Cardiovascular Applications of Stem Cells 9819907217, 9789819907212

Citation preview

Khawaja H. Haider   Editor

Cardiovascular Applications of Stem Cells

Cardiovascular Applications of Stem Cells

Khawaja H. Haider Editor

Cardiovascular Applications of Stem Cells

Editor Khawaja H. Haider Department of Basic Sciences Sulaiman Alrajhi University Al Bukayriyah, Saudi Arabia

ISBN 978-981-99-0721-2 ISBN 978-981-99-0722-9 https://doi.org/10.1007/978-981-99-0722-9

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 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

Dedicated to my two sisters, Chano and Gud and my wife DIYA who mothered our two sons: Mowahid who is the love of my life and Anas “the angel of paradise” whose departure from my life is a constant source of inspiration for me to do science.

v

Foreword

Science and technology revolutionize the development of world economy. As the WWII baby boomers get old and sick, coupled with the much under-reported 7 million Covid-19 deaths, human health and biosafety have gained top priority in our daily life. Regenerative Medicine technologies of cell therapy and gene therapy published since 1990 (Law et al. Lancet 336:114–115) have revolutionized how medicine will be practiced. They prolong life and improve quality of life, by replenishing live cells to degenerative organs, with long-lasting benefits and no side effects. In allografts, they offer the normal genome for genetic complementation treatment, replenishing missing structural and/or regulatory protein(s). They regenerate tissues and organs, thus conveying great social and economic values. Cardiovascular diseases collectively have been the number one killer of mankind with 523 million sufferers worldwide. Ultimately, heart muscle cell degeneration and/or cardiovascular dysfunction are the common pathway leading to death. In 2000, human myoblasts were implanted into porcine myocardium using the NOGA endovascular catheter delivery system. Since then, heart muscle regeneration with myoblasts has now been developed into EMA- and FDA-approved Phase II/III clinical trials. In 2004, Professors KhH Haider, PK Law and colleagues published the mechanisms of engraftment of human myoblast allografts and their pioneering studies of therapeutic angiomyogenesis to provide safe and efficacious concomitant regeneration of human muscle and capillaries of the left ventricular myocardium in mammals. The exponential development of regenerative medicine to treating heart failure deems every effort be exhausted in the selection, manufacturing, and testing of the most eligible cell type; be it natural or manipulated; be it myoblast, mesenchymal stem cell, induced pluripotent stem cell, or cardiac progenitor cell. It is in these arenas that Professor Haider has devoted his eighth stem cell volume to. The information contained in the 17 chapters demands his breadth and depth of knowledge and expertise to edit, and undoubtedly will expand the knowledge of its readers. Professor Haider has devoted more than two decades in these arenas, publishing cutting-edge research with complete dedication and passion.

vii

viii

Foreword

This book primarily focuses on cardiovascular applications of stem cells. With basic/clinical scientists, pharmaceutic companies, and governmental regulating agencies working in harmony, the world of science and medicine will look forward to conquering heart diseases with genetic cell therapy of regenerative medicine within this decade. Cell Therapy Institute Wuhan, China

Peter K. Law Ph.D.

Contents

1

2

3

4

Recent Advances in In Vitro Generation of Mature Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saravanan Ramesh, Kavitha Govarthanan, Arthi Sunil Richard, Siva Chander Chabattula, and Khawaja H. Haider Cardiac Reprogramming with Stem Cells: An Advanced Therapeutic Strategy in Advanced Heart Failure . . . . . . . . . . . . . . Alexander E. Berezin and Alexander A. Berezin Induced Pluripotent Stem Cells and Allogeneic Mesenchymal Stem Cell Therapy in Cardiovascular Diseases . . . . . . . . . . . . . . . . Bjarke Follin, Guido Caluori, Magdalena M. Dobrolinska, Jarek Stachura, Hassan Muzzamil, Wojciech Wojakowski, Abbas Ali Qayyum, and Tomasz Jadczyk “Heart Cells” Derived from Pluripotent Stem Cells and Therapeutic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushmita Roy, Eric G. Schmuck, and Amish N. Raval

1

23

65

97

5

Stem Cells and Regenerative Medicine in Valvulopathies . . . . . . . . 119 Marisa Jaconi and Michel Puceat

6

Rejuvenation and Regenerative Potential of Heart Stem Cells . . . . . 129 Moussa Ide Nasser, Han Zhongyu, Deng Gang, Massood Muqadas, Salah Adlat, Chi Liu, and Ping Zhu

7

Atrial Appendage-Derived Cardiac Micrografts: An Emerging Cellular Therapy for Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . 155 Esko Kankuri, Pasi Karjalainen, and Antti Vento

8

Cardiac Progenitor Cells in Cardiac Tissue Repair . . . . . . . . . . . . . 183 Adegbenro Omotuyi John Fakoya, Martin Tarzian, Mariana Ndrio, and Khawaja H. Haider

9

Cardiac Tissue Regeneration Based on Stem Cell Therapy . . . . . . . 207 Elham Afjeh-Dana, Behnaz Ashtari, Masoud Akhshik, Mohsen Akbari, and Khawaja H. Haider ix

x

Contents

10

Stem Cell Applications in Cardiac Tissue Regeneration . . . . . . . . . 243 Elsa N. Garza-Treviño, Adriana G. Quiroz-Reyes, Jorge A. Roacho-Perez, and Jose Francisco Islas

11

Therapeutic Targeting of Epicardial and Cardiac Progenitors in the Heart Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Fatih Kocabaş

12

Human Myoblast Genome Therapy and the Regenerative Heart . . . 307 Peter K. Law, Lei Ye, Wenbin Li, Leo A. Bockeria, Ilia I. Berishvili, Vadim S. Repin, Margarita N. Vakhromeevarant, Tea Kukachaya, Khawaja H. Haider, Nabil Dib, Weyland Cheng, Ping Lu, and Danlin M. Law

13

The Effect of Time of Cell Delivery on Post-MI Cardiac Regeneration: A Review of Preclinical and Clinical Studies . . . . . . . 349 Yazan M. Kalou, Abdullah Murhaf Al-Khani, and Khawaja H. Haider

14

Avant-Garde Hydrogels as Stem Cell Niche for Cardiovascular Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 Hilal Deniz Yilmaz and Yavuz Emre Arslan

15

Human Stem Cell-Derived Cardiac Organoid-Like Structures: Generation and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Marie-Noelle Giraud, Shaista Ahmed, and Nina D. Ullrich

16

Cardiovascular Stem Cell Applications in Experimental Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Jennie N. Jeyapalan, James Cockcroft, Albert A. Rizvanov, Khawaja H. Haider, and Catrin S. Rutland

17

Stem Cells in Heart Failure: Future Perspective . . . . . . . . . . . . . . . 491 Sabina Frljak, Roksana Gozdowska, Dominika Klimczak-Tomaniak, Magdalena Kucia, Marek Kuch, Tomasz Jadczyk, Bojan Vrtovec, and Ricardo Sanz-Ruiz

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. Before his current assignment, he served in several prestigious institutions in various parts of the world. He has also served as Principal Investigator (PI) and Co-PI on multiple National Institute of Health (NIH) funded stem cell research projects. He has been on the editorial boards of various research journals and an invited reviewer for several respected international journals. His research focuses on using DNA, miRNAs, and stem cells as “drugs,” a topic that has gained popularity in regenerative medicine. He has published nearly 325 book chapters, abstracts, and research papers in various books and leading research journals, including Circulation, Circulation Research, Cardiovascular Research, JCMM, the Journal of Biological Chemistry, Cell Cycle, Basic Research in Cardiology, and Antioxidant Redox Signaling. He has also given numerous presentations and edited seven books covering various facets of stem cells and their applications from drug to drug development.

xi

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes Saravanan Ramesh, Kavitha Govarthanan, Arthi Sunil Richard, Siva Chander Chabattula, and Khawaja H. Haider

Abstract

Human adult cardiomyocytes are one of the most sought-after and on-demand appropriate model systems to understand cardiomyocyte development and decipher the pathophysiological mechanisms underlying various diseases of the heart. Hitherto, a more suitable humanized model depicting the mature cardiomyocyte profile has not yet been reported due to its complex structural and physiological features. With the recent technological advancements in cell culture systems, conventional 2D culture systems have been replaced by more advanced and physiologically relevant 3D platforms, which can potentially modulate multitudinous culture conditions manifesting more maturation and biological characteristics in vitro. This chapter precisely narrates the up-to-date advances in state-of-the-art infrastructure developed to improve the maturation aspects in induced pluripotent stem cells (iPSCs)-derived cardiomyocytes. A robustly defined scaffold-based cell culture system employing perfectly tailored biomaterials for mimicking in vivo architecture will also be focused on. Furthermore, this chapter also will provide in-depth information about the fast-emerging organoid platform approach, its prospects in engineering human heart tissue using iPSCs-derived cardiomyocytes, and its emerging role in surrogating for drug discovery and clinical applications.

