating the rapid entry of quiescent HSCs into the cell cycle during hematopoiesis stress [83]. Cyclin E1-null HSCs display an increased proportion of quiescent HSCs, which, fortuitously, provides better protection from HSC exhaustion [83]. By generating hepatocyte-specific cyclin E1 gene deleted transgenic mice, a recent study demonstrated that cyclin E1 is essential for mouse liver regeneration. Combined inactivation of cyclin E1 and Cdk2 (Cdk2ΔhepaCcnE1−/−) but not cyclin E2 and Cdk2 (Cdk2ΔhepaCcnE2−/−) significantly impaired liver reconstitution after partial hepatectomy because of loss of hepatocyte S-phase progression [84].

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CYCLIN B1 Two types of mitotic cyclins, B1 and B2, have been identified in mammals. Cyclin B1 is an essential gene and its deletion leads to mouse embryonic lethality, whereas cyclin B2 null mice develop normally [85]. From “knock-out” studies, it has been shown that deficiency of the mitotic cyclin B1 or Cdk1 results in embryonic lethality, but deficiency of other interphase cyclins or Cdks show a compensatory function except cyclin A2 [38, 85, 77], as homozygous loss of cyclin A2 leads to embryonic lethality [77]. These observations suggest that the “core” components of the cell-cycle machinery, Cdk1, cyclins B and A, represent absolutely fundamental elements of the cell cycle engine for development [78]. Moreover, cyclin B is expressed in both germline and somatic lineage cells in Drosophila [86]. Although cyclin B mutation does not have an effect on somatic development of Drosophila, it is essential for germline stem cell self-renewing division, which cannot be functionally substituted by other B-type cyclins such as cyclin A and B3 (cyclin A, B, and B3 are three B-cyclins in Drosophila). It is possible; therefore, that cyclin B1 may play an important role in pluripotent determination. In summary, self-renewal and differentiation of stem cells are intrinsically associated with cell cycle regulators, through controlling cell cycle progression or regulatory functions, independent of cell cycle regulation. CONFLICT OF INTEREST The author confirms that author has no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2] [3] [4] [5]

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CHAPTER 8

Effect of Microenvironment Modulation on Stem Cell Therapy for Peripheral Nerve Injury Sufang Liu1 and Feng Tao2,* 1

Department of Physiology, Zhengzhou University School of Medicine, Zhengzhou, Henan Province, China; 2Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, Texas, USA Abstract: Previous studies have demonstrated that nerve damage results in rapid disruption of nerve function. Neural stem cells (NSCs) from spinal cord have been shown to promote peripheral nerve regeneration. However, the stem cell therapy still shows low efficiency in vivo and some miscommunications after nerve recovery. Our studies found that chemotactic factor stromal-cell derived factor 1α and hypoxia may improve the survival of the transplanted NSCs and their function. Here we discuss the effects of microenvironment on neural stem cell therapy for peripheral nerve injury and recent progresses in this research field.

Keywords: Hypoxia, microenvironment, Neural stem cells, neurotrophic factor, pain, Peripheral nerve injury, Stromal cell derived factor. 1. INTRODUCTION Peripheral nerve injury (PNI) is defined and categorized according to nerve damage and the surrounding environment alterations. PNI is accompanied by Wallerian degeneration at the start of nerve injury. And then the local damaged debris around the nerve transection will be cleared to promote the axon regeneration and nerve re-innervation. Even though peripheral axons after injury may regenerate and reconnect with the targets, it is hard to achieve therapeutic effects if the transection gap is too long more than 15mm [1]. Therefore, it is imperative to develop therapeutic strategies to minimize the extent of neurological disabilities and to promote recovery of function after PNI. Because functional loss of PNI results from the initial direct damage of the nerve demyelination and a secondary cascade to form the scar and then worsens the extent of damage and cell loss. So treatment of PNI should include preventing further damage and inflammation, stimulating growth factors and other molecules, and transplanting *Corresponding Author Feng Tao: Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, Texas, USA; Tel: 1-214-828-8272; Fax: 1-214-874-4538; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds.) All rights reserved-© 2016 Bentham Science Publishers

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neural stem cells or Schwann cells to promote remyelination and nerve recovery. Currently, peripheral nerve repair involves many complete factors including stem cell origins, extracellular vesicle growth, inflammatory factor infiltration, and nutrition support reconstruction. The main standard method for PNI therapy is to use suture even though many side effects are coming with the nerve repair progress, such as inflammation, scar formation, axonal misdirection and so on [2]. And this is limited by the nerve gaps between the distal and proximal nerve ends after PNI. Regarding this, conduits or acellular nerve allografts were used to repair PNI [3]. However, deficiency of extracellular matrix (ECM) and necessary nutrition makes this method for PNI therapy to be limited while helping axon extension. Functional re-innervation of injured neurons after neurological damage of PNI is an essential requirement of potential therapies. This can be achieved by regenerating the injured axons and by promoting the microenvironment reconstruction. Recently, stem cells are regarded as a novel method to repair damaged nerve and may have clinical implications. Stem cell-based therapy has been applied in animal models of PNI, and successful functional recovery has been reported. Given that neurological deficits after PNI are mainly attributable to a degeneration of axons and chronic progressive loss of the myelin that ensheaths the axons, the goals of stem cell transplantation should be guided to achieve reconnection of axons and promotion of remyelination of spared axons. 2. NEURAL STEM CELLS AND SCHWANN CELLS Stem cells are of multiple differentiation, self-renewal, and unlimited proliferation. This makes it possible to have the potential to repair damaged neurons or Schwann cells, thus promotes the axon growth to pass through the damaged area and reduces the scar formation. Fairbairn NG et al. summarized the different origins of stem cells for the axon repair after sciatic nerve or facial nerve transection [2].Generally speaking, stem cells used for PNI therapy are mainly focusing on the origins like embryonic stem cells (ESCs), spinal cord, the induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). Li et al. used different conduits seeded with aligned NSCs and found that a 15mm critical gap defect was bridged in a sciatic nerve injury animal model. Axon regeneration and nerve remylination were shown better in the conduits therapy with seed NSCs than the empty conduits, by detecting the axon connection and animal behavior [4]. In addition, NSCs from the spinal cord of P14 Sprague-Dawley (SD) rat embryos were separated and seeded into tibial nerve transection. And NSCs enhanced the nerve repair by expressing neurotrophin-3 (NTF3) gene [5]. Xu and colleagues seeded NSCs into a silicone tube that was used to bridge the two

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stumps of a transected sciatic nerve, and found that NSCs could increase the number of nerve fibers and improve the functional recovery of the injured sciatic nerve [1]. And they also found NSCs may remarkably increase the recovery of motor function during nerve repair period [1]. By using short-term MRI tracking, Cheng et al. found that the NSCs grafted to acutely distract peripheral nerves could enhance nerve regeneration and repair during recovery time [6]. Schwann cells (SCs) are the main glial cells and exert myelinating axons for peripheral nerve. Besides that, SC can provide some trophic support for the axon growth and regeneration after PNI. SCs are critically important for responding to PNI in the first step that is Wallerian degeneration. Then some macrophages infiltrate into the damaged area to clear the debris [7]. Some SCs may dedifferentiate into immature SCs and stimulate axon extension to pass through the stump area. Currently, SCs separated from peripheral nerve are hardly cultured in vitro. Most of researchers tried to get SCs from ESCs, iPSCs or even mesenchymal stem cells (MSCs) for the PNI therapy. 3. NEUROTROPIC FACTORS 3.1. Stromal Cell-Derived Factor During the development of the peripheral nervous system, one of chemokines that is closely related to neuron destinations is stromal cell derived factor-1 or SDF1 (CXCL12). This small protein is secreted and discovered as a kind of chemotactic cytokine to regulate cell trafficking and migration. Recently, it is demonstrated that it can also modulate the neurogenesis of peripheral nervous development and differentiation of neural stem cells. SDF1 is a very small molecular protein, 810kDa, with four conserved cysteine residues. According to the amino acid sequence around the first two cysteine residues, chemokines are divided into four subfamilies, CXC chemokines or α-chemokines, CC chemokines or βchemokines, CX3C or γ-chemokines, and the C or δ-chemokines [8, 9]. Receptors for this chemokine family are all of G protein coupled receptors (GPCRs). They are divided into four types, CXCR, CCR, CX3CR and CR, binding to different ligands of the four subfamilies described above. Interactions between ligands and receptors thus activate cellular signaling pathways like ERK, MAPK, PI3K, and AKT. In our previous study, SDF1 α has been shown to increase phosphorylation of ERK1/2 in the cultured hippocampal neurons [10]. During these receptors binding to chemokine SDF1 α, CXCR4 is considered recently the main receptor to modulate neuron function, even it is always thought as a vital factor in HIV development. It has been shown that SDF1/CXCR4 axis have an important role in

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the hippocampal dentate gyrus (DG) development. The most important is that DG is one of the critical areas for stem cell initiation and neurogenesis in the adult brain [11]. In our previous studies, we found that SDF1α was highly up-regulated in cultured hippocampal cells after hypoxia. Furthermore, 24h application of SDF1α increased neurite outgrowth and neural network damaged by hypoxia. Actin filament polymerization was also enhanced under the condition of SDF1α treatment wherever in hypoxia or normoxia microenvironment. Moreover, SDF1α exposure promoted the migration of progenitor cells, thus may have the potential to migrate into damaged area in the brain. Besides that, cell get a high sensitivity to respond to SDF1α at early stages after hypoxia. As far as the underlying mechanisms are considered, CXCR4 and CXCR7 are involved in this process significantly. Especially, we found that CXCR7, a new novel receptor of SDF1α, plays important role in neurogenesis and cell migration. Hypoxia enhanced CXCR7 expression and increased the numbers of CXCR7 positive neurons cultured in vitro. By constructing CXCR7 shRNA and transfecting it into hippocampal neurons, we failed to find any significant difference between untreated cells and CXCR7 shRNA transfected cells with acute stimulation of SDF1α. However, 24 hours later of SDF1α exposure, cell migration shows different rate between the two groups described above. This suggests that CXCR7 may be necessary, but not sufficient for cell migration, as pre-treatment of CXCR7 shRNA could not block the time-dependent promotion of SDF1α until 24 h later. Our observations showed that CXCR7 silencing caused neurite outgrowth deficiency and migration capacity decline significantly [10]. Taken together, SDF1α is a novel factor to promote neuron capacity thus has the potential to enhance neural progenitor cell capacity and neurogenesis. Interesting, Dubový et al. used an animal model of unilateral chronic constriction injury (CCI) in sciatic nerve to demonstrate that SDF1/CXCR4 protein alterations are not limited to DRG associated with injured nerve but that they also spread to DRG nonassociated with such nerve [12]. 3.2. Neuregulin-1 During the development of the peripheral nervous system (PNS), Schwann cells always play important role in forming myelin sheath around the axons. Recently Schwann cells have been shown to be regulated by axons that need different numbers of Schwann cells to make different myelin. This suggests that diameter of a critical axon triggers Schwann cell myelination reflecting either the size of the axon or the quantity of signaling molecules on its surface [13].

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Proteins encoded by the neuregulin-1 (NRG1) gene are candidate axonal signals for regulating Schwann cell differentiation [14]. And NRG1/ErbB signaling is necessary for normal myelination and sensory function. This signaling depends on ErbB4 interaction with PSD-95 in neurons. The NRG1 family comprises more than 15 membrane-associated and secreted proteins [15]. These are derived from one of the largest mammalian genes (on human chromosome 8p22 and mouse chromosome 8A3) and are generated by use of multiple transcription sites and by extensive alternative RNA splicing [16]. All NRG1 isoforms share an epidermal growth factor (EGF)-like signaling domain that is necessary and sufficient for activation of their receptors. NRG1 isoforms are subdivided into several subtypes on the basis of their distinct amino-termini [17]. NRG1 types I and II have Nterminal immunoglobulin-like domains. Transmembrane forms of NRG1 undergo proteolytic cleavage by metalloproteinases, including tumor-necrosis factor-αconverting enzyme. As a consequence, NRG1 types I and II are shed from the neuronal cell surface and function as paracrine signaling molecules. NRG1 type III is defined by its cysteine-rich domain, which functions as a second transmembrane domain. Consequently, NRG1 type III remains tethered to the cell surface after cleavage and functions as a juxtacrine signal [18]. In addition, exons encoding shorter amino termini of NRG1 have been identified by sequence analysis (referred to as types IV–VI), but these isoforms have not been further characterized. Within the nervous system, NRG1 types I and III are the most abundant forms and have been detected in many projection neurons, but also in glia. In addition to the axon–glia signaling, the proposed functions of NRG1 include the development of motor endplates, migration of interneurons, and synaptogenesis and synaptic plasticity in the CNS. NRG1 isoforms mediate their effects by binding to ErbB receptors, members of the EGF receptor superfamily. NRG1 binds to either ErbB3, which lacks an active kinase domain, or ErbB4, which has such a kinase domain; each receptor in turn can heterodimerize with ErbB2, which cannot bind NRG1 directly but has an active kinase domain. ErbB receptors dimerize not by virtue of a bridging effect of NRG1, but following a ligand-activated conformational change in the ectodomain of ErbB3 or ErbB4[19]. Whereas Schwann cells principally express ErbB2 and ErbB3, cells in the oligodendrocyte lineage express all three ErbB receptors in a developmentally regulated manner, indicating significant complexity of potential ErbB receptor heterodimers and downstream signaling events in these cells. Moreover, ErbB4 receptor co-localizes with N- methyl-Daspartate (NMDA) receptors at synaptic sites in the CNS by interacting with NMDA receptor scaffolding protein PSD-95, which is involved in the spinal

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mechanism of chronic neuropathic pain [20, 21]. The association of ErbB4 with PSD-95 is important for NRG1-induced neuronal differentiation. Myelinated axons are surrounded by a very large multilayered sheet of specialized plasma membrane that is elaborated by Schwann cells in the PNS and oligodendrocytes in the CNS. These glial cells repeatedly wrap and then tightly compact myelin around axons. Two studies that examined myelination in the PNS demonstrated that a specific isoform of the growth factor NRG1, expressed on the surface of axons, binds to ErbB receptor tyrosine kinases expressed by Schwann cells and serves as the inducing trigger for myelination [13, 22]. These studies reveal that the strength of NRG1/ErbB signaling determines whether Schwann cells elaborate myelin, and if so, how much myelin they produce. And Taveggia et al. found that the dual regulation of Schwann cell generation and differentiation by NRG1 type III coordinates Schwann cell numbers to their alternative phenotypes, which suggests that axons that express high level of NRG1 type III may stimulate more Schwann cells to produce myelination around nerve fibers [13]. In our previous study, we observed the effects of NRG1/ErbB signaling in mouse embryonic stem cell-derived oligodendrocyte progenitor cells (OPCs) to treat spinal cord injury (SCI). Demyelination (loss of myelin) and dysmyelination (abnormal myelination) are important contributors to behavioral deficits associated with SCI. Remyelination of the injured spinal cord is one of the key elements for functional recovery from SCI. Our data illustrate that SCI inhibits spinal NRG1/ErbB signaling, reduces myelination in the injured spinal cord, and induces chronic neuropathic pain. Our study also shows that spinal transplantation of mouse ES cell-derived OPCs counteracts the effect of SCI on NRG1expression, enhances remyelination in the injured spinal cord, and alleviates SCI pain. By knocking down NRG1 using an siRNA strategy, we further revealed that NRG1/ErbB signaling contributes to the effects of transplanted OPCs on remyelination and SCI pain. These findings not only provide new insight into the molecular mechanism by which stem cell transplantation can treat SCI-induced chronic neuropathic pain, but also provide information for the stem cell therapy to PNI [23]. 3.3. Nerve Growth Factor Previous studies have shown that some trophic molecules are released from the damaged area after PNI. This includes ciliary neurotrophic factor (CNTF), neurotrophin 3 (NT3), fibroblast growth factor (FGF) and nerve growth factor

