Stem cells: From Potential to Promise 9811603006, 9789811603006

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Khawaja Husnain Haider  Editor

Stem cells: From Potential to Promise

Stem cells: From Potential to Promise

Khawaja Husnain Haider Editor

Stem cells: From Potential to Promise

Editor Khawaja Husnain Haider Molecular and Cellular Pharmacology (Stem cells and Gene Therapy), Department of Basic Sciences Sulaiman AlRajhi Colleges Al Bukairiyah, Kingdom of Saudi Arabia

ISBN 978-981-16-0300-6 ISBN 978-981-16-0301-3 https://doi.org/10.1007/978-981-16-0301-3

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

To my father, who taught me to believe in hard work and dedication.

Contents

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Imaging Stem Cell-Based Myocardial Vasculoprotection . . . . . . . . Dean P. J. Kavanagh, Adam Lokman, and Neena Kalia

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Stem Cells of the Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentin P. Shichkin

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Characteristic and Regenerative Potential of Human Endometrial Stem Cells and Progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Azin Ghamari, Faezeh Daghigh, Ali Mohebbi, Yekta Rahimi, Layla Shojaie, and Masoumeh Majidi Zolbin

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Ovarian Stem Cells and Progenitors and Their Regenerative Capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Masoumeh Majidi Zolbin, Fereshteh Aliakbari, Shayesteh Mehdinejadiani, Seyedeh Sima Dayabari, Layla Shojaie, Khawaja Husnain Haider, and Joshua Johnson

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Genomic Instability in Stem Cells: The Basic Issues . . . . . . . . . . . . E. A. Prieto González and Khawaja Husnain Haider

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Mesenchymal Stem Cell-Based Heart Cell Therapy: The Effect of Route of Cell Delivery in the Clinical Perspective . . . . . . . . . . . . Yazan M. N. Kalou, Ammar S. A. Hashemi, Rayan M. Joudeh, Beatrice Aramini, and Khawaja Husnain Haider

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Researches and Applications of Stem Cell Secretome . . . . . . . . . . . Jufeng Xia, Shunichi Arai, and Khawaja Husnain Haider

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Therapeutic Application of Perinatal Stem Cells in Cardiovascular Diseases: Current Progress and Future Prospects . . . . . . . . . . . . . . Renata Szydlak

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Chromatin Remodeling and Cardiac Differentiation of Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mukhtar Ullah, Hana’a Iqbal, Kanwal Haneef, Irfan Khan, and Asmat Salim

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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About the Editor

Khawaja Husnain Haider is currently a Professor of Cellular and Molecular Pharmacology (Stem Cells and Gene Therapy) and Chairman of the Basic Sciences Department (Medical Program) at Sulaiman AlRajhi University. He has also served on the editorial boards of various research journals and as an invited reviewer for several respected international journals. His research focuses on the use of DNA and stem cells as “drugs,” a topic that has gained popularity in regenerative medicine. He has over 270 publications to his credit including book chapters, and abstract and research papers in various leading international journals. He has also given numerous presentations and recently edited four books covering various facets of stem cell biology.

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Imaging Stem Cell-Based Myocardial Vasculoprotection Dean P. J. Kavanagh, Adam Lokman, and Neena Kalia

Abstract

Treatment of myocardial infarction (MI) focuses on the rapid re-establishment of regional myocardial perfusion following a blockage in one or more of the coronary arteries. Although the culprit artery can be re-perfused, a significant proportion of patients still incur extensive myocardial damage resulting in heart failure. This is partly due to reperfusion paradoxically leading to additional tissue damage, a condition called ischaemia-reperfusion (IR) injury that has been ascribed to inadequate coronary microcirculatory perfusion. Nevertheless, the degree of microvascular perfusion is unknown, limited by an inability to clinically image coronary microvessels. Haematopoietic stem/progenitor cells (HSPCs) offer a potential vasculoprotective therapy but little is known about their homing dynamics and whether they do indeed protect coronary microvessels. We have recently reported that after systemic infusion, individual HSPCs were readily observed trafficking through the murine injured heart albeit with poor local retention. Despite this, remarkable anti-thrombo-inflammatory effects were observed, consequently improving microvascular perfusion as observed by intravital microscopic imaging, thus resulting in attenuated infarct size. This chapter assesses in-depth the coronary vasculoprotective potential of exogenous HSPCs and its assessment by imaging with primary focus on novel intravital microscopy technique applied to the beating heart.

D. P. J. Kavanagh · A. Lokman · N. Kalia (*) Microcirculation Research Group, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 K. H. Haider (ed.), Stem cells: From Potential to Promise, https://doi.org/10.1007/978-981-16-0301-3_1

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Keywords

Coronary microcirculation · Haematopoietic stem cells · Infarction · Intravital · Microscopy · Myocardial · Progenitor cells · Vasculoprotection

Abbreviations BM CMR CVD EC(s) ECG G-CSF GM-CSF HSC(s) HSPC(s) IL-1β/2/3/5/6/10/12(p40)/13/18 IR IVM KC LVEF MACE MCP-1 MI MIP-1β MSC(s) NF-κβ PCI PET RANTES ROS TNF-α TSG-6 VCAM-1

Bone marrow Cardiac magnetic resonance Cardiovascular disease Endothelial cell(s) Electrocardiographic Granulocyte colony-stimulating factor Granulocyte-macrophage colony-stimulating factor Haematopoietic stem cell(s) Haematopoietic stem/progenitor cell(s) Interleukin-6. . . Ischaemia-reperfusion Intravital microscopy Keratinocyte (derived) chemokine Left ventricular ejection fraction Major adverse cardiovascular event Monocyte chemoattractant protein-1 Myocardial infarction Macrophage inflammatory protein-1β Mesenchymal stem cell(s) Nuclear factor-κβ Percutaneous coronary intervention Positron emission tomography Regulated upon activation, normal T cell expressed and secreted Reactive oxygen species Tumour necrosis factor-α Tumour stimulated gene/protein-6 Vascular cell adhesion molecule-1

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1.1

Introduction

1.1.1

Myocardial Infarction Is a Significant Global Burden

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Cardiovascular diseases (CVD), including myocardial infarction (MI) and strokes, account for the majority of non-communicable diseases worldwide, with an estimated 17.8 million deaths occurring in 2017 alone (World Health Organisation 2019). Just over half of CVD-related deaths are due to MI. The majority of MI cases occur due to the rupture or erosion of an atherosclerotic plaque leading to thrombus formation within a major epicardial coronary artery. The lumen of the affected coronary artery becomes significantly narrow or fully obstructed, thereby preventing downstream blood flow (Heusch and Gersh 2017). Tissue affected by this blockage becomes infarcted, consisting of a well-demarcated lesion of damaged and necrotic cells. Current treatment strategies look to rapidly dissolve the thrombus using thrombolytic therapy or involve a primary percutaneous coronary intervention (PCI) to place a stent in the blocked artery (Bulluck et al. 2016). PCI is routinely combined with pharmacological therapy such as anti-platelets, anti-hypertensives, and lipid-lowering drugs to prevent the occurrence of another MI event.

1.1.2

The Coronary Microcirculation in Myocardial Infarction: Culprit and Victim

Despite these revolutionary treatments, a significant proportion of MI patients still incur extensive muscle damage. This loss of functioning cardiomyocytes reduces pumping efficiency and subsequently leads to the development of heart failure, a major driver of morbidity, mortality, and healthcare cost (Cahill and Kharbanda 2017). This damage is partly due to PCI-induced reperfusion, which is the only way to salvage the ischaemic myocardium, paradoxically leading to additional tissue damage, a condition termed ischaemia-reperfusion (IR) injury (Yellon and Hausenloy 2007). Indeed, it is estimated that IR injury is responsible for approximately 50% of the final necrotic infarct size. Clinically, restoration of normal epicardial coronary artery blood vessel flow, but with sub-optimal myocardial perfusion, can be observed in as many as 50% of PCI patients, leading to worse outcomes than in patients with full perfusion recovery (Bolognese et al. 2004; Camici and Crea 2007; De Maria et al. 2015; Niccoli et al. 2016). Indeed, in a clinical study of 126 patients, echocardiography revealed that 37% of treated MI patients had reduced myocardial perfusion which proved to be a prognostic indicator for the development of early congestive heart failure (Ito et al. 1996). This suggests that myocardial tissue damage likely occurs after inadequate perfusion at the level of the coronary microcirculation (Bolognese et al. 2004; McAlindon et al. 2018). Indeed, no ‘re-flow’ is often noted post-PCI, likely brought about by targeted damage of the tiniest blood vessels of the heart as a result of the ischaemic insult. However, as well as being a ‘victim’, the re-perfused coronary microcirculation can also contribute to tissue damage. This can be mediated by

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multiple microcirculatory perturbations, including endothelial cell (EC) damage and loss of integrity, neutrophil and platelet activation and aggregation, microvascular red blood cell congestion, capillary haemorrhage, reactive oxygen species production (ROS), and oedema formation to list but a few (Fig. 1.1). Impaired vasodilation of coronary arterioles, which physiologically modulate local blood flow, also impacts detrimentally on cardiac perfusion (Hausenloy and Yellon 2013). These various microvascular perturbations contribute to surrounding tissue damage through a reduction in the density of coronary functional capillaries or thromboinflammatory pathways and ultimately determine infarct size. It is therefore clear that myocardial IR injury can be considered a multi-faceted microvascular pathology which explains why targeting just one microvascular disturbance, such as the thrombotic events using anti-platelet strategies, has not always been a successful therapeutic option. As such the coronary microcirculation, a ‘culprit and victim’ of MI, has been identified as a prime target for cardioprotection (Hausenloy et al. 2019). This increased clinical recognition of the importance of the coronary microcirculation has meant identifying effective strategies to improve perturbations and restore microvascular integrity has gained recent attention (Sambuceti et al. 2000; Crea et al. 2014; Pries and Reglin 2017; Lüscher 2017). Surprisingly, however, clinical research into the role of the coronary microcirculation in CVD has been limited. Several challenges present themselves making it difficult to investigate dynamic events within the coronary microcirculation clinically. Primarily, the microvessels of interest are small at less than 200 μm in diameter making imaging modalities such as cardiac magnetic resonance (CMR) and positron emission tomography (PET) unsuitable due to their limited spatial resolution (Pries et al. 2008; Crea et al. 2014). This inability to directly image coronary microvessels has led to cardiologists focusing their efforts on improving flow post-MI within the angiographically visible part of the coronary circulation. Hence, little is known about the full range of coronary microcirculatory responses to reperfusion-mediated injury in the clinical setting. Nevertheless, the progressive loss of distal microvessel perfusion has been clinically demonstrated recently in patients post-MI using PET scanning (Thackeray et al. 2015). Furthermore, CMR imaging has demonstrated a dark hypo-intense core within the infarct zone representing microvascular obstruction in MI patients, which when persistent was indicative of patients more likely to develop adverse ventricular remodelling (Bulluck et al. 2018). Functional readouts, such as local blood flow and pressure, determined clinically using a Doppler flow wire inserted into the coronary artery, have also indicated a lack of perfusion in microvessels downstream of an opened epicardial artery. The index of microcirculatory resistance, a pressuretemperature sensor guidewire-based measurement, has also been proposed for quantitatively assessing coronary microvessel functional status indirectly post-PCI in the territory surrounding the artery from which the measurement is made (Fearon and Kobayashi 2017). However, these various anatomical and functional assessments are mainly of the larger coronary vessels. Hence, actual visualization of the coronary microcirculation, let alone any perturbations within them or the vasculoprotective

Imaging Stem Cell-Based Myocardial Vasculoprotection

Fig. 1.1 Poor prognosis of a significant proportion of treated MI patients may be linked to damage to the coronary microcirculation. Although technological advances in PCI combined with anti-platelet therapies have revolutionized the treatment of MI patients, further improvements to patient outcome will depend on addressing problems in the coronary microcirculation that arise immediately after the blocked artery is opened. Indeed, reperfusion paradoxically leads to greater damage and infarct formation mediated through a diverse array of microcirculatory structural and functional disturbances. Therefore, effective cellular therapies need to be able to prevent as many of these as possible in order to restore microvascular integrity and ensure adequate myocardial perfusion. (Image taken from: https://drvalentino.com/heartattack/)

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effects of novel therapies, remains impossible to ascertain with current clinical imaging tools.

1.2

Experimental Imaging of the Cardiac Microcirculation: 70 Years in the Making

Imaging the multitude of dynamic thrombo-inflammatory and vasoactive events that take place in the coronary microcirculation in real time is clearly key to understanding the mechanisms that underpin myocardial IR injury. Furthermore, identifying whether novel therapeutic interventions are beneficial through direct vasculoprotective actions on coronary microvessels is important. The lack of clinical methodologies capable of visualizing coronary microcirculation has contributed to a research ‘black box’ with regard to our understanding of the dynamic changes that occur in the microcirculation of the IR injured heart (Pries and Reglin 2017). This gap in our current knowledge can be met through experimental studies. Indeed, most of our understanding of microvascular dysfunction post-reperfusion injury has been obtained from heart tissue imaged histologically for morphological deterioration, inflammatory cell infiltration, and infarct size. However, these static snapshots provide no indication of the real-time kinetics of deleterious thrombo-inflammatory cell recruitment in the presence of pathophysiological flow, nor information on microvessel integrity post-injury. Recently, the cardiovascular research community suggested creating an international ‘coronary microcirculatory observatory’, an EU-wide virtual facility, utilizing in vivo imaging techniques such as intravital microscopy (IVM) to properly interrogate coronary microvascular dysfunction (Pries and Reglin 2017). IVM is used extensively to experimentally image in real time the microcirculation of solid organs (liver, kidney, gut) or transparent tissues (mesentery, cremaster muscle) in anaesthetised rodents. Indeed, much of our existing knowledge of the impact of IR injury on the microcirculation per se has been obtained using transparent preparations such as the cremaster muscle or gut mesentery (Kalia et al. 2002; Gavins and Chatterjee 2004). However, the application of this powerful technique to the beating heart in rodents has been challenging primarily due to cardiac cycle and respiratory-related movement of the heart in all three dimensions, thus imposing a practical limitation on the imaging resolution of the coronary microvasculature. Indeed, when compared to other organs, the heart has one of the highest maximal displacements of any organ (19.9 mm/s), and this displacement more than doubles during ventilation (47.8 mm/s) which is essential for opened chest procedures (Lee et al. 2017). As such, other surrogate tissue beds, particularly the cremaster, have been used as models of the cardiac microcirculation (Pries and Reglin 2017). However, the coronary microcirculation is unique in both its anatomical design and physical properties, particularly its contractile activity which compresses coronary capillaries during systole reducing their diameter up to 20% (Kassab et al. 1993; Yada et al. 1993; Toyota et al. 2002). As such, the impact of IR injury, and the

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mechanisms by which therapeutic vasculoprotective strategies act, may vary in a site-specific manner. The earliest experimental studies to image the cardiac microcirculation were performed around the 1950s. These studies were predominantly pre-occupied with the identification of vascular structure and function but were relatively inefficient as they did not control for movement (Martini and Honig 1969). Imaging studies developed over the subsequent 30 years and introduced stabilization and electrocardiographic (ECG) gating as a means to counteract the physical movement of the heart (Tillmanns et al. 1974; Nellis et al. 1981; Nellis and Leidtke 1982; Chilian et al. 1986; Chilian 1991; Chilian and Defily 1991). These studies were effective and helped provide significant information around the physical properties of the cardiac microcirculation. However, these techniques were not suitable for imaging at a cellular resolution, and the lack of fluorescent tools and camera technology available at the time limited the information which could be obtained on cellular trafficking in the heart. It is only over the last 10 years that suitable techniques capable of imaging cellular trafficking have been developed and combined with ECG gating (Lee et al. 2012), pacing (Aguirre et al. 2014), stabilizers (Vinegoni et al. 2015; Jones et al. 2018; Kavanagh et al. 2019b), and advanced software tools (Soulet et al. 2013; Gómez-Conde et al. 2015; Kavanagh et al. 2019a). The main commonality between these studies is the use of a tissue stabilizer which is attached to the heart using an appropriate veterinary glue (Lee et al. 2012; Jones et al. 2018; Kavanagh et al. 2019b) or is held in place by suction (Vinegoni et al. 2012; Jung et al. 2013). The stabilizer secures a small portion of the heart ventricle in space and permits imaging of the epicardial surface through a central ring and, importantly, without impeding its physiological role in blood circulation. As a result of this developmental work, several recent studies have investigated cellular trafficking in the context of pathological cardiac microcirculatory conditions (Jung et al. 2013; Matsuura et al. 2018; Kavanagh et al. 2019b).