S. Ramesh · K. Govarthanan (✉) Centre for Cardiovascular Biology and Disease, inStem, Bengaluru, Karnataka, India e-mail: [email protected] A. S. Richard · S. C. Chabattula Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India K. H. Haider Department of Basic Sciences, Sulaiman Alrajhi University, Al Bukayriyah, Saudi Arabia # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 K. H. Haider (ed.), Cardiovascular Applications of Stem Cells, https://doi.org/10.1007/978-981-99-0722-9_1

1

2

S. Ramesh et al.

Keywords

Cardiomyocytes · 3D bioprinting · Differentiation · Hydrogel · Maturation · Molecular · Myocardial · Stem cells

Abbreviations APA APD bFGF BMPs BPM cACT CMs CTD cTn-I CVD DKK EBs ECs ERR ESCs FGF2 GM-CSF hESCs hiPSCs hPSC-CMs hPSCs iPSCs LQTS MSCs PAA PDMS PPAR RMP SDF-1α sEVs VEGF

Action potential amplitude Action potential duration Basic fibroblast growth factor Bone morphogenetic proteins Beats per minute Cardiac α-actinin Cardiomyocytes Calcium transient duration Cardiac troponin I Cardiovascular diseases Dickkopf-related protein Embryoid bodies Endothelial cells Estrogen-related receptor gamma Embryonic stem cells Fibroblast growth factor 2 Granulocyte-macrophage colony-stimulating factor Human embryonic stem cells Human induced pluripotent stem cells hPSC-derived cardiomyocytes Human pluripotent cells Induced pluripotent stem cells Long QT syndrome Mesenchymal stem cells Polyacrylamide Polydimethylsiloxane Peroxisome-proliferator-associated receptor Resting membrane potential Stromal cell-derived factor-1alpha Small extracellular vesicles Vascular endothelial growth factor

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

1.1

3

Introduction

Human origin cardiomyocytes (CMs) grasp a prodigious promise in exemplifying the physiology of the heart and the pathology of cardiovascular diseases. It is well established that homeostasis in the heart is controlled by the CMs. The shape of the CMs and their number are vital for the heart’s normal function. Any abnormality in the shape and deficit in the number adversely affect the mechanism of myocardial homeostasis and generally lead to various cardiovascular pathologies (Azevedo et al. 2016; Ranek et al. 2022). Nowadays, partially supporting palliative care-mediated therapeutic strategies are being established to treat cardiovascular diseases (CVD) and disorders. These strategies often slow the disease progression while potentially increasing various comorbid conditions in patients with CVD. The shortage of organ donors has led researchers globally to look for an alternative source of morphofunctionally competent CMs for cell-based therapy to repopulate the injured myocardium. Given the prominence of human CMs, it is indispensable to understand the orchestral events and molecular pathways happening during the CMs’ development and their maturation in vitro. It is widely known that CMs’ development and maturation is an intricate, harmonized, and multifactorial process; hitherto, the molecular mechanisms of CMs’ maturations are yet to be elucidated (Govarthanan et al. 2022). Nevertheless, the current differentiation protocols merely yield a fetal analog of CMs, constraining the translational potential of the in vitro-generated CMs (Heng et al. 2004). Therefore, it is essential to optimize the manipulation of the stem cells biochemically and mechanically (i.e., preconditioning, genetic manipulation, electrical stimulation, etc.) before subjecting them to the differentiation protocols to generate cells with competent and mature microscopic structure and functional readouts of an adult CM, which holds massive potential clinical applications. The prospects for using human pluripotent cells (hPSCs), mainly human induced pluripotent stem cells (hiPSCs), in disease modeling, drug screening, and regenerative therapies have been significantly boosted by the capacity to differentiate hiPSCs in the population of CMs with high purity and sub-type specificity (UçkanÇetinkaya and Haider 2021). Still, the immature, fetal-like phenotype of hPSCderived cardiomyocytes (hPSC-CMs) produced by the currently available guided differentiation protocols restricts their potential for both in vitro and in vivo applications (Ullah et al. 2021; Tsvelaya et al. 2018). Since these biological processes must be recapitulated and expedited in any realistic hPSC-CM maturation technique, it is essential to have in-depth knowledge of CMs’ biology and differentiation to create fully functional CMs. Thus, unveiling such emphasized complementary signals and induction factors necessary for the successful differentiation into mature CM phenotype is considered as a leap forward toward effectively using stem cells for damaged or worn-out myocardial tissue engineering applications. The current chapter will be focused on highlighting the contemporary advanced technological platforms developed for facilitating the maturation aspects of the CMs.

4

1.2

S. Ramesh et al.

Mature Cardiomyocyte Internal Complexity

Since hPSCs-derived CMs vary in maturity, they can be broadly classified into two categories: first early phase CMs, with contractile cells holding limited proliferating property and electrophysiology akin to an embryo (i.e., small negative membrane potential and small action potential amplitude), and the second category of late phase terminally differentiated CMs defined by the loss of proliferative potential and having more adult-like electrophysiological property. Therefore, the hPSC-derived CMs typically exhibit early phase characteristics during the first month following the start of a contraction before achieving late phase characteristics (Gherghiceanu et al. 2011; Yokoo et al. 2009; Zhang et al. 2009), which is influenced by multiple factors, i.e., time in culture (Zhang et al. 2009; Sartiani et al. 2002), co-cultured cells (Kim et al. 2010; Bauwens et al. 2008), and culture conditions (Bauwens et al. 2008; McDevitt et al. 2005). These parameters impact different maturity levels, although the factors influencing maturity warrant in-depth investigation to understand their underlying molecular-level involvement. The structural similarity of hPSC-derived CMs to embryonic or fetal CMs has been widely recognized (Mummery et al. 2003; Fijnvandraat et al. 2003). However, when these cells are juxtaposed with embryonic or adult CMs, striking differences may be observed in the discrepancy in size. With approximately 150 μm/10 μm for ventricular myocytes, the size of adult CMs is large and cylindrical (Lieu et al. 2009), whereas fetal and embryonic CMs are smaller in size (Smolich 1995). Like early CMs, early hPSC-derived CMs are smaller, round to slightly oblong, and have a diameter of 5–10 μm (Gherghiceanu et al. 2011; Gepstein et al. 2003). Although they are smaller compared to mature hPSC-derived CMs (30 μm × 10 μm), late hPSCs-derived CMs (>35 days) develop a more oblong shape (Snir et al. 2003). Additionally, like early embryonic CMs, hPSCs-derived CMs are mononuclear cells whereas most adult CMs are bi- or multinucleated. Both hPSCs-derived CMs and embryonic CMs lack the large t-tubule network found in adult ventricular CMs (Lieu et al. 2009). As a result, calcium enters the cell primarily through the sarcolemma as instead of being released from the sarcoplasmic reticulum (SR), and the excitationcontraction coupling is slower (Binah et al. 2007; Dolnikov et al. 2006; Fu et al. 2006; Itzhaki et al. 2006). Early hPSCs-derived CMs thus share structural similarities with embryonic CM. On the contrary, late hPSCs-derived CMs exhibit a more adult-like shape as their culture duration increases; however, they don’t seem to generate t-tubules or multi-nucleation. In conclusion, whereas late hPSCs-derived CMs are non-proliferating cells, early hPSCs-derived CMs do proliferate at a slower rate than their pluripotent progenitors. Two-thirds of the cytoplasmic volume in adult CMs is made up of contractile machinery and mitochondria (myofibril cell area ranges from 40 to 52% and mitochondria range from 15 to 25%) (Fig. 1.1). In contrast, the sarcomeric area in hPSCs-derived CMs and embryonic CMs is smaller, and the mitochondria are more evenly distributed (Porter Jr et al. 2011). Similar to fetal (20 weeks) or adult cardiomyocytes (more than 20 weeks), the expression of contractile and cytoskeletal genes is significantly reduced in hPSCs-derived CMs (unknown age). Adult CMs

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

5

Fig. 1.1 Infographics illustrating the structural complexity of adult cardiomyocytes

have a high metabolic rate and primarily rely on oxidative metabolism to produce ATP (acetyl-CoA is produced in 90% of adult CMs through fatty acid oxidation). Contrarily, embryonic and fetal CMs depend on glycolysis for ATP synthesis (fatty acid oxidation accounts for just 15% of acetyl-CoA synthesis) (Lopaschuk and Jaswal 2010), which results in a phenotype that is comparatively hypoxia resistant and provides substrates for protein synthesis (Porter Jr et al. 2011).