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(NGF). They exert specific functions and responses during PIN development. Tong et al. found that axon regeneration and remylination in motor and sensory functional recovery after PNI is different in male and female rats with sciatic nerve crush, by morphometric analysis of electron microscopic images on the basis of nerve fiber classification [24]. A recent study [25] showed that artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs), and trkA, a neurotrophin receptor for NGF, may have different function on neurite initiation, elongation and branching. They compared neurite outgrowth of unmyelinated (Cfiber) sensory DRG neurons that were uninjured or axotomised in a somatic peripheral nerve injury model. And they found NGF and artemin can play different roles in neurite initiation and elongation [25]. NGF, as the first neurotrophic factor discovered, is effective for PNI therapy. Recently Payne et al. used a special visceral nerve injury model in Wistar rats to determine the capacity of injury nerve to reinnervate into organs [26]. In this study, an activating transcription factor-3 (ATF3) was shown to have the ability to enhance regeneration responses after visceral nerve injury. This suggests that different microenvironment is involved in different peripheral nerve injury area [26]. Recently a study on NGF that affects peripheral nerve regeneration has shown that adding NGF combined with nerve fragments into the autologous epineurium small gap can enhance autonomic activities and root ulcers recovery. And this also increased the regeneration of nerve fibers and conduction velocity during nerve repair process [27].Recently some researchers focus on the NGF-microspheres that can release under control so as to apply in clinical PNI. In this study, the data has shown that intraperitoneally administrated both NGF and NGF-microspheres may enhance peripheral nerve recovery and regeneration after sciatic nerve crush injury. And NGF-microspheres showed better effects than NGF administered alone. This suggests that NGF may be considered as a potential agent for the treatment of PNI [28]. PNI therapy is not only involved in nerve regeneration and axon elongation, but also involved in microenvironment improvement, such as microvessel formation and reconstruction during nerve recovery. Asanome et al. found that NGF not only enhanced peripheral nerve innervation but also microvessel maturation and stabilization around injury area during pathological angiogenesis [29]. This suggests that NGF may improve microenvironment in the damaged area thus to fasten nerve recovery after PNI [29]. Jin et al. used a nanofiber poly-L-lactide-cocaprolactone (PLCL) scaffold that was filled with collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to observe neurite out-growth after PNI [30]. They loaded a hydrogel consisting of 4 mg/mL collagen and 4 mg/mL hyaluronan (4C+4H) with 100 ng/mL of the NGF, and then

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tested the kinetics of NGF release. The data showed that NGF release was up to 20% during the first 3 days and additional 6% from day 4 until day 7. In vitro studies showed that NGF in a dose-dependent manner enhanced neurite outgrowth. However, in vivo studies failed to show significant effect on neurite extension. Effects of 500 ng/mL of NGF on neurite out-growth was equivalent to 20 ng/mL of the NGF and less than that with the 50 and 100 ng/mL NGF concentrations. And this study suggests that NGF combined with hydrogels that was filled into tubular nerve conduits may contribute to assisting restoration of protective sensation following peripheral nerve injury [30]. Neural stem cell therapy is considered an effective approach to treat PNI. NGF has been demonstrated to contribute to the proliferation and differentiation of NSCs. Liu et al. found that NGF may induce NSC differentiation, and increase β-tubulin III and phosphorylated ERK expression. Thus NGF may be a potential agent to induce NSC differentiation to treat clinical PNI. And in this study, they found NGF combined with BDNF may enhance the function of NSCs obtained from an adherent monolayer culture [31]. Besides that, NGF has been demonstrated to have the effect on restoring and maintaining neuronal function during neurodegenerative disorders. Razavi et al. described that NGF combined with immune system function can contribute to recovery of multiple sclerosis [32]. Mouse-derived NGF and human NGF have 92% sequence homology. Previous studies have shown that NGF administration is closely linked to peripheral nerve development, axonal branching, dendritic and terminal arborization, and even synaptic plasticity [33]. And most of the developing sensory DRG neurons are dependent on NGF for survival. NGF knockout mice showed cell loss in the sensory and sympathetic neurons in DRG. In addition, NGF has shown to be transported retrogradely from peripheral dendrite to cell body. Therefore, NGF release and transport may regulate DRG and peripheral nerve development. Thus it may be considered as a clinical trophic factor to treat specific peripheral neuropathies [34]. NGF receptors include p75 and trkA that express in many sensory DRG neurons. A significant increase of NGF and its receptors were shown in the sciatic nerve transection. However, NGF and its receptor p75 may be disappeared after reinnervation and regeneration. So NGF may be linked to peripheral nerve recovery process. These receptors expressed in different area and pattern in the normal and injured nerve [34]. 3.4. Ciliary Neurotrophic Factor Kamei et al. [35] co-cultured the cortex and spinal cord slices in vitro to determine the mechanism of corticospinal axon regeneration following

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transplantation of neural progenitor cells (NPCs) in the injured spinal cord. They found axon growth promoted by NPCs was significantly suppressed by neutralizing antibodies against BDNF, NT-3, and NGF. However, axon outgrowth was not inhibited significantly by neutralizing antibodies against ciliary neurotrophic factor (CNTF). This suggests BDNF, NT-3 and NGF were involved in axon extension, but CNTF failed to participate in this process. The underlying mechanisms involved in this process still remain uncertain. Some researchers think CNTF is related to axon growth but some not. Bregman and colleagues found that CNTF contributed to peripheral nerve axon outgrowth but failed to enhance axon extension in the spinal cord [36]. 3.5. Growth Hormone Growth hormone (GH) is playing an important role in the regeneration of neural cells and nerve regeneration. Pablo Devesa and colleagues found that GH could enhance the function and regeneration of sciatic nerve after transection and repair. In their study, they observed the ability of GH on the promotion of axon regeneration after surgical section and repair in the sciatic nerve in rats. Histological analyses data showed that the sciatic section in the GH-treated rats illustrated more abundant immunoreactive axons and linearly ordered structures. Whereas in the saline-treated rats, the section site of sciatic nerve only showed little regeneration, presenting limited and dispersed immunoreactive axons and disorganized instruction. Moreover, at week 8 after surgery, the GH-treated sciatic nerve showed more expression of Schwann cell marker S100 with increase of axonal diameter and thickness of myelin sheath. By electrophysilogical recording on the compound muscle action potential (CMAP), the data showed a decrease in CMAP latency and an increase in CMAP amplitude [37]. Both these demonstrated that growth hormone has the potency to treat sciatic nerve injury in the coming future. In addition, Javier Saceda and colleagues found that human growth hormone could enhance the ulnar nerve regeneration after section in 18 specimens of Wistar rats. In the human growth hormone treated group, conduction velocity and actin potential amplitude were increased significantly. This further demonstrated the effective influence of growth hormone on peripheral nerve injury repair [38]. 4. PAIN INDUCED BY PERIPHERAL NERVE INJURY Pain is a hallmark of tissue damage and inflammation, which promotes tissue protection and thereby contributes to repair. Many persons who have sustained PNI experience clinically significant pain after injury. Post-PNI pain can increase

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with time and is often refractory to conventional treatment approaches. Over the past decade, clinical studies have shown that post-PNI pain may transit from acute pain to chronic pain or neuropathic pain. Ko and colleagues found that metabotropic glutamate receptor subtype 5 (mGluR5) is closely linked to nociceptive transmission and peripheral hypersensitivities after chronic constriction injury (CCI) in rats [39]. They used sciatic nerve with loose ligatures to address the hypothesis that mGluR5 may be involved in the process of CCI. The data showed that CCI animal model exhibited neuropathic pain symptoms, such as allodynia and hyperalgesia. And an inhibitor of mGluR5, 2-methyl-6(phenylethynyl)-pyridine (MPEP), may decrease the pain induced by CCI. Furthermore, mGluR5 expression increased sharply in the injured primary nerves with demyelination. That is to say, peripheral nerve injury like CCI may result in neuropathic pain with the underlying mechanism of alterations of mGluR5. Moreover, Kambiz et al. found that thermal hypersensitivity induced by transected and reconstructed sciatic nerve is linked to epidermal peptidergic fibers innervation in the glabrous skin of rats’ hind paws [40]. Thus, pain induced by peripheral nerve injury is not only related to some special receptors described above, but also related to some peptidergic fibers [40]. Another study [41] also showed the epidermal changes induced by peripheral nerve injury like gene transcription and translation in the DRG. In this study, a transcription factor, myeloid zinc finger protein 1 (MZF1), was up-regulated in the injured DRG. To address the effect of MZF1 in the injured DRG, they down-regulated the MZF1 by injecting siRNA into the injured DRG or upregulated it by injecting adenoassociated virus 5 expressing full-length MZF1 into the DRG. The data shown that this transcription factor, MZF1, is related to voltage-gated potassium 1.2 (Kv1.2) and neuropathic pain [41]. A recently report [42] by Casals-Diaz showed that voltage-gated sodium channels (VGSCs) α-subunits were involved in the process or neuropathic pain in the sciatic nerve crush animal model. Thy determined the VGSCs α-subunits including Nav1.3, Nav1.7, Nav1.8 and Nav1.9. Different expression patterns were shown among these VGSCs α-subunits in the DRG after sciatic nerve crush and spared nerve injury. Besides that, spared nerve injury and crush animal model showed different expression of VGSCs α-subunits in the DRG. This study provided more evidence that channel expression and pain process may be related to the development of PNI during degeneration and regeneration time [42]. Another study about sodium channel, Nav1.6, showed that local knockdown of Nav1.6 by injecting siRNA attenuated mechanical pain induced by SNL, and decreased sympathetic sprouting. And blockade of Nav1.6 also reduced pain behaviors in animal model with chronic constriction of the sciatic nerve [43]. Besides sodium channels and potassium channels, calcium

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channels are also involved in neuropathic pain induced by PNI. Murali et al. used peripheral nerve ligation model to address the effects of calcium channels in the DRG [44]. They prepared partial sciatic nerve ligation animal model and whole patch clamp recording to observe the currents mediated by voltage-gated calcium channels (VGCCs) in the DRG neurons of C57BL/6 mice. It was N-type not P/Qtype that increased in the medium and large diameter neurons in the DRG. This study provides strong evidence that alteration of N- and P/Q-type VGCCs is involved to PNI and different neurons in injured DRG [44]. Pinheiro et al. found that transient receptor potential ankyrin 1 (TRPA1) is related to CCI-mediated neuropathic pain in mice [45]. The data showed that TRPA1 may sensitize mouse nociception by injecting TRPA1 agonist. However, its antagonist HC-030031 could reverse the allodynia induced by CCI of sciatic nerve. Moreover, the antagonist HC-030031 injection may also reduce CCI-induced norepinephrine hypersensitivity [45]. Berger et al. also studied effects of calcium channel in the pain process induced by sciatic nerve injury. The data showed that a novel T-type channel inhibitor (NMP-7) treatment by intraperitoneal (i.p.) or intragastric (i.g.) routes reduced mechanical hyperalgesia in mice with complete Freund’s adjuvant (CFA)-induced inflammatory pain and sciatic nerve injury-induced neuropathic pain [46]. Moreover, a recent study showed that the effect of NMP-7 on CFA-induced inflammatory pain is inhibited in CaV3.2-null mice, suggesting CaV3.2 is the key target of pain induced by CFA [46]. Nishinaka et al. studied the nerve-injury-induced thermal and mechanical hypersensitivity following the early life stress [47]. Early life stress was induced by maternal separation and social isolation (MSSI). They found MSSI resulted in anxiety-like behaviors in female mice and enhanced thermal and mechanical hypersensitivity after sciatic nerve injury [47]. In our previous study, we found that stress can prolong pain behavior induced by planter incision and thus transit it from acute to chronic pain. Our study demonstrates that by releasing stress hormones, stress regulates AMPA receptor phosphorylation and trafficking, which leads to a change in synaptic AMPA receptor subunit composition and causes AMPA receptor subunit switch from Ca2+impermeable (containing GluA2) to Ca2+ permeable (lackingGluA2). This switch will enhance Ca2+influx and further activate Ca2+-dependent protein kinases, thereby promoting AMPA receptor phosphorylation and other phosphorylation-triggered activities. This positive feedback loop may contribute to the molecular mechanisms that underlie stress-induced pain transition after surgery [48]. Another study showed that

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BDNF contributes to neuropathic pain by activating GluN2B-NMDA receptors viaSrc homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) phosphorylation in rats [49]. This study suggests that neurotrophins may participate in the pathogenesis of neuropathic pain induced by PNI. Some neuropoietic factor can be also involved in neuropathic pain induced by peripheral nerve injury (CCI of sciatic nerve). Van Steenwinckel et al. found that CCL2 chemokine and its cognate receptor CCR2 participate into the process of pain hypersensitivity and PNI [50]. By using irradiated bone marrow-chimeric CCI mice, they found CCL2/CCR2 axis plays important role in the neuropathic pain development and inflammatory cell recruitment into the lesion [50]. 5. EFFECT OF MICROENVIRONMENT MODULATION ON NEURAL STEM CELL DEVELOPMENT Neural stem cells (NSCs) are considered as an effective resource for PNI therapy. However, many factors may affect the NSCs development including differentiation, proliferation, and maturation. So improvement on microenvironment of NSCs development is such important for its application in the PNI therapy. Usually NSCs line in the subventricular zone (SVZ) and dentate gyrus of the hippocampus in the CNS. And some researchers focus on NSCs derived from embryonic stem cells or iPSCs. In our previous study, we tried to get NSCs from the embryonic spinal cords to address our experiment aim. After injury, the extracellular microenvironment changes and thus influences the development of NSCs in the injury area. Our previous review summarized the effect of transplantation of human embryonic stem cell-derived neural progenitor cells on adult neurogenesis in aged hippocampus. In this review, we focus on effect of special microenvironment on neurogenic niches. Multiple cell types, including endothelial cells, astroglia, ependymal cells, immature progeny of neural stem cells, and mature neurons, comprise the neurogenic niche. Differentiation of embryonic stem cells towards the neural lineage results in the generation of different neuronal subtypes and non-neuronal cells (mainly astrocytes). Therefore, it is reasonable to hypothesize that transplantation of human embryonic stem cell-derived neural progenitor cells can be used to modify neurogenic niches for facilitating adult neurogenesis [51]. Our previous study investigated the role of neuregulin-1/ErbB signaling pathway in the stem cell therapy for SCI-induced chronic pain [23]. In this study, we prepared a rat contusion SCI model and tested the chronic neuropathic pain for 56 days. We found that spinal transplantation of mouse embryonic stem cell-derived oligodendrocyte progenitor cells (OPCs) enhances remyelination in the injured