1.3

Haematopoietic Stem/Progenitor Cells: Recruitment and Repair Mechanisms

Haematopoietic stem/progenitor cells (HSPCs) are rare multipotent cells, normally found in the bone marrow (BM), responsible for the maintenance of homeostasis through the generation of red blood cells, leukocytes, and platelets. BM transplants, which essentially seek to repopulate the HSC compartment in patients suffering from various cancers or blood disorders, are now routine medical interventions and so the safety of this procedure has been established. The frequency of serious adverse events during BM transplantation is exceptionally low (Bosi and Bartolozzi 2010), fuelling optimism that any other therapeutics which rely on their administration is likely to be safe. HSPCs are typically isolated based on their surface markers, which include c-Kit/Sca-1 in mice and CD34 in humans (Wognum et al. 2003). These are combined with negative staining for several lineage markers (Lin ) for differentiated progenitor cells. HSPCs can mobilize from the BM and egress into

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the peripheral circulation in response to the release of soluble mediators from remote sites of tissue injury (Dalakas et al. 2003; Kale et al. 2003; Lemoli et al. 2006). After homing to the injured tissue, HSPCs have been shown to aid its repair and regeneration. However, the level of HSPCs in the peripheral circulation following mobilization is thought to be very low. Indeed, even with robust mobilization strategies, only 0.2–0.5% of circulating leukocytes are CD34+. Once in the circulation, endogenous HSPCs, as well as those exogenously administered for therapeutic purposes, use many different surface adhesion molecules to engage with the microvasculature of the injured organ. HSPC-EC interactions take place in response to the activation of both cell types by locally released inflammatory cytokines and chemokines. Not surprisingly, HSPCs have a similar repertoire of surface adhesion molecules to other circulating blood cells. Indeed, the mechanisms governing their homing to injured tissue and subsequent adhesive interactions with ECs closely match those used by inflammatory and immune leukocytes (Kobayashi et al. 1994; Perlin et al. 2017). HSPC surface adhesion molecules include selectins (CD62L), integrins (CD11a/CD18, CD49d/ CD29), immunoglobulin superfamily members (CD31), and other glycoproteins (CD44, HCELL) (Kavanagh and Kalia 2011). While we have a significant understanding of how mobilized or transplanted HSPCs home to the BM (Frenette et al. 1998; Mazo et al. 1998; Peled et al. 2000), we have fewer data around how these cells are recruited to extra-medullary sites of injury such as the heart. Intravital studies of non-cardiac tissue show recruitment to be primarily dependent on interactions of the surface α4β1 integrin with vascular cell adhesion molecule-1 (VCAM-1) located on inflamed endothelium (Frenette et al. 1998; Mazo et al. 1998; Kavanagh et al. 2010, 2013a). Of note, the recruitment of HSPCs to the heart has also been shown to be dependent on α4β1 (Zhang et al. 2007). In addition to ‘traditional’ adhesion molecule interactions, other more novel recruitment mechanisms have been postulated to regulate HSPC recruitment to extra-medullary tissues. For example, adherent platelets, present in abundance in ischaemically injured tissue microvasculature, are a viable substrate upon which HSPCs can bind (De Boer et al. 2006; Langer et al. 2006; Massberg et al. 2006). Historically, the vast majority of published literature which investigates HSPC activity and function has focused primarily on their haematopoietic activities. However, it is now clear that these cells possess reparative capabilities which can be leveraged as a therapeutic tool following injury. HSPCs can promote tissue repair through mediating angiogenesis and neovascularization, particularly in ischaemic tissue. Furthermore, their ability to protect the smallest vascular structures is being increasingly postulated through anti-oxidant, anti-inflammatory, and anti-thrombotic effects (Li et al. 2010; Kavanagh et al. 2013b; Bijkerk et al. 2014; Abdelmawgoud and Saleh 2018). For example, in a model of murine renal IR injury, mobilization of HSPCs using microRNA-126 led to a higher density of peritubular capillaries than in control mice (Bijkerk et al. 2014). The ability to confer vasculoprotection through the release of paracrine anti-inflammatory and pro-growth mediators by HSPCs has arguably received the most attention in recent years. This phenomenon was observed in an experimental model of rheumatoid arthritis where HSPCs were able to reduce

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paw swelling by significantly decreasing the levels of pro-inflammatory cytokines such as TNF-α and IL-6 as well as reduce oxidative stress injury as measured by glutathione levels (Abdelmawgoud and Saleh 2018). A similar reduction in paw weight, lymphatic organ weight, and TNF-α release were observed by intravenous administration of HSPCs in a murine model of contact hypersensitivity (Biziuleviciene et al. 2007). Evidence also suggests that HSPCs can be beneficial following ischaemic insults. Indeed, following transient cochlear ischaemia in rodents, HSPC administration significantly reduced hearing loss by stimulating the expression of glial cell line-derived neurotrophic factor (Yoshida et al. 2007). Interestingly, HSPCs have also been shown to decrease the release of inflammatory cells from the spleen in response to cerebral ischaemia (Schwarting et al. 2008). For HSPC-based therapy to be efficacious, it seems logical that recruitment of these trafficking cells by local injured tissue microvessels, and their subsequent retention in the inflamed/injured milieu, is a prerequisite. Many experimental studies seem, on the surface, to support this claim. Interventions that increase the migratory and adhesive potential of HSPCs have been shown to significantly improve their ability to drive reparative responses (Sasaki et al. 2006). Examples of such interventions include integrin pre-activation using activating antibodies which have been shown to increase both the homing and subsequent angiogenic potential of HSPCs in a model of hind limb ischaemia (Chavakis et al. 2005). More recently, Ziegler and colleagues demonstrated significantly improved homing of Sca-1+ peripheral blood mononuclear cells, pre-treated with an engineered tandem singlechain antibody, to the hearts of mice undergoing myocardial IR injury. These pre-treated cells had a high affinity for binding to the platelet surface glycoprotein GPIIb/IIIa. Since platelets are found in abundance in injured hearts, targeted and increased delivery of therapeutic cells to the injured heart was noted, which concomitantly resulted in a greater reduction in inflammatory cell infiltration and fibrosis and an increased capillary density as assessed histologically (Ziegler et al. 2017). Since HSPCs appear to confer benefit primarily through the release of paracrine mediators, their local retention dependence is not as critical as these ‘goodies’ could simply be released into the peripheral circulation from any site in the body. Indeed, an increasing number of stem cell populations have been shown to mediate a therapeutic paracrine effect while being positioned outside of the injured tissue. Roddy and colleagues showed that systemic administration of mesenchymal stem cells (MSCs) reduced corneal inflammation through release of the potent antiinflammatory agent, namely TNF-α stimulated gene/protein-6 (TSG-6), without actual engraftment in the eye itself (Roddy et al. 2011). Similarly, MSCs administered intraperitoneally were unable to attenuate peritoneal adhesions, whereas MSCs administered intravenously were beneficial despite not being present locally (Wang et al. 2012a). This effect was again driven through the paracrine release of TSG-6 from MSCs retained in the pulmonary microcirculation (Wang et al. 2012b). The importance of TSG-6 as an important paracrine factor utilized by HSPCs was demonstrated after acute lung injury was induced in rats. In this model, freshly isolated human CD34+ HSPC cells were anti-inflammatory at least in part,

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by activation of CD34+ cells expressing the TSG-6 gene (Abdallah et al. 2015). In a cardiovascular context, it has been shown that the administration of vascular progenitor cells was beneficial following IR injury as evidenced by decreased fibrosis and infarct size and improved function, despite the majority of cells being trapped in the pulmonary microcirculation (Ziegler et al. 2019). Also, the administration of MSCs into the intrascapular region of mice undergoing myocardial IR significantly improved functional cardiac outcomes despite no evidence of their migration out of the injected site to the heart (Preda et al. 2014).

1.4

Haematopoietic Stem/Progenitor Cells for Myocardial Infarction

In the specific setting of cardiac injury and dysfunction, the evidence that HSPCs have the inherent ability to repair the infarcted myocardium has been around for almost two decades. In 2001, Jackson and colleagues reconstituted the murine BM of lethally irradiated mice with labelled CD34 /c-kit+/Sca-1+ ‘side population’ cells, a population highly enriched for HSCs. They noted the incorporation of these cells in both myocytes and vascular (mainly capillary) ECs in the postischaemic heart 2 weeks after coronary ligation was performed (Jackson et al. 2001). This elegant work demonstrated for the first time that HSPCs could migrate from the BM to the heart and localize in newly forming vessel structures and regenerate healthy muscle. However, it was not clear whether it was HSCs that contributed to repair or their progeny. Later work by Orlic and colleagues examined the role of c-kit+/Lin HSCs directly administered into an area of cardiac muscle infarction and found they improved myocardial regeneration and cardiac function (Orlic et al. 2001a). The same group further demonstrated a therapeutic benefit from using HSPC mobilizing agents and suggested that the presence of HSPCs locally in the injured tissue was required for therapeutic effectiveness (Orlic et al. 2001b). Since these initial experimental studies, many publications have continued to support a potential role for HSPCs as a therapeutic intervention in CVD. However, controversy surrounded the therapeutic mechanisms employed by HSPCs in these early experimental studies, particularly with regard to their ability to contribute to cells of the cardiac parenchyma via transdifferentiation. Some argued that the presence of donor HSPC antigens in newly formed cardiac tissue was a result of donor-host cell fusion rather than transdifferentiation (Wagers et al. 2002; Quijada and Sussman 2015). Importantly, concerns were raised as to the exact phenotype of the effective cells and whether they were indeed purely HSPCs since the infusions used were more heterogeneous than originally thought (Cook et al. 2009). The lack of consensus around the basic biology of HSPCs did not prevent their use in clinical trials for cardiovascular disease, particularly for MI patients. The first such trial, TOPCARE-AMI, administered CD34+/CD45 cells into the infarct artery at 4–5 days post-MI (Assmus et al. 2002). Patients receiving HSPCs demonstrated improved left ventricular ejection fractions (LVEF) at both 4 months and 1-year follow-up when compared to control patients receiving no cellular

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therapy (Schachinger et al. 2004). The BOOST trial injected a single dose of undifferentiated BM-derived nucleated cells 5 days post-MI and examined patients at 6 months and 5 years. Although improvements were seen in LVEF at 6 months, this improvement was lost at 5 years (Wollert et al. 2004). Furthermore, no difference in major adverse cardiovascular events (MACEs) was noted between patients receiving the cell injection and those receiving vehicle. REPAIR-AMI used cells isolated by density centrifugation of BM aspirates, which even the authors stated contained ‘other mononuclear cells’, and administered them to patients 3–7 days post-MI and monitored recovery at 4 months and 1 year. Again, improved functional readouts, namely LVEF and contractility, was noted (Schachinger et al. 2006a, 2006b). Interestingly, follow-up work from this study suggested that these cells elicited an improvement in microvascular function as measured by improvements in coronary flow reserve, an indirect surrogate marker of microcirculation function (Erbs et al. 2007). The major issue raised following publication of these studies was the fact that the BM aspirates, although likely contained HSPCs, were a hotchpotch of different cell types. Therefore, subsequent clinical trials began to take a more refined approach to cell isolation. The SCIPIO trial, which utilized Lin /c-kit+ cells post-coronary artery grafting, initially showed positive results (Bolli et al. 2011). However, the study has since been retracted by editors over concerns surrounding the reliability of some of the experimental data (The Lancet 2019). The PreSERVE-AMI study examined the role of purified CD34+ cells as a therapeutic post-MI (Quyyumi et al. 2017). Patients received a bolus dose of CD34+ cells at between 4 and 11 days after presenting with an MI and were followed up at 12 months. Although there were no functional improvements identified at 1 year in broad terms, when controlling for the initial period of ischaemia, the authors identified a significant improvement in LVEF, infarct size, and number of MACEs (Quyyumi et al. 2017).

1.5

HSPCs for Myocardial Infarction: The Debate Goes on!

Despite experimental and clinical studies providing strong evidence that BM-derived HSPCs are beneficial in models of ischaemic heart injury, just as many clinical trials have either failed to replicate these findings or have shown minor or temporary benefit (Madigan and Atoui 2018; Leri and Anversa 2013). This may in part be since there is no consistency amongst the various studies with regard to the phenotypic nature of the cells used, route of delivery, number of cells injected, the timing of delivery post-MI, etc. Indeed, there remains no consensus on which route of delivery is best for cellular therapy to be effective in the heart. Although cells can be delivered transendocardially, others have shown this route is not necessarily superior to intravascular delivery in the ischaemic heart (van der Spoel et al. 2012; Fukushima et al. 2008). Furthermore, electromechanical mapping guidance, required to guide the surgeon to the site where stem cells should be injected transendocardially, is challenging and can lead to incorrect injection sites. There is also an additional and significant risk of perforation and cardiac tamponade.

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Besides, intramyocardial or transendocardial delivery clusters stem cells at the injection site from where they do not necessarily migrate, making that region of the heart prone to arrhythmias (Fukushima et al. 2008). In contrast, intravascular delivery is believed to better distribute stem cells throughout the heart via the coronary microcirculation, particularly to areas where microvascular damage, and subsequent development of infarction, is prominent. However, whether this is the case is difficult to substantiate without directly imaging the beating heart coronary microcirculation at a cellular level and identifying the exact location of fluorescently labelled cells? Importantly, intramyocardial delivery is primarily aimed at targeting improvements in the muscle itself and hence appears the most obvious route of delivery to achieve this outcome. Although this is important, the critical role of the coronary microcirculation in the pathology of MI suggests cellular therapies also need to prevent the multiple vascular perturbations and improve myocardial perfusion in the immediate aftermath of reperfusion. Indeed, any form of cellular therapy for the heart must not only create healthy and functional muscle but also preserve microvascular integrity (Lerman et al. 2016). Most of the clinical trials to date have used a variety of unpurified or crudely purified BM mononuclear cells aspirates rather than a more homogeneous population of HSPCs. This inconsistency in the cell population used, and whether they even are HSPCs, is possibly one of the main contributors to poor clinical success (Muller et al. 2018). While there exists a consensus amongst the research community on how other stem cell populations should be defined [e.g. the International Society for Cellular Therapy’s MSC definition (Dominici et al. 2006)], no such criteria have been established for HSPCs.

1.6

HSPCs for Coronary Vasculoprotection: Missing a Trick?

Since coronary microcirculatory dysfunction is increasingly identified as a critical pathological component in ischaemic heart disease, understanding whether HSPCs or indeed any form of cellular therapy can protect these delicate structures is important. However, this has received very little attention with pertinent questions remaining unanswered. For example, do systemically infused HSPCs actually traffic to the injured heart and subsequently make firm adhesive interactions with the coronary microvasculature in the presence of pathological flow, or do they simply passage through? Which anatomical branches of the microcirculation mediate this capture—arterioles, capillaries, post-capillary venules—and are these interactions short-lived? Most importantly, does HSPC therapy preserve the integrity of the coronary microvessels and ensure flow is sustained through the heart—do they only modify one or the many different microvascular disturbances? Furthermore, are potential vasculoprotective effects mediated in vivo without the need for local retention? Research using conventional immunohistochemistry of tissue sections has tried to answer some of these questions but the information they provide is limited as the assays used simply to provide a ‘static’ snapshot of events at a particular moment in time. Indeed, such studies do not inform us whether HSPCs are genuinely

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adherent in the heart or simply caught in time passing through it. Ultimately, restoration of blood flow and ventricular muscle perfusion is key to the survival of the myocardium, yet static assays cannot ‘show’ dynamic events such as blood flow. As such, they do not indicate whether injected stem cells themselves can occlude blood flow within the tiny coronary vessels. If this does occur, and it is a significant event, it may cause more damage than good and impact negatively on their therapeutic effectiveness. It is noteworthy that clinical studies have monitored the therapeutic effectiveness of HSPCs many weeks or months after their administration, focusing on functional improvements or changes in infarct size. However, damage to the coronary vasculature post-reperfusion injury is usually mediated within minutes, with the development of tissue infarction observed experimentally within hours. Whether HSPCs have the potential to modify these rapid microvascular perturbations has not previously been investigated but may prove critical to improving outcomes for MI patients in the acute period post-PCI. However, to even begin understanding the potential vasculoprotective efficacy of HSPCs, a detailed understanding of the exact damage the coronary microcirculation undergoes following IR injury in vivo is needed. Currently, little is known about the full range of dynamic microcirculatory responses to MI. We have been the first to apply powerful spinning Nipkow-based confocal intravital imaging to the beating mouse heart (Kavanagh et al. 2019b). Our stabilization method, which involved vet-bonding a 3D printed in-house designed stabilizer to the left ventricle, sufficiently reduced motion in a small region of the beating heart to permit intravital imaging of the coronary microcirculation (Fig. 1.2a). Additional assessment of regional ventricular blood flow was also determined using laser speckle imaging, applied for the first time to the beating mouse heart. Using these combined methods, we characterized thrombo-inflammatory and perfusion events taking place in the beating mouse heart for 2 h post-IR injury. A reversible left anterior descending (LAD) coronary artery ligation injury model was used as this closely resembles the catheter lab scenario of reperfusion of an occluded artery post-PCI (Fig. 1.2b). Laser speckle imaging of a large region of the injured left ventricle identified a sustained hyperaemic response throughout reperfusion. On its own, this could have been interpreted as flow being adequately re-established after ischaemia. However, mice still developed infarcts within hours of injury, a scenario akin to that seen clinically after stent implantation (Kavanagh et al. 2019b). However, it was clear from the intravital studies that this was poorly transmitted to coronary capillaries and thus did not correspond to adequate perfusion at a microvascular level. Within minutes of reperfusion, rapid neutrophil adhesion took place primarily within coronary capillaries. Furthermore, multiple platelet-rich aggregates formed, often elongated as they followed the contours of coronary capillaries. Although individual adherent neutrophils did not always hinder flow, the high burden of platelet microthrombi were significant contributors to poor perfusion, occluding blood flow within downstream capillaries. Indeed, multiple regions became devoid of functional capillaries as evidenced by patchy areas ‘empty’ of fluorescently labelled albumin perfused capillaries (Fig. 1.3a–c). Although individual neutrophils did not appear to hinder flow, their increased presence is detrimental. Since activated neutrophils generate ROS, their rapid and high capillary presence increases the