1.3

CM Differentiation Protocols

In one of the differentiation protocols, the cells are subjected to two to three passages for expansion to achieve sufficient cell number using Versene supplemented with 0.02% EDTA solution. Before differentiation, the final passage cells are seeded on 1% Matrigel-coated six-well cell culture plates with glass coverslips. iPSCs were cultured until they reached 80–90% confluence in an incubator with humidified conditions at 37 °C and 5% CO2 before proceeding with the differentiation procedure. The differentiation protocol is started on day 0 using RPMI-1640 media supplemented with 2% B27 without insulin and 6 μM CHIR99021 to induce mesodermal differentiation (Lopaschuk and Jaswal 2010). However, this primary step essentially remains the same in all differentiation protocols with slight modifications. For example, as reported by Lian et al., on day 3 of differentiation, RPMI-1640 medium with 2% B27 supplement without insulin and IWP2 (5 μM) was added to the cells for 48 h (Lian et al. 2013). Cells were then cultured in RPMI-1640 cell culture medium with 2% B27 supplement with insulin (Burridge et al. 2014). Another protocol described by Burridge et al. (2014) employed on day 2 of

6

S. Ramesh et al.

differentiation cells was cultured in RPMI-1640 medium with 2% B27 supplement without insulin and 2 μM Wnt-C59 for the next 48 h (Porter et al. 2011). The cells were subsequently cultured in RPMI-1640 medium with 2% B27 supplement without insulin. From day 0 of differentiation, cells were cultured in an incubator with a humidified atmosphere at 37 °C and 5% CO2. Cells were cultured until D20 of differentiation before performing further experiments. For RNA isolation and singlecell patch-clamp recordings, dissociation of iPSC-CMs was performed using Accumax or TrypLE according to the manufacturer’s protocol. Un-dissociated cells were fixed on days 20–25 of differentiation and observed using immunofluorescence staining for cardiac-specific markers [cardiac α-actinin (cACT), cardiac troponin I (cTn-I), and Nkx2.5], according to the well-defined established protocol. Analysis of iPSC-derived CMs quality was performed based on the expression of cardiac-specific markers on RNA level, and the expression and organization of the sarcomeric proteins based on immunofluorescence staining. Electrophysiological properties were characterized from AP/CT recordings to investigate signal propagation in iPSC-CMs and their general electrophysiological activity. Obtaining tissue-specific cells for in vitro modeling presents a significant challenge for current cardiac research. The ground-breaking discovery of somatic cell reprogramming has opened new avenues of theranostic applications of stem cells (Ibrahim et al. 2016). Since the inception of this technology, iPSCs have become a desirable alternative source of pluripotent stem cells without ethical and moral restrictions for cell-based therapy, patient-specific, and disease-specific modeling and drug testing (Buccini et al. 2012; Ahmed et al. 2011a, b). The use of embryonic stem cells (ESCs)-derived cardiomyocytes for myocardial repair has always been an area of interest (Heng et al. 2005). Human embryonic stem cells (hESCs) were initially attempted to culture in a serum-supplemented suspension condition to form embryoid bodies (EBs) (Kehat et al. 2001), with a yield of 5–10% of hESC-derived CMs obtained. Zhang et al. developed a protocol based on culturing in serum-free conditions, supplementing with the essential growth factors that were known to play a critical role in the cardiac developmental process. The supplemented factors in the culture medium included the widely used ones as cues in the differentiation protocols, i.e., activin A, bone morphogenetic proteins (BMPs), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), Dickkopf-related protein (DKK), and the Wnt agonists and antagonists (Lian et al. 2013; Yazawa et al. 2011; Yang et al. 2014; Burridge et al. 2011; Minami et al. 2012). These growth factors and chemical cues supplementation have remarkably increased the rate of cardiomyogenic differentiation, yielding 10% to roughly around 90% CMs. Even though the growth factor supplementation strategy significantly increased CM yield, the 3D EB-mediated mass culture system severely hampered the growth factor diffusion and oxygen distribution, leading to reproducibility concerns (Correia et al. 2014). As an alternative strategy to the suspension culture, adherent monolayer protocols were proposed with a reported efficacy of 80–99% cardiomyogenic differentiation rate (Burridge et al. 2014; Lian et al. 2013; Bhattacharya et al. 2014; D’Amour et al. 2005; Ren et al. 2011). The aforementioned protocols determined the CMs differentiation efficacy using the reported CM-specific markers based on

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

a

Day of differentiation

-3 -2 Y-27 Small molecules 632

0

1

b

3

5

RPMI+B27(ins-)

50 µm

Day 0

10

15

RPMI+B27(ins+)

100 µm

Day 0

200 µm

Day 0 50 µm

Day 1

7

IWP2

CHIR

E8

Basal medium

MYL7 MYL2 TNNT2

NKX2-5 ISL1

MESP

POU5F1

7

50 µm

Day 8

Fig. 1.2 Schematic outline of the standard protocol for differentiating iPSCs into cardiomyocytelike cells. (Adapted from Balafkan et al. 2020)

the percentage of troponin-T or cardiac actinin-positive cells during flow cytometric analysis. The reported protocols have been tested for their reproducibility in various laboratories and proven for their efficiency. Overall, the optimized differentiation protocol widely used for cardiomyogenic differentiation of iPSCs is depicted in Fig. 1.2. Interestingly, various researchers have demonstrated the iPSCs-derived disease model using the abovementioned protocol by employing the cells from the patients and reprogramming them into iPSCs, followed by induction studies. Based on the conventional reprogramming methods, the first published arrhythmia model using iPSC-derived CMs from Long QT syndrome (LQTS) patients (Moretti et al. 2010a, b). The disease features were demonstrated by comparative mode between normal and patient-specific iPSC-derived CMs. The study by Moretti et al. extensively used variations in action potential duration and pattern with the downregulation of potassium current density to demonstrate the arrhythmia model. In the patch-clamping technique, researchers are currently employing more versatile data acquisition platforms using fluorescent conjugates recording intracellular calcium surges (Nijak et al. 2021; Hwang et al. 2015; Balafkan et al. 2020). Presently, studies are performed in microfluidic conditions with multiple options for compartmentalization. These models of micro-compartmentalization will

8

S. Ramesh et al.

ultimately aid in understanding the pathological sequences under more realistic and dynamic culture conditions. The fast-emerging bioprinting technology is considered the most advanced tool vibrantly used to mimic the cardiac microtissue architecture under the 3D culture system. To underscore the efficacy of the 3D system in comparison with the conventional culture model, the important feature action potential (AP) are analysed, which include action potential duration (APD), action potential amplitude (APA), resting membrane potential (RMP), and beating rate (beats per minute, BPM), during the patch-clamp investigations. Additionally, we used optical dyes to describe the CT’s time of rise and decay, beating rate, and calcium transient duration (CTD).

1.3.1

Hormones

The thyroid hormone plays a vital function in upholding the homeostasis level of cardiovascular physiology and cardiac development (Klein and Ojamaa 2001), and studies have also demonstrated its significant role in the maturation of hiPSCderived CMs (Yang et al. 2014). Analysis of culture followed by thyroid treatment revealed larger sarcomeres, elongated cell shape, and high levels of contractile force with calcium transients, with increased mitochondrial activity. For the fetal heart’s anatomical and functional development and maturity, glucocorticoids are critical (Rog-Zielinska et al. 2013). In fetal CMs, endogenous glucocorticoids act on and stimulate the glucocorticoid receptors to support Z-disc and myofibril construction and boost mitochondrial activity (Rog-Zielinska et al. 2015). According to studies, T3 is insufficient on its own to produce mature hiPSCderived CMs. Dexamethasone and T3 work synergistically to improve the hiPSCderived CMs’ maturation. Using Dexamethasone and T3 hormones throughout the 15-day culture of the hiPSC-CMs promoted T-tubule growth, raised the calcium transient level, and improved excitation-contraction coupling (Parikh et al. 2017). The formation of t-tubules was significantly more extensive in the cells receiving combined treatment than those receiving either dexamethasone or T3 alone. Confocal images also revealed spatially and temporally uniform Ca2- release, which indicated characteristic excitation/contraction coupling in the differentiated cells.