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spinal cord and reduces SCI-induced chronic neuropathic pain [23]. Moreover, we found that SCI reduces the protein level of neuregulin-1 and ErbB4 in the injured spinal cord and that OPC transplantation enhances the spinal expression of both proteins after SCI [23]. Finally, intrathecal injection of neuregulin-1 siRNA, but not the control non-target RNA, diminishes OPC transplantation-produced remyelination and reverses the antinociceptive effect of OPC transplantation [23]. Our findings suggest that the transplantation of embryonic stem cell-derived OPCs is an appropriate therapeutic intervention for treatment of SCI-induced chronic neuropathic pain, and that neuregulin-1/ErbB signaling plays an important role in central remyelination under pathological conditions and contributes to the alleviation of such pain [23]. Another study investigated the intrinsic microenvironment of the neural precursor cells (NPCs) for SCI therapy and showed that matrix chondroitin sulfate proteoglycans (CSPGs) plays important role in the SCI repair [52]. Removing CSPGs from the SCI environment enhances the potential of adult NPCs for spinal cord repair [52]. And CSPGs significantly decrease NPCs growth, attachment, survival, proliferation and oligodendrocytes differentiation in the adult spinal cord [52]. Besides that, several signaling pathways like receptor protein tyrosine phosphate sigma (RPTPσ), leukocyte common antigen-related phosphatase (LAR), Rho/ROCK, AKT and Erk1/2 pathways are also involved. Thus, CSPGs as key factor in the microenvironment of NPCs development is considered as potential target for SCI repair [52]. Another source of NSCs is from the neural crest like cells that have been identified in the dermis of human skin with self-renewal capacity and differentiate into neural crest derivatives. Environment for the neural crest like cells-derived NSCs can affect the proliferation and differentiation. Fukunaga-Kalabis et al. found that Notch signaling is involved in the development of NSCs [53]. The data showed that Notch signaling is the key target for the differentiation and motility of the neural crest like cells-derived NSCs. Besides that, they also found that Notch signaling is crossing talk with Wnt5a in the process of NSCs maturation. These two pathways are both involved in the controlling skin microenvironment in stem cell fate decision [53]. 5.1. Three-Dimensional Microenvironment To further investigate the effect of microenvironment on stem cell development, Sart et al. used three-dimensional (3D) aggregate-like structure mimicking embryonic development [54]. They found intact embryoid bodies showed better

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survival ability and recovery from thaw than dissociated embryoid bodies, suggesting that microenvironment including extracellular matrix, cell-cell contacts, and F-actin organization is important in neural differentiation postcryopreservation [54]. Other factors that affect microenvironment of NSCs development include cadherin and some adhesion molecules. Cherry et al. focused on the design of neurotrophic biomaterial constructs for human NSCs and found that N-cadherin and L1 molecular are involved in the development of NSCs [55]. In this study, two-dimensional (2-D) films or three-dimensional (3-D) microfibrous scaffolds were used to compare the function of N-cadherin and L1 molecular on the neuronal differentiation and neurite outgrowth of the humanembryonic-stem-cell-derived neural stem cells. In addition, 3-D fibrous scaffolds showed better microenvironment than 2-D films for the NSCs development [55]. Moreover, another study also described the importance of 3D microenvironment on the stem cell development [56]. In this study, Park et al. used 3D collagen hydrogel to culture the umbilical cord blood cells and found that microenvironment in 3D status is better than that in 2D culture, showing more expression of a series of neurotrophic factors, including neurotrophins, nerve growth factor, brain-derived neurotrophic factor, and ciliary neurotrophic factor as verified by the gene and protein analysis. And they also found co-cultured with human neural precursor cells may enhance the neurite outgrowth of neural precursor cells [56]. Faghihi et al. also studied the effects of 2D and 3Ddimensional Culture Systems on the differentiation of chorion-derived mesenchymal stem cells that can differentiate into motor neuron like cells [57]. Even they used different cell resource for the neural stem cell differentiation, it still demonstrated that 3D microenvironment is important for maintain the characters and maturation of NSCs [57]. Kothapalli and colleagues cultured embryoid bodies of ESCs on 2D substrates or within 3D scaffolds to observe the differentiation of stem cells under the condition of retinoic acid (RA) and sonic hedgehog (Shh). They found that neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1μM RA. Moreover, 3D matrigel contributed to dopaminergic neuron differentiation from ESCs and improved motor neuron formation when RA and Shh were added into the culture system [58].Yang et al. focused on the hypoxic microenvironment of stem cells transplanted in vivo [59]. They mimicked the NSC niche conditions with hypoxia in 3D extracellular matrices. Since 3D extracellular matrices is easier to provide similar microenvironment of NSCs in vivo and thus it would be reasonable to observe the morphology, structure, gene expression, neurite outgrowth, axon

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elongation, differentiation, proliferation and maturation. And this paper provides us a new thought on physiologically microenvironment similar to NSC niches in vivo [59]. Shao et al compared the characters of human pluripotent stem cells in the 2D and 3D culture system in a recent review. And they summarized that 3D cell microenvironment is reasonable and similar to that in vivo. Cell fate and differentiation are due to microenvironment where the cells live and the neurotrophines are released [60]. Another review also concludes the influence of microenvironment where stem cells survive, proliferate, differentiate and selfrenewal. Hazeltine et al. firstly described the factors that influence and maintain hPSC pluripotency. One kind of the factors is the soluble one, including bFGF, IGF, GSK3 and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). When these factors described above are added into the culture system, hESCs show different protein expression and self-renewal. They also paid attention on neural like stem cells from hPSC and the underlying mechanisms. Neuroepithelial commitment is one of the important one to affect neurogenesis from hESCs. Different labs used different induction method to get NSCs with or without bFGF. Some of the researchers used to add RA to induce ESCs into NSCs. And some searchers changed the culture microenvironment by altering the substrate of ECM like laminin-coated plate. In this review, culture architecture is also summarized to explain the influence of microenvironment on stem cell self-renewal [61]. Axon elongation and re-myelination are important for the NSCs transplanted to treat PNI. Many factors influence axon guidance, axon elongation, neurite outgrowth and innervation. Xu et al. reviewed the myelin-associated inhibitors including NogoA, myelin-associated glycoprotein (MAG), and oligodendrocytemyelin glycoprotein (OMgp), and the axon guidance molecules like semaphorins, netrin, slit/robo and ephrin/eph. Sema4C is the most important factor that affects NSCs differentiation in the semaphorins family. Effects of slit/robo signaling still remain unclear. And the ephrin/eph signaling pathway has been studied for a long time and shown to play important role during neurogenesis and development of NSCs. Thus the axon guidance and innervation must be considered during the PNI repair so as to improve nerve elongation and prevent misdirection [62]. Other factors like vascular microenvironment can also affect NSC niche. NSCs delivered into the injury area has been significantly influenced by micro-vessels in the damaged area where is abnormal for the stem cell survival. Thus one of the key approaches to improvement therapeutic effects is to increase blood vessel reconstruction and regeneration, which is important for maintaining homeostasis. 3D vasculature and extracellular matrix microenvironment provide NSCs suitable physiological environment that includes chemical materials, and some effective

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factors. This special niche allows NSCs to improve self-renewal, proliferation, maturation and axon guidance with similar vascular microenvironment in vivo [63]. Studies have shown that neurogenesis and angiogenesis occur in the injured area. And the NSCs activation is followed by angiogenesis since the NSC maturation and migration are dependent on the chemokine and neurotrophine release from the microenvironment. A recent review described the vascular regulation of adult neurogenesis under physiological and pathological conditions [64]. 5.2. Matrix & Neurogenic Niches Neural stem cells induced from other stem cells need to experience four stages: neural commitment, immature neurons, mid-neurons, and mature neurons. Engler et al. [65] studied the effects of matrix elasticity on stem cell lineage specification and found that only cells on the softest matrices express protein markers for neuronal commitment (nestin), immature neurons (β3-tubulin), mid/lateneurons (microtubule associated protein 2, MAP2), and even mature neurons (NFL, NFH, and P-NFH). Besides that, soluble agonists, such as RA and dimethylsulfoxide (DMSO) have been reported to induce reversible branching in stem cells [65]. Mohyeldin et al. reviewed the effect of oxygen in stem cell biology and thought that oxygen is a critical component of the stem cell niche [66]. In adult tissues, oxygen tensions are different from the inhaled ambient oxygen tension of 21%. It ranges from 0.55% in the mesencephalon to 8% at the surface of the brain. Neurogenic niches in the brains have the ability to maintain NSCs in their undifferentiated state. NSCs implanted into damaged areas may keep survival by HIF1a mediated secretion of vascular endothelial growth factor. Hypoxia could contribute to NSCs in an undifferentiated state. And oxygen tension also affects proliferation and multipotentiality of NSCs. In this review, the authors think that oxygen tension in the neural niche functions to maintain stem cell self-renewal and keep in undifferentiated state. Hypoxia could also influence migration of NSCs [58]. 5.3. Cell-Cell Interactions and Cell-Matrix Interactions Microenvironment for the neurogenesis and stem cell differentiation is to provide special niches to improve their characters. For the neural stem cells, it is very important to keep in quiescence or activity during neurogenesis after PNI. Regarding the therapeutic effect of stem cell transplantation, we need to know how many and how long the stem cell may differentiate and maturate into target

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cells. Thus NSCs transplanted need to be activated and keep their pluripotent characters so as to treat PNI. Direct cell-cell interactions mediated by integral membrane proteins in the microenvironment of the damaged area need to be considered in transplanted-stem cell activities. Ottone et al. [67] studied the cellcell contact with vascular niche and found that these interactions keep stem cells to be quiescent. Direct cell–cell interactions with the endothelium regulate neural stem cell behavior in culture and in the SVZ where new neurons are continuously produced throughout life. They co-cultured SVZ postnatal neural precursor cells (NPCs) or adult neural stem cells (aNSCs) with endothelial cells that includes primary brain microvascular endothelial cells (bmvECs), the brain microvascular endothelial cell line bEND3.1 (bENDs) or conditionally immortalized pulmonary endothelial cells (pECs). The data showed that endothelial cell contact switched NPC migration from repulsion to attraction, indicative of a change in the adhesive properties of the neural progenitor cells. Moreover, interactions with endothelial cells induced to stem cell to be quiescent with an increase in G0 –G1 phase. In this study, Eph and Notch signaling pathways were also involved in stem cell differentiation under the condition of cell-cell interactions. Exposure with ephrinB2 or/and Jagged 1, one of the highly expressed ligands of Notch, changed NPC differentiation and neurogenesis. Stimulation of NPCs with ephrinB2 and Jagged1 did not induce identity genes to a greater extent than individual ligands. This study provides strong evidence that cell-cell contact may be critical player in stem cell differentiation and fate decision. Although further studies still need to be done, people have to consider this factor to improve microenvironment for transplanted NSC differentiation, maturation and synaptic plasticity [67]. Lane et al. [68] reviewed the treatment in regenerative medicine about modulating the stem cell niche for tissue regeneration. One of the key factors they mentioned in this paper is the cell-cell contact. Direct cell-cell contact includes interactions with many adhesion molecules and receptors in the membrane. Notch and Wnt signaling pathways are considered to be critical in this complicated process. Besides that ECM, secreted factors and hypoxia are also regarded as important factors and involved in stem cell development. Thus by regulating the microenvironment, stem cell therapy to PNI may be more reasonable for clinical medicine [68]. Another review summarized the control factors of stem cell fate by physical interactions with the extracellular matrix. “Cell shape is a regulator of stem cell fate”, is described in this review. That is to say, some intrinsic and extrinsic factors may influence cell shape thus to influence its function. Cell shape plays important role in interacting with other cells and matrix. And this review also

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mentions the effect of the extracellular matrix on the stem cell differentiation, cell fate decision and lineage commitment. Matrix may provide some contractile forces, resulting in stresses in the cytoskeleton. Thus stiffness of the extracellular matrix plays important influence on cell migration, apoptosis and differentiation [69]. Besides that, extracellular matrix may influence receptor expression during stem cell development and maturation. So it may be helpful to improve cell-cell interactions and cell-matrix interactions during stem cell therapy to disease of nervous system. For PNI therapy, improvement of cell-microenvironment interactions is very important for neural stem cell survival, differentiation and maturation. 5.4. Reprogramming Induced pluripotent stem cells (iPSCs) can differentiate into any cell type in the body. Yamanaka and Takahashi reprogrammed skin fibroblast cells into iPSCs and provide potential for disease treatment [70]. Lucas et al. summarized the effects of reprogramming for stem cell niche, including the experiment described above [71]. Mariano et al. [72] summarized the current options, limitations and perspectives of adult stem cells in neural repair. They describe one of the important features of stem cells as the following: “Another important feature of stem cells is their immunomodulatory power, which allows them to “feel” the environment and change their patterns of migration, interaction and survival. When transplanted into the lesion site, some stem cells interact with the proinflammatory environment, migrate to the site of injury and differentiate into the required cell type in order to execute repair. Moreover, some stem cells play an important role in the proliferation and migration of new cells to the site of injury. This helper profile has been observed in neural stem cells and mesenchymal stem cells. In the case of mesenchymal stem cells, this function can play an important role in changing the macrophage proinflammatory profile into a proregenerating role, once there is no longer an urgent need for a robust immune response” [72]. This provides us a new concept that microenvironment and stem cell transplanted can affect each other. For PNI therapy, stem cells transplanted in the lesion site need to reconstruct the microenvironment, and the microenvironment in the damaged area needs to be improved for stem cell survival and differentiation. Jaenisch and Young reviewed “Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming” and summarized the strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming [73]. Moreover, several lines have also illustrated the possibility of reprograming the