Fig. 1.2 Intravital imaging of the mouse beating heart in vivo to ascertain the effects of myocardial injury and subsequent vasculoprotective effects of cellular therapy at the level of the coronary microcirculation. (a) An in-house designed 3D-printed stabilizer is vet-bonded to the ventricle of the anaesthetized mouse beating heart allowing Spinning Nipkow confocal-based intravital imaging in its centre. (b) To induce myocardial ischaemia-reperfusion injury, the left anterior descending (LAD) artery is reversibly suture ligated against plastic tubing to prevent muscle damage. Note the pale left ventricle as a consequence of this. The ligature is removed after 45 min to initiate reperfusion. In LAD ligated and re-perfused hearts, the stabilizer is attached downstream of the ligated site (Kavanagh et al. 2019a, b)

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Fig. 1.3 Multiple coronary microcirculatory perturbations imaged in vivo in the ischaemiareperfusion (IR) injured beating heart intravitally. (a) Left panels: IR injury increases neutrophil adhesion primarily within coronary capillaries. Middle panels: Adherent neutrophils in healthy hearts do not compromise blood flow as widespread FITC-BSA perfused capillaries are visible. However, blood flow is compromised in injured hearts as evidenced by not all capillaries being filled with FITC-BSA—a large perfused blood vessel can be seen in the image but note fewer perfused capillaries. Neutrophils are not necessarily occlusive. Right panels: Neutrophil adhesion, platelet aggregation, and microthrombus formation increased in injured hearts. Microthrombi occupy and follow the contours of significant lengths of some capillaries. Co-localization (yellow) indicates aggregates can be comprised of both neutrophils and platelets. (b) Snapshots from an IR injured beating heart—circles show a circulating neutrophil (green) unable to move downstream of a capillary occluded by a platelet thrombus (red). (Asterisk) circulating neutrophils seen in one

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susceptibility of these delicate structures to early and significant oxidative damage. Indeed, our flow cytometry data demonstrated coronary endothelium, rather than myocytes, was the primary and initial target of oxidative injury (Kavanagh et al. 2019b). These are the first detailed in vivo descriptions of the extent of myocardial microcirculatory disturbances at a cellular level. Clearly, these experiments show that a mismatch exists in vivo between the global hyperaemic response during reperfusion and microcirculatory flow heterogeneity, with platelets playing a critical part in the latter. Our beating heart model was further used for the first time to image stem cell trafficking in the heart in vivo at a cellular level and, importantly, ascertain whether vasculoprotection was a critical therapeutic mechanism. Although clinical studies have primarily used total un-fractionated BM mononuclear cells (comprising HSPCs, MSCs, lymphocytes, and monocytes), we imaged myocardial homing and trafficking of an exogenous population of pure BM-derived HSPCs. A fourfold increase in circulating HSPCs was identified in injured hearts with 25–30 circulating cells consistently observed throughout reperfusion. This was in contrast to other IR injured organs we have previously imaged where HSPCs were only observed on the ‘first pass’ immediately after infusion and not thereafter (Kavanagh et al. 2013a, b; White et al. 2013). Surprisingly, these homing events did not result in any dramatic myocardial HSPC retention, which remained low. Poor retention is a well-described phenomenon. Pulmonary entrapment is a common feature of cellular therapy and was also noted in our current study. Even though HSPCs were not retained locally, they did lead to a rapid and potent reduction in both neutrophil and platelet recruitment in injured hearts (Fig. 1.4a, b). As previously mentioned, it has been suggested that local retention of cellular therapeutics is not an essential prerequisite to their beneficial function. The amelioration of injury could, therefore, result from a number of mechanisms. For example, HSPCs retained remotely in the body could exert benefit through circulating paracrine factors, or as they passaged through the heart. Additional data we generated suggested the former was the most likely method. Serum was analysed for a plethora of pro- and anti-inflammatory cytokines (Table 1.1). Unsurprisingly, we noted that IR injury led to increases in the concentrations of many pro-inflammatory agents (Kavanagh et al. 2019b). However, significant reductions in IL-1β, IL-2, IL-3, IL-5, IL-6, IL-12(p40), IL-13, Eotaxin, G-CSF, GM-CSF, KC (IL-18), MCP-1, MIP-1β, RANTES, and TNF-α was noted within a couple of hours in mice receiving HSCPs. It is likely, therefore, that their administration leads to an amelioration in neutrophil and platelet recruitment via the release of reparative agents into the circulation. Interestingly, we also show that HSPCs also induced a significant increase in circulating IL-10 levels in injured mice.

Fig. 1.3 (continued) frame but not the next. (c) In healthy mice, an extensive network of FITCBSA perfused capillaries can be observed. IR injury is associated with multiple areas in which FITC-BSA does not perfuse, resulting in visualization of patchy areas devoid of any vasculature (asterisk). In some fields of view, at least half the imaged area appears non-perfused. Green: neutrophils (PE + anti-Gr-1ab); Red: FITC-BSA or endogenous platelets (APC + anti-CD41ab)

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Imaging Stem Cell-Based Myocardial Vasculoprotection

Fig. 1.4 Despite poor HSPC retention within ischaemia-reperfusion (IR) injured hearts, rapid vasculoprotective effects are identified. (a) Systemic infusion of HSPCs (100 μL of 2  106) resulted in 20–30 free flowing HSPCs circulating through myocardial microvessels at all time points in healthy hearts in vivo. An almost fourfold increase was noted at 30 min post-reperfusion in injured hearts. HSPC adhesion was not increased as a result of injury but gradually rises in all groups. Interestingly, pre-treatment with H2O2 does not enhance their retention in the injured heart, something which we have previously observed in IR injured gut intravitally (Kavanagh et al. 2013a). Representative intravital images show a similar retention of intra-arterially injected exogenous CFSE-labelled HSPCs in

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D. P. J. Kavanagh et al.

IL-10 is a naturally occurring, potent anti-inflammatory cytokine that can suppress the secretion of various pro-inflammatory cytokines both in vitro and in vivo via inhibition of NF-κβ (Saxena et al. 2015). Several immunohistochemical studies have demonstrated that exogenous IL-10 can suppress myocardial inflammation as evidenced by decreased neutrophil infiltration in similar injury models (Krishnamurthy et al. 2009; Jung et al. 2017). Furthermore, genetically engineering MSCs to overexpress IL-10 has been shown to better reduce infarct size and cardiac impairment compared to individual treatments of MSC or IL-10 administrations (Meng et al. 2018). It is therefore likely that increased serum levels of IL-10 in mice receiving HSPCs mechanistically explain the vasculoprotective effects observed in the current study. The direct or indirect functional consequence of this amelioration in inflammatory factors and increased IL-10 was an increase in functional capillary density in the injured beating heart in vivo. Flow cytometric analysis also demonstrated decreased oxidative damage specifically of the coronary ECs. The ability of HSPCs to reduce ROS generation and EC oxidative stress was further confirmed during in vitro studies. Overall, our study clearly shows that HSPCs can ameliorate the rapid microcirculatory damage and thrombo-inflammatory events that occur in the immediate aftermath of reperfusion injury. The consequence of preserving coronary microcirculatory perfusion was a significant reduction in the necrotic damage to the heart muscles as evidenced by a reduced infarct size in vivo (Fig. 1.4b–e). From a clinical perspective, our protocol of systemically infusing cells alongside reperfusion would be relatively easy to perform at any hospital catheter lab as no specialist equipment would be required. Nevertheless, some would argue that the use of HSPCs in conditions involving cardiac inflammation is superfluous and that the administration of one of the many available anti-inflammatory drugs alone would suffice. However, it is important to reiterate that the microcirculatory response to injury is multi-faceted, involving changes to the activity of platelets, neutrophils, monocytes, vessel tone, permeability, and more molecular changes leading to endothelial oxidative damage and in the levels of soluble inflammatory cytokines and chemokines. Repurposing of existing anti-inflammatory drugs would likely target just the leukocytes which are just one of the many problem cells. Similarly, anti-platelets would modify mainly the platelet responses. Indeed, they are already

Fig. 1.4 (continued) both groups. (b) Upper and Middle panels: HSPCs systemically injected within 5 min of reperfusion (100 μL of 2  106) significantly reduced both neutrophil adhesion and the presence of platelet aggregates and microthrombi. Bottom panel: Flow cytometric analysis of digested hearts showed a significant reduction in oxidative damage of coronary endothelial cells as determined using an anti-8-OHdG antibody. (c) Improvement in functional capillary density in mice receiving HSPCs was observed as determined using FITC-BSA perfusion. (d) Importantly, this resulted in an improvement in infarction. (e) Anti-oxidant effect of HSPCs was confirmed in vitro. Both generation of reactive oxygen species and their ability to induce oxidative damage within vena cava endothelial cells (VCECs) was decreased within 24 h of HPSC co-incubation with VCECs. This was tested by staining VCECs using DHE staining (red) or an anti-8-OHdG antibody (red) respectively. CFSE-labelled HPSCs appear yellow. ****p < 0.0001

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Table 1.1 Heatmap showing HSPC (HPC-7) therapy in myocardial IR injury significantly reduces plasma levels of multiple pro-inflammatory cytokines but significantly increases levels of IL-10. Using a Luminex cytokine array kit, a number of pro-inflammatory cytokines in the plasma were significantly increased following myocardial IR injury. Of these, IL-6, IL-12, G-CSF, CXCL1, CCL2, and CCL4 showed greater than sixfold increases. HSPC therapy significantly reduced 9 of the 10 cytokines increased in the IR injury group. IL-10 was the only one not increased by injury and further increased with therapy. */# p < 0.05; ## p < 0.01; ### p < 0.001 Cytokines

Sham (pg/mL)

IR injury (pg/mL)

IR injury + HSCs (pg/mL)

Interleukin-1α

14.07 ± 2.90

18.62 ± 3.95

10.63 ± 0.67#

Interleukin-1β

37.18 ± 5.06

42.84 ± 4.37

24.94 ± 2.40##

Interleukin-2

14.92 ± 3.00

11.65 ± 0.86

8.17 ± 0.48##

Interleukin-3

17.13 ± 2.45

16.46 ± 1.67

10.93 ± 0.93##

Interleukin-4

13.61 ± 3.48

10.27 ± 2.77

6.76 ± 4.29

Interleukin-5

32.63 ± 11.95

31.56 ± 5.69

17.78 ± 2.91#

Interleukin-6

316.138 ± 118.21

1993.53 ± 841.45*

425.07 ± 78.19#

Interleukin-9

51.29 ± 7.54

45.76 ± 4.83

34.22 ± 2.64#

Interleukin-10

143.57 ± 9.06

222.70 ± 45.84*

452.48 ± 41.06###

Interleukin-12(p40)

2253.75 ± 350.50

14 588.86 ± 5889.30*

2424.66 ± 179.39#

Interleukin-12(p70)

501.84 ± 85.43

555.36 ± 57.31

350.24 ± 38.07##

Interleukin-13

278.182 ± 51.10

337.87 ± 38.67

159.26 ±25.87###

Interleukin-17

18.70 ± 2.68

14.50 ± 1.22

11.36 ± 1.06#

CCL11

992.67 ± 113.83

1607.58 ± 50.43*

1020.15 ± 90.44###

G-CSF

395.23 ± 97.79

2720.72 ± 1272.80*

200.29 ± 36.04#

GM-CSF

54.52 ± 7.25

61.95 ± 6.50

41.09 ± 4.75##

IFNγ

46.44 ± 7.28

42.25 ± 4.50

30.55 ± 2.20#

CXCL1

326.15 ± 93.22

1769.48 ± 783.47*

227.52 ± 28.93#

CCL2

638.92 ± 121.67

5016.62 ± 2117.35*

704.97 ± 93.02#

CCL3

21.84 ± 8.95

48.51 ± 21.69

37.30 ± 10.35

CCL4

169.46 ± 56.60

1545.83 ± 736.81*

250.28 ± 58.80#

CCL5

108.06 ± 15.03

248.96 ± 79.09*

85.75 ± 10.29#

TNFα

191.95 ± 29.16

369.49 ± 95.21*

154.62 ± 19.28#

0

Cytokines (pg/mL)

5000+

given at the time of PCI yet 50% of these patients still progress to heart failure. The ‘beauty’ of cellular therapy is that it appears to have multiple vasculoprotective actions at the level of the coronary microcirculation as demonstrated in our beating heart study (Fig. 1.5). Indeed, HSPCs inhibited thrombo-inflammatory events,

Fig. 1.5 Mechanism by which systemically injected exogenous HSPCs may confer vasculoprotection in the ischaemia-reperfusion injured coronary microcirculation. Beating heart intravital imaging reveals that although HSPCs (green cells) can traffic through the injured heart after systemic infusion, limited numbers actually adhere within the coronary microvessels. Most pass through the heart and become entrapped within the pulmonary microvasculature (dotted arrow). However, even from these remote sites, HSPCs possess a remarkable and rapid ability to inhibit the high burden of thrombo-inflammatory cells normally identified in injured hearts that would otherwise have occluded the tiniest coronary capillaries heart to the detriment of myocardial perfusion. We and others have shown that this may be linked to the paracrine release (solid arrow) of potent anti-inflammatory cytokines such as interleukin-10 (IL-10) and factors such as TNF-α stimulated gene/protein-6 (TSG-6), although many other soluble factors may mediate this effect. Additional vasculoprotective pathways are indicated that ultimately lead to coronary microvascular patency and perfusion being maintained

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improved ventricular myocardial blood flow, lowered serum levels of potent inflammatory mediators, protected the endothelium from oxidative damage, and also raised serum levels of potentially anti-inflammatory cytokines such as IL-10. This does not take into account any non-vascular benefits that may have been conferred on cardiomyocytes!

1.7

Conclusions

Although technological advances in clinical PCI combined with anti-platelet therapies have revolutionized the treatment of MI patients, further improvements to the patient outcome will critically depend on addressing problems in the coronary microcirculation that arise immediately after reperfusion. Successful therapies that can prevent reperfusion-related injury have not yet been identified and thus remain one of the top ten unmet clinical needs in cardiology. We applied novel state-of-theart intravital imaging to the beating mouse heart to directly quantify a high burden of thrombo-inflammation which occluded multiple coronary capillaries immediately post-reperfusion. To be able to directly image at a cellular level the trafficking of individual HSPCs through the heart and their vasculoprotective benefits was gamechanging and remarkable, not least due to the speed of their response. Importantly, this offers the potential to upgrade our options in relation to targeted treatment of coronary microvascular pathologies in ischaemic heart diseases. Unlike pharmacological drugs, HSPCs appear to combine a number of paracrine protective mechanisms to simultaneously resolve multiple microvascular cellular and molecular perturbations. In recent years, the shift towards MSCs has taken the focus away from HSPCs, most notably due to the former cells displaying anti-inflammatory potential than appears to exceed that of HSPCs. However, there is still hope for HSPCs in the setting of cardiovascular disease, and their specific ability to protect the heart’s delicate and precious microvessels should be pursued further. Indeed, if we could identify the mechanisms underlying the functional response of these cells when in the coronary microcirculation, it would be possible to drive therapeutic benefit without needing the cellular components at all. Whether the final therapeutic product for mediating coronary microvasculoprotection is HSPCs or their releasate remains to be seen. Nevertheless, there is a bright future for the use of HSPCs in the management and treatment of cardiovascular disease, which has been pushed on significantly by advances in cardiac intravital imaging. When looking at these intricate cellular processes, ‘seeing is believing’.