1.3.2

Energy Source

The metabolic switch from the glucose source is a prominent signature of mature CMs. During the initial days of differentiation, and around the days between nine and twelve, the newly differentiated CMs were immature, and glucose was sufficient to support their normal contractile activity. However, during further development and maturation, which necessitated a higher rate of ATP consumption, the maturing CMs gradually shifted their metabolism from glucose to fatty acids utilization to meet their energy requirements (Yang et al. 2019). However, an in vitro investigation showed that glycolysis rather than fatty acid metabolism was used as the energy

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

9

source for iPSC-derived CMs generation. The iPSCs-derived CMs were subjected to glucose-free media containing insulin and fatty acids for 3 days to acquire the mature CMs phenotype. Resultantly, iPSCs-derived CMs primarily relied on fatty acid metabolism for their energy needs, leading to improved electrophysiological properties with long sarcomere (Drawnel et al. 2014). Galactose and fatty acids, more especially carnitine (Nakano et al. 2017), oleic acid (Correia et al. 2017), palmitate (Horikoshi et al. 2019), and linoleic acid (Feyen et al. 2020), increased the development of iPSCs-derived CMs when glucose was substituted as the energy source. Additionally, growing hiPSC-derived CMs in a high-glucose environment impairs their structural and functional maturation by increasing nucleotide production, lowering myocardial glucose absorption, and raising nucleotide deprivation (Nakano et al. 2017). Changes in the cellular energy source place more emphasis on the maturation of iPSCs-derived CMs, enhancing not only the metabolic flow and mitochondrial activity but also the shape, structure, and physiologic stage of maturation. Feyan et al. designed maturation media that combine glucose with oxidative substrates like calcium, taurine, and L-carnitine to facilitate fatty acid oxidation. They successfully cultured human iPSCs-derived CMs using a medium that enhanced mechanical, structural, and electrophysiological parameters of physiological maturity (Feyen et al. 2020). In a recent study, Walter et al. reported that hiPSC-derived CMs under micropatterned and hypoxic culture conditions expressed adult-like CM phenotypes, including sarcomeric maturity, well-expanded cell size, and enhanced myofibril contractile force and activity (Knight et al. 2021). Furthermore, by switching their metabolism from glycolysis to fatty acid oxidation, the peroxisome proliferatorassociated receptor (PPAR) delta and estrogen-related receptor gamma (ERR) signaling pathways are involved in the maturation of hiPSC-derived CMs (Miki et al. 2021; Wickramasinghe et al. 2022). This metabolic activity switching results in elongated cell morphology, myofibril organization, longer sarcomere, and enhanced contractile and electrical coupling.

1.3.3

Prolonged Culture Period

Since it takes in vivo CMS years to reach their fully developed and mature structural and functional characteristics (Vreeker et al. 2014), it is hypothesized that long-term culture will aid in the maturation of hiPSC-derived CMs. For several reported research studies, hiPSC-derived CMs were cultivated for an extended duration to assess the features of mature CMs. HiPSC-derived CMs showed mature phenotypic traits, such as elongated cell shape, fully developed sarcomeres, high myofibril density, and calcium handling (Lundy et al. 2013). It is pertinent to mention that hiPSC-derived CMs produced mature cardiac genes with isoform switching, including MYH7 (Lewandowski et al. 2018). When the culture period was increased to 180 days, mature H-band, Z-band, I-band, and A-band but not M-bands were generated in more densely packed myofibrils. M-band, a crucial element of

10

S. Ramesh et al.

sarcomere structure, eventually developed after 360 days of continuous culturing (Kamakura et al. 2013).

1.3.4

Cellular Interactions

Through insoluble (i.e., small extracellular vesicles, sEVs) and soluble paracrine factors (bioactive molecules) produced by the neighboring heart cells, cellular interactions may improve the maturation of CMs (Talman and Kivelä 2018). Given that mesenchymal stem cells (MSCs) and endothelial cells (ECs) release a copious amount of bioactive molecules and sEVs as a part of their paracrine activity, Yoshida et al. in one of their recent studies co-cultured hiPSC-derived CMs with non-cardiac cells, MSCs and ECs, to study their effect on CMs (Yoshida et al. 2018). They observed significant maturation of the CMs in terms of structure and functionality. Profiling of the conditioned medium revealed stromal cell-derived factor1alpha (SDF-1α), VEGF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and basic fibroblast growth factor (bFGF) were among the cytokines and sEVs produced by MSCs that mediated the maturation of hiPSC-derived CMs (Yoshida et al. 2018). Abecasis et al. also observed that the sarcomere length of hiPSC-derived CMs was increased by ECs co-culture through the production of ECM, such as collagens I and III, thrombospondin-4, and fibronectin (Abecasis et al. 2019). Other studies have also shown that the ECM can greatly aid hiPSC-derived CMs’ structural and functional maturation (Chun et al. 2015; Ogasawara et al. 2017).

1.3.5

Biophysical Stimulation

Biophysical stimulation of cells is lacking in traditional cultural conditions. The CMs frequently experience mechanical stress and electrical stimulation in their native cardiac. Continuous electric stimulation may cause hiPSC-CMs to mature into rod-like structures with greater cellular alignment and better organization (Chan et al. 2013). In addition, hiPSC-CMs stimulated electrically and mechanically showed increased N-cadherin localization to the cell membrane and decreased transmembrane calcium current, suggesting a more mature phenotype (Kroll et al. 2017).

1.3.6

Substrate Stiffness

The ECM affects tissue stiffness, and collagen buildup significantly increases myocardial stiffness. The process of rendering the heart stiffer increases the heart’s ability to pump blood (Jacot et al. 2010). Given that cell culture dishes are considerably stiffer (1 MPa) than hearts (10 kPa), it was proposed that soft matrices would be advantageous for hiPSC-derived CMs maturation (Ogasawara et al. 2017). Conditions for in vitro cell culture are typically maintained using

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

11

polydimethylsiloxane (PDMS) and polyacrylamide (PAA). HiPSC-derived CMs on soft surfaces (6–10 kPa) produced higher force (0.1 N) than those on hard surfaces (35 kPa, 0.01–2 N) (Ribeiro et al. 2015). Soft surfaces also change sarcomere tension and contractility. When hiPSC-derived CMs are controlled into a maturation state, PDMS and PAA are employed to modify the topology of cell morphology (McCain et al. 2014).

1.3.7

Micropatterning

Micropatterning is one of the techniques which uses a soft lithography platform for generating microcontact protein printing on the hydrogel, thus guiding topological cues for the cells in vitro. Standard protocols for differentiating iPSCs into CMs primarily result in heterogeneous cell populations with small, non-aligned, immature CMs of diverse shapes, lacking well-formed myofibrils and T-tubules, polyploidy, polarized intercalated discs, or abundant mitochondria. Ideally, the adult CM phenotype stretches the cell’s structural framework with well-aligned contractile fibrils and registered Z-lines and directly establishes critical functional properties of the cells, such as electrophysiology and contractility (Henderson et al. 2017; Tu et al. 2018; Marzuca-Nasri et al. 2018). Previous studies have demonstrated that CMs’ membrane capacitance is directly proportional to cell surface area; hence, elongated, an anisotropic shape resulting in the presence of long myofibrils with laterally organized sarcomeres, permitting efficient cardiac contractility (Karbassi et al. 2020). Concisely, numerous studies have shown the successful differentiation of iPSCs to beating CMs under the 2D platform; however, the generated CMs exhibit a profile analogous to fetal or primitive CMs. Consequently, an immature CM phenotype is not an appropriate model for various preclinical and clinical studies. Alterations in the structure between immature and mature CMs lead to functional variances restraining the potential of hPSCCMs to recapitulate normal development or model human disease, thus limiting its clinical and research applications. Although advanced 3D organoid culture systems obtained from native decellularized ECM frameworks were developed, still limited success was achieved due to reproducibility concerns. Therefore, developing a chemically defined hydrogel-based scaffold would be an efficient option to induce CMs’ maturation using an organoid-based CM system. These organoids are a microphysiological system having the miniature versions of the organs that support the cells cultured on a scaffold, recapitulating in vivo cell microenvironment, thus facilitating the amenable heterogeneous culture systems with long-term viability, preservation of the anisotropic rod-shaped structure via micropattern culture system, and enabling the highest uniform elongated shape of the CMs and their function. In such culture systems, the CM maturation is influenced by the addition of long and well-organized specialized myofibrils, forming the contractile apparatuses of CMs. The complexity in the matured CMs is primarily due to the proper assembly and alignment of multimeric protein units in a well-organized manner (Feaster et al. 2015). Any defects or improper alignment in the arrangement of these protein units

12

a

S. Ramesh et al.

NRVMs Serum Free

b

ARVM

d

iPSCs

NRVMs PE

c

HL-1

Immature CMs

Adult CMs α-Actinin/DAPI

HL-1 20 µm

20 µm

Fig. 1.3 Structural comparison between fetal and adult cardiomyocytes (CMs). (Adapted from Peter et al. 2016, Ahmed et al. 2020)

often lead to the generation of morphofunctionally incompetent CMs. Therefore, it is apparent that the shape of the CM correlated directly to its functional maturity and the striated self-beating rod-shaped human ventricular CMs were 100–150 by 20–35 μm in size (Severs 2002). “The CMs are roughly cylindrical and measure 17–25 μm in diameter and 60–140 μm in length,” giving length to diameter ratio of about 5 to 1 (Tracy and Sander 2011). Studies have been carried out with different length-by-width ratios and demonstrated that the 7:1 aspect ratio was best suited for the purpose of guiding the shape of the CMs using micropatterning on the polyacrylamide soft gel base (Ribeiro et al. 2015) (Fig. 1.3).