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ganglionic microenvironment in PNS to harvest local pluripotent cells. Czaja K and colleagues studied capsaincin induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats. By determining cleaved caspase3,TUNEL, BrdU, the neuron-selective marker PGP9.5, and neurofilament-M-immunoreactivity. They found that neuronal death after 30 days post-capsaicin treatment. However, no significant of difference was shown in the total numbers of neuraonal nuclei after 60 days postcapsaicin, suggesting new neurons were proliferated and added into the ganglia [74]. This research group further studied the neuronal proliferation of satellite glial cells after capsaicin induced neuronal death. The data showed that a progenitor cell marker nestin was increased at the early time points following capsaicin administration followed by recovery of neuronal numbers in the dorsal root ganglia [75]. Furthermore, this group also found that restoration of spinal afferent projections and signaling pathways compensate after capsaicin induced peripheral nerve injury, by determining NR1 and Nav1.8 in the fast blue e labeled neurons [76]. Besides that, this group also reviewed neurogenesis in the adult peripheral nerve system and revealed the evidence on the under estimated potential for generation of new neurons [77]. Geuna S and colleagues reviewed and provided a basic framework information on nerve morphology in many experimental studies on peripheral nerve repair and regeneration [78]. Muratori L and colleagues studied the neurogenesis of dorsal root ganglia in adult rats after peripheral nerve crush injury. The data showed that there was no relation between neuraonal loss and nerve injury. However, by detecting Ki67, nanog, nestin and sox-2, they found posttraumatic neurogenesis in dorsal root ganglia. Moreover, the satellite glial cell proliferation was shown after nerve crush injury, suggesting new neurons were generated after nerve injury [79]. Another paper illustrated the hippocampal neurogenesis after vagus nerve injury induced by capcaicin and vagotomization. And the data suggested microglia activation in the granular cell layer and long-lasting changes in dentate gyrus after vagus nerve injury [80]. In addition, by comparing the neuronal restorative capacities induced by vagotomy and capsaicin administration, Ryu V and colleagues found that nodose ganglion neurons showed plasticity after peripheral nerve injury, and the capsaicin treatment may result in more significant restorative capacities than that of vagotomy when innervating into the stomach [81]. Besides that, Czaja K and colleagues reviewed the DNA replication and neural proliferation after peripheral nerve injury, aiming to explaining the mechanisms of DNA replication in neuron division and differentiation during neurogenesis in the nervous system. In this review, not only central nervous system injury but also peripheral nerve injury was reviewed to highlight the effect of DNA replication after injury. Even many

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studies showed different results, there are still some papers illustrated DNA replication may lead to new neuron proliferation and differentiation, thus lead to neurogenesis after PNI [82]. Even nuclear reprogramming plays important role in stem cell differentiation, some questions still need to be solved. For example, how to prevent the risk of mutations that is induced by the transfected virus? And how to keep stem cell characters after transplantation but not to be changed into cancer? 5.5. Electric Stimulation During the stem cell development, some researchers found that electric stimulation may change cell fate and differentiation. Thrivikraman et al. cultured human mesenchymal stem cells (hMSCs) in the absence of biochemical growth factors [83]. They used electric field stimulation with a parameter of 100mV/cm in a stimulation cycle of 10min/day. And they found if the electric stimulation strength was too high, the cell death increased. However, if the stimulation strength is too low, it couldn’t alter stem cell behavior. By stimulation of electricity, hMSCs differentiated into neural like cells with neural morphology and specific gene expression. But they did not determine the action potential of the induced neuron-like cells. Despite neuron-like cells-induced by electric field stimulation were failed to demonstrate their physiological functions, this paper demonstrated that conditioned electricity stimulation may be a kind of potential factor to decide cell fate and differentiation [83]. Gu et al. [84] summarized that “the interaction between neural cells and electrically conductive biomaterials may arise from an increased adsorption of positively charged matrix proteins onto the negatively charged surface of biomaterials”. And low intensity direct electric stimulation enhanced nerve regeneration in a sciatic nerve crush injury animal model. The researches described above provide us that electric stimulation may be a potential therapeutic approach for stem cell therapy to PNI. Because stimulation can change both stem cell fate and nerve elongation. Currently, the parameters of electric stimulation should be difficult factor to select during stem cell therapy [84]. Keuters et al. used transcranial direct current stimulation (tDCS) to improve neurorehabilitation and observed a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex [85]. The data showed that migratory activity of NSCs almost doubled after anodal tDCS, which increased the undirected migratory activity of implanted NSCs [85]. Moreover, a recent study found low-frequency electrical stimulation induces the proliferation and differentiation of peripheral blood stem cells into Schwann cells [86]. This may contribute to study the underlying mechanisms of stem cell differentiation

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during stem cell therapy to PNI [86]. Furthermore, by using balanced biphasic electric fields stimulation, subependymal neural precursor cells (NPCs) were induced to proliferate and migrate toward the lesion site where they differentiate into neural cells. And NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation. This paper suggests that balanced biphasic stimulation represents a clinically viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neuro-repair [87]. Kobelt et al. [88] cultured neural stem progenitor cells (NSPCs) from 6-week old rats and expanded them into neurospheres under the neural stem cell culture medium. The stimulation chambers consisted of a 63mm long non-conductive open silicone rectangular box with a glass slide bottom. The stimulation parameters are 0.53 or 1.83v/m for 10 min/days for 2 days. The length, polarity and differentiation of NSPCs are calculated by analyzing the images of neurons by Image J software. The data showed that Stimulated NSPCs showed lengths that were over five times longer than un-stimulated controls (112.0 ± 88.8 μm at 0.53 V/m vs. 21.3 ± 8.5 μm for 0 V/m with IFN-γ) with the longest neurites reaching up to 600 µm. However, the maximum length of neurites in the control group is no more than 200 µm. The data also showed that electric stimulation increased intracellular Ca2+ concentration. Besides that, electric stimulation contributed to neuron maturation by expressing neural markers like βIII tubulin, neuronal nuclei (NeuN), and filamentous-actin. However, in this study, neuron metabolism and activities were not affected by electric stimulation [88]. Therefore, this study provides us evidence that electric stimulation could change neural morphology and differentiation by altering their axon-elongation, neurite outgrowth and specific markers. However, this study mainly focused on effects of electric stimulation on neural stem cells in vitro. Further study still needs to be done in vivo. Tandon et al. [89] also used electrical stimulation to study neuron maturation and differentiation in early retinal development in vitro. They found that biomimetic electrical stimulation could result in mouse retinal progenitor cell gene expression and morphology alterations. Moreover, this stimulation can also contribute to neuron maturation and functional organization [89]. Li et al. found that steady direct current electric fields influenced neural precursor cell migration, which were derived from embryonic stem cells. And this alteration was dependent on the field strength [90]. Another study [91] also considered the effects of electric stimulation on neuron migration. In this study, motor cortex was stimulated with electrodes implanted in the rat brain, which increased cell proliferation in the SVZ, where neural progenitor cells produce after some injury. And this study also showed cell migration increase under the electric stimulation

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[91]. Taken together, electric stimulation can induce stem cell maturation, expressing some specific neural markers and reorganizing cell morphology. And it can also contribute to neuron migration so as to play potential role in stem cell therapy to nervous system diseases. 6. SIGNALING PATHWAYS 6.1 cAMP Signaling PNI results in nerve degeneration and demyelination. Neurotrophins that affect nerve regeneration can bind to their receptors and then activate cellular signaling pathways to respond to PNI. Previous studies have shown that cAMP signaling cascades play important role in nerve regeneration and neural stem cell maturation. Intracellular cAMP can be increased during peripheral nerve injury, with the underlying mechanism that is linked to stimulating adenylatecyclase with forskolin. Forskolin can increase the rate of regeneration for transected axons in the sciatic nerve [92]. Chan et al. also found that axon outgrowth was enhanced by increasing cAMP level induced by trk receptor signaling [93]. Ying et al. studied the cellular mechanisms after sensing nerve injury at the axonal endoplasmic reticulum [94]. They found that luman/cAMP response element binding protein 3 was closely linked to axon length elongation. Axotomy induces axonal luman synthesis and also release from the axonal ER of Luman's transcriptionally active amino terminus, which is transported to the cell body in an importin-mediated manner [94]. However, a previous study has shown that dibutyryl cAMP (db-cAMP) contributes to central branches to regenerate in the spinal cord. And conditioning lesion can increase intracellular cAMP level and the expression of growth-associated tubulin isotypes to promote regeneration. But db-cAMP fails to increase intrinsic axon growth capacity enough to raise the rate of regeneration of peripheral branches in the sciatic nerve or enable central branches to elongate long distances in an environment free of all CNS inhibitors of elongation [95]. Because peripheral nerve injury always accompanies with neuropathic pain, some researchers focused on the effects of cAMP and cAMP responsive element binding protein (CREB) on neuropathic pain. Three weeks following partial sciatic nerve ligation (PSNL), daily intrathecal injection of antisense CREB oligodeoxynucleotide (ODN, 20 µg/day) for 5 days significantly attenuated tactile allodynia. These data suggest that phosphorylation of CREB is an important contributing event in the central plasticity of nerve injury and in the pathogenesis of neuropathic pain [96]. A recent study has shown that cAMP signaling is involved in neural stem cell differentiation and maturation [97]. Lepski et al. [98] found that cAMP agonist forskolin or IBMX(3-isobutyl-1-

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methylxantine) promoted neuronal functional maturation. Besides that, IBMX can increase channel expressions including calcium channel and fire activities in the neurons. And nifedipine completely blocks IBMX-induced CREB phosphorylation [98]. Previous studies have shown that cAMP signaling is involved in differentiation of NSCs. Iguchi et al. also studied the effects of CREB on the cell proliferation of NSCs isolated from the SVZ of adult mice. The data showed that protein kinase A (PKA) inhibitors H89 and KT5720 inhibited epidermal growth factor (EGF)stimulated NSC proliferation [99]. Li et al. also found that CREB is closely linked to proliferation of NSCs [100]. And N-methyl-D-aspartate receptors (NMDARs) contribute to NSC proliferation, which may be attenuated when CREB phosphorylation was decreased. This suggests that cAMP signaling pathway is involved in the proliferation process induced by NMDARs [100]. Zhang et al. found that cAMP initiated neuron-like morphology changes early and neural differentiation much later [101]. They focused on the morphological alterations of neuron like cells derived from mesenchymal stem cells (MSCs) and found that cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. Besides that, this study also suggests that cAMP is able to direct MSCs towards neural differentiation, but the neuron like cells cannot achieve terminal differentiation [101]. Dworkin et al. reviewed the CREB function in the development of neurogenesis including expansion and ultimately homeostasis of neural cell number [102]. In the previous study of this research group, the data showed that cAMP response element binding protein is required for mouse neural progenitor cell survival and expansion. In this study, they generated CREB-/- mice and found that CREB-deficient embryos are smaller and exhibit defects in brain and retina. Besides that, CREB loss leads to abnormal Nestin immunoreactive cell morphology. Wild-type mouse brains exhibited nestin-positive cells with highly branched and long dendrite-like nestinimmunoreactive projections; however, CREB-/- embryo brains exhibited shorter nestin immune-reactive projections and less complex branching. To determine the effects of CREB on the neural stem cell survival and growth, terminal deoxynucleotidyl transferase Biotin dUTP Nick End Labeling assays were performed on CREB+/+ and CREB-/- brains. The data showed that CREB is related to NSC survival and growth, although the survival defect is only seen when NSCs are moved from their in vivo niche. Moreover, NSC self-renewal and survival were also determined in the CREB+/+ and CREB-/-embryos. Both neurosphere formation and cell viability were reduced in the CREB-/- cells compared to

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control. Even though CREB played influence on NSC proliferation, survival and self-renewal, it failed to alter cell cycle progression in vitro and glial-neuronal fate [103]. This study provides evidence that CREB is an important signaling regulator within the developing neurogenic niche. 6.2. Wnt Signaling (Wingless-Related MMTV Integration Site) Schwann cells (SCs) are the glial cells of the peripheral nervous system essential for nerve ensheathment and myelination. Grigoryan et al. demonstrated that canonical Wnt signals play a critical role in radial sorting [104]. They tested the expression of Wnt signaling components in the developing SCs and neurons in the DRG, spinal and sciatic nerves, and primary embryonic SCs. The data showed that Wnt/Rspondin/β-catenin signals control axonal sorting and lineage progression in Schwann cell development [104]. Tawk et al. demonstrated that Wnt/β-catenin pathway was an essential driver of myelin gene expression that contributes to development of nervous system and neuronal plasticity [105]. Primary mouse Schwann cell cultures were obtained from postnatal day 0 (P0) to P2 sciatic nerves. The data showed Wnt/β-catenin signaling components were expressed in Schwann cells and peripheral myelin. And Wnt/β-catenin pathway was involved in the regulation of myelin gene promoter activity in Schwann cells. Moreover, endogenous Wnt was sufficient for myelin gene expression in Schwann cells, and it contributed to oligodendrocyte maturation and myelination. This suggests that Wnt signaling pathway is involved in the development of peripheral nerve system and the regeneration process after PNI [105]. Innervation is an important role during the regeneration after PNI. Bodmer et al. found that NGF can promote neuronal survival and axon elongation, which is closely linked to Wnt signaling pathway [106].Wnt5a, a member of the Wnt family of secreted growth factors, is a key downstream effector of NGF in mediating sympathetic axonal branching and growth in developing sympathetic neurons. In vitro study showed that Wnt5a induced axon branching and axon extension. However, in vivo study showed that Wnt5a was involved in neuronal apoptosis by using Wnt5a (-/-) mice [106]. Fancy et al. also found that Wnt signaling played important role in the process of myelination of CNS [107]. Dysregulation of the Wnt pathway inhibited timely myelination and remyelination in the mammalian CNS [107]. Besides that, frizzled 9 protein, one of members of Wnt receptor family, was found in the growth cones of adult spiral ganglion neurons that were regenerating neurites in culture [108]. Thus, Wnt signaling, in particular, emerges as a candidate pathway for guiding neurite outgrowth towards a cochlear implant after sensorineural hearing loss [108]. Frizzled3 (Fz3), another member of Wnt receptor family, is

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required for neurogenesis and target innervation during sympathetic nervous system development. Fz3 (-/-) mice was used to observe the effects of Fz3 on sympathetic neuroblasts. Fz3 acts at early developmental stages to maintain a pool of dividing sympathetic precursors, likely via activation of β-catenin, and Fz3 functions at later stages to promote innervation of final peripheral targets by postmitotic sympathetic neurons [109]. Neuropathic pain is a challenge for PNI treatment. Wnt signaling not only participates in the process of peripheral nerve development, but also in the development of neuropathic pain. Shi et al. [110] studied the effects of Wnt signaling pathway during the development of acute and chronic pain process, by hind-paw injection of capsaicin, intrathecal (I.T.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). They found that Wnt3a and β-catenin were increased in the spinal cord dorsal horn of mouse pain models described above, suggesting that Wnt signaling pathways are regulated by nociceptive input during acute and chronic pain development [110]. Moreover, during the development of neuropathic pain after sciatic nerve injury and bone cancer in rodents, Wnt/frizzled/β-catenin signaling were activated and increased. Spinal blockade of Wnt signaling pathways inhibited the production and persistence of neuropathic pain and the accompanying neurochemical alterations without affecting normal pain sensitivity and locomotor activity. However, Wnt signaling activation stimulated production of the proinflammatory cytokines IL-18 and TNF-α and regulated the NR2B glutamate receptor and Ca2+-dependent signals through the β-catenin pathway in the spinal cord [111]. And Wnt3a recruited the Wnt-calcium signaling pathway and the Wnt planar cell polarity pathway in peripheral nerves to alter pain sensitivity in cancer-associated pain in vivo [112]. A recent study showed that Wnt/β-catenin signaling ablation leads to premature NPC differentiation. Wnt/β-catenin signaling ablation leads to premature NPC differentiation. Thus, β-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stagespecific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain [113]. Another study showed that persistent Wnt/βcatenin signaling determines dorsalization of the postnatal SVZ and neural stem cell specification into oligodendrocytes and glutamatergic neurons [114].Van Camp reviewed the “Wnt signaling and the control of human stem cell fate” and discussed the response to Wnt signal activation in embryonic stem cells and human, adult stem cells of mesenchymal, hematopoetic, intestinal, gastric, epidermal, mammary and neural lineages [115].Yang et al. used an animal model of optic nerve crush to observe the effects of Wnt3a on the transplanted-NSC