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Peled A, Kollet O, Ponomaryov T et al (2000) The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95:3289–3296 Perlin JR, Sporrij A, Zon LI (2017) Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment. J Mol Med (Berlin, Germany) 95:809–819 Preda MB, Ronningen T, Burlacu A et al (2014) Remote transplantation of mesenchymal stem cells protects the heart against ischemia-reperfusion injury. Stem Cells 32:2123–2134 Pries AR, Reglin B (2017) Coronary microcirculatory pathophysiology: can we afford it to remain a black box? Eur Heart J 38:478–488 Pries AR, Habazettl H, Ambrosio G et al (2008) A review of methods for assessment of coronary microvascular disease in both clinical and experimental settings. Cardiovasc Res 80:165–174 Quijada P, Sussman MA (2015) Circulating around the tissue: hematopoietic cell-based fusion versus transdifferentiation. Circ Res 116:563–565 Quyyumi AA, Vasquez A, Kereiakes DJ et al (2017) PreSERVE-AMI: a randomized, double-blind, placebo-controlled clinical trial of intracoronary administration of autologous CD34+ cells in patients with left ventricular dysfunction post STEMI. Circ Res 120:324–331 Roddy GW, Oh JY, Lee RH et al (2011) Action at a distance: systemically administered adult stem/ progenitor cells (MSCs) reduce inflammatory damage to the cornea without engraftment and primarily by secretion of TNF-α stimulated gene/protein 6. Stem Cells 29:1572–1579 Sambuceti G, L’Abbate A, Marzilli M (2000) Why should we study the coronary microcirculation? Am J Physiol Heart Circ Phys 279:H2581–H2584 Sasaki K-I, Heeschen C, Aicher A et al (2006) Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy. Proc Natl Acad Sci 103:14537–14541 Saxena A, Khosraviani S, Noel S et al (2015) Interleukin-10 paradox: a potent immunoregulatory cytokine that has been difficult to harness for immunotherapy. Cytokine 74:27–34 Schachinger V, Assmus B, Britten MB (2004) Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI trial. J Am Coll Cardiol 44:1690–1699 Schachinger V, Erbs S, Elsasser A et al (2006a) Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 355:1210–1221 Schachinger V, Erbs S, Elsasser A (2006b) Improved clinical outcome after intracoronary administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J 27:2775–2783 Schwarting S, Litwak S, Hao W et al (2008) Hematopoietic stem cells reduce postischemic inflammation and ameliorate ischemic brain injury. Stroke 39:2867–2875 Soulet D, Paré A, Coste J et al (2013) Automated filtering of intrinsic movement artifacts during two-photon intravital microscopy. PLoS One 8:e53942 Thackeray JT, Derlin T, Haghikia A et al (2015) Molecular imaging of the chemokine receptor CXCR4 after acute myocardial infarction. JACC Cardiovasc Imaging 8:1417–1426 The Lancet (2019) Retraction-Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial [retraction of: Lancet. 2011 378:184757]. Lancet 393:1084 Tillmanns H, Ikeda S, Hansen H et al (1974) Microcirculation in the ventricle of the dog and turtle. Circ Res 34:561–569 Toyota E, Fujimoto K, Ogasawara Y et al (2002) Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts. Circulation 105:621–626 van der Spoel TI, Vrijsen KR, Koudstaal S et al (2012) Transendocardial cell injection is not superior to intracoronary infusion in a porcine model of ischaemic cardiomyopathy: a study on delivery efficiency. J Cell Mol Med 16:2768–2776 Vinegoni C, Lee S, Gorbatov R et al (2012) Motion compensation using a suctioning stabilizer for intravital microscopy. Intravital 1:115–121

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Vinegoni C, Aguirre AD, Lee S et al (2015) Imaging the beating heart in the mouse using intravital microscopy techniques. Nat Protoc 10:1802–1819 Wagers AJ, Sherwood RI, Christensen JL et al (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297:2256–2259 Wang N, Li Q, Zhang L et al (2012a) Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6. PLoS One 7:e43768 Wang N, Shao Y, Mei Y et al (2012b) Novel mechanism for mesenchymal stem cells in attenuating peritoneal adhesion: accumulating in the lung and secreting tumor necrosis factor alphastimulating gene-6. Stem Cell Res Ther 3:51 White RL, Nash G, Kavanagh DPJ et al (2013) Modulating the adhesion of haematopoietic stem cells with chemokines to enhance their recruitment to the ischaemically injured murine kidney. PLoS One 8:e66489 Wognum AW, Eaves AC, Thomas TE (2003) Identification and isolation of hematopoietic stem cells. Arch Med Res 34:461–475 Wollert KC, Meyer GP, Lotz J et al (2004) Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364:141–148 World Health Organisation (2019) World Health Organization cardiovascular disease risk charts: revised models to estimate risk in 21 global regions. Lancet Glob Health 7:e1332–e1345 Yada T, Hiramatsu O, Kimura A et al (1993) In vivo observation of subendocardial microvessels of the beating porcine heart using a needle-probe videomicroscope with a CCD camera. Circ Res 72:939–946 Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury. N Engl J Med 357:1121–1135 Yoshida T, Hakuba N, Morizane I et al (2007) Hematopoietic stem cells prevent hair cell death after transient cochlear ischemia through paracrine effects. Neuroscience 145:923–930 Zhang S, Shpall E, Willerson JT et al (2007) Fusion of human hematopoietic progenitor cells and murine cardiomyocytes is mediated by alpha 4 beta 1 integrin/vascular cell adhesion molecule-1 interaction. Circ Res 100:693–702 Ziegler M, Wang X, Lim B et al (2017) Platelet-targeted delivery of peripheral blood mononuclear cells to the ischemic heart restores cardiac function after ischemia-reperfusion injury. Theranostics 7:3192–3206 Ziegler M, Haigh K, Nguyen T et al (2019) The pulmonary microvasculature entraps induced vascular progenitor cells (iVPCs) systemically delivered after cardiac ischemia-reperfusion injury: indication for preservation of heart function via paracrine effects beyond engraftment. Microcirculation 26:e12493

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Stem Cells of the Thymus Valentin P. Shichkin

Abstract

The thymus is an essential organ of the immune system since it is the main site of T-lymphocyte development as well as in the regulation of adaptive immunity. The thymus has a highly complex structure that includes the thymic stroma and developing thymocytes. The thymic stroma contains dendritic cells, macrophages, epithelial, mesenchymal and vascular elements. This multicellular environment houses several minor stem cell populations including thymic epithelial progenitor cells/thymic epithelial stem cells, mesenchymal stem cells, and lymphoid progenitor/stem cells. The chapter discusses the role of these intrathymic stem cell populations for thymic cell architecture generation and function, and for the thymic regeneration after injury. Classical transplantation and modern bioengineering approaches and strategies for reconstitution of thymic structure and function using stem cell-based technologies have also been discussed in the chapter in the context of their foreseen perspectives and restrictions for different groups of immunocompromised patients. These groups include patients, which were thymectomized during heart surgery, chemotherapytreated cancer patients, elderly people with age-related thymus involution and impaired thymic function, and in some cases, patients with removed thymoma and thymus-associated autoimmune diseases. Understanding the role of each thymic stem cell population for supporting the thymus structure and function is a significant factor for choosing a promising thymus reconstitution strategy for these specific groups of patients. In this chapter, the author revises his long-time

V. P. Shichkin (*) Open International University of Human Development “Ukraine”, Kiev, Ukraine Enamine-Bienta Ltd, Kiev, Ukraine e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 K. H. Haider (ed.), Stem cells: From Potential to Promise, https://doi.org/10.1007/978-981-16-0301-3_2

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bench experience of the thymic stem cells in the context of current advancements in this cutting area of research. Keywords

Intrathymic T-cell progenitors · Stem cells · Thymus · Thymic stem cells · Thymic epithelial cells · Thymic mesenchymal cells · Thymus regeneration · Radioresistant thymic cells

Abbreviations Con A cTECs ESCs Gy HSCs IL iPSCs LPCs MCs MSCs mTECs NK PHA SCF Sv TECs TEPS TESCs THGF TLPs TLSCs

2.1

Concanavalin A Cortical TECs Embryonic stem cells Gray Hematopoietic stem cells Interleukin Induced pluripotent stem cells Lymphoid progenitor cells Mesenchymal cells Mesenchymal stem cells Medullary TECs Natural killer Phytohemagglutinin Stem cell factor Sievert Thymic epithelial cells Thymic epithelial progenitor cells Thymic epithelial stem cells Thymocyte growth factor T-lymphocyte progenitors Thymic lymphoid stem cells

Introduction

The thymus is one of the central organs of the immune system, and it is the major site of T-lymphocyte generation and regulation of adaptive immunity. Significance of the thymus for development and function of the immune system is the subject of hot discussions since 1961 when thymus function was first reported by Jacques Miller (Miller 1961). Early studies on transplantation of syngeneic thymus to thymectomized mice had shown the important role of the organ (Dalmasso et al. 1964). It was shown that the impaired thymus function might have several dramatic

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consequences including increased susceptibility to infection and autoimmunity, high risk of cancer development, as well as decreased response to vaccines with age (Mancebo et al. 2008; Prelog et al. 2009; Kurobe et al. 2013; Stosio et al. 2017). Patients undergoing complete thymectomy as neonates are more likely to suffer from age-related diseases, such as atherosclerosis, autoimmune or neurodegenerative diseases, and they have persistent imbalance of naïve T cells in the periphery (Prelog et al. 2009; Afifi et al. 2010; Kurobe et al. 2013; Stosio et al. 2017). Current epidemiological data indicate that almost 1 in 100 children is born with a heart defect, and they are potential patients for heart surgery and partial or total thymectomy that may have severe consequences, especially if thymectomy was done at the age below 1 year (Kurobe et al. 2013; Stosio et al. 2017; Shichkin and Antica 2020). According to other data, thymectomy has no critical clinical effects, if performed in the postinfant period (Roosen et al. 2015). However, most data were collected during a short follow-up time after thymectomy thus not allowing relating to the onset of age-related diseases in the thymectomized group. Besides, the inclusion of individuals with residual thymic tissues might cause an underestimation of the impact of thymectomy. Since thymectomy is a part of standard surgical procedure for congenital heart diseases, the thymus is a medical waste and may be used at least as an alternative source of autologous tissue-specific stem cells for personalized treatment of thymectomized infants. In relation to this, actual challenges are the optimization of thymectomy procedure in infants, thymic tissue collection, and cryopreservation, as well as preparation of thymic stem cells, their clonal expansion, and development of stem cell-based therapy for thymus regenerative therapy for infections, allergies, autoimmune, oncological and other diseases associated with the impaired thymic function.

2.2

Cell Architecture of Thymus

The thymus has a highly complex structure that is comprised of thymic stroma (about 5%) and the developing T cells (thymocytes), which constitute over 95% of the organ (Fig. 2.1). Two distinct layers, cortical and medullary, form the intrathymic structure. Each layer contains thymocytes at a discrete stage of maturation and a small number of B cells, natural killer (NK) cells, dendritic cells, fibroblasts, macrophages, as well as epithelial, mesenchymal, and vascular elements forming a thymic stromal-epithelial microenvironment for the development of T cells (Spits 1994; Manley et al. 2011; Abramson and Anderson 2017; Muсoz and Zapata 2019; Shichkin and Antica 2020). Moreover, several minor stem cell populations can be found in the thymus, in particular thymic epithelial progenitor cells/thymic epithelial stem cells (TEPCs/TESCs) (Bennett et al. 2002; Ucar et al. 2014; Ulyanchenko et al. 2016; Abramson and Anderson 2017), mesenchymal stem cells (MSCs) (Siepe et al. 2009; Iacobazzi et al. 2018; Wang et al. 2018), and multipotent progenitor cells/lymphoid progenitor cells (LPCs) (Palacios and

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Fig. 2.1 Human thymus cell architecture. The human thymus is located in the upper anterior part of chest behind sternum between lungs and lies on top of the heart along the trachea. The thymus reaches maximum weight (about 28 g) during puberty. This is pinkish-gray organ consisted by two lobes. The thymic lobes separated by connective tissue strands (trabeculae) on lobules. Each thymic lobule consisted by cortex and medulla. Cortex contains CD34+ uncommitted pluripotent hematopoietic precursor cells (HPC) entering the thymus through postcapillary venules located at cortico-medullary junction (CMJ) and migrating to the capsule, committed CD4 CD8 T precursor cells (TPC) located in the subcapsular region, and immature CD4+CD8+ cortical thymocytes migrating through the cortex and CMJ to the medullar zone. Medulla contains CD4+ and CD8+ naïve thymocytes migrating to the periphery after maturing. Stromal-epithelial compartment of the thymus is submitted by minor populations of EpCam+(CD326+)Foxn1+ bipotent thymic epithelial precursor cells/thymic epithelial stem cells (TEPC/TESC) and mesenchymal stem cells (MSC) located probably into the thymic parenchyma close to the CMJ region, as well as EpCam+CD205+ cortical thymic epithelia cells (cTEC) located in the cortex and EpCam+Air+ medullary thymic epithelial cells (mTEC) located in the medulla. Moreover, cortex and medulla contain also macrophages, fibroblasts, and dendritic cells (DC) that together with cTEC and mTEC participate in differentiation, maturation, and positive and negative selection of thymocytes. HPC generate the all thymocyte populations and alternatively may generate macrophages and DC; TEPC/TESC generate cTEC and mTEC lineages depending of local microenvironment and cross-talk with cortical or medullary thymocytes; MSC generate thymic fibroblasts and adipocytes. BV blood vessel, DT dead thymocyte, HC Hassall’s corpuscle. (Reproduced from Shichkin and Antica 2020)

Pelkonen 1988; Shichkin 1990; Wu et al. 1991; Antica et al. 1993). Of these, thymic epithelial cells (TECs) presented by cortical (c) and medullary (m) TECs that are morphologically and functionally distinct, provide most of the specialized organ functions, mediating different aspects of T-cell development (Calderon and Boehm 2012; Abramson and Anderson 2017). cTECs are required

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for commitment of early thymocyte precursors to a T-cell lineage through the provision of Notch ligand Dll4 (Garcia-Peydro et al. 2006; Koch et al. 2008; García-León et al. 2018), and to drive the expansion of thymocytes at several stages of development through the provision of growth factors and cytokines including IL-7, IL-22, SCF, and a few others (Rodewald et al. 1997; Manley et al. 2011; Dudakov et al. 2012; Abramson and Anderson 2017). They also regulate the positive selection of T-cell repertoire through the provision of a unique set of peptides generated by a thymus-specific proteasome subunit, β5t (Murata et al. 2008; Takahama et al. 2012). mTECs regulate migration of positively selected thymocytes from the cortex into the medulla, via expression of CCL19 and CCL21, and they also regulate the accumulation and positioning of dendritic cells in the medulla via secretion of a chemokine XCL1 (Lei et al. 2011). Both processes are also regulated by thymus resident dendritic cells, which are critical hematopoietic components of the thymic microenvironment (Martin-Gayo et al. 2010, 2017). Among the thymic cells, intrathymic LPCs are also an important component for the maintenance, differentiation, and regeneration of the thymic tissue (Wu et al. 1991; Këpuska and Sempowski 2011; Muсoz and Zapata 2019). Thus, the production of a functional, self-tolerant T-cell repertoire requires interactions between developing thymocytes and a variety of cTECs and mTECs types derived from TESCs/TEPSs as well as with other stromal and lymphoid components of the thymus.

2.3

The Thymic Stem Cells

2.3.1

Thymic Epithelial Stem Cells

Analysis of thymus development has established that cTECs and mTECs can originate from a common epithelial precursor in both fetal and postnatal thymus (Bennett et al. 2002; Bleul et al. 2006; Wong et al. 2014; Ulyanchenko et al. 2016). Transplantation of TEPSs/TESCs is sufficient for the direct establishment of an entire thymus, including thymocytes and all stromal elements (Bleul et al. 2006; Wong et al. 2014; Bredenkamp et al. 2015). In mice, the TECs express the cell surface protein Plet1 (unexpressed in human TECs) that marks TESCs generating all epithelial cell types of the mature thymus (Depreter et al. 2008; Nowell et al. 2011). Plet1+ TEPCs express EpCam (CD326) surface protein (Ulyanchenko et al. 2016) that is also expressed in early human fetal thymus, and therefore EpCam in combination with other markers can be used to identify human TEPCs/TESCs populations (O’Neill et al. 2016; Shichkin et al. 2017, 2018). The epithelial component of the postnatal thymus also is maintained by TEPCs/TESCs mediated by Foxn1 and Notch signaling (Nowell et al. 2011; Vaidya et al. 2016; Žuklys et al. 2016; Liu et al. 2020). In mice, these TEPCs/TESCs comprise 1–2% of the total TECs, located into the thymic parenchyma at the cortico-medullary junction (Fig. 2.1), express Plet1 and Ly51 surface proteins and can generate both cTECs and mTECs (Ulyanchenko et al. 2016; Liu et al. 2020). Fetal human thymus expresses CD326, and therefore in combination with Foxn1, this marker was used to identify human

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TEPCs/TESCs populations in human fetal (O’Neill et al. 2016) and postnatal thymus (Shichkin et al. 2017, 2018). Under non-adhesive conditions, TESCs are able to form thymospheres, which in the mouse thymic cultures were described as Foxn1 epithelial cells (Ucar et al. 2014). According to other data, these thymospheres are formed by Foxn1 EpCAM mesenchymal cells with the potential to generate only adipocytes, but not epithelial cells (Sheridan et al. 2017), and therefore this should be investigated more carefully in human thymus.