1.4

3D Culture System

During the decade of research in the field, 3D cultures (organoids) have been exploited for diverse transdisciplinary tissue engineering applications. Because the typical 2D monolayer culture cannot accurately reproduce the intricate network of in vivo cellular phenotype 3D culturing approach has emerged as an intriguing alternative. Furthermore, 3D cultures allow equitable distribution of potential chemical and biological inducers to reach the cells, closely match the in vivo ECM, and encourage better cell interaction. Compared to the 2D cultures, hiPSCs-derived CMs express a higher structural, functional, and metabolic maturity in the 3D cultures. According to the published data, the 3D culture system enhances the hiPSC-derived CMs’ metabolic maturation by elevating oxidative phosphorylation-related genes while downregulating genes linked to glycolysis and lipid synthesis. The transcriptome analysis also showed that hiPSCs-derived CMs in the 3D culture

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

13

matured more quickly than in 2D cultures, which slows or prevents maturation (Ahmed et al. 2020). Even though hiPSCs-derived CMs in the 3D cultures can create highly developed CMs, they are insufficient on their own. In a 3D culture system, a mixture of the 2D maturation-promoting strategies—hormones, cell-cell contact, electrical stimulation, etc.—should be applied (Murphy et al. 2021). For instance, exposing the biowire for electrical stimulation after seeding hiPSCs-derived CMs with collagen polymer to create a wire-like structure results in improved calcium and electrophysiological management and greater myofibril organization (Xia et al. 1997). Additionally, carbon nanotubes or silicon nanowires were placed into hiPSC-derived CMs spheroids to create an electrically conductive environment. This environment was further enhanced by exogenous electrical stimulation. The hiPSCs-derived CMs showed high sarcomere length, calcium handling, and noticeably enhanced contraction after exposure to electric stimulation (Radisic et al. 2004). Mechanically, hiPSCs-derived CMs’ structural and functional maturation could be aided by applying passive stretch or afterload to 3D-created cardiac tissue. By upregulating mature cardiac genes, such as sarcomeric protein troponin-T, -adrenergic receptors, and t-tubule protein caveolin-3, as well as increasing calcium dynamics, a passive stretch may help hiPSCs-derived CMs mature. Similar to how hiPSC-CMs express characteristics of maturation such as cell elongation, high-rate calcium handling, and increasing the expression of key markers of cardiac maturation under moderate afterload conditions, high afterload may have detrimental effects and induce pathological changes (Nunes et al. 2013). Additionally, the synergistic effects of electrical and mechanical stimuli, such as cyclic stretch, may help the HiPSC-CMs mature more quickly in these conditions than in just one stimulus [Boudou et al. 2012]. Thyroid hormone, dexamethasone, and insulin-like growth factor-1 may help hiPSCCMs mature in the 3D cardiac microtissue.

1.5

Bioprinting

Recent methods for creating 3D cardiac tissue have produced encouraging results, suggesting its potential for developing alternatives to heart transplants. These techniques combine biomaterials, including decellularized extracellular matrix (ECM), alginate, gelatin, and collagen with bioprinting techniques like extrusion, inkjet, laser-assisted stereolithography, or scaffold-free approaches. Specific combinations of biomaterials and bioprinters are chosen according to the type of cardiac tissue generated, for example, myocardium, valves, blood vessels, and connective tissue. Some studies that successfully recreated the shape, mechanical parameters, protein expression, and electrical properties of adult natural cardiac ECM have been documented, each with its unique advantages and limitations. These four broad categories are further subdivided to discover the most efficient approaches since the in vitro tissue culture-developed myocardial tissue closely approximating the native cardiac environment is likely to recapitulate the normal myocardial function in vivo. For instance, contractile force and conduction velocity

14

S. Ramesh et al.

are mechanical qualities, whereas resting membrane potential and upstroke velocity are electrophysiological properties. Here, the emphasis is on the technology that successfully produced constructs of adult native cardiac tissue that mimics natural heart tissue.

1.6

Microelectrode Cultured CM

It is difficult to create sophisticated electrically active cell models. Internal electrical signaling systems in cardiomyocytes cause myocytes to contract and relax synchronized and pulsatile, resulting in effective blood pumping. It has been investigated how electrical stimulation affects mature CMs. As an illustration, 80V stimulation was applied to neonatal rat cardiomyocytes cultivated on polystyrene culture plates for 15 min to 72 h (Xia et al. 1997). The peak expression of COXVa and -MHC mRNA was observed 24 h after electrical stimulation. After 24 h, hypertrophy and enhanced mitochondrial contents and activity were also generated. To mature CMs, newborn rat CMs were grown on collagen sponges and subjected to electrical pulses (rectangular, 2 ms, 5 V/cm, 1 Hz) for 5 days. The cells also displayed greater contractility and mitochondrial contents in addition to higher expression of -MHC, CK-MM, Cx43, and TnI (Radisic et al. 2004). Neonatal rat CMs cultured on micro-grooved polystyrene showed evidence of maturation and cells grew elongated with gap junctions positioned at the cell-cell contact area (Heidi Au et al. 2009). This was accomplished by combining topography and electrical stimulation. 3D biowires consisting of hPSCs, supporting cells, and collagen sutures were electrically stimulated (Nunes et al. 2013). The stimulated constructions had higher conduction speed, expression of heart contractile proteins, and improved Ca2+ handling abilities. As previously indicated, the cardiac microtissue investigation exposed the tissues to electrical stimulation for 4 days (Boudou et al. 2012). In addition to experiencing more dynamic contraction stress, the CMs exposed to electrical stimulation arrived at cellular alignment around 2 days earlier than unexposed constructions. According to these observations, electrical stimulation is a valuable and effective strategy for CMs maturation. Interestingly, the commercialization of microelectrode array plates has also been available in the market, which can also be used to test the efficacy of the electrical influence as a critical parameter in adult CM-related research. Maestro Pro and Edge MEA systems are the currently available platforms designed specifically for the real-time readouts of formation and maturation of the syncytium, analyzing beat characteristics quantitatively and categorizing cell behavior, etc. (Kussauer et al. 2021). The Maestro MEA platform offers unprecedented access to heart activity across minutes or months to noninvasively track the development of a cell’s distinct phenotype in culture.