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proliferation and differentiation [116]. A recombinant lentivirus (Lenti-Wnt3a) was engineered to express Wnt3a. NSCs infected with control lentivirus (LentiGFP) or Lenti-Wnt3a was transplanted into the subretinal space immediately after the optic nerve crush. Overexpression of Wnt3a in NSCs induced activation of Wnt signaling, promoted proliferation, and directed the differentiation of the NSCs into neurons both in vitro and in vivo [116]. Even though, Heavner et al. found that establishment of the neurogenic boundary of the mouse retina requires cooperation of SOX2 and WNT signaling [117]. And they also found a Wntindependent role for SOX2 in maintaining retinal progenitor cell proliferation [117]. Shin et al. found that miR-29b was important for neurogenesis during mouse development and this effect was dependent on Wnt/β-catenin signaling induced by inhibitor of β-catenin and T cell factor [118]. Rharass et al. studied the effects of reactive oxygen species (ROS) and its underlying mechanisms on neural differentiation [119]. The data showed that increased ROS level enhanced neural differentiation by activating Wnt/β-catenin signaling. A previous study has shown that Wnt signaling includes canonical and noncanonical pathways [120]. Most researches focus on canonical Wnt pathways in regulating neural differentiation and proliferation. However, Bengoa-Vergniory et al. studied the differentiation of human neural stem cells by activating canonical and noncanonical Wnt pathways [121]. The data showed that neuronal differentiation was accompanied by a reduction in β-catenin/Tcf-dependent transcription and target gene expression, increased levels and/or phosphorylation of activating transcription factor 2 (ATF2), cyclic AMP response element-binding protein, and c-Jun, and increased AP-1-dependent transcription. Inhibition of Wnt secretion using the porcupine inhibitors IWP-2 and Wnt-C59 blocked neuronal differentiation, while activation or inhibition of Wnt/β-catenin signaling had no effect. Neuronal differentiation increased expression of several Wnt genes, including Wnt3A, silencing of which reduced differentiation. And a switch from canonical to noncanonical Wnt signaling mediates early differentiation of human neural stem cells [121]. Qu et al. studied the effects of Wnt7a, a member of the Wnt family of signaling molecules, on regulating the multiple steps of neurogenesis in adult mouse brains [122]. The data showed that loss of Wnt7a affected neural progenitor cell cycle progression, including cell proliferation and self-renewal by BrdU staining and clonal analysis. Wnt7a-/- neural stem cells showed decreased neurosphere size and neurosphere formation rate. Moreover, loss of Wnt7a inhibited neuronal differentiation and maturation. NSCs were induced by retinoic acid (RA) and fetal bovine serum to be differentiated into neurons, astrocytes and oligodendrocytes, respectively. The data showed that Wnt7a-/- neural stem cells could differentiate into three cell lines. However, Wnt7a-/- neural stem cells showed decreased Tuj1+

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neurons compared to wild type NSCs. And GFAP positive cells increased in Wnt7a-/- NSCs compared to control. No significant difference of oligodendrocytes was shown between two kinds of NSCs. In this study, the data also showed that Wnt signaling could regulate neural differentiation, proliferation and maturation by activating β-catenin-cyclin D1 and β-catenin-neurogenin 2 pathways. Wnt7a mainly expressed in hippocampal dentate gyrus which is one of the areas that NSCs are produced, so this study may provide evidence that Wnt7a could be a potential target for regulation NSCs function in adult brain [122]. Besides hippocampus, cerebellar stem cell growth and differentiation were also studied to determine the effects of Wnt signaling and its underlying mechanisms. The data showed that cerebellar progenitor cells from embryonic day (E) 13.5 following infection with β-catenin-encoding viruses demonstrated to have more proliferation than that from E17.5, suggesting Wnt-catenin signaling was mainly involved during early cerebellar stem cell proliferation. Moreover, activation of β-catenin expanded NSCs in the cerebellar ventricular zone. However, continued expression of β-catenin disrupted cerebellar development and overexpression of β-catenin impaired NSC self-renewal and differentiation in vitro. This study provides evidence that Wnt signaling could induce and increase NSC proliferation, but it may repair the function of NSC differentiation and self-renewal [123]. 6.3. Notch Signaling Previous studies have shown that Notch signaling plays important role in NSC differentiation and maturation. Notch functions as a receptor, and mammals have four Notch receptors (Notch1, Notch2, Notch3 and Notch4) and many ligands, including jagged 1 (JAG1) and JAG2(homologues of serrate), and delta-like proteins [124]. Ben-Shushan et al. studied the effects of Notch signaling on differentiation of human embryonic stem cell-derived motor neurons [125]. The Notch receptors, including Notch1, Notch2 and Notch3, were determined in the cultured human embryonic stem cells. And the data showed similar level of three Notch receptors and low level of Hes1 and Hes5 expressions. Hes5 was identified as Notch target during spinal cord development. Thus in this study, a lentiviral vector was used to over-express Flag-tagged Hes5 in the progenitor cells prior to differentiation into neural cells. Notch was inhibited by DAPT and thus decreased Hes5 expression and increased progenitor cell differentiation into motor neurons. Notch was upregulated by Lentiviral-Hes5 transfection into the progenitor cells. The data showed that over-expression of Hes5 inhibited progenitor cell differentiation into motor neurons. This study provides evidence that Notch signaling pathway is closely involved in stem cell differentiation into motor

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neurons. And it inhibits progenitor cell maturation into specific fate [125]. Besides that, Notch3 plays different role during brain and spinal cord development. In Notch 3 knockout mice, mature inhibitory interneurons were reduced and excitable immature neurons in spinal cord were increased. Notch3 was expressed in the spinal cord and progenitor cells. Notch3 is necessary and sufficient for neuronal differentiation. Moreover, Notch3-dependent neuronal maturation in the adult spinal cord modulates pain behavior [126]. Notch has several key functional regions that control different neurogenesis, angiogenesis and hematopoiesis, respectively. Jang J studied the effects of Notch intracellular domain on the NSC characters and neurogenesis in the brain. The data showed that Notch intranuclear localization is not essential to the oncogenesis of Notch1 in certain types of cells [127]. Shimojo et al. reviewed the function of Notch signaling on the maintenance of NSCs and the Notch gene expression during NSC proliferation and differentiation [128]. Hes1, a transcriptional repressor, was able to change the expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand gene Delta-like1. After Hes1 expression is repressed, Ngn2 is expressed in a sustained manner, promoting neuronal differentiation. Oscillatory versus sustained Hes1 expression leads to different outcomes in neural stem/progenitor cells. So this review suggests that different gene expression results in different outcomes in NSCs [128]. During peripheral nerve system development, Sox9 supports glial development and is necessary to initiate the induction of embryonic and adult neural stem cells. Notch1 could up-regulate Sox9 expression and induce neural differentiation. The lineage decision between neuronal and glial differentiation induced by Notch1 was mediated by Sox9. Knockdown of the Notch1-induced Sox9 expression reversed Notch1-induced astroglial cell differentiation, increase in neural stem cells, and the decrease in neurons, whereas the Notch1 effects on NC development were hardly affected by knockdown of Sox9 expression.Notch1 signaling induces glial cell differentiation of ESCs and neural stem cell self-renewal via the direct up-regulation of the transcription factor Sox9[129]. 6.4. Other Factors that Influence Characters of NSCs Neurotrophins [such as neurotrophin 3 (NT3) and brain-derived neurotrophic factor (BDNF) play key roles in OPC proliferation and myelin formation. D15A is a multineurotrophin that binds to neurotrophin receptors trkB and trkC and has both NT3 and BDNF activities. NT3 and BDNF regulate neuronal development and axonal regeneration. They are also important mediators of myelination. Mice that lack functional trkC or NT3 are deficient in both mature oligodendrocytes

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and OPCs. NT3 enhances the survival and proliferation of OPCs in vitro and in vivo. Myelination by oligodendrocytes is also enhanced by NT3 in cultured neurons and the injured CNS. BDNF is known to be important for myelin formation during development because inactivation of BDNF signaling by deletion of trkB receptors causes myelin deficits both in vivo and in vitro. CONCLUSION Even though stem cells can be potentially developed as a useful therapy for the treatment of PNI, there are still some obstacles in clinical application. Based on current studies, NSCs need to combine environment improvement so as to increase the efficiency of PNI treatment. It is important to study the properties of NSCs and the regulation of microenvironment in the injured areas. Further studies on the survival, differentiation and maturation of NSCs after transplantation should be conducted to fine-tune stem cell therapy for PNI treatment. In addition, how we can prevent misdirection during nerve recovery still remains to be illustrated. Moreover, preventing pain induced by PNI also needs further studies. CONFLICT OF INTEREST The author confirms that author has no conflict of interest to declare for this publication. ACKNOWLEDGMENTS This work was supported by US National Institutes of Health grants DE022880 and DE023551 to F.T. REFERENCES [1] [2] [3] [4] [5]

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[121] Bengoa-Vergniory N, Gorroño-Etxebarria I, González-Salazar I, Kypta RM. A switch from canonical to noncanonical Wnt signaling mediates early differentiation of human neural stem cells. Stem Cells 2014; 32(12): 3196-208. [122] Qu Q, Sun G, Murai K, et al. Wnt7a regulates multiple steps of neurogenesis. Mol Cell Biol 2013; 33(13); 2551-9. [123] Pei Y, Brun SN, Markant SL, et al. WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum. Development 2012;139(10); 1724-33. [124] Ables JL, Breunig JJ, Eisch AJ, Rakic P. Not(ch) just development: Notch signalling in the adult brain. Nat Rev Neurosci 2011; 12(5): 269-83. [125] Ben-Shushan E, Feldman E, Reubinoff BE. Notch signaling regulates motor neuron differentiation of human embryonic stem cells. Stem Cells 2015;33(2): 403-15. [126] Rusanescu G, Mao J. Notch3 is necessary for neuronal differentiation and maturation in the adult spinal cord. J Cell Mol Med 2014; 18(10): 2103-16. [127] Jang J, Byun SH, Han D, et al. Notch intracellular domain deficiency in nuclear localization activity retains the ability to enhance neural stem cell character and block neurogenesis in mammalian brain development. Stem Cells Dev 2014; 23(23); 2841-50. [128] Shimojo H, Ohtsuka T, Kageyama R. Dynamic expression of notch signaling genes in neural stem/progenitor cells. Front Neurosci 2011; 5: 78. [129] Martini S, Bernoth K, Main H, et al. A critical role for Sox9 in notch-induced astrogliogenesis and stem cell maintenance. Stem Cells 2013;31(4);741-51.

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CHAPTER 9

Transplantation of Umbilical Cord Blood Cells for Patients with Neonatal Hypoxic-Ischemic Encephalopathy and Cerebral Palsy: From Preclinical Studies to Ongoing Clinical Trials Pedro M. Pimentel-Coelho1,*, Paulo H. Rosado-de-Castro2, Fernanda Gubert1, Rosalia Mendez-Otero1 1

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio Janeiro, Rio de Janeiro, Brazil and 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Abstract: Neonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of longterm neurological disability in children, being responsible for at least 14% of the cases of cerebral palsy. Despite the moderate protection provided by therapeutic hypothermia, a significant number of infants would still benefit from an adjuvant therapy. Since the first report showing the beneficial effect of umbilical cord blood cells (UCBCs) transplantation in a rat model of HIE in 2006, a growing number of studies have improved our understanding of the mechanisms underlying the neuroprotective action of transplanted cells in animal models of HIE, intrauterine hypoxia and neonatal stroke. The aim of this book chapter is to summarize these findings and to discuss recent data from several clinical trials and case reports that have evaluated the safety and feasibility of UCBCs therapy in newborns with HIE and in children with cerebral palsy.