2.3.2

Thymic Mesenchymal Stem Cells

Mesenchymal cells (MCs) are major producers of the extracellular matrix, which provides a structural framework for cell migration, and serves as a reservoir for cytokines and growth factors used by epithelial and hematopoietic parenchymal cells. In adults, MCs were found in all tissues and organs, where they play mechanical and metabolic roles. Among these, bone marrow MCs are the most studied because they are key components of the hematopoietic stem cells (HSCs) niche, and they contain mesenchymal stem cells (MSCs) that display the ability to regulate immune responses and coordinate tissue regeneration (Mendez-Ferrer et al. 2010; Morrison and Scadden 2014). However, little is known about the thymic MCs and especially about thymic MSCs. Thymic MCs regulate the proliferation of thymic epithelial cells through production of fibroblast growth factor (FGF)-7 and -10, insulin-like growth factor (IGF)-1 and -2, and retinoic acid (Jenkinson et al. 2007; Sitnik et al. 2012). During the postnatal life, thymic CD248+ MCs play a significant role for revascularization of the thymus during infection-dependent regeneration, and thymic FSP1+ MCs are essential for the maintenance of the medullary thymic epithelium (Lax et al. 2012; Sun et al. 2015). Thymic MCs express the differentially expressed genes (DEGs) that are connected to organ-specific features such as clearance of apoptotic cells in the thymus (thymic MCs), osteoclastogenesis (bone marrow MCs), and hair follicle homeostasis (skin MCs) (Patenaude and Perreault 2016). According to recent data, mouse Foxn1 EpCAM thymic MCs may form thymospheres in non-adhesive cultures in vitro, which have the potential to generate adipocytes (Sheridan et al. 2017). In autologous co-cultures, thymic MCs support the viability and differentiation of thymocytes through direct contact (Azghadi et al. 2016). However, in allogeneic co-cultures, human thymic MCs only slightly enhance the proliferation of responding cells, and reduce the proliferation of already activated lymphocytes by about 50% thus highlighting the immunomodulating property of human thymic MSCs (Siepe et al. 2009). The neonatal human thymus contains MCs fulfilling the minimal criteria postulated for MSCs: adherence to plastic surface under standard in vitro culture conditions, MSC-like surface marker expression, and tri-lineage differentiation potential in vitro into osteogenic, chondrogenic, and adipogenic mesenchymal cell lineages. Additionally, neonatal thymic MSCs possess both the immunomodulatory characteristics and potential to differentiate into a cardiomyogenic lineage (Siepe et al. 2009; Wang et al. 2018). Moreover, neonatal thymic MSCs express and secrete

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in vitro the proangiogenic and cardiac regenerative morphogen sonic hedgehog (Shh) more than bone-derived MSCs, and organoid cultures of neonatal MSCs stimulate Shh expression providing cytoprotective effect for neonatal cardiomyocytes (Wang et al. 2018). Thymic MSCs are negative for the immunologically relevant surface antigens HLA-DR, CD40, CD40L, CD80, and CD86, and have phenotype as CD34 , CD38 , CD44+, CD45 , anti-HLA-DR , anti-HLAABC , CD80 , CD66 , CD86 , CD13+, CD29+, CD73+, CD90+, CD105+, CD106 , and CD166+ (Siepe et al. 2009). Thus, human MSCs might be considered for a concomitant cell therapy to thymus regenerative therapy with TESCs for patients with congenital heart diseases.

2.3.3

Thymic Lymphoid Stem Cells

Thymic lymphoid stem cells (TLSCs) are presented by CD34+CD4 CD8 TCRα/β pluripotent bone marrow migrants, which are committed to the development of lymphoid lineages in the thymus that include NK cells, T cells, B cells, and intrathymic dendritic cells (Martin-Gayo et al. 2010, 2017; Raghav and Gangenahalli 2018). The stem cell proliferation and cell fate in the bone marrow are maintained by stem cell factor (SCF) and its c-kit receptor as well as different morphogen factors such as Notch ligands, in particular Wnt, Hedgehog, TGFβ, and BMP (Sharma et al. 2006; Raghav and Singh 2018; Raghav and Gangenahalli 2018). These factors provide signals that regulate their self-renewal and differentiation into multiple lineages (Raghav and Gangenahalli 2018). The early TLSCs migrated from the bone marrow get lodged around the cortico-medullary junction close to entering into the thymus, and later they migrate to the subcapsular zone of the thymus. From this location, TLSCs consequently migrate to cortical and medullary zones undergoing the differentiation in immature CD4+ CD8+ double-positive thymocytes and then in naive single-positive CD4+ and CD8+ thymocytes (Fig. 2.1), subsequently undergoing positive and negative selection (Wu et al. 1991; Antica et al. 1993; Muсoz and Zapata 2019; Shichkin and Antica 2020). Direct cross-talk of thymocytes with stromal-epithelial cells as well as chemokine, hormonal, and cytokine signals provide the necessary conditions for these intrathymic events (Palacios and Pelkonen 1988; Martin-Gayo et al. 2010; Manley et al. 2011; Kasai et al. 2014; Sekai et al. 2014). The early phase of T-cell development mainly occurs in the thymic cortex that is mediated by the contact with cTECs and high expression of chemokines, CCL25, CXCR4, CXCL12, DLL4, and cytokines, IL-7, and c-kit, by these TECs. Chemokines CCL25 and CXCL12 contribute to the accumulation and survival of T-lymphoid progenitor cells in the cortex (Trampont et al. 2010; Commins et al. 2010), the Notch ligand DLL4 contributes to the differentiation of T-lymphoid progenitors into T cells (García-León et al. 2018), and IL-7 and c-kit contribute to the proliferation of immature T cells (Rodewald et al. 1997; GarciaPeydro et al. 2006; Commins et al. 2010). As a consequence of T-cell lineage determination, cells undergo V(D)J rearrangement of a T-cell antigen receptor (TCR) loci in the nucleus and express TCR on the cell surface (Palacios and

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Pelkonen 1988). Immature T cells also begin the expression of coreceptors CD4 and CD8, thereby becoming CD4 and CD8 double-positive thymocytes that express the TCRαβ–CD3 complex (Kasai et al. 2014). Finally, they undergo positive and negative selection and become single-positive naïve CD4 and CD8 thymocytes (Takahama et al. 2012), which migrate from medullar zone of the thymus to lymphoid organs. Besides the T-cell lineage differentiation, TLSCs may contribute also to the development of the thymic B cells, dendritic cells, NK cells, and macrophages (Ardavin et al. 1993; Marquez et al. 1998) confirming their pluripotent stem cell potential.

2.4

Intrathymic Cytokine Network

The process of thymocyte proliferation and maturation is in consequence of direct contact of thymocytes with stromal-epithelial elements and in response to various cytokines, that are secreted both by lymphoid and stromal cells of the thymus. The most important of them is IL-7, which regulates intrathymic maturation, differentiation, and survival of T cells in the early stages of their development (Costello et al. 1993; Kim et al. 1998; Shitara et al. 2013). However, several other cytokines that are normally present in periphery can also be found in the thymus. Since the thymus is a relatively closed organ for macromolecular migration into/from the organ, this situation necessitates the existence of thymus’s own cytokine network that is adapted for utilization inside the thymus and does not secrete these cytokines to periphery, though for many intrathymic cytokines their biological role is still unclear. Many cytokines including IL-1, TNFα, GM-CSF, G-CSF, M-CSF, SCF, IL-3, IL-6, IL-7, IL-8, IL-12, IL-15, and TFRβ that are spontaneous products of TECs and other stromal thymic cells, as well as IL-1, IL-2, IL-3, IL-4, IL-5, IL-10, IL-22, IL-23, and IFN-γ that are spontaneous products of CD4 CD8 TLPs or/and activated CD4+ thymocytes have been identified in the thymus and their role for development of T cells was analyzed (Moore et al. 1993; Rich 1997; Yarilin and Belyakov 2004; Dudakov et al. 2012; Lowe et al. 2017). Though all types of thymic cells are able to produce cytokines either spontaneously or after stimulation, the main producers of cytokines in the thymus are TECs and thymocytes that consist of two major populations of thymic cells. The subcapsular and mTECs are more active cytokine producers than cTECs, and the cytokine spectrum of TECs is very close to the spectrum of cytokines produced by peripheral macrophages and monocytes. Thymocytes constitute the most abundant thymic cell population that consists about 95% of thymic cells, and though they are relatively weak cytokine producers as compared to non-lymphoid thymic cells, their contribution to intrathymic cytokine network is essential. The ability of thymocytes to produce cytokines and express cytokine receptors is gradually reduced as they mature from the stage of CD44+CD3 CD4 CD8 precursor cells to the stage of immature CD3loCD4+CD8+ cortical thymocytes and become completely blocked in the latter CD4+CD8+ stage. The capacity of thymocytes to produce cytokines and respond to their action is restored at the monopositive CD4+ and CD8+ stage of thymocytes after the

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completion of the selection process (Yarilin and Belyakov 2004). The triggering factors that induce intrathymic cytokine production are still remaining relatively contentious and require further elucidation. In several model experiments in vitro, thymocytes have been shown to produce cytokines in response to binding of TCR-CD3 receptor complex or alternative mitogenic stimulation (Park et al. 2010). An important inducing or modulating role for cytokine production in the thymus has been assigned to the intercellular contacts especially between thymocytes and epithelial cells; the peptide hormones and cytokines are produced in response to this stimulation (Yarilin and Belyakov 2004; Lowe et al. 2017). Thymic cytokines act inside the thymus as short distance factors and the spectrum of their biological effects is determined by the expression of cytokine receptors on thymic cells. Some thymic cytokines act in a paracrine manner while the others act as autocrine factors. Examples of paracrine thymic cytokines are IL-7 and SCF that are produced by TECs and thymic MCs and induce the growth and differentiation of CD4 CD8 TLPs (Shitara et al. 2013; Politikos et al. 2015). Another example is INFγ that is produced by thymocytes and induces the TECs activation (Yarilin and Belyakov 2004). Examples of autocrine action are IL-2 and IL-4, for which the producers and targets are thymocytes, and THGF, for which the producers and targets are radio-/cortisol-resistant intrathymic CD4 CD8 precursors (Shichkin et al. 1988b; Protsak et al. 1989b; Shichkin 1990, 1992). While SCF, IL-7, and THGF are sufficient to promote the proliferation and survival of CD4 CD8 intrathymic precursors, IL-2 and IL-4 are more specific towards the final stages of T-cell development and differentiation to single-positive CD4+ and CD8+ thymocytes (Kim et al. 1998; Yarilin and Belyakov 2004; Park et al. 2010). On the other hand, some CD4 CD8 stages of intrathymic TLPs are also sensitive to IL-2 and IL-4 action (Raulet 1985; Lowenthal et al. 1988), while IL-7 together with IL-12, IL-22, IL-23, and IFN-γ actively contribute to the final stages of thymocyte differentiation and their negative selection (Lowe et al. 2017). SCF plays an important role in hematopoiesis and lymphopoiesis (Broudy 1997; Politikos et al. 2015). SCF is produced by thymic stromal cells, and its receptor c-kit is highly expressed by the early thymic progenitors (Godfrey et al. 1992; deCastro et al. 1994). Besides IL-7, the SCF/c-kit complex is essential during the early stages of thymopoiesis (Godfrey et al. 1992; Morrissey et al. 1994; Rodewald et al. 1995; Massa et al. 2006). Besides cytokine signaling, which is mediated through the Janus kinase signal transducer and activation of transcription (JAK-STAT) pathway, steroid hormone receptors, especially the orphan steroid receptors, play a key role in thymocyte development, selection, and survival (Akdis et al. 2011; Lowe et al. 2017).

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Radioresistant Thymic Cells and Thymocyte Growth Factor (THGF)

The pioneering findings that the thymus of adult mice contains a population of radioresistant cells, which are capable of temporarily restoring of T-cell populations in the thymus after sublethal total body irradiation were first reported by Kadish et al. (Kadish and Basch 1975). These radioresistant thymic cells constituted only a minor population of CD4 CD8 intrathymic TLPs (previously known as L3T4 Lyt2 ) located presumably in the subcapsular zone of the thymic cortex (Kadish and Basch 1975; Fredrickson and Basch 1994). While the main populations of thymic cells have been extensively studied and well characterized, the biological role and significance of radioresistant intrathymic TLPs for thymic function remain less wellunderstood. Nevertheless, several research groups, including Prof. Yarilin’s and Dr. Shichkin’s, have made sterling contributions in this exciting area of research (Shichkin et al. 1987a, b, 1988a, b; Protsak et al. 1989a, b; Shichkin 1990, 1992; Yarilin et al. 1990; Talaev et al. 1991a, b, 1992; Yarilin 1997).

2.5.1

Producers of THGF

We have previously established two stable transformed CD4 CD8 murine thymic cell lines TC.SC-1/2.0 and TC.SC-1/1.0, which were induced by multifold injections of human IL-2 to BALB/c mice (Shichkin et al. 1987a). These cell lines were intensively analyzed and characterized to establish their CD4 CD8 T-cell origin (Shichkin et al. 1987b, 1988a, b). These cell lines were rich in IL-2 receptor expression and were responding to IL-2 in the in vitro culture conditions. In culture, these cells spontaneously differentiated in CD4+ and CD8+ T cells while maintaining the main pool of CD4 CD8 precursors. During single-cell cloning, the clones included four cell populations: CD4 CD8 (about 75%), CD4+CD8+ (about 15%), CD4+ (about 7%), and CD8+ (about 3%), thus demonstrating the stem cell potential of CD4 CD8 cells (Shichkin et al. 1988a). During the early period of the establishment, two antigens SC-1 and Thy-1 were simultaneously expressed on the cell surface of the lines, which are peculiar to intrathymic TLPs. The cells also expressed a receptor for peanut agglutinin (PNA), the expression level of which decreased significantly upon treatment of the cells with α-1 thymosin. No markers specific for B cells or macrophages were found (Shichkin et al. 1988a). Following the gammairradiation, these cell lines secreted thymocyte growth factor, THGF, which acted in autocrine manner (Shichkin et al. 1988b). At this, in a diapason of the tested doses 3–24 Gy (Sv), only 12 Gy dose could strongly activate THGF production (Shichkin et al. 1988b; Protsak et al. 1989a). Moreover, our experiments also showed that TC. SC-1/2.0 cell line, in addition to THGF, could show IL-2 and IL-3 activities in lower concentration either spontaneously or after gamma-irradiation (Shichkin 1992). We also detected THGF-like activity in the supernatants of 28-h cultures of thymocytes obtained from mice on days 2, 5, or 12 after the sublethal irradiation

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and also in supernatants of freshly isolated thymocytes after 12 Gy gammairradiation in vitro (Shichkin, 1992). Interestingly, after the intraperitoneal injection of TC.SC-1/2.0 cells into syngeneic BALB/c mice, some of the cells formed local solid and ascitic tumors, while the remaining cells migrated into the thymus to form thymomas. Furthermore, these tumors cell lines showed contradicting properties, in particular the cell line established from a thymoma was THGF producing, actively proliferating and non-responding to exogenic THGF, while the cell line established from an ascytic tumor was THGF non-producer, slowly proliferating and responding to exogenic THGF (unpublished data). Since the thymus is open for access to committed CD34+CD4 CD8 bone marrow HSCs only but not to the mature T cells, this observation also supports the primary stem cell status of the THGF-producing thymic cell lines.