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

1.7

15

Conclusion and Future Challenges

Despite recent efforts, there is still no widely acknowledged cut-off for classifying completely mature CMs produced from stem cells (Pasqualini et al. 2015). There is a continuous discussion about whether naïve stem/progenitor cells are superior to their pre-differentiated or partially differentiated derivative cells. And if the latter is used for cell-based therapy, then what level of pre-differentiation is optimal to achieve the best results (Haider and Ashraf 2005)? Furthermore, even on a small scale, maturation protocols and techniques are less than optimal and lack reproducibility. The pharmaceutical industry mainly relies on large-scale, high-content screens in a human-relevant model to discover new drugs and their development. This could be achieved by CMs produced from stem cells that closely resemble the electrical, chemical, and physical traits of adult CMs. Successful cell therapy for heart failure has been severely hampered by stem cell retention, survival, and long-term engraftment (Sanganalmath and Bolli 2013); however, different strategies have been developed to overcome these issues in genetic modulation of the cells with pro-survival genes, growth factor treatment, pharmacological and physical manipulation, electrical stimulation, etc. (Lu et al. 2009; Kim et al. 2009, 2012a, b; Haider et al. 2010; Suzuki et al. 2010; Lai et al. 2012; Lu et al. 2012a, b). Stem cell therapy has additional difficulties because of several serious risk issues, including the possibility of tumorigenesis (Herberts et al. 2011; Ahmed et al. 2011a, b) and arrhythmia creation (Chong et al. 2014). These problems may be resolved by mature CMs produced from stem cells by minimizing the discrepancies between native and transplanted CMs. Combining in vitro and in vivo approaches, the personalization of medicine is a novel and exciting application. Modeling uncommon electrophysiological abnormalities using patient-sourced iPSCs-derived CMs and individually patient-based therapy screening have both been done using these cells (Kim et al. 2013; Moretti et al. 2010a, b). iPSCs obtained from patients have been utilized to create a disease-specific model of Barth syndrome, a rare genetic condition of cardiomyopathy, used to investigate potential treatments (Wang et al. 2014; Cagavi et al. 2018). Personalized disease models can also be created using a patient’s cells, reducing the requirement for mass manufacture and solving the cell source problem. Cell-based therapies in regenerative medicine would significantly improve if it were possible to create healthy, mature CMs from a patient’s cells. However, this method of understanding and treating disease represents a significant departure from the dominant paradigm and would necessitate substantial speculation in infrastructure for drug discovery and development. Therefore, maturated stem cell-derived CMs will be a useful tool to spot and treat druginduced cardiotoxicity when the maturation process’ efficiency is increased. CMs produced from pluripotent stem cells (PSC-CMs) are the viable hope of cellular source for the supply of cardiac cells. Having abundant availability, they can be used to generate better disease models and drug testing platforms, as well as the potential for cell-based treatments and tissue transplantation (manipulated and un-manipulated in vitro) as observed in the experimental animal models (Rufaihah et al. 2007, 2010). Therefore, PSC-derived CMs purity and production should be

16

S. Ramesh et al.

Fig. 1.4 A summary of the overall cardiomyocyte maturation-inducing factors

significantly improved with the prerequisite of morphological and functional maturity strictly in place. To enhance the maturation process, a wide range of bioengineering techniques have been investigated along with the parameters such as long-term culture, co-culture, exposure to mechanical stimuli, 3D cultures, cellmatrix interactions, and electrical stimulation, etc. (Fig. 1.4). Despite modest gains, there is still a wide gap between the phenotype that must be obtained and the state of maturation that is now attainable. Additionally, there is a dire need for a standard metric to reproducibly assess the maturity of CMs because the methods used to quantify maturation are highly diverse. Increased functional maturation and the generation of better methods for measuring functional parameters warrant additional research. Future differentiation and maturation protocols will probably integrate some of the strategies discussed in this analysis to devise a biomimetic microenvironment that is conducive to the desired morphofunctionally competent CMs phenotype. Dynamic microenvironmental cues, such as substrate elastic moduli, can be further improved to reproduce the cardiogenic events better. It is necessary to generate the predicted values for the functional assessment of mature CMs concurrently with the development and combining of the microenvironmental cues to produce mature CMs. Thereby, substantial progress can be achieved in developing a bench-to-bedside therapy for CVDs.

References Abecasis B, Gomes-Alves P, Rosa S, Gouveia PJ, Ferreira L, Serra M, Alves PM (2019) Unveiling the molecular crosstalk in a human induced pluripotent stem cell-derived cardiac model. Biotechnol Bioeng 116(5):1245–1252. https://doi.org/10.1002/bit.26929. Epub 2019 Jan 30. PMID: 30659585

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

17

Ahmed RPH, Haider KH, Buccini S, Shujia J, Ashraf M (2011a) Reprogramming of skeletal myoblasts for induction of pluripotency for tumor-free cardiomyogenesis in the infarcted heart. Circ Res 109:60–70 Ahmed RPH, Ashraf M, Buccini S, Shujia J, Haider KH (2011b) Cardiac tumorigenic potential of induced pluripotent stem cells in an immunocompetent host: a note of caution. Regen Med 6: 171–178 Ahmed RE, Anzai T, Chanthra N, Uosaki H (2020) A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes. Front Cell Dev Biol 8:178 Azevedo PS, Polegato BF, Minicucci MF, Paiva SA, Zornoff LA (2016) Cardiac remodeling: concepts, clinical impact, pathophysiological mechanisms and pharmacologic treatment. Arq Bras Cardiol 106(1):62–69. https://doi.org/10.5935/abc.20160005 Balafkan N, Mostafavi S, Schubert M et al (2020) A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates. Sci Rep 10: 18498. https://doi.org/10.1038/s41598-020-73656-2 Bauwens CL, Peerani R, Niebruegge S et al (2008) Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26(9): 2300–2310 Bhattacharya S, Burridge PW, Kropp EM, Chuppa SL, Kwok WM, Wu JC, Boheler KR, Gundry RL (2014) High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry. J Vis Exp (91):52010 Binah O, Dolnikov K, Sadan O et al (2007) Functional and developmental properties of human embryonic stem cells-derived cardiomyocytes. J Electrocardiol 40(6 Suppl):S192–S196 Boudou T, Legant WR, Mu A, Borochin MA, Thavandiran N, Radisic M et al (2012) A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. Tissue Eng Part A 18:910–919 Buccini S, Haider KH, Ahmed RPH, Jiang S, Ashraf M (2012) Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol 107(6):301–314 Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, Mahairaki V et al (2011) A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6(4):e18293. https://doi.org/10.1371/ journal.pone.0018293 Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F et al (2014) Chemically defined generation of human cardiomyocytes. Nat Methods 11(8):855–860. https://doi.org/10. 1038/nmeth.2999 Cagavi E, Akgul Caglar T, Soztekin GI, Haider KH (2018) Patient-specific induced pluripotent stem cells for cardiac disease modelling. In: Haider KH, Aziz S (eds) Stem cells: from hype to real hope. Medicine & Life Sciences, De Gruyter, Berlin Chan YC, Ting S, Lee YK, Ng KM, Zhang J, Chen Z, Siu CW, Oh SK, Tse HF (2013) Electrical stimulation promotes maturation of cardiomyocytes derived from human embryonic stem cells. J Cardiovasc Transl Res 6(6):989–999. https://doi.org/10.1007/s12265-013-9510-z Chong JJ, Yang X, Don CW, Minami E, Liu YW, Weyers JJ et al (2014) Human embryonic-stemcell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510:273–277 Chun YW, Balikov DA, Feaster TK et al (2015) Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes. Biomaterials 67: 52–64. https://doi.org/10.1016/j.biomaterials.2015.07.004 Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y et al (2014) Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes. Stem Cell Rev Rep 10(6):786–801. https://doi.org/10.1007/s12015014-9533-0 Correia C, Koshkin A, Duarte P et al (2017) Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Sci Rep 7:8590. https://doi.org/10.1038/s41598-017-08713-4

18

S. Ramesh et al.

D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23:1534–1541. https://doi.org/10.1038/nbt1163 Dolnikov K, Shilkrut M, Zeevi Levin N et al (2006) Functional properties of human embryonic stem cell derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction. Stem Cells 24(2):236–245 Drawnel FM, Boccardo S, Prummer M, Delobel F, Graff A, Weber M, Gérard R et al (2014) Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells. Cell Rep 9(3):810–821. https://doi.org/10.1016/j.celrep.2014. 09.055 Feaster TK, Cadar AG, Wang L, Williams CH, Chun YW, Hempel JE, Bloodworth N, Merryman WD et al (2015) Matrigel mattress: a method for the generation of single contracting humaninduced pluripotent stem cell-derived cardiomyocytes. Circ Res 117(12):995–1000. https://doi. org/10.1161/CIRCRESAHA.115.307580 Feyen DAM, McKeithan WL, Bruyneel AAN, Spiering S, Hörmann L, Ulmer B, Zhang H et al (2020) Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes. Cell Rep 32(3):107925. https://doi.org/10.1016/j.celrep.2020.107925 Fijnvandraat AC, van Ginneken AC, de Boer PA et al (2003) Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube. Cardiovasc Res 58(2):399–409 Fu JD, Li J, Tweedie D et al (2006) Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells. FASEB J 20(1):181–183 Gepstein L, Snir M, Kehat I et al (2003) Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 285(6):H2355–H2363 Gherghiceanu M, Barad L, Novak A et al (2011) Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure. J Cell Mol Med 15(11): 2539–2551 Govarthanan K, Gupta PK, Zipporah BE, Sharma V, Rajasundari M, Haider KH (2022) Molecular signature of stem cells undergoing cardiomyogenic differentiation. In: Haider KH (ed) Handbook of stem cell therapy. Springer, Singapore. https://doi.org/10.1007/978-981-166016-0_25-1 Haider KH, Ashraf M (2005) Pre-differentiation of stem cells for transplantation: to be or not to be that is the question. ASIAO J 51:301–302 Haider KH, Lee Y-J, Jiang S, Ahmad RPH, Ahn MK, Ashraf M (2010) Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells. Am J Physiol Heart Lung Physiol 299:H1395–HH404 Heidi Au HT, Cui B, Chu ZE, Veres T, Radisic M (2009) Cell culture chips for simultaneous application of topographical and electrical cues enhance phenotype of cardiomyocytes. Lab Chip 9:564–575 Henderson CA, Gomez CG, Novak SM, Mi-Mi L, Gregorio CC (2017) Overview of the muscle cytoskeleton. Compr Physiol 7(3):891–944. https://doi.org/10.1002/cphy.c160033. PMID: 28640448; PMCID: PMC5890934 Heng BC, Cao T, Haider KH, Sim EKW, Cao T, Ng SC (2004) Strategies for directing the differentiation of stem cells into the cardiomyocytes lineage in vitro. Cardiovasc Res 62:34–42 Heng BC, Haider KH, Sim EKW, Cao T, Tong GQ, Ng SC (2005) Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage. Acta Cardiol 60:7–12 Herberts CA, Kwa MS, Hermsen HP (2011) Risk factors in the development of stem cell therapy. J Transl Med 9:29 Horikoshi Y, Yan Y, Terashvili M, Wells C, Horikoshi H, Fujita S, Bosnjak ZJ et al (2019) Fatty acid-treated induced pluripotent stem cell-derived human cardiomyocytes exhibit adult