Keywords: Astrocytes, brain repair, cell therapies, cerebral palsy, hematopoietic stem cells, immunomodulation, microglia, mononuclear cells, neonatal encephalopathy, neonatal hypoxia-ischemia, neuroprotection, regeneration, stem cells, umbilical cord blood. INTRODUCTION It has been estimated that 43% of the deaths in children under five years of age occur in the neonatal period (i.e., within the first 28 days of life) [1]. In addition, 31% of the infants who survive intrauterine and neonatal insults, such as *Corresponding author Pedro M. Pimentel-Coelho: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio Janeiro, Rio de Janeiro, Brazil; Tel: (+5521) 3938-6554; Fax: (+5521) 22808193; E-mail: [email protected]

Atta-ur-Rahman & Shazia Anjum (Eds.) All rights reserved-© 2016 Bentham Science Publishers

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congenital and neonatal infections, neonatal hypoxic-ischemic encephalopathy (HIE) and neonatal jaundice, will develop long-term neurological impairments. Learning difficulties, developmental delay and cerebral palsy (CP) are amongst the most prevalent sequel observed in the surviving infants [2]. According to Mutch et al. [3], CP is defined as “an umbrella term covering a group of non-progressive, but often changing, motor impairment syndromes secondary to lesions or anomalies of the brain arising in the early stages of development”. This definition was further expanded by a report that highlighted the importance of nonmotor impairments, including behavioral and cognitive disturbances, epilepsy, and secondary musculoskeletal problems, in patients with CP [4]. The prevalence of CP is 2-3 cases per 1000 live births [5], although it can be as high as 39.5 per 1000 live births in very low birth weight infants (those weighting less than 1500 g at birth) [6]. Indeed, low birth weight is one of the potential risk factors associated with CP. Other risk factors include prematurity, maternal infection and inflammation, chorioamnionitis, maternal thyroid disease, iodine deficiency and multiple pregnancy. However, in many cases, the etiology of CP cannot be determined [7, 8]. Among the identifiable causes of CP, arterial ischemic stroke in the fetal or perinatal period can account for up to 22% of the cases in term and near-term infants, whereas intrapartum hypoxia-ischemia is responsible for up to 14.5% of the cases in term infants [9, 10]. Intrapartum hypoxia-ischemia is an important cause of HIE, a clinical syndrome characterized by the appearance of neurological symptoms in the first days of life of term newborns [11]. In 2010, the prevalence of HIE was 8.5 cases per 1000 live births. It was also estimated that, from the 125 million infants born in 2010, 413.000 would have neurodevelopmental impairments related to HIE [12]. Currently, hypothermia is the standard treatment for HIE. Therapeutic hypothermia initiated within the first 6 hours of life decreases the risk of death or major neurodevelopmental disability at 18-24 months in term and late preterm infants (≥35 weeks) with moderate or severe HIE. Moreover, the follow-up study of one large clinical trial showed that therapeutic hypothermia decreased the rate of cerebral palsy from 29% to 17%, at 6-7 years [13]. However, data compiled from six clinical trials and one pilot study indicate that a significant number of infants would still benefit from an adjuvant therapy. Remarkably, 6-7 infants need to be treated to observe the neuroprotective effect of hypothermia in one infant [14, 15]. Patients with CP are usually treated by a multidisciplinary team. Treatment includes physical, occupational and speech therapies, management of neurological

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complications and orthopedic care, as reviewed by Aisen et al. [16]. Difficulties regarding the early diagnosis of CP in young children, together with the fact that, in many cases, the brain insult occurs in the fetal life, make it more complex to implement neuroprotective strategies in CP. For this reason, rehabilitation strategies are primarily focused on taking advantage of neuroplasticity. Other possible therapeutic targets are the so-called tertiary mechanisms of brain damage, such as chronic inflammation, long-lasting epigenetic changes and the chronic inhibition of endogenous mechanisms of brain repair and regeneration [17]. Cell therapies represent a promising option for the treatment of neurological diseases. While pluripotent stem cells and neural stem cells/progenitors have the potential to replace lost neurons and glia, non-neural stem cells/progenitors are thought to exert their therapeutic action through paracrine mechanisms [18]. Umbilical cord blood (UCB) has emerged as a rich source of hematopoietic stem cells (HSCs) and progenitors for transplantation in hematopoietic disorders, as well as for regenerative medicine applications. This book chapter aims to provide an overview of the use of umbilical cord blood cells (UCBCs) in HIE and CP. UMBILICAL CORD BLOOD AS A SOURCE OF HEMATOPOIETIC STEM CELLS FOR TRANSPLANTATION UCB can be collected from the umbilical cord vein, before or after placental expulsion, into a sterile collection bag containing anticoagulant, immediately after birth. It has been estimated that more than 600.000 UCB units have been stored in at least 36 public banks and 150 private banks worldwide [19-21]. The landmark study of Søren Knudtzon and the early studies of Hal Broxmeyer et al. had laid the ground for the recognition of UCB as a rich source of transplantable HSCs, readily available for use [22, 23]. Moreover, there is now evidence that cryopreserved long-term repopulating and self-renewing UCB HSCs can be retrieved for up to two decades after storage [24]. A child with Fanconi anemia was the first recipient of a UCB HSCs transplantation in 1988. Since then, there have been more than 30,000 transplants in children and adults [19, 21, 25]. Currently, UCB HSCs transplantation is indicated for the treatment of several hematological and non-hematological diseases, such as hemoglobinopathies, acute and chronic myeloid and lymphoid leukemias, myelodysplastic syndromes, primary immunodeficiencies and inborn errors of metabolism. UCB HSCs elicit less acute and chronic graft-versus-host

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disease (GVHD), compared to bone marrow-derived HSCs. Therefore, it is possible to transplant UCB units that are mismatched at one or two HLA-loci. Other advantages of using UCB HSCs are the reduced risk of transmitting infection and the fact that UCB collection is simple and noninvasive, compared to bone marrow aspiration. However, there are still challenges regarding the transplantation of UCB HSCs in adult patients [19, 21, 25]. The first experimental demonstration of the therapeutic potential of transplanting UCBCs in a neurological disease came from the work of Norman Ende and collaborators [26, 27]. They have shown that human UCBCs transplantation was able to increase the life span of SOD mice, a model of amyotrophic lateral sclerosis. Best results were obtained when a large dose of UCB mononuclear cells (7x107 cells) was injected following sublethal irradiation. These studies were followed by numerous investigations showing the neuroprotective and neurorestorative effects of intravenously injected UCBCs in rodent models of stroke, without the need of irradiation [28-32]. Given that UCB can be easily collected and processed immediately after birth, it would be possible to prospectively collect and store UCBCs from infants at risk of HIE and CP. This, in addition to the increased number of parents who choose to store their newborn's UCB in private banks, would permit the autologous transplantation of UCBCs at anytime in the child's life. However, is there evidence that UCBCs transplantation improve the neurological function in models of HIE and CP? PRECLINICAL STUDIES: TESTING THE EFFICACY OF UCBCs TRANSPLANTATION IN ANIMAL MODELS OF HIE AND CP Meier et al. were the first to report the beneficial effects of transplanting human UCB mononuclear cells in the Rice-Vannucci animal model of HIE [33]. In their study, postnatal day 8 rats were treated with an intraperitoneal injection of 1 x 107 cells, 24 hours after the hypoxic-ischemic insult. Footprint analysis showed an alleviation of HIE-induced deficits in the contralateral hind paw, 13 days upon transplantation. Further studies from this group have shown that the therapeutic effects persisted for up to 43 days after transplantation. In addition, the treatment restored the spontaneous use of the contralateral forepaw to control levels and improved the muscle strength of the contralateral hind limb, as assessed by the

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cylinder test and the rope suspension test, respectively [34, 35]. Interestingly, there was no difference in the outcome, when the intraperitoneal delivery route was compared to the intrathecal injection of cells into the cerebellomedullary cistern [35]. Our group has also provided evidence for the therapeutic potential of intraperitoneally injected UCB mononuclear cells in the rodent model of HIE. In our study, 2 x 106 cells were transplanted 3 hours after hypoxia and the treated group exhibited a better performance in two developmental sensorimotor reflexes (negative geotaxis and cliff aversion reflex) in the first week after the insult [36]. In contrast, a recent study has shown that the intraperitoneal transplantation of 1 x 105 UCB CD34+ cells 48 hours after neonatal stroke could not improve the mice's performance on an open-field test. However, the authors did find sex-dependent effects of the treatment on the neural and glial responses to stroke [37]. The intravenous delivery of UCB mononuclear cells was also effective in protecting hypoxic-ischemic rats from developing neurological deficits. Yasuhara et al. injected 1.5 x 104 cells via the jugular vein, 7 days after neonatal hypoxiaischemia in rats [38]. The Rotarod performance test and the elevated body swing test were performed 7 and 14 days after transplantation, showing that the treatment was able to improve the motor coordination and to attenuate the motor asymmetry. However, the best results were obtained when mannitol was injected immediately after transplantation, to increase the permeability of the blood-brain barrier. Using a similar protocol (4 x 105 cells were intravenously injected, 7 days after hypoxia-ischemia in neonatal rats) a recent study reported that the cell therapy improved the performance in the limb placing test and in the corner turn test at 7 and 14 days after transplantation [39]. In contrast, a dose-response study showed that a higher dose (1 x 108 cells, intravenously injected 24 hours after the insult) was necessary to protect against long-term spatial memory deficits after HIE, as evaluated by the Morris water maze, 8 weeks after transplantation [40, 41]. Accordingly, Bae et al. have shown that this high dose of UCB mononuclear cells (1 x 108 cells, intravenously transplanted 24 hours after the insult) can exert long-lasting beneficial effects on cognitive and motor functions after HIE. Treated animals had a better performance in several tests (cylinder test, passive avoidance, forced swim test and light/dark exploration) for up to 10 weeks after the insult [42]. In this study, animals were treated daily with the immunosuppressive drug cyclosporine A until the end of the protocol, to overcome the potential immune response to xenotransplantation. Indeed, the fact that human cells could elicit undesirable immune responses in rodents is an important limitation that has been

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overlooked by nearly all the preclinical studies in the field, probably because UCB HSCs are considered less immunogenic than HSCs from other sources. In a recent study, Tsuji et al. [43] employed another strategy to minimize the unwanted immune responses to xenotransplantation. Human UCB CD34+ cells were intravenously transplanted via the femoral vein 48 hours after middle cerebral artery occlusion in severe combined immunodeficiency (SCID) postnatal day 12 mice, which lack functional B and T lymphocytes. They found that, unlike vehicle-treated mice, the animals that received CD34+ cells exhibited no significant differences to sham-operated mice in the Rotarod performance test, at 6 weeks post-insult. The intra-arterial route has also been used to deliver UCB mononuclear cells in the animal model of HIE. A minimum dose of 1 x 107 cells administered into the contralateral common carotid artery 24 hours after the insult was capable of improving learning and long-term spatial memory in the Morris water maze paradigm, at 9 weeks following the insult in rats [44]. Besides the studies in rodents, a recent report has employed a model of CP in rabbits to investigate the therapeutic effects of human UCBCs [45]. Fetal hypoxia-ischemia was induced by uterine ischemia at 22 days of gestation (E22). Newborn kits received an intravenous injection of either a high dose (5 × 106 cells) or a low dose (2.5 × 106 cells) of UCBCs at 4 h after birth on E31, via the external jugular or anterior abdominal vein. Neurobehavioral examination revealed that both doses could alleviate several sensorimotor dysfunctions, such as postural and locomotor deficits and dystonia, although the higher dose was more efficient in reducing such deficits. The authors have used the forced swimming test to show that, compared to control rabbits, the treated animals had a different pattern of joint movements, suggesting that the motor improvements could be explained by compensatory mechanisms. Taken together, these studies (which are summarized in Table 1) have clearly demonstrated that, irrespective of the route of administration, UCB mononuclear cells promote neurological recovery or prevent the appearance of long-lasting neurological deficits, when transplanted within the first 7 days after the hypoxicischemic insult. The intravenous and the intraperitoneal routes were the most extensively studied and the therapeutic effect was dose-dependent. The putative mechanisms underlying such effect will be discussed in the next session.

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Table 1. Preclinical studies.

Cell Type

Dose, Route, Timing

Animals (Age; Sex)

Model

Engraftment (days after transplantation)

HUCBCs

1 x 107 cells, IP, 24 hours after HI

Wistar rats (P7; NA)

Hypoxia-Ischemia (80 min)

Many cells in the ischemic hemisphere (13d)

Hypoxia-Ischemia (150 min)

Few cells in the ipsilateral hippocampus (14d)

HUCBCs 1,5 x 104 cells, or IV (via the HUCBCs jugular vein), 7 + days after HI Mannitol

SpragueDawley rats (P7; NA)

Improved Increased levels of GDNF, motor BDNF and NGF in the outcome brain. Increased (rotarod and hippocampal CA1 elevated body dendritic density. swing test)

[38]

Decreased striatal neuronal death. Decreased microglial activation in the cerebral cortex.

[36]

Hypoxia-Ischemia (120 min)

Few cells in the brain (24h, 1 and 3 weeks)

No effects in spatial memory deficits (Morris water maze)

No effect in the infarct size.

[41]

Wistar rats (P7; NA)

Hypoxia-Ischemia (80 min)

Many cells in the ischemic hemisphere (1d and 13d)

NA

Increased SDF-1 expression in the ipsilateral hemisphere.

[46]

Hypoxia-Ischemia (80 min)

Cells in the ischemic hemisphere (41d)

Improved motor outcome (footprint analysis and cylinder test)

Restoration of the dimensions of cortical maps and receptive fields. The lesion-induced hyperexcitability was prevented.

[34]

Many cells in the ischemic hemisphere, but it decreased with time (1 to 10 weeks)

Improved functional outcome (cylinder test, passive avoidance, forced swim test and light/dark exploration task)

Increased number of neurons in the neocortex. Increased number of microglial cells in the periventricular striatum. Increased number of neuroblasts in the subventricular zone. Increased levels of MIP1 mRNA in the brain.

[42]

Decreased cerebral atrophy, but only with medium and high doses.

[40]

Decreased number of apoptotic cells and

[55]

HUCBCs

HUCBCs

1 x 107 cells, IP, 24 hours after HI

HUCBCs

[33]

Wistar rats (P7; NA)

1 x 107 cells, IV; 24 hours after HI

1 x 107 cells, IV, 24 hours after HI

NA

Few cells in the ipsilateral cerebral cortex and striatum (2d)

ListerHooded rats (P7; male)

HUCBCs

Reference

Hypoxia-Ischemia (90 min)

2 x 106 cells, IP; 3 hours after HI

1 x 10 cells, IP, 24 hours after HI

Cellular/Molecular Effect (treated versus control)

Improved performance in two neonatal reflexes (cliff aversion and negative geotaxis)

HUCBCs

7

Functional Outcome (treated versus control) Improved motor outcome (footprint analysis)

Wistar rats (P7; equal numbers of male and female)

SpragueDawley rats (P7; NA)

Hypoxia-Ischemia (90 min)

HUCBCs

1 x 106, 1 x 107 or 1 x 108 cells, IV; 24 hours after HI

Wistar rats (P7; NA)

Hypoxia-Ischemia (120 min)

Cells in the cerebral cortex and hippocampus (7d)

Improvement of spatial memory deficits, but only with the highest dose (Morris water maze)

HUCBCs

1 x 107 cells, IP, 24 hours

Wistar rats (P7;

Hypoxia-Ischemia (80 min)

NA

NA

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Table 1: contd…

after HI

HUCBCs

1 x 107 cells, IP or IT, 24 hours after HI

106, 3 x 106 or 107 cells, IP or IV, 1 or 24 HUCBCs hours following ibotenate injection

HUCBCs

HUCBCs

HUCBCs

HUCBCs (CD34+)

1 x 107 cells, IP, 24 hours after HI

3 x 106 cells, ICV, 24 hours after HI

1 x 106 or 1 x 107, IA, 24 hours after HI

1 x 105 cells, IV, 48 hours after stroke

increased number of neurons in the ipsilateral hemisphere. Increased expression of Tie-2 and occludin in the ipsilateral hemisphere. Increased expression of BDNF and VEGF in the ipsilateral hemisphere.

NA)

Wistar rats (P7; both genders)

Hypoxia-Ischemia (80 min)

Cells in the ischemic hemisphere (13d)

Decreased microglial activation and decreased Improved astrocytic activation in the motor ipsilateral hemisphere. outcome (toe Reduced expression of distance, rope connexin 43 in the suspension ipsilateral hemisphere. test and Reduced expression of cylinder test) SHP-1 in the injured hemisphere in the chronic phase.

[35]

[47]

SpragueDawley rats (P5; both genders

Excitotoxic brain lesion (intracerebral injection of ibotenate)

Cells detected in the brain after IV injection (at 2 hours, but not at 24 hours), but not after IP injection in control pups

NA

Decreased injury in the cortex (only with 106 cells, IP, 1 hour following ibotenate injection). Increased injury in the white matter (only with 107 cells, IP, 1 or 24 hours after ibotenate injection). Increased number of macrophages/microglia (only with 107 cells, IP). The intravenous injection of HUCBCs had no effect on excitotoxic brain lesions.