2.5.2

Biology of THGF

Biological activity of THGF in culture supernatants of the CD4 CD8 cell lines was discovered by its direct action to promote the proliferation of freshly isolated thymocytes in a 5-day test culture without mitogens or in the presence of co-stimulating agents using 3H-thymidine as a reporter system. The classical mitogens such as concanavalin A (Con A) and phytohemagglutinin (PHA) or phorbol esters such as phorbol myristate acetate (PMA) did not increase the action of THGF on thymocytes (Shichkin et al. 1988b). These observations clearly demonstrated the difference of the biological activity of THGF as compared to the other known cytokines, especially IL-1, IL-2, IL-3, and IL-4 that were described as co-stimulatory molecules for the proliferative action of mitogens on thymocytes (Yarilin and Belyakov 2004). THGF was isolated and purified from the TC.SC-1/2.0 serum-free cell culture supernatant as a two-component glycoprotein with a molecular weight 22 kDa (THGF/gp22) and 15 kDa (THGF/gp15) (Talaev et al. 1991a). Both glycoprotein fractions showed the specific THGF activity in the standard test cultures of mouse thymocytes but the maximal proliferative response was observed on day 3 for THGF/gp15 and day 5 for THGF/gp22. Together, these fractions showed a synergistic effect. Similar to the TC.SC-1/2.0 cell culture supernatant, pure THGF fractions did not increase thymocyte proliferation in the presence of mitogens. The optimal stimulating dose of the purified THGF was 8–16 pg/ml which was equivalent to 12.5–25% concentration of TC.SC-1/2.0 supernatant, and the dose of 1–2 pg/ ml was enough to stimulate the thymocyte proliferation (Talaev et al. 1991b, 1992). Similarly, the optimal dose for most of the other thymus-related cytokines tested in analogous conditions was in the range of 50–100 μg/ml (unpublished data). Akin to the most mouse growth factors, THGF action was species-specific; the factor did not stimulate proliferation of rat and human thymocytes; however, THGF was active on the allogeneic mouse thymocytes (Shichkin et al. 1988b; Talaev et al. 1992).

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It is pertinent to mention that only 30–60 min of pre-incubation with THGF was required to activate the proliferation of freshly isolated thymocyte. However, the full proliferative effect of THGF was realized during 24-h pre-incubation. The prolonged presence of THGF in the culture medium did not increase the proliferative activity of thymocytes (Shichkin et al. 1988b; Shichkin 1990; Talaev et al. 1992). For supporting long-lived THGF-dependent cultures of thymocytes, replacement of the culture medium with a fresh portion of the THGF-containing supernatant was required after every 10–15 days since THGF in the culture medium was completely inactivated at 37  C in 25–30 days (Shichkin 1990). These data suggest that THGF possesses both the mitogenic and growth-supporting properties. On the contrary, a relatively long latent phase in the development of a proliferative response to THGF is required while activation period requirement is relatively short (about 1 h) which is suggestive of the involvement of some secondary growth factors induced by THGF. These secondary growth factors thus released may be responsible for the post-activation proliferation of the target cells.

2.5.3

Target Cells for THGF

It is now well established that the population of mouse thymocytes consists of immature double-positive CD4+CD8+ cortical thymocytes (80–90%) that express PNA receptors and are negative for IL-2 receptors (IL-2R or CD25). On the other hand, mature single-positive CD4+ and CD8+ medullar thymocytes (about 10–15%) express IL-2R but are negative for PNA while the intrathymic TLPs are PNA+CD25+CD4 CD8 and constitute 0.5–3% of thymocytes only (Spits 1994). The early intrathymic PNA+CD25+CD4 CD8 population also expresses the stem cell-1 (SC-1) antigen that is well presented on bone marrow derived HSCs (Kadish and Basch 1975; Yarilin et al. 1990), and this antigen probably is an analog of the SCF-specific receptor c-kit (CD117) (Blechman et al. 1993). On the same note, intrathymic TLPs are most resistant thymocytes to the action of corticosteroid hormones and gamma-irradiation (Kadish and Basch 1975; Yarilin 1997). Some important discoveries clarifying the nature of thymic target cells for THGF have been reported. We have reported that these cells are sensitive to the treatment with the intrathymic hormone α-1 thymosin that downregulates the expression of SC-1 on bone marrow derived SC-1+HSCs, which do not respond to THGF and shifts from a THGF-unresponsive stage to a THGF-responsive SC-1+ stage. At the same time, the treatment of intrathymic THGF-sensitive SC-1+ cells with α-1 thymosin shifts the cells to the THGF-unresponsive SC-1 stage (Protsak et al. 1989b). Furthermore, THGF-responsive thymocytes are resistant to the sublethal gamma-irradiation. Treatment of mice with hydrocortisone as well as exposure to gamma-radiation of their thymocytes in vitro in a dose range of 10–50 Gy did not alter their viability and responsiveness to THGF during long-term culture without exogenous cytokines, including THGF (Shichkin 1990, 1992). Pre-incubation of survived cells or freshly isolated thymocytes with THGF enhanced their sensitivity to IL-2, IL-3, and IL-1. Contrarily, pre-incubation of thymocytes with IL-2 led to

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accumulation of an IL-2-responsive cell population that was unresponsive to THGF (Shichkin et al. 1988b; Shichkin 1990). Injection of THGF to the sublethally irradiated mice promoted the accumulation of immature thymocytes in the thymus with SC-1+PNA+CD4 CD8 phenotype and their responsiveness in vitro to THGF, IL-2, and IL-3 (Shichkin 1990, 1992). IL-2-sensitive thymocytes are the activated mature T cells and CD4 CD8 TLPs (Yarilin and Belyakov 2004). TLPs, however, express low-affinity receptors for IL-2 (IL-2R) and may respond to IL-2 only after activation by mitogens (Raulet 1985) or in the presence of IL-1 (Howe et al. 1986; Dinarello 1994). On the other hand, THGF-responsive target cells are PNA+SC-1+CD4 CD8 intrathymic TLPs (Shichkin et al. 1988b; Protsak et al. 1989b; Shichkin 1990, 1992). Therefore, THGF-responsive TLPs probably express low-affinity IL-2R. Pre-incubation of these cells with THGF, as well as pre-injection of THGF into mice, stimulates the expression of the high-affinity IL-2R similar to the IL-1 effect. Indeed, blocking IL-2R with CD25-specific monoclonal antibodies recognizing α-chain of IL-2R (IL-2Rα) significantly inhibited thymocyte’s response to THGF (Talaev et al. 1992). There are three different IL-2R chains that together generate low-, intermediate-, and high-affinity IL-2R complex (Liao et al. 2011). The ligand-specific IL-2Rα (CD25), which is expressed specifically on the activated lymphocytes, binds with IL-2 with low-affinity; the combination of IL-2Rβ (CD122) and IL-2Rγ (CD132) together forms an IL-2Rβ/γ complex that binds with IL-2 with intermediate affinity. When all three receptor chains are co-expressed on the activated T cells, it favors their high-affinity binding with IL-2 (Liao et al. 2011). The intermediate- and highaffinity receptor forms are functional and may transduce IL-2 signals (Liao et al. 2011). Together, these data suggest that IL-2 and/or other cytokines such as IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21, which bind with IL-2Rγ (Liao et al. 2011), may act as the secondary signal or co-stimulators in the development of the total response to THGF. Probably, THGF can also use the IL-2Rα for own signal transduction. Presumably, THGF-specific receptor components may also exist. Thus, these data propose that cortisol/radioresistant SC-1+PNA+CD4 CD8 intrathymic TLPs that constitutively express the low-affinity receptors for IL-2 are the target cells for the proliferative action of THGF, and THGF uses IL-2Rα to induce expression of highaffinity IL-2R complex. Furthermore, these data provide ample evidence about the specificity of THGF-dependent proliferation of thymocytes and successive change of THGF-sensitive stage to THGF/IL-2-sensitive stage. While the proliferative response to THGF was usually registered in a 5-day test culture, the maximal response was reached only on days 9–11. At the same time, the number of viable thymocytes in the 11-day cell culture was decreased to 2–5%, and it slowly increased again during the prolonged cell culture for up to 25–30 days but not exceeding 25–30% of the primary seeding (Shichkin 1990). It was interesting to note that maximum proliferative response to THGF corresponded to the minimal concentration of viable thymocytes in the culture, while the period of increase in cell number correlated with THGF-independent and weak spontaneous proliferation (Shichkin 1990). Furthermore, we observed that colchicine, an inhibitor of cell mitosis had no influence on THGF-dependent proliferation of thymocytes in a

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5-day test culture, but prevented ConA- and IL-2-induced proliferation that was evaluated by the rate of DNA synthesis in the cells with 3H-thymidine (Shichkin 1992). An assumption that the plasma membrane of THGF-responding cells is impenetrable for colchicine, and the THGF-induced DNA synthesis is not accompanied by increased cell division at least for the first 5–10 days of thymocyte culturing with THGF, may explain these novel observations. In other words, THGF primarily induces multifold accumulation of nuclear material in the cells or forming of multinucleated cells without mitosis, thus showing the peak of proliferative response on days 5–10, followed by the formation of cell clones or cell clusters, which supports the increase in cell number in the extended cultures.

2.5.4

Long-Lived TGHF-Dependent Thymic Cell Cultures

The mouse THGF-dependent long-lived thymic cell culture/line with unique growth properties was established and morphological and growth properties of the cells were observed for more than 1 year (Shichkin 1990). It was observed that the addition of the fresh THGF-supernatant promoted the formation of rosette-like thymic cell clusters. The cell clusters contained two morphologically distinct cell types, a central large cell population and the small cells attached to the central cell population. The cell clusters may be found both in the adherent and suspension cell cultures in vitro (Fig. 2.2a–c) (Shichkin 1990). The appearance of the small cells was observed only over 15–20 days after cell seeding and selection of the thymic cells in the presence of THGF-supernatant. Moreover, the cluster-forming process at the level of the single large cells was cycled. The cycle had a 25–30-day period and finally ended by death of most of the small cells (Shichkin 1990). This was similar to the two waves of thymus recovery and depletion in vivo in mice after the sublethal gamma-irradiation (Kadish and Basch 1975; Ayukawa et al. 1990; Këpuska and Sempowski 2011). Unfortunately, the data from this experiment neither allowed us to draw a definite inference about the origin of both the small and large cells in the clusters nor their sensitivity to THGF. Nevertheless, morphological comparison of the cell growth in culture with the dynamics of proliferative response of thymocytes to THGF allowed us to propose that the large cells were acceptors of the first activating signals of THGF. Moreover, the population of small cells presumably was formed inside the large cells as a result of this activation. Similar observations have been previously reported by other research groups which showed that the thymic epithelial nurse cells absorb immature CD4+CD8+ thymocytes and provided the intracellular sites for the functional maturation of TCRγ/δ T cells (Kyewski 1986; García and Tamayo 2013; Shitara et al. 2013). Other similar examples include the formation of thymic rosettes by the thymocytes associated with the intrathymic adherent macrophages and non-adherent dendritic cells (Shortman et al. 1989; Shortman and Vremec 1991). Although there is a possibility that the rosettes/clusters in the long-lived THGF-dependent thymic cultures may be formed by macrophages or/and dendritic cells (the central cells in the rosettes), the question about the origin and type of the small cells in the clusters

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Fig. 2.2 (a–c) Specific features of the morphology and growth of mouse thymic cells in presence of THGF, 320–410 days of observation, 700 magnification. (a, b) Adhesive growth, (c) suspension growth (modified from Shichkin 1990). (d, e) 90-day culture of gamma-irradiated by a dose of 50 Sv mouse thymocytes, 200 magnification. (d) Non-stained lifetime culture, (e) eosinhematoxylin stained fixed lifetime culture (unpublished data)

remains contentious. Native CD4+CD8+ or CD4+ and CD8+ thymocytes are not long-lived in cell cultures and die within 15–30 days of culture in vitro in the absence of exogenous stimuli (Shichkin 1990, 1992). However, they may appear spontaneously, if CD4 CD8 TLPs are present in the long-lived cultures. We identified these cell populations in a culture of mouse thymocytes that were gamma-irradiated with a dose 50 Gy and supported in vitro for more than 90 days (unpublished data). On the contrary, we were not able to find similar rosettes/clusters in spontaneous thymocyte cultures as well as in the long-lived thymocyte cultures in the presence of recombinant IL-2, IL-3, IL-4, IL-6, IL-7, SCF, and GM-CSF either alone or in the different combinations (unpublished data). Furthermore, we may assume that the large cells in our described long-lived THGF-dependent cell culture/line were self-renewing intrathymic CD4 CD8 stem cells, which were activated by THGF and autocrine growth factors. It is also possible that small cells were their daughter cells with

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similar phenotype. Among these daughter cells may also exist the immature CD4+CD8+ thymocytes, which require other cytokines and signals for their further development such as IL-7, besides the need for direct contact with the stromalepithelial microenvironment. This property of THGF together with its ability to increase the formation of hemopoietic colonies in the spleen (Yarilin et al. 1990) supports the idea that THGF is a member of the SCF family, thus bringing together but not identifying THGF with IL-7, SCF, and GM-CSF. The proliferative response of the long-lived THGF-dependent thymic culture/line to the treatment with THGF, interleukins, and mitogens, in principle, does not differ from the response given by the freshly isolated thymocytes, irradiated thymocytes, or the thymocytes pre-incubated in the absence of growth factors. However, their response was time-dependent after the addition of a fresh portion of THGF. As rule, by 10–15 days after the addition of THGF, the level of THGF-induced proliferation in the long-lived culture practically was reduced to as low as spontaneous proliferation of the naïve untreated thymocytes. In this case, the THGF-dependent cells did not respond to the presence of IL-1, IL-2, IL-3, Con A, PHA or their combinations in terms of enhanced rate of proliferation. However, the combination of THGF with these cytokines (but not with mitogens) provided the co-stimulatory effect (Shichkin 1990, 1992). These data supported our assumption that THGF induces the expression of high-affinity receptors for other cytokines. We completed these investigations between 1985 and 1990 at the Institute of Immunology, Moscow, Russia in Prof. Yarilin’s laboratory. Unfortunately, subsequent dramatic events in the former USSR did not allow us to complete the investigations and THGF-producing cell lines were lost. Later, a unique experiment was carried out by the author (Dr. Shichkin) in 1999 at Dr. J. Oppenheim and Dr. S. Durum Laboratories (NIH, NCI-FCRDC, Frederick, MD, USA) to simulate the irradiation-induced growth of mouse thymocytes and to analyze their supernatants for a wide panel of cytokines and their biological activities. However, the novel data from these experiments still remains unpublished and are briefly presented in the following sections.