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

19

cardiomyocyte-like energy metabolism phenotypes. Cell 8(9):1095. https://doi.org/10.3390/ cells8091095 Hwang HS, Kryshtal DO, Feaster TK, Sanchez-Freire V, Zhang J, Kamp TJ, Hong CC et al (2015) Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories. J Mol Cell Cardiol 85:79–88. https://doi.org/10.1016/j.yjmcc.2015.05.003 Ibrahim AY, Mehdi Q, Abbas AO, Alashkar A, Haider KH (2016) Induced pluripotent stem cells: next generation cells for tissue regeneration. J Biomed Sci Eng 9(4):226–244 Itzhaki I, Schiller J, Beyar R et al (2006) Calcium handling in embryonic stem cell-derived cardiac myocytes: of mice and men. Ann N Y Acad Sci 1080:207–215 Jacot JG, Martin JC, Hunt DL (2010) Mechanobiology of cardiomyocyte development. J Biomech 43(1):93–98. https://doi.org/10.1016/j.jbiomech.2009.09.014 Kamakura T, Makiyama T, Sasaki K, Yoshida Y, Wuriyanghai Y, Chen J, Hattori T et al (2013) Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture. Circ J 77(5):1307–1314. https://doi.org/10.1253/circj.cj-12-0987 Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X, Murry CE (2020) Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol 17(6):341–359. https://doi.org/10.1038/s41569-019-0331-x Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A et al (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108(3):407–414 Kim HW, Haider KH, Jiang S, Ashraf M (2009) Ischemic preconditioning augments survival of stem cells via miR-107 and miR-210 expression. J Biol Chem 284:33161–33168 Kim C, Majdi M, Xia P et al (2010) Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells Dev 19(6):783–795 Kim HW, Malik F, Durrani S, Ashraf M, Jiang S, Haider KH (2012a) Concomitant activation of mir-107/pdcd10 and hypoxamir-210/casp8ap2 and their role in cytoprotection during ischemic preconditioning of stem cells. Antioxid Redox Signal 17(8):1053–1065 Kim HW, Ashraf M, Jiang S, Haider KH (2012b) Stem cell based delivery of hypoxamir-210 to the infarcted heart: implications on stem cell survival and preservation of the infarcted heart function. J Mol Med 90(9):997–1010 Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S et al (2013) Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494:105–110 Klein I, Ojamaa K (2001) Thyroid hormone and the cardiovascular system. N Engl J Med 344:501– 509 Knight WE, Cao Y, Lin YH, Chi C, Bai B, Sparagna GC, Zhao Y et al (2021) Maturation of pluripotent stem cell-derived cardiomyocytes enables modeling of human hypertrophic cardiomyopathy. Stem Cell Rep 16(3):519–533. https://doi.org/10.1016/j.stemcr.2021.01.018 Kroll K, Chabria M, Wang K, Häusermann F, Schuler F, Polonchuk L (2017) Electro-mechanical conditioning of human iPSC-derived cardiomyocytes for translational research. Prog Biophys Mol Biol 130(Pt B):212–222. https://doi.org/10.1016/j.pbiomolbio.2017.07.003 Kussauer S, David R, Lemcke H (2021) Micro-electrode-arrays: a valuable tool to analyze stem cell derived cardiomyocytes. In: Haider KH (ed) Stem cells; latest advances, chap 1. Springer, Switzerland, pp 1–21 Lai VK, Afzal MR, Ashraf M, Jiang S, Haider KH (2012) Non-hypoxic stabilization of Hif-1α during coordinated interaction between Akt and angiopoietin-1 enhances endothelial commitment of bone marrow stem cells. J Mol Med 90(6):719–730 Lewandowski J, Rozwadowska N, Kolanowski TJ, Malcher A, Zimna A, Rugowska A, Fiedorowicz K (2018) The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin. Cell Transplant 27(7):1047–1067. https://doi.org/10.1177/0963689718779346 Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP (2013) Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating

20

S. Ramesh et al.

Wnt/β-catenin signaling under fully defined conditions. Nat Protoc 8(1):162–175. https://doi. org/10.1038/nprot.2012.150. Epub 2012 Dec 20. PMID: 23257984; PMCID: PMC3612968 Lieu DK, Liu J, Siu CW et al (2009) Absence of transverse tubules contributes to non-uniform Ca (2+) wavefronts in mouse and human embryonic stem cell-derived cardiomyocytes. Stem Cells Dev 18(10):1493–1500 Lopaschuk G, Jaswal J (2010) Energy metabolic phenotype of the cardiomyocyte during development, differentiation and postnatal maturation. J Cardiovasc Pharmacol 56(2):130–140 Lu G, Haider KH, Jiang S, Ashraf M (2009) Sca-1+ stem cell survival and engraftment in the infarcted heart: dual role for preconditioning-induced Connexin-43. Circulation 119(19): 2587–2296 Lu G, Ashraf M, Haider KH (2012a) IGF-1 preconditioning accentuates intrinsic survival mechanism in stem cells to resist ischemic injury by orchestrating PKCα-MAPK activation. Antioxid Redox Signal 16(3):217–227 Lu G, Jiang S, Ashraf M, Haider KH (2012b) Mitochondria specific connexin-43 transgene expression protects stem cells in the ischemic heart via shifting the balance between Bak and Bcl-xL. Regen Med 7(3):323–334 Lundy SD, Zhu WZ, Regnier M, Laflamme MA (2013) Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev 22(14):1991–2002. https://doi.org/10.1089/scd.2012.0490. Epub 2013 Apr 5. PMID: 23461462; PMCID: PMC3699903 Marzuca-Nasri GN, Vitzel KF, Mancilla-Solorza E, Márquez JL (2018) Sarcomere structure: the importance of desmin protein in muscle atrophy. Int J Morphol 36(2):576–583 McCain ML, Yuan H, Pasqualini FS, Campbell PH, Parker KK (2014) Matrix elasticity regulates the optimal cardiac myocyte shape for contractility. Am J Physiol Heart Circ Physiol 306(11): H1525–H1539. https://doi.org/10.1152/ajpheart.00799.2013. Epub 2014 Mar 28. PMID: 24682394; PMCID: PMC4042196 McDevitt TC, Laflamme MA, Murry CE (2005) Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway. J Mol Cell Cardiol 239(6):865–873 Miki K, Deguchi K, Nakanishi-Koakutsu M et al (2021) ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes. Nat Commun 12(1):3596. https:// doi.org/10.1038/s41467-021-23816-3 Minami I, Yamada K, Otsuji TG, Yamamoto T, Shen Y, Otsuka S, Kadota S et al (2012) A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine-and xeno-free conditions. Cell Rep 2(5):1448–1460 Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L et al (2010a) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363:1397–1409 Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L, Dorn T, Goedel A, Höhnke C, Hofmann F et al (2010b) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363:1397–1409. https://doi.org/10.1056/NEJMoa0908679 Mummery C, Wardvan Oostwaard D, Doevendans P et al (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107(21):2733–2740 Murphy SA, Chen EZ, Tung L, Boheler KR, Kwon C (2021) Maturing heart muscle cells: mechanisms and transcriptomic insights. In: Seminars in cell & developmental biology, vol 119. Academic Press, London, pp 49–60 Nakano H, Minami I, Braas D, Pappoe H, Wu X, Sagadevan A, Vergnes L et al (2017) Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis. Elife 6:e29330. https://doi. org/10.7554/eLife.29330. PMID: 29231167; PMCID: PMC5726851 Nijak A, Saenen J, Labro AJ, Schepers D, Loeys BL, Alaerts M (2021) iPSC-Cardiomyocyte models of brugada syndrome - achievements, challenges and future perspectives. Int J Mol Sci 22:2825. https://doi.org/10.3390/ijms22062825