Wistar rats (P7; NA)

Hypoxia-Ischemia (80 min)

NA

NA

Reduced serum levels of IL-1. Decreased microglial activation.

[57]

[56]

SpragueDawley rats (P7; both genders)

Wistar rats (P7; NA)

Hypoxia-Ischemia (120 min)

NA

NA

Increased proliferation of endogenous progenitors in the subventricular zone. Increased expression of Shh and Gli1 in the ischemic hemisphere. Decreased neuronal loss in the hippocampus and cerebral cortex.

Hypoxia-Ischemia (120 min)

Human β-actin gene detected up to 30d in the ischemic hemisphere.

Improvement of spatial learning and memory, only with the highest dose (Morris water maze)

No effect on cerebral atrophy.

[44]

Few cells in the brain (24 hours)

Compared to the shamoperated group, the Rotarod performance was impaired only in the untreated mice

Increased brain perfusion. Increased vessel diameter in the peri-stroke region. Decreased stroke volume.

[43]

Neonatal Stroke SCID (electrocoagulation mice (P12; both of the left middle genders) cerebral artery)

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Table 1: contd…

6

HUCBCs

3 x 10 cells, ICV, 24 hours after HI

SpragueDawley rats (P7; both genders)

5 x 106 cells or 2.5 x 106 cells, IV (via the jugular or anterior New abdominal Zealand vein), 4 hours white after birth rabbits HUCBCs (E31/P0) or 5 x (E22; NA; 106 feridex- or and E29; gadofluorineNA) labeled cells, IV, after Csection at E30 (for in vivo MRI)

5

2 x 10 cells, HUCBCs IV, 7 days after HI

HUCBCs (CD34+)

HUCBCs

5

1 x 10 cells, IP, 48 hours after ischemia

1 x 107 cells, IP, 6 hours after HI

Wistar rats (two weeks old; NA)

[58]

NA

[45]

Improved motor outcome (limb- placing test and corner turn test)

Decreased lesion volume.

[39]

No effect (open field test)

Increased proliferation of endogenous progenitors of the subgranular zone of the hippocampal dentate gyrus (only in males). Higher GFAP expression in the contralateral subventricular zone (only in females). No effect on hemispheric or hippocampal atrophy.

[37]

The tests were not able to detect differences Decreased apoptosis, between oxidative stress and shammicroglia activation in the operated and granule cell layer of the injured hippocampal dentate animals (gait gyrus (24 hours after HI). analysis and active avoidance test)

[54]

Hypoxia-Ischemia (120 min)

NA

NA

Improved motor outcome

Intrauterine Ischemia (40 min at E22 and 32 min at E29)

Some feridexlabeled cells could be detected in the choroid plexus, but not in the brain parenchyma (24h). Human DNA detected in the cerebral cortex and thalamus of P5 kits.

Hypoxia-Ischemia (60-120min).

+

BrdU cells in the ischemic region (14d)

CD1 mice Non-specific (P12; Neonatal Stroke labeling with equal (unilateral common antibodies against numbers carotid artery HuNu and human of male ligation) CD34 (9d) and female)

Wistar/ST rats (P7; NA)

Increased neurogenesis. Increased expression of Shh, Gli1 and Ngn1 in the ischemic hemisphere. Decreased expression of BMP4 in the ischemic hemisphere.

Hypoxia-Ischemia (60 min)

NA

(locomotion, tone, posture, righting reflex and dystonia). Milder improvement with the low dose.

BrdU: 5-bromo-2'-deoxyuridine; GFAP: glial-fibrillary acidic protein; HI: hypoxia-ischemia; HUCBCs: human umbilical cord blood cells; ICV: intracerebroventricular; IP: intraperitoneal; IT: intrathecal; MNCs: mononuclear cells; NA: not available;

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PRECLINICAL STUDIES: INVESTIGATING THE DISTRIBUTION OF TRANSPLANTED CELLS Conflicting results have been reported regarding the distribution of intraperitoneally injected UCBCs in animal models of HIE and neonatal stroke. Meier et al. have shown that large numbers of donor-derived cells could be found in the brain, up to 13 days after transplantation. Human cells were identified by the expression of HLA-DR and were localized in the injured regions, where there was a strong astroglial response. These areas also corresponded to sites with high stromal-derived factor-1 (SDF-1) expression and it was shown that a large fraction of UCBCs expressed CXCR4, one of the receptors for this chemokine. Moreover, the administration of anti-SDF-1 neutralizing antibodies reduced the migration of transplanted cells to the hypoxic-ischemic brain [33, 35, 46]. In contrast, in our study, very few UCBCs were found in the injured striatum and cerebral cortex, 2 days after intraperitoneal transplantation, as assessed by immunohistochemistry with an anti-human nuclei antibody [36]. Among the possible explanations for these discrepant results are the differences in the number of administered cells, the timing of transplantation and the methodology used for the identification of human cells. Recently, the non-specific labeling of rodent cells by anti-HLA and anti-human nuclei antibodies has been reported, indicating that previous studies may have overestimated the number of human UCBCs in the rodent brain [37, 47]. Furthermore, one study has shown that intraperitoneally transplanted UCBCs fail to enter the systemic circulation of control rat pups. In this study, polymerase chain reaction (PCR) for a human-specific gene fragment of CEP68 was performed on blood and lung samples, from 5 minutes to 5 days after transplantation. Nevertheless, elevated serum levels of several cytokines were observed 24 hours after transplantation, suggesting that the cells could induce a systemic immune response [47]. There are convincing evidence that intravenously transplanted UCBCs can home to the brain, although in very low numbers. Small amounts of UCBCs were transiently detected in the brain within the first two hours after injection into the right jugular vein of control rat pups [47]. In three other studies, very few human UCBCs could be detected by the anti-human nuclei antibody in the hypoxicischemic rat brain, for up to 10 weeks after transplantation [38, 40, 41, 42]. Even when human CD34+ UCBCs were transplanted in SCID mice, the number of cells labeled by the anti-human nuclei antibody in the ischemic brain was very low [43]. This suggests that there may be other factors that limit the

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homing/engraftment of UCBCs into the brain, besides the immune response against xenografts. By PCR analysis it was also possible to detect the human βglobin and the human growth hormone 1 genes in the hypoxic-ischemic rat brain, 7 and 13 days after UCBCs transplantation, respectively [40, 46]. In addition, small amounts of human DNA (a portion of the human mitochondrial cytochrome b region) were found in samples from the cortex and thalamus of P5 kits, a few days after human UCBCs transplantation in hypoxic-ischemic near-term rabbits. The high sensitivity of the PCR assay was highlighted by the fact that as little as 40 human cells could be identified in 50 mg of rabbit brain tissue. This study has also used magnetic resonance imaging to track UCBCs labeled with superparamagnetic iron oxide particles or gadofluorine M, following injection either at 24 or 72 hours after hypoxia-ischemia in E29 rabbits. Intravenously transplanted cells were detected in brain blood vessels and in the choroid plexus, but not in the brain parenchyma, 24 hours after injection in kits [45]. Nested PCR has also been employed to detect the human β-actin gene in the injured brain hemisphere for up to 30 days following the intra-arterial transplantation of UCBCs in hypoxic-ischemic rats [44]. However, it is still unclear whether the intra-arterial route is capable of improving the homing of UCBCs to the brain and current evidence favors the use of the intravenous route of administration. Taken together, these studies suggest that UCBCs can improve the neurological function after HIE, despite the low number of transplanted cells found in the brain parenchyma. Similarly, the neuroprotective action of intravenously injected UCB mononuclear cells following stroke occurs independently of cell infiltration in the ischemic brain parenchyma [30]. Such results exclude the possibility that UCBCs were replacing lost neurons. Indeed, although it has been proposed that HSCs could differentiate into neuronal cells [48, 49], it soon become evident that cell fusion rather than terminal differentiation could explain these early findings [50]. Also, it is still unclear whether the small number of cells that enter the brain parenchyma could modulate the local environment through paracrine mechanisms. Alternatively, the therapeutic effects could be mediated by the release of neurotrophic factors, immune mediators and/or extracellular vesicles in the brain circulation or by the transient integration of transplanted cells in other organs. For instance, it has been reported that a large number of UCBCs migrate to the lungs and liver within the first hours after their intravenous administration

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in neonatal rats [47]. A similar pattern of cell distribution has been observed following the systemic injection of bone marrow-derived mononuclear cells in experimental animals and in patients with stroke [51, 52]. In this regard, it has been shown that the activation of intravenously injected mesenchymal stem cells in the lungs is a crucial step for the healing of the infarcted myocardium [53] and that UCBCs can exert a potent immunomodulatory effect in the spleen after ischemic stroke [32]. PRECLINICAL STUDIES: INVESTIGATING THE MECHANISMS OF ACTION OF TRANSPLANTED CELLS Several studies have demonstrated the neuroprotective potential of UCBCs. We have observed a decreased number of degenerating neurons and of cells dying via the caspase-3 pathway in the striatum of treated rat pups, 2 days after the hypoxicischemic insult [36]. Recently, the neuroprotective effect afforded by the early treatment with UCBCs has also been reported in the subgranular layer of the hippocampal dentate gyrus. The number of apoptotic cells (expressing activated caspase-3 or apoptosis-inducing factor) was lower in the treated group, 24 hours after the injury [54]. Furthermore, one study has observed a significant reduction in the number of activated caspase-3 expressing cells in ten regions of the ipsilateral hemisphere at a later time point (13 days after transplantation), suggesting that the treatment could decrease the delayed cell death that occurs in the hypoxic-ischemic brain [55]. Conflicting results have been reported regarding the effect of UCBCs on preserving brain volume in rodent models of neonatal hypoxia-ischemia and neonatal stroke. While some studies have found no differences in brain volume after the treatment [33, 34, 37, 42, 44, 54], other investigators have observed a less pronounced tissue loss [39, 40, 43]. However, Bae et al. showed that, despite the lack of change in brain volume loss, the cell therapy increased the number of surviving neurons in the hypoxic-ischemic brain [42]. In this context, Wang et al. have shown that the intracerebroventricular administration of UCBCs increased the number of surviving neurons in the cerebral cortex and in the hippocampal CA1 region in a model of HIE. This effect was abrogated by the coadministration of cyclopamine, an antagonist of sonic hedgehog signaling [56]. Besides their neuroprotective action, human UCBCs seem to improve neural function by reestablishing the sizes of cortical maps and receptive fields in the

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primary somatosensory cortex [34]. Although the cellular and molecular basis of this effect still needs to be characterized, it has already been shown that the treatment with UCBCs partially restores the dendritic density in the CA1 hippocampal region [38]. These beneficial effects on neuronal survival and functioning are probably partially mediated, or at least facilitated, by the action of UCBCs on glial cells. Intraperitoneally injected UCBCs attenuated the chronic activation of astrocytes in response to the hypoxic-ischemic insult and affected the formation of the glial scar [35]. Treatment with UCBCs also modulated the activity of microglial cells, the resident cells of the innate immune system in the central nervous system. The cell therapy decreased the number of CD68-positive macrophages/activated microglia in the cerebral cortex [36] and in the hippocampal cell layer [54], as well as reduced the expression of CD68 in brain homogenates [57], in the acute phase of the injury. Interestingly, the intrathecal and intraperitoneal routes of cell administration were equally effective at decreasing the area covered by CD68positive cells in the subacute and chronic phases of the injury [35]. In contrast, Bae et al. found an increase in the number of microglia/macrophages in the striatum, at 1 week, but not at 10 weeks, after transplantation [42]. Taken together, these results indicate that UCBCs transplantation affects the cellular immune response and the composition of the glial scar in the hypoxic-ischemic brain, although it is still uncertain how these changes contribute to the functional outcome. In addition, there are scarce data regarding how UCBCs modulate the systemic inflammatory response in neonatal animals. While Dalous et al. [47] have shown that the intraperitoneal administration of UCBCs in control rat pups led to an increase in serum levels of several pro-inflammatory and antiinflammatory cytokines (including IL-1α, IL-1β, IL-6, IL-4 and IL-10), Rosenkranz et al. have demonstrated that intraperitoneally transplanted UCBCs decreased the serum levels of IL-1α in hypoxic-ischemic rat pups [57]. In both cases, the analyses were performed 1 day after transplantation. Human UCBCs might also act on endothelial cells and perivascular cells. In a recent study, the cell therapy increased the area with adequate cerebral blood flow in the peri-infarct region at 24 hours after transplantation, but not at other time points, in a model of neonatal stroke. This effect was accompanied by a longlasting change on the mean diameter of cerebral blood vessels in the peri-infarct region, which were significantly larger in the treated group [43]. Furthermore, another study has observed that the treatment with UCBCs increased the expression of the tight junction protein occluding and of the tyrosine kinase with

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immunoglobulin-like and EGF-like domains (Tie)-2, one of the angiopoietin receptors, in the hypoxic-ischemic hemisphere [55]. Thus, the pro-angiogenic effect of UCBCs and their potential to regulate the blood-brain barrier are topics that warrant further investigation. Treatment with UCBCs may stimulate the proliferation of neural stem cells/progenitors and the generation of new neurons in the subventricular zone (SVZ) in the animal model of HIE [56, 58]. The SVZ is one of the neurogenic niches that persist throughout life, generating new neurons that migrate to the olfactory bulb, where they replace local interneurons in rodents. In humans, despite the presence of neural stem cells in the adult SVZ, the number of newly generated neurons that migrate to the olfactory bulb and to the ventromedial prefrontal cortex is drastically reduced within the first 6 months of postnatal life [59]. Upon a hypoxic-ischemic brain injury in rats, a large number of SVZ neuroblasts migrate to the striatum and to the cerebral cortex. Interestingly, this regenerative process persists for up to 5 months, although only 15% of these neuroblasts survive and mature [60]. However, these newly formed striatal neurons seem to be committed to a very specific phenotype, differentiating into calretinin-positive interneurons in the damaged striatum [61]. These and other studies point to the increased regenerative capacity of the neonatal brain, which represents a promising therapeutic target for HIE and CP. For this reason, the mechanisms by which UCBCs affect the generation of new neurons, their survival and integration into functional neural circuits, still need to be characterized. As suggested by Taguchi et al., it is possible that UCBCs could provide a favorable environment for this regenerative response, at least partially, by acting on the cerebral vasculature [29]. It is conceivable that UCBCs could exert these therapeutic effects by the secretion of growth factors and inflammatory mediators. Soluble factors released by UCB mononuclear cells were shown to protect neurons from hypoxia-induced apoptosis in vitro [62]. Three independent studies have shown that IL-6, IL-8 and CCL2 were the most abundant cytokines/chemokines found in the culture supernatant of UCB mononuclear cells [42, 63, 64]. The secretome of cultured UCBCs also includes several growth factors, such as brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), epidermal growth factor (EGF), granulocytecolony stimulating factor (G-CSF), platelet-derived growth factor subunit B (PDGF-B) and hepatocyte growth factor (HGF) [62, 64, 65]. Additionally, intravenously or intraperitoneally transplanted UCBCs increased the expression of BDNF and other growth factors in the brain of hypoxic-ischemic rats, although it