2.5.5

Radiation-Induced Growth of Thymic Cells

Since THGF-producing and THGF-sensitive cells presumably present the same population of radioresistant intrathymic stem cells, and gamma-irradiation acts as an activating factor for them, CBA mice were sublethally irradiated to delete the radiation-sensitive cells in the thymus. Two days later, thymic cells were isolated and irradiated in vitro with gamma-radiation doses of 12 Gy (Sv) and 50 Gy (Sv). The cells were seeded in culture chambers with the regular culture medium and observed for more than 90 days. As controls, we used non-irradiated mice and their derivative non-irradiated thymic cells, besides thymic cells derived from the irradiated mice without any additional irradiation in vitro. During the 30th day of culturing, we observed a dramatic reduction in the number of alive cells in the all cell culture chambers. The radioresistant thymic cells (irradiated in vivo and in vitro by a dose 50 Gy), which survived in the culture, formed visible clones to day 30. When

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the clones attained a sufficient size (day 90), they were fixed and stained in the same culture chamber where they were primarily seeded (Fig. 2.2d, e). Although this cell culture was grown without treatment with exogenous THGF, the rosette/cluster structures similar to the long-lived thymocyte culture grown in the presence of THGF were observed. The conditioned medium of this culture was tested for a panel of cytokines (IL-2, IL-4, IL-7, IL-9, GM-CSF, and SCF) using cytokine-dependent cell lines, using recombinant cytokines as controls. The THGF-like activity was tested in their supernatants using the standard 5-day test culture of mouse thymocytes. These assays showed that the supernatant samples received from the irradiated thymocytes had THGF-like activity, unlike the other tested cytokines. Moreover, these supernatants were not active for IL-7-, SCF, and GM-CSF-dependent cell lines. On the other hand, IL-7 and SCF showed the same effect on thymocytes in the 5-day test culture as the supernatants of irradiated thymocytes (induction of proliferation without PHA and absence of stimulating effect with PHA). These data demonstrated the presence of a THGF-like activity in the supernatants of irradiated thymocytes that was not identical to IL-2, IL-4, IL-7, IL-9, GM-CSF, and SCF. The 50 Gy irradiated and non-irradiated long-lived thymic cells were analyzed for proliferative response to these cytokines besides the expression of surface markers using monoclonal antibodies specific for CD4, CD8, CD44, and CD25 antigens in a two-color FACS assay. Cells were analyzed on days 5, 10, 25, and 90 after seeding in the culture chambers. A number of viable thymocytes in both irradiated and non-irradiated cell cultures progressively decreased as low as 10–15% by the day 25 in culture. By day 90 in the culture, the number of viable cells was nearly 95%. Irradiated survived thymic cells showed low rate of proliferation, and day 90 culture was not responding to IL-2, IL-4, IL-7, IL-9, SCF, and GM-CSF. These cells however were responding to IL-7 and SCF as well as to the supernatant of the irradiated thymocytes until day 25 in culture. The response of non-irradiated 5-day and 10-day cultures was similar to the response of a 5-day culture of irradiated thymocytes. These data suggest that long-lived thymocytes self-supported their proliferation in an autocrine manner, and the analyzed cytokines did not play any significant role in their survival and proliferation. By day 10, most of the cells were CD4+CD8+ and CD25+CD44 . The cell populations expressing CD4 CD8 , CD4+CD8 , CD8+CD4 and CD25 CD44 , CD44+CD25+, and CD44+CD25 were presented as minor. On the other hand, by day 90, most of the cells were CD4 CD8 and CD25 CD44 . Minor cell populations were observed, which expressed CD4+CD8 , CD4+CD8+, CD8+ CD4 and CD44+CD25 , CD44+CD25+, CD25+CD44 . These data suggest that most cells in the clones of 90-day cultures of irradiated thymocytes contained CD4 CD8 CD25 CD44 cells (74–76%) (Table 2.1). This cell population is the most early intrathymic TLPs (Liu et al. 2014), which are the direct target cells for TGHF. The most important index of radioresistance for lymphoid cells is their stability to interphase death, which is an important characteristic of the resting cells, and observed during the early days after irradiation. It is now well established that D0 (dose causing 63% cell death) for lymphocytes lies in the range of 1–10 Gy and

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Table 2.1 Surface marker expression profile of mouse thymocytes either irradiated with a dose of 50 Gy (Sv) or non-irradiated cultured for 5, 10, 25, and 90 days without cytokines Marker CD4 CD8 CD4+CD8+ CD4+CD8 CD8+CD4 CD25 CD44 CD25+CD44+ CD25+CD44 CD44+CD25

Irradiated thymocytes (%) 5 days 10 days 25 days 4 1 27 79 86 31 11 10 19 6 3 23 25 9 64 2

90 days 76 18 3 3 74 22 3 3

Non-irradiated thymocytes (%) 5 days 10 days 4 0.5 80 80 10 18 6 1.5 42 7 50 1

Bold values denote p < 0.05

varies with the maturation stage of the cells and type of the sub-population. However, it should be kept in consideration that a small population of intrathymic TLPs represents the most radioresistant thymocytes, which has special role in the postradiation restoration of the thymus (Ayukawa et al. 1990; Yarilin 1997). The results of our experiments show that target cells for THGF belong to the population of radioresistant intrathymic precursors (intrathymic stem cells), for which D0 is more than 50 Gy. At doses of radiation more than 15 Gy, these cells are preserved over a long time in an inactive state. However, exogenic THGF or its combination with IL-2 may activate their proliferation. The dose of 12 Gy gamma-irradiation may induce the secretion of endogenous THGF by the radioresistant cells, and which are then responsible for the autocrine regulation of their proliferation. On the other hand, some authors have shown that the intrathymic precursors possess pluripotentiality and may generate not only a T-cell lineage but also NK cells (Lee et al. 1999), dendritic cells (Lucas et al. 1998), macrophages, and B cells (Peault et al. 1994). These cells could in turn secrete IL-7, SCF, and other cytokines and thereby support T-cell development during the reconstitution of the irradiated thymus.

2.5.6

THGF and Intrathymic Cytokines

As we have shown earlier that the cells responding to THGF are the cortisol/ radioresistant CD4 CD8 intrathymic TLPs expressing PNA receptors and stem cell antigen SC-1. Many cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-15, IL-21, SCF, and GM-CSF may directly induce the thymocyte proliferation, but contrary to the THGF, this proliferation is significantly increased in the presence of mitogens. In most of cases, the target cells for the proliferative action of these cytokines are mature CD4+ and CD8+ thymocytes, and in some cases, immature CD4+CD8+ thymocytes. However, IL-1, IL-2, IL-3, IL-4, IL-7, SCF, and GM-CSF may also act on some sub-populations of CD4 CD8 intrathymic TLPs (Yarilin and Belyakov 2004; Akdis et al. 2011). In particular, CD4 CD8 intrathymic TLPs may respond to IL-2, if they have been preliminarily activated by treatment with a

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mitogen or IL-1 (Raulet 1985; Howe et al. 1986; Dinarello 1994). It is generally believed that IL-4 may appear as an autocrine growth factor for self-renewing CD4 CD8 intrathymic TLPs (Lowenthal et al. 1988). Table 2.2 provides a direct comparison of THGF with other most relevant cytokines. The data show that THGF/ gp15 is associated with some cytokines, such as IL-3, with the same molecular weight, and similar direct activating action on thymocytes, while none of the known cytokines has similarities with THGF/gp22. The nearest to THGF/gp22 are IL-7 and SCF, which in the thymus are preferably secreted by TECs and may directly stimulate the proliferation of CD4 CD8 precursors (Rich 1997; Chung et al. 2011). Together, IL-7, SCF, and THGF have possibly a functional relationship.

2.6

Thymus Reconstitution Strategies and Key Challenges

The potential of tissue-specific stem cells for treating incurable diseases and conditions is widely recognized through their capacity to restore tissue function by either cell transplantation or regenerative therapies. Stem cells underpin a number of modern therapies; however, all rely on transplantation of cells harvested and expanded ex vivo. The limited capacity to achieve robust expansion of tissuespecific stem cells in vitro is recognized as a basic limitation for the development of new stem cell-based therapies. Furthermore, some human tissues, including the thymus, are not amenable to harvesting stem cells for autologous therapy either due to the tissue inaccessibility or the meagre availability of stem cells. Therefore, strategies for clinical use of thymic stem cells depend on their ability to generate or propagate undifferentiated TESCs in vitro, and to control their differentiation in order to generate transplantable functional thymic organoids or to support thymus regeneration in vivo for a complete recapitulation of thymus function. They further require strong medical-grade procedures for thymic epithelial cell lines and cultures, including protocols for cryopreservation of cultured cells and ex vivo tissue. Finally, they depend on the capacity to translate the experimental developments and instruments from mouse models to humans. Current approaches for restoring of the thymic function in patients are based on increasing regeneration of the endogenous thymus by cytokines and drugs (Alpdogan et al. 2006; Këpuska and Sempowski 2011; Dudakov et al. 2012; Garfin et al. 2013; Liu et al. 2016), transplantation of autologous thymus (Davis et al. 2017), transplantation of thymic organoids generated from ESCs or iPSCs (Bredenkamp et al. 2014; Deng et al. 2016), transplantation of pluripotent TESCs/TEPSs that generate thymic microenvironment in vivo (Bredenkamp et al. 2015) or the creation of a transplantable synthetic thymus (Tajima et al. 2016). However, the main obstacles for translation of these technologies in medical practice are the small numbers of TESCs in the human thymus, difficulties of their isolation, purification, and especially expansion in vitro. The having of effective instruments for maintenance of their undifferentiated status and function in vitro, as well as inhibition the preferential growth of fibroblasts in long-lived cultures in vitro still represent a major challenge for study and possible application of the cryopreserved TESCs (Shichkin et al. 2017, 2018; Villegas et al. 2018). So far, these problems remain unresolved. Current approaches

Monomer 15 kDa

gp15 and gp22 monomer forms

IL-4

THGF

Receptor IL-7R and sIL-7R c-kit GM-CSF α/β receptor IL-2R (α + β + γ) IL-3Rα+β chain IL-4R type I, IL-4R type II THGFRα (gp15) THGF-R (gp22) +

+

+++

++

++

+ +

+

+ 

TECs, MCs, DCs, macrophages Macrophages, T cells, mast cells, endothelial and NK, fibroblasts CD4+ and D8+ activated Th, DCs, NK, NKT Activated T cells, macrophages, NK cells Th2 cells, basophils, mast cells, eosinophils, NKT cells, γ/δ T cells c-kit+CD34+CD4 CD8 intrathymic TLPs

Producers TECs, MCs, DCs

CD4 CD8 TLPs, CD8+Th, CD4+Th, B cells c-kit+CD34+CD4 CD8 intrathymic TLPs and c-kit+ CD34+CD4 CD8 HSCs

activated CD4 CD8 TLPs, CD8+Th, D4 +Th c-kit+CD34+CD4 CD8 HSC

Targets CD4 CD8 TLPs, CD4+Th, CD4+CD8 +Th, CD8+Th c-kit+CD34+CD4 CD8 HSC and TLPs Neutrophils, macrophages, eosinophils

PHA phytohemagglutinin, w/o without, Th thymocytes, TECs thymic epithelial cells, MCs mesenchymal cells, DCs dendritic cells, NK natural killers, NKT natural killer T cells, HSCs hematopoietic stem cells, TLPs T-lymphocyte progenitors

IL-3

Dimer 18.5 kDa Glycopolypeptide 14–34 kDa Monomer 15.5 kDa Monomer 15 kDa

Structure and molecular weight Monomer 25 kDa

SCF GMCSF IL-2

Cytokine IL-7

Proliferative response (5-day test culture) w/o With PHA PHA +

Table 2.2 THGF versus the most related cytokines in the thymus

46 V. P. Shichkin

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47

Fig. 2.3 Thymic epithelial stem cell/small chemical compound (TESC/SCC)-based strategy for thymus regenerative therapy should include the development of clinical grade protocols for the collection, preparation and cryopreservation of primary thymic tissue and TESC-enriched samples. These TESCs could be used further to screen SCC that regulates differentiation and proliferation of human TESCs. The selected compounds should be tested for clonal expansion of TESCs and for supporting of thymic tissue growth in vitro as well as for reconstitution of thymic function in terms of maturation, differentiation, and tolerance of autologous T cells. Finally, full pharmacological evaluation of the properly selected and optimized compounds should be performed for high efficacy and low toxicity and further drug development. The use of microfluidic chips in combination with by human 3D thymic organ cultures (Thymus-on-Chip) for screening of target-specific compounds and assessment of their specificity and toxicity could significantly accelerate drug development for thymus-compromised patients. An actual challenge is the optimization of thymectomy procedure in patients to preserve a thymic fragment for consequent postsurgical thymus regenerative therapy. An additional impact on the efficacy of the postsurgical rehabilitation may provide the quality life monitoring of thymectomized patients in relation to their resistance to infections, allergies, autoimmune, oncological and other diseases associated with the impaired thymic function. (Modified from Shichkin and Antica 2020)

that are exploring how to reach a substantial TESCs growth in vitro include the use of serum-free culture media with TESCs-supporting growth factors and other supplements inhibiting the growth of other cell types (Garcia-Peydro et al. 2006; Brizzi et al. 2012) as well as the use of low/non-adhesive materials and matrices for 3D cultures (Simons and Clevers 2011; Tajima et al. 2016). A new promising approach is the use of small chemical compounds blocking or enhancing signaling mediated by specific protein kinases and thus regulating differentiation and clonal expansion of stem cells (Liu et al. 2016; Clarke et al. 2018; Yasuda et al. 2018). A number of such target-specific compounds were already screened and tested using high throughput screening assays with human pluripotent ESCs, iPSCs, and HSCs (Clarke et al. 2018). These studies provided highly promising results validating the use of compounds in regenerative medicine both for tissue engineering in vitro and for boosting regenerative potential of stem cells in vivo. However, optimal compounds for TESCs have yet to be identified and structurally optimized to achieve adequate efficiency and low toxicity in vitro and in vivo, and other benefits for the patients and the pharmaceutical industry (Fig. 2.3).

48

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Shichkin VP, Gorbach OI, Zuieva OA, Martsenyuk OP (2018) Optimization of quality parameters for human thymic cell samples stored in liquid nitrogen. Trends Transplant 10(5):1–11. https:// doi.org/10.15761/TiT.1000244 Shitara S, Hara T, Liang B (2013) IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRδγ+ intraepithelial lymphocytes. J Immunol 190:6173–6179. https://doi.org/10.4049/jimmunol.1202573 Shortman K, Vremec D (1991) Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes. Dev Immunol 1:225–235. https://doi.org/10.1155/1991/49025 Shortman K, Vremec D, D’Amico A, Battye F, Boyd R (1989) Nature of the thymocytes associated with dendritic cells and macrophages in thymic rosettes. Cell Immunol 119:85–100. https://doi. org/10.1016/0008-8749(89)90226-8 Siepe M, Thomsen AR, Duerkopp N et al (2009) Human neonatal thymus–derived mesenchymal stromal cells (nTMSC): characterization, differentiation, and immunomodulatory properties. Tissue Eng Part A 15(7):1787–1796. https://doi.org/10.1089/ten.tea.2008.0356 Simons BD, Clevers H (2011) Strategies for homeostatic stem cell self-renewal in adult tissues. Cell 145:851–862. https://doi.org/10.1016/j.cell.2011.05.033 Sitnik KM, Kotarsky K, White AJ et al (2012) Mesenchymal cells regulate retinoic acid receptordependent cortical thymic epithelial cell homeostasis. J Immunol 188:4801–4809. https://doi. org/10.4049/jimmunol.1200358 Spits H (1994) Early stages in human and mouse T-cell development. Curr Opin Immunol 6:212–221 Stosio M, Ruszkowski J, Mikosik-Roczyńska A, Haponiuk I, Witkowski JM (2017) The significance of neonatal thymectomy for shaping the immune system in children with congenital heart defects. Kardiochirurgia I Torakochirurgia Polska 14:258–262. https://doi.org/10.5114/kitp. 2017.72231 Sun L, Sun C, Liang Z et al (2015) FSP1(+) fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells. Sci Rep 5:14871. https://doi.org/10. 1038/srep14871 Tajima A, Pradhan I, Trucco M, FanY (2016) Restoration of thymus function with bioengineered thymus organoids. Curr Stem Cell Rep 2(2):128–139. https://doi.org/10.1007/s40778-0160040-x Takahama Y, Takada K, Murata S, Tanaka K (2012) beta5t-containing thymoproteasome: specific expression in thymic cortical epithelial cells and role in positive selection of CD8+ T cells. Curr Opin Immunol 24:92–98. https://doi.org/10.1016/j.coi.2012.01.006 Talaev VY, Shuvaeva TM, Lipkin VM, Yarilin AA, Shichkin VP (1991a) Purification and characterization of a thymocyte growth factor.1. Purification. Biomed Sci 2:511–514. https:// www.researchgate.net/publication/21314823 Talaev VY, Yarilin AA, Sharova NI, Shichkin VP, Shuvaeva TM (1991b) Purification and characterization of a thymocyte growth factor. 2. Biological activity of the thymocyte growth factor (1991). Biomed Sci 2:515–519. https://www.researchgate.net/publication/21314824 Talaev VY, Shichkin VP, Yarilin AA, Shuvaeva TM (1992) Characterization of mitogenic activity of purified thymocyte growth factor. Byulleten Eksperimentalnoi Biologii i Meditsini 5:516–518. (Russ) Trampont PC, Tosello-Trampont A-C, Shen Y (2010) CXCR4 acts as a costimulator during thymic beta-selection. Nat Immunol 11(2):162–170. https://doi.org/10.1038/ni.1830 Ucar A, Ucar O, Klug P, Matt S et al (2014) Adult thymus contains FoxN1 epithelial stem cells that are bipotent for medullary and cortical thymic epithelial lineages. Immunity 41(2):257–269. https://doi.org/10.1016/j.immuni.2014.07.005 Ulyanchenko S, O’Neill KE, Medley T et al (2016) Identification of a bipotent epithelial progenitor population in the adult thymus. Cell Rep 14:2819–2832. https://doi.org/10.1016/j.celrep.2016. 02.080

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Vaidya HJ, Leon AB, Blackburn CC (2016) FOXN1 in thymus organogenesis and development. Eur J Immunol 46:1826–1837. https://doi.org/10.1002/eji.201545814 Villegas JA, Gradolatto A, Truffault F et al (2018) Cultured human thymic-derived cells display medullary thymic epithelial cell phenotype and functionality. Front Immunol 9:1663. https:// doi.org/10.3389/fimmu.2018.01663 Wang S, Huang S, Gong L et al (2018) Human neonatal thymus mesenchymal stem cells promote neovascularization and cardiac regeneration. Stem Cells Int 2018:8503468. https://doi.org/10. 1155/2018/8503468 Wong K, Liser NL, Barsanti M et al (2014) Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus. Cell Rep 8:198–1209. https://doi.org/ 10.1016/j.celrep.2014.07.029 Wu L, Antica M, Johnson GL, Scollay R, Shortman K (1991) Developmental potential of the earliest precursor cells from the adult mouse thymus. J Exp Med 174:1617–1627. https://doi. org/10.1084/jem.174.6.1617 Yarilin AA (1997) Radiation-induced damage to thymocytes and thymic stromal cells. Manifestations and after-effects. Physiol Gen Biol Rev 10(4):3–57 Yarilin AA, Belyakov IM (2004) Cytokines in the thymus: production and biological effects. Curr Med Chem 11(4):447–464. https://doi.org/10.2174/0929867043455972 Yarilin AA, Miroshnichenko IV, Sharova NI et al (1990) Bone marrow and intrathymic precursors of T-cells produce a factor, which enhances colony formation in the spleen. Biomed Sci 1 (2):133–138. https://www.researchgate.net/publication/21052809 Yasuda S, Ikeda T, Shahsavarani H et al (2018) Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells. Nat Biomed Eng 2:173–182. https://doi.org/10.1038/s41551-018-0200-7 Žuklys S, Handel A, Zhanybekova S et al (2016) Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells. Nat Immunol 17(10):1206–1215. https:// doi.org/10.1038/ni.35

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Characteristic and Regenerative Potential of Human Endometrial Stem Cells and Progenitors Azin Ghamari, Faezeh Daghigh, Ali Mohebbi, Yekta Rahimi, Layla Shojaie, and Masoumeh Majidi Zolbin

Abstract

The endometrium has stem cell niches that contribute to the regeneration of the endometrial tissue following menstruation. These cells potentially could serve as a foundation of adult stem cells aimed at autologous stem cell-based therapies. A mechanistic understanding of these cells and their role in endometrial regeneration may aid to the intentional stimulation of a patient’s endometrial stem cell niche for regenerative therapies. The first evidence reporting their presence described endometrial stem cell as a clonogenic cell population in the human epithelial and stromal endometrium. Clonogenicity of epithelial cells was well supported by transforming growth factor-α (TGFα), epidermal growth factor (EGF), and platelet-derived growth factor-BB (PDGF-BB). Leukemia-inhibitory factor, hepatocyte growth A. Ghamari Growth and Development Research Center, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran F. Daghigh · A. Mohebbi Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran Growth and Development Research Center, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Y. Rahimi · M. M. Zolbin (*) Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran L. Shojaie Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran Division of GI/Liver, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 K. H. Haider (ed.), Stem cells: From Potential to Promise, https://doi.org/10.1007/978-981-16-0301-3_3

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factor (HGF), stem-cell factor (SCF), and insulin-like growth factor-I (IFG-I) were inadequately supportive and basic fibroblast growth factor (bFGF) did not affect clonogenicity of epithelial cells. About 3% of epithelial and 6% of stromal cells in mouse endometrium are label-retaining cells (LRCs). Moreover, CD146+ PDGFRβ+ human endometrial stem cell shows MSCs-like properties. Stromal cells are abundant in colony-forming cells that undergo differentiation into mesenchymal lineages and express MSC-specific surface markers CD29, CD44, CD73, CD90, and CD105. Further investigations have corroborated the evidence for MSC presence in endometrial stroma. The book chapter will provide an in-depth insight into the importance of uterine stem cell and their role in the pathogenesis of female reproductive tract diseases. Keywords

Differentiation · Endometrium · Endometrial stem cells · Menstruation · Myometrium · Regeneration · Reproductive system · Stem cell

Abbreviations ABC ABCG2 BCRP1 bFGF BMI-1 CD CFU CSCs DAPT EC EGF eMSCs EndoSCs ER-Alpha GE Gli GRO HER2 HGF HIF-1 HSCs IGF-1 LIF LRCs MCP-1 MenSCs

ATP-binding cassette ATP-binding cassette subfamily G member 2 Breast cancer resistance protein Basic fibroblast growth factor (bFGF) B-cell-specific Moloney leukemia virus insertion site 1 Cluster of differentiation Colony-forming units Cancer stem cells Dipeptide γ-secretase inhibitor Endometrial cancer Epidermal growth factor Endometrial mesenchymal stem cells Endometrial stem cells Estrogen-receptor alfa Glandular epithelium Glioma-associated oncogene Growth regulated oncogene Human epidermal growth factor Hepatocyte growth factor Hypoxia-inducible factor-1 Hematopoietic stem cells Insulin-like growth factor-1 Leukemia-inhibitory factor Label-retaining cells Monocyte chemoattractant protein-1 Menstrual blood-derived stem cells

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Characteristic and Regenerative Potential of Human Endometrial Stem Cells. . .

MMP MSCs n-cadherin NK PDGF PDGF-Rβ PgR SCF SDF-1 SSEA-1 SUSD2 TGFα/β uNK VEGF

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Matrix metallopeptidases Mesenchymal stem cells Neural cadherin Natural killer Platelet-derived growth factor Platelet-derived growth factor receptor beta Progesterone receptor Stem-cell factor Stromal derived factor Stage-specific embryonic antigen-1 Sushi domain containing 2 Transforming growth factor-α/β Uterine natural killer Vascular endothelial growth factor

3.1

Introduction

3.1.1

Uterine Stem Cell History

The extraordinary progress in stem cell biology has offered several opportunities for researchers working in the fast-emerging and rapidly developing field regenerative medicine. Regenerative medicine strategy is special in terms of its reconstructive approach for tissue and organ repair rather than symptomatic relief for the patients. Stem cells possess an inherent potential to differentiate into several specialized cell lineages in different organs in response to the biological cues from their microenvironment. Although stem cells have been classified based on the source of their origin, potentiality, surface marker expression, etc., they may be classified into two main sub-types. First, the embryonic stem cells are derived from the inner cell mass of the blastocyst and have the ability to differentiate into any cell type of the three germ layers. The second main broad category includes the adult stem cells (or somatic stem cells), which are derived from post-embryonic cell lineages and their presence has been reported in almost every tissue/organ in the body including the hematopoietic system (Shostak 2006; Gargett 2007). It is now generally believed that the existence of these stem cells contributes towards the regeneration and repair of the tissue/organ to which they belong in the event of injury. This special population of resident stem cells are tissue-specific and reside in a niche with a specific microenvironment that is conducive for their survival, undifferentiated self-renewal, and clonogenicity. The human uterus is an organ that is characterized by the remarkable regenerative capacity that is supportive of incessant and intense physiological changes that ensue during a female’s reproductive life. Endometrium which is the inner layer of the uterus is regulated to monthly dynamic remodeling that is characterized by extensive

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but controlled/regulated proliferative activity, differentiation, and shedding (menstruation) to sustain a dynamic environment for possible implantation of an embryo to support pregnancy. Both the regenerative potential and the remodeling process in uterine cells allude to the fact that endometrium and myometrium consist of a remarkable population of progenitor cells (Maruyama 2014). The endometrium has two layers: the upper two-thirds, which is shedding during each menstrual cycle and is defined as the functional layer, and the basalis layer, which remains during menstruation and is responsible for producing new functionalis layer. The dynamic characteristics of the endometrium are afforded by the rich presence of adult uterine stem cells population that is placed in the basalis layer. The existence of this stem/progenitor cells was first reported in 1978 by Prianishnikov (1978) while these adult stem cells were first isolated from endometrium in 2004. Subsequent researches showed that stem cells were present in both the functionalis and basalis layers (Chan et al. 2004) and the endometrial stem cells in the basalis layer niche are identified by CD146 and CD140b expression. The existence of endometrial somatic stem cells (SSCs) has been postulated, and several studies have confirmed the existence of SSC-like cells in the human endometrium (Chan et al. 2004; Schwab et al. 2005; Tsuji et al. 2008; Maruyama et al. 2010; Verdi et al. 2014; Birajdar et al. 2017). The basal part of the endometrial lining is characterized by the presence of stem cells. The human endometrium also contains epithelial cells (luminal and glandular) and mesenchymal cells (stromal cells) as well as leukocytes and endothelial cells (Kayisli et al. 2004; Spencer et al. 2005). Cervello et al. have reported the presence of the side population (SP) in the human endometrium in both the stromal and epithelial compartments which incidentally showed the typical functional and phenotypic features of SSCs (Cervello et al. 2010). The authors successfully isolated the side population cells from the two compartments, characterized them in vitro for clonogenicity and differentiation potential to adopt adipogenic and osteogenic phenotypes and subsequently showed their ability to form human endometrium post-transplantation in NOD-SCID mice. Side population (SP) cells (identified by their ability to extrude Hoechst 33342: a DNA-binding dye) which are assumed to function like stem or progenitor cells in several tissues, have also been reported and isolated from the normal human endometrium (Kato et al. 2007). They indicated that these cells constituted only 0.01–3% of whole live endometrial cells and were enriched in the CD9(
)CD13(
) fraction with the ability to repopulate and produce gland (CD9+), stroma (CD13+)like cells. Tsuji et al. demonstrated that these cells expressed the endothelial cellspecific markers (CD31, CD34), the epithelial membrane antigen, MSCs-specific markers (CD105, CD146), BCRP1/ABCG2 that is highly expressed in the vascular endothelium and the epithelium of the endometrial basalis layer (Tsuji et al. 2008). These cells have long-term proliferation capability and multipotential differentiation ability to various tissues and they are considered as the adult endometrial stem cells. Additionally, fetal stem cells have been extensively discussed in the literature for their presence in the adult uterus for the replacement of stroma and glandular epithelium (Sasson and Taylor 2008). Using a single-cell study approach, a recent study has reported that the presence of a bipotent cell population is present in the

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Characteristic and Regenerative Potential of Human Endometrial Stem Cells. . .

59

uterine epithelium of mice (Jin 2019). These uterine epithelial stem cells contribute to the regeneration of endometrial epithelium while they manage to survive during the cyclical flushing-off of the epithelium. Pulse-chase experiments with 5-ethynyl20 deoxyuridine revealed that these stem cells are residing between the luminar and epithelial layers. This chapter aims to review and summarize recent evidence for a putative reserve of stem cells in the uterus, which would represent a population of adult stem cells.

3.1.2

Classification of Stem Cells in the Uterus

The endometrium can grow from 0.5 to 1 mm in thickness at the initial stage of proliferation to 5–7 mm in the course of each menstruation cycle (McLennan and Rydell 1965). The role of endometrial stem cells (EndoSCs), including stromal, epithelial, and endothelial cells, is important in cyclic endometrial regeneration (Santamaria et al. 2018) and these cells are primarily housed in the perivascular areas of the basalis and functionalis layers of the endometrium (Fayazi et al. 2016). Using human endometrial tissue samples, Fayaziet et al. have carried out quantification of the stem cell population to show that nearly 12% of the cells were CD146+ while 89% and 85% cells expressed CD90 and CD105 on their surface. OCT4 and Nanog expressing cells were counted as 1.4% and 0.4% only. The propensity of these cells was more in the endometrial basalis layer. Evans et al. consider that the human endometrial stem cells have side population cells (SP), epithelial progenitor cells, and eMSCs (Evans et al. 2016). It is pertinent to mention that recent reports are attributing a significant role for SP cells in imparting chemoresistance and radioresistance in endometrial cancers (Liu et al. 2020). On the same note, Gargett et al. have introduced the presence of endometrial MSCs (eMSCs), epithelial progenitor cells, and endothelial progenitor cells (Gargett and Masuda 2010). The presence of eMSCs, besides their other functions, possess immunomodulatory properties similar to MSCs from other sources (Darzi et al. 2018). The extensive plasticity and the regenerative capacity of myometrium have been attributed to the presence of potential progenitor cells. For example, experimental studies in the mouse model have shown the presence of Sma+CD34+Klf4+ stem cells and their contribution to endometrial epithelium while at the molecular level, this regenerative capacity is ascribed to the involvement of SNEP1-mediated Era suppression (Yin et al. 2019). The increase in the number of myometrial cells during the early stages of pregnancy which is characterized by myometrial hyperplasia also points to the existence of stem cells in the myometrial tissue (Teixeira et al. 2008). Although there is consensus on the existence of stem cells, the location of these cells remains contentious and is suggested that the endometrial regeneration process is mediated more by stem cells located in the basal layer rather than the functionalis layer (Padykula 1989; Padykula et al. 1989). The epithelium of endometrium is composed of glandular epithelia (GE) and luminal epithelia (Chan et al. 2004). Epithelial stem-like cells in the luminal epithelium are known to be responsible for the growth of endometrial glands in the adult mouse. Luminal epithelial cells have an

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essential role in the development of luminal epithelium, which bears an exceptional proliferative and apoptotic role throughout endometrial regeneration. Molecular studies have revealed significantly elevated gene and protein level expression of p53 during the proliferative phase of the estrous cycle in an experimental rodent model (Mendoza-Rodríguez et al. 2002). Large epithelial cells can differentiate into glandular-like structures (Gargett et al. 2009). Although menstrual blood is a good source of pluripotent stem cells with immunoprivileged properties and differentiation potential (Faramarzi et al. 2016), epithelial progenitor cells have not been identified in menstrual blood. It was reported that the niche of epithelial stem cells might be present in the basal layer of the endometrium (Gargett et al. 2016). Interestingly, the epithelial progenitor cells have been detected in the basal layer of the endometrium in menopausal women, thus suggesting that these cells may serve as the potential source of stem cells in these populations (Nguyen et al. 2012). In non-menstruating species, such as laboratory rodents, endometrium undergoes proliferation and apoptosis instead of shedding, which confirms the presence of stem cells in the endometrium. eMSCs, another group of compact and small cells, identified in the perivascular area in both basalis and functionalis layer and they are currently being extensively assessed for their regenerative capacity. Additionally, these multipotential progenitor cells are widely present in shedding scrapes of the menstrual bleeding (Gargett et al. 2009).

3.2

Characterization of Stem Cell Populations

Various techniques and stem cell markers have been used to identify human endometrial stem cells during the last two decades. These cells possess unique functional characteristics including clonogenicity, long-term culturing capability, multilineage differentiation potential, plasticity, and expression of the stem cell markers (Santamaria et al. 2018). As discussed earlier, the endometrial side population cells include MSCs, epithelial and endothelial progenitor cell populations, which are located at both the basalis and functionalis layer and around the vessels and are identified by CD105, CD146, CD31, CD34, CD144, CD90, BCRP1, MDR1, c-kit, and Oct-4 expression (Tsai et al. 2002; Chan et al. 2004; Kato et al. 2007; Cervello et al. 2010, 2012; Nguyen et al. 2017; Campo et al. 2020). Endometrial stromal/MSCs include MSCs and endothelial progenitor stem cells, which are located in the basalis and functionalis layer and around the vessels, similar to side population cells. CD146, PDGFRB, W5C5-SUSD2, CD31, CD29, CD44, CD73, CD90, and CD105 are among the known markers of these cells (Fujii et al. 1989; Chan et al. 2004; Masuda et al. 2012; Gurung et al. 2018; Campo et al. 2020). Endometrial epithelial stem cells are located in the glands in the basalis layer, the markers of these cells include SSEA-1, Nanog, Oct-4, SOX9, N-cadherin, and Lgr5 (Kim et al. 2005; Schwab and Gargett 2007; Spitzer et al. 2012; Valentijn et al. 2013; Campo et al. 2020). As extensively reviewed by Tempest et al., and listed in Table 3.1, several markers have been

2

2

2

2

FUNC

BAS

Perivascular

Stromal

+

+

FUNC

BAS

+

+

+

+

+

UD

+

+

LRCs

BAS

+

UI

UI

UI

SP

FUNC

UD

+

2

2

LUM

Epithelial

SUSD2

Location

Cell type

CD146/ PDGFR β

++

2

+

SSEA1

++

2

+

SOX9

+

2

2

Nuclear β-Catenin

++

2

2

N-Cadherin

+

OCT4

UI

+

+

+

Musashi1

+

+

+

Notch/ Numb

+

2

2

+

MSCA1

+

2

++

LGR5

+

+

Telomerase

+

+

NTPDase2

Table 3.1 Different endometrial stem cell markers according to Tempest et al. (2018). BAS basalis, FUNC functionalis, LRC label-retaining cell, LGR5 leucine-rich repeat-containing G-protein-coupled receptor 5, NTPDase2 ectonucleoside triphosphate diphosphohydrolase-2, SP side population, UI under investigation, UD undefined, UI under investigation

3 Characteristic and Regenerative Potential of Human Endometrial Stem Cells. . . 61

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identified as specific for endometrial stem cells present in the epithelium, stroma, and perivascular regions (Tempest et al. 2018).

3.2.1

Regenerative Characteristic of Uterus Stem Cells

The clonogenic cell population in the inactive endometrium maintains the presence of endometrial stem cell niches in the basalis layer of the endometrium. The earlier reports about their clonogenic potential in human endometrial tissue showed that the formation of large colonies was rare, whereas these cells were able to develop into typical small colonies consisting of