1

Recent Advances in In Vitro Generation of Mature Cardiomyocytes

21

Nunes SS, Miklas JW, Liu J, Aschar-Sobbi R, Xiao Y, Zhang B et al (2013) Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat Methods 10:781–787 Ogasawara T, Okano S, Ichimura H, Kadota S, Tanaka Y, Minami I, Uesugi M et al (2017) Impact of extracellular matrix on engraftment and maturation of pluripotent stem cell-derived cardiomyocytes in a rat myocardial infarct model. Sci Rep 7(1):8630. https://doi.org/10.1038/ s41598-017-09217-x. PMID: 28819182; PMCID: PMC5561148 Parikh SS, Blackwell DJ, Gomez-Hurtado N, Frisk M, Wang L, Kim K et al (2017) Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Circ Res 121:1323–1330. https://doi.org/10.1161/ CIRCRESAHA.117.311920 Pasqualini FS, Sheehy SP, Agarwal A, Aratyn-Schaus Y, Parker KK (2015) Structural phenotyping of stem cell-derived cardiomyocytes. Stem Cell Rep 4:340–347 Peter AK, Bjerke MA, Leinwand LA (2016) Biology of the cardiac myocyte in heart disease. Mol Biol Cell 27(14):2149–2160 Porter GA Jr, Hom J, Hoffman D et al (2011) Bioenergetics, mitochondria, and cardiac myocyte differentiation. Prog Pediatr Cardiol 31(2):75–81 Radisic M, Park H, Shing H, Consi T, Schoen FJ, Langer R et al (2004) Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci 101:18129–18134 Ranek MJ, Berthiaume JM, Kirk JA, Lyon RC, Sheikh F, Jensen BC, Hoit BD et al (2022) Pathophysiology of heart failure and an overview of therapies. Cardiovasc Pathol 1:149–221 Ren Y, Lee MY, Schliffke S, Paavola J, Amos PJ, Ge X, Ye M, Zhu S, Senyei G, Lum L et al (2011) Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells. J Mol Cell Cardiol 51:280–287 Ribeiro AJ, Ang YS, Fu JD, Rivas RN, Mohamed TM, Higgs GC, Srivastava D, Pruitt BL (2015) Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proc Natl Acad Sci U S A 112(41):12705–12710. https://doi.org/10.1073/pnas.1508073112. Epub 2015 Sep 28. PMID: 26417073; PMCID: PMC4611612 Rog-Zielinska EA, Thomson A, Kenyon CJ, Brownstein DG, Moran CM, Szumska D et al (2013) Glucocorticoid receptor is required for foetal heart maturation. Hum Mol Genet 22:3269–3282. https://doi.org/10.1093/hmg/ddt182 Rog-Zielinska EA, Craig M-A, Manning JR, Richardson RV, Gowans GJ, Dunbar DR et al (2015) Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α. Cell Death Differ 22:1106–1116. https://doi.org/10.1038/cdd.2014.181 Rufaihah AJ, Haider KH, Heng BC, Tian XF, Lei Y, Ge R, Cao T (2007) Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing VEGF165 gene. J Gene Med 9(6):452–461 Rufaihah AJ, Haider KH, Heng BC, Ye L, Tan RS, Toh WS, Tian XF et al (2010) Therapeutic angiogenesis by transplantation of human embryonic stem cell-derived CD133+ endothelial progenitor cells for cardiac repair. Regen Med 5:231–244 Sanganalmath SK, Bolli R (2013) Cell therapy for heart failure: a comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circ Res 113:810– 834 Sartiani L, Bochet P, Cerbai E et al (2002) Functional expression of the hyperpolarization-activated, non-selective cation current if in immortalized HL-1 cardiomyocytes. J Physiol 545(Pt1):81–92 Severs NJ (2002) Gap junctions and connexin expression in human heart disease. In: De Mello WC, Janse MJ (eds) Heart cell coupling and impulse propagation in health and disease. Kluwer Academic, Boston, pp 321–334 Smolich JJ (1995) Ultrastructural and functional features of the developing mammalian heart: a brief overview. Reprod Fertil Dev 7(3):451–461

22

S. Ramesh et al.

Snir M, Kehat I, Gepstein A et al (2003) Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 285:H2355–H2363 Suzuki Y, Kim HW, Ashraf M, Haider KH (2010) Diazoxide potentiates mesenchymal stem cell survival via NF-κB-dependent miR-146a expression. Am J Physiol 299:H1077–H1082 Talman V, Kivelä R (2018) Cardiomyocyte—endothelial cell interactions in cardiac remodeling and regeneration. Front Cardiovasc Med 5:101 Tracy RE, Sander GE (2011) Histologically measured cardiomyocyte hypertrophy correlates with body height as strongly as with body mass index. Cardiol Res Pract 2011:658958. https://doi. org/10.4061/2011/658958. Epub 2011 Jun 8. PMID: 21738859; PMCID: PMC3123935 Tsvelaya VA, Gams A, Aziz J, Efimov IR (2018) Induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs): novel diagnostic platform. In: Haider KH, Aziz S (eds) Stem cells: from hype to real hope. Medicine & Life Sciences, De Gruyter, Berlin Tu C, Chao BS, Wu JC (2018) Strategies for improving the maturity of human induced pluripotent stem cell-derived cardiomyocytes. Circ Res 123(5):512–514. https://doi.org/10.1161/ CIRCRESAHA.118.313472. PMID: 30355143; PMCID: PMC6392006 Uçkan-Çetinkaya D, Haider KH (2021) Induced pluripotent stem cells in pediatric research and clinical translation. In: Haider KH (ed) Stem cells, chap 13. Springer, Cham, pp 203–214. https://doi.org/10.1007/978-3-030-77052-5_13 Ullah M, Iqbal H, Haneef K, Khan I, Salim A (2021) Chromatin remodeling and cardiac differentiation of stem cells. In: Haider KH (ed) Stem cells: from potential to promise, chap 9. Springer, Singapore, pp 251–273. https://doi.org/10.1007/978-981-16-0301-3_9 Vreeker A, Van Stuijvenberg L, Hund TJ, Mohler PJ, Nikkels PG, Van Veen TA (2014) Assembly of the cardiac intercalated disk during pre-and postnatal development of the human heart. PLoS One 9(4):e94722 Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A et al (2014) Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat Med 20:616–623 Wickramasinghe NM, Sachs D, Shewale B, Gonzalez DM, Dhanan-Krishnan P, Torre D, LaMarca E et al (2022) PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 29(4):559–576.e7. https://doi.org/ 10.1016/j.stem.2022.02.011. Epub 2022 Mar 23. PMID: 35325615 Xia Y, Buja LM, Scarpulla RC, McMillin JB (1997) Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation. Proc Natl Acad Sci 94:11399–11404 Yang X, Rodriguez M, Pabon L, Fischer KA, Reinecke H, Regnier M, Sniadecki NJ et al (2014) Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. J Mol Cell Cardiol 72:296–304. https://doi.org/10.1016/j.yjmcc.2014.04. 005. Epub 2014 Apr 13. PMID: 24735830; PMCID: PMC4041732 Yang X, Rodriguez ML, Leonard A, Sun L, Fischer KA, Wang Y et al (2019) Fatty acids enhance the maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cell Rep 13: 657–668. https://doi.org/10.1016/j.stemcr.2019.08.013 Yazawa M, Hsueh B, Jia X et al (2011) Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471:230–234. https://doi.org/10.1038/nature09855 Yokoo N, Baba S, Kaichi S et al (2009) The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells. Biochem Biophys Res Commun 387:482– 488 Yoshida S, Miyagawa S, Fukushima S, Kawamura T, Kashiyama N, Ohashi F, Toyofuku T, Toda K (2018) Maturation of human induced pluripotent stem cell-derived cardiomyocytes by soluble factors from human mesenchymal stem cells. Mol Ther 26(11):2681–2695. https://doi.org/10. 1016/j.ymthe.2018.08.012. Epub 2018 Aug 