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was unclear whether the transplanted cells secreted these growth factors or induced endogenous cells to produce and release them [38, 55]. Recent evidence indicates that the intercellular communication between transplanted UCBCs and host cells might also involve the release of extracellular vesicles, such as exosomes and microvesicles. Extracellular vesicles could act as carriers for the transfer of functional mRNAs, miRNAs, proteins and bioactive lipids to nearby cells or even to distant cells through the blood circulation [66]. It has been demonstrated that UCB CD133+ cell-derived microvesicles exert a strong proangiopoietic effect on human umbilical vein endothelial cells in vitro and stimulate angiogenesis in vivo. These microvesicles expressed the mRNA of numerous cytokines, chemokines and growth factors, including IL-8, HGF and vascular endothelial growth factor (VEGF) [67]. Finally, it remains to be determined whether the transfer of UCBCs organelles (e.g., mitochondria and ribosomes) to damaged cells might occur and contribute to brain repair [68, 69]. In conclusion, several groups have demonstrated that UCBCs can induce neuroprotection and stimulate mechanisms of brain repair, plasticity and regeneration. However, there is still a lack of understanding about how the transplanted cells exert these effects at the molecular level. CLINICAL TRIALS: TESTING THE FEASIBILITY, SAFETY AND EFFICACY OF UCB CELL THERAPY The promising results observed in the animal models of HIE, intrauterine hypoxia and neonatal stroke and the accumulated experience with UCB HSC transplantation in pediatric patients have stimulated the initiation of several clinical trials. Phase I studies, testing the feasibility and the safety of UCB cell therapy in patients with CP or HIE, were initiated at the end of the last decade and the number of clinical trials had increased in the last years. Table 2 summarizes the main characteristics of ongoing clinical trials using UCBCs in patients with CP or HIE, registered at ClinicalTrials.gov. Different groups are testing distinct therapeutic approaches, using autologous or allogeneic cells and combining the cell therapy with other treatments, such as erythropoietin administration and hypothermia. Some of these studies have been completed and have shown the safety and feasibility of the cell therapy. Preliminary evidence for the beneficial effects of UCBCs transplantation has also been reported.

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Table 2. Ongoing clinical trials registered at ClinicalTrials.gov. Pathology

First received

Location/Country

Age

Procedure

Status

Phase

Reference

HIE

2008

Duke University Medical Center, USA

Newborns (up to 14 days after birth)

Recruting

I

NCT00593242 [74]

HIE

2012

Hospital Universitario Dr. Jose E. Gonzalez, Mexico

Unknown 37 to 42 Intravenous infusion of Weeks freshly isolated CD34+ cells (within the (autologous) first 48 hours after birth)

-

NCT01506258

HIE

2012

National University Hospital, Newborns Infusion of freshly isolated Singapore (up to 3 days cells (autologous) after birth)

Recruting

I

NCT01649648

HIE

2014

Neonatal Encephalopathy Consortium, Japan

Newborns 3 infusions of freshly (up to 3 days isolated UCB (volume- and after birth) red blood cell-reduced UCB)

Recruting

I

NCT02256618

CP

2010

Georgia Regents University, USA

1 to 12 Years 1 X 107 cells per kg body weight. (autologous)

Recruiting

I/II

NCT01072370

CP

2010

Duke University Medical Center, USA

12 Months to ≥ 1 x 107 cells/kg body 6 Years weight (autologous)

Ongoing

II

NCT01147653

CP

2010

Sung Kwang Medical Foundation, Republic of Korea

10 Months to Intravenous infusion of > 3 10 Years x 107 celss/kg UCB (allogeneic) Erythropoietin Injection

Completed

-

NCT01193660 [75]

CP

2012

Bundang CHA Hospital, Republic of Korea

6 Months to 20 Years

Intraveously or intraarterially infusion of UCB (allogeneic)

Completed

-

NCT01639404

CP

2013

Bundang CHA Hospital, Republic of Korea

Up to 15 Years

≥ 3 x 107 cells/kg (allogeneic)

Recruiting

-

NCT02025972

CP

2013

Bundang CHA Hospital, Republic of Korea

10 Months to ≥ 3 x 107/kg (allogeneic) 6 Years Erythropoietin Injection

Recruiting

-

NCT01991145

CP

2013

Charles Cox, The Children's Fund Distinguished Professor, Department of Pediatric Surgery,The University of Texas Health Science Center, Houston, USA

2 Years to 10 < 10 million cells/kg Years (autologous)

Recruiting

II

NCT01988584

CP

2014

Bundang CHA Hospital, Republic of Korea

19 Years or over

Recruiting

-

NCT02236065

1-4 infusions of 5 x 107 cells/kg (autologous)

Infusion of UCB (allogeneic) G-CSF injection

CP: cerebral palsy; HIE: hypoxic-ischemic encephalopathy; UCB: umbilical cord blood

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We found 9 articles in the English language reporting on 8 trials, 7 of them of UCB cell therapy for CP and 1 of UCB cell therapy for HIE, with a total of 318 treated patients (Table 3). Seven trials carried out intravenous injections and 1 trial carried out intravenous or intra-arterial injections. Table 3. Clinical trials with published results. Study Country Study design Route Cell type Reference

[70]

United States

Nonrandomized, open-label

Autologous IV UCBCs

No. of patients (No. of controls)

Patients’ Administration of Type of lesion Age range immunosuppressants (mean) Different neurological 6 Days to disorders, 9.5 Years including cerebral palsy (76%)

No

No. of Followinjected up cells

0.1 – 184 (140 13.3 x with cerebral 7 10 /kg (2 palsy) x 107/kg)

-

Thailand Case report

IV

Autologus UCBCs

Cerebral palsy

19-32 Months

No

2 (no controls)

5.8 x 108 7-28 - 6.6 x months 108

Nonrandomized, open-label

IV

Autologous UCBCs

Cerebral palsy

23-91 Months (55 Months)

No

20 (no controls)

5.5 x 6 107/kg months

[73-74] Germany Case report

IV

Autologous UCBCs

Cerebral palsy

2.5 Years

No

1 (no controls)

5.7 x 108 7 years

Cerebral palsy

10 Months to 10 Years (39.8 Months)

Yes

No

23 (no controls) 80 (no controls)

[71]

[72]

South Korea

[76]

Double-blind, randomized, South placeboIV Korea controlled trial

[75]

United States

Nonrandomized, open-label

IV

34-40 Autologous Hypoxic-Ischemic Weeks (38 UCBCs Encephalopathy Weeks)

[78]

Russia

Nonrandomized, open-label

IV

Allogeneic UCBCs

Cerebral palsy

1-12 Years

No

[77]

South Korea

Double-blind, randomized, IV, Allogeneic placeboIA UCBCs controlled trial

Cerebral palsy

6-216 Months

Yes

Allogeneic UCBCs

96 patients (33 received UCB-MNCs 3x + rhEPO; 31 107/kg only rhEPO and 32 placebo) 1-5 x 107/kg

1 year

1 year

3 x 108 – 3-36 4 x 108 months

18 patients 1.0-7.1 x 6 (18 controls) 107/kg months

IA, intra-arterial; IV, intravenous; MNCs, mononuclear cells; rhEPO, recombinant human erythropoietin; UCBCs, umbilical cord blood cells

PUBLISHED THERAPY

CLINICAL

TRIALS:

AUTOLOGOUS

UCB

CELL

Sun et al. from Duke University (USA), reported on a safety and feasibility study of autologous UCB cell infusions for patients with acquired neurological diseases [70]. A total of 184 children age 6 days-9.5 years with acquired neurologic diseases were included, 140 patients (76%) of which had CP. Pre-cryopreservation total nucleated

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cell count (TNC) had to be more than 1 x 107 cells/kg. Only 14 patients received two infusions. No neurodevelopmental results were formally evaluated, but the infusions were feasible and safe. Infusion reactions were observed in 3 patients, but were responsive to medical therapy and to the discontinuation of the infusion. Papadopoulos et al. reported the transfusion of autologous UCB cells into 2 toddlers with CP in the Vejthani Hospital in Thailand [71]. The first patient, who was unable to stand or walk at 19 months of age and was diagnosed with spastic diplegia, received an intravenous infusion of 6.6 x 108 TNCs over 30 minutes. Twelve months after UCB cell infusion, G-CSF was administered subcutaneously for a total of 10 days, for the mobilization of bone marrow HSCs. The second patient had a diagnosis of spastic diplegia. At 32 months of age, he received a transfusion of autologous UCBCs (5,0 × 108 TNCs). In the five days prior to the UCBCs transfusion, as well as in the five days following UCBCs infusion, the patient received subcutaneous low-doses of G-CSF. The authors reported that both patients had an improvement on their Gross Motor Function Classification System from level III at baseline to level I at the end of follow-up. No significant side effects were observed, except for mild and transient nausea lasting less than 1 minute during the infusion. Lee et al. from the Hanyang University Medical Center in South Korea, have performed a non-randomized, open-label trial of autologous UCBCs transplantation for patients with CP [72]. Twenty patients aged 2-10 years and diagnosed with CP were included. Patients with epilepsy were excluded. The patients were followed up for 24 weeks after transplantation, but additional medication or rehabilitation programs were not provided. Approximately 5.5 ± 3.8 × 107 TNCs/kg were intravenously infused over 10-20 minutes. Minor side effects, such as temporary nausea, hemoglobinuria and urticaria, were reported in 5 patients, but were easily controlled. Brain perfusion single-photon emission computed tomography (SPECT) with Technetium-99m labeled ethyl cysteinate dimer (99mTc-ECD), and brain brain magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) were carried out at baseline and after 24 weeks to evaluate functional changes in the brain. Only five patients exhibited improvements in the neurodevelopmental tests. In two of these patients, who had mild forms of hemiplegia, the perfusion of the left thalamus was improved. However, the reduced perfusion of the unilateral thalamus was expanded to the contralateral thalamus in 6 patients. Neuroimaging findings revealed an overall increase of the fractional anisotropy value in 3 regions of interest (right temporal,

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corpus callosum, and right periventricular white matter), compared to the pretreatment value. Jensen and Hamelmann, from Ruhr-University Bochum (Germany), published a case report of autologous UCBCs transfusion in a patient with CP due to a global ischemic brain damage at the age of 2.5 years [73]. The child received an intravenous injection of 5.7 × 108 UCB mononuclear cells, nine weeks after the cardiac arrest, and was followed-up for 40 months. The authors concluded that, even though causality could not be established, it was difficult to explain the patient’s recovery by intense active rehabilitation alone (physio- and ergotherapy). More recently, Jensen published a review on autologous UCB therapy for infantile cerebral palsy in which he mentioned that the patient described in the case report had entered primary school and was still using a posterior gait trainer for ambulation, 4.5 years after transplantation [74]. Cotten et al., from Duke University (USA), published a phase 1, non-randomized, open-label trial using UCBCs therapy for infants with HIE [75]. 23 infants were enrolled, cooled and received cells, while 82 infants did not have cells collected but were cooled. Up to 4 infusions of 1-5 x 107 cells/kg were performed. The first infusion was performed 3.9 to 220 hours after UCB collection, but 6 (26%) infants received the first injection within 6 hours. During weekdays, the average time was lower, ranging from 3.9 to 12 hours. Only 2 infants were enrolled in the first year of the study. In the second year, the authors successfully implement a new policy, in which UCB collection at delivery was considered for all obstetric emergencies. Infusion reactions were not observed. None of the patients that received UCBCs died prior to discharge, while 11 (13%) patients of the concurrent cooled group died, although this difference was not statistically significant. Of the 18 UCBCstreated infants with developmental outcome at 1 year, 13 (72%) had Bayley scores ≥85 in all test domains, whereas 19 (41%) patients of the concurrent cooled group had Bayley III scores ≥85 in all 3 domains. At 14 months, two patients who were treated with UCBCs died. One of them, who was diagnosed with cytomegalovirus infection during his 5th postnatal week, died of an acute gastroenteritis with hypovolemic shock. The other patient, who was diagnosed with a chromosome 17p12 deletion as well as with Wolf–Parkinson–White syndrome, died of respiratory syncytial virus pneumonia. Neither of them could be scored on the Bayley III at 1 year, due to their severe impairments. These results indicate that

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UCBCs transplantation in infants with HIE is feasible and safe. The authors are planning a phase II randomized trial. PUBLISHED CLINICAL TRIALS: ALLOGENEIC UCB CELL THERAPY Min and collaborators, from the CHA University in South Korea, published a paper reporting on a double-blind, randomized, placebo-controlled clinical trial investigating the effects of allogeneic UCBCs therapy for CP [76]. 105 children with CP aged between 10 months and 10 years were enrolled and randomly assigned to one of the experimental groups. The first group was treated with recombinant human erythropoietin (rhEPO) and UCBCs (potentiated UCB, or pUCB group). The second group received rhEPO, as well as autologous peripheral blood as placebo (EPO group). The control group was treated with autologous peripheral blood and normal saline as placebos. The pUCB group was also treated with the immunosuppressant cyclosporine for 4 weeks, while the other groups received intravenous albumin and orally given orange juice as placebos. UCBCs transplantation consisted of a single intravenous injection of at least 3 x 107 TNCs/kg, matched for at least four of six HLAs. All patients participated in a rehabilitation program for 6 months and were followed up for 1year. 18F-fluorodeoxy-glucose positron emission tomography (18F-FDG-PET/CT) and brain MRI with DTI were performed at baseline and at 2 weeks and 6 months following the therapy. The authors stated that nine patients presented serious adverse events, although they were equally distributed among the groups. One patient of the pUCB group died at 14 weeks after the treatment, but the authors considered that the death was not related to the therapy. Serious side effects were not observed, but pneumonia and irritability were more common in the pUCB group and hemoglobin levels were elevated in children that received rhEPO. The pUCB group showed greater improvements in several functional assessments. Higher TNC and a higher number of infused CD34+ cells were associated with better outcomes. Neuroimaging findings revealed significant fractional anisotropy increments at six regions of interest in the pUCB group, which were not observed in the other groups. Kang et al., from CHA University in South Korea, the same group that published the previous report, performed a second clinical trial using UCBCs for CP [77]. A total of 18 subjects (aging 6 months to 216 months) with CP received UCBCs therapy, 16 of them by intravenous and 2 by intra-arterial injection. UCBCs treated patients received 1.0-7.1 x 107 TNC/kg. The authors reported that the

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intra-arterial delivery was carried out by transfemoral cerebral angiography and was favored for cord blood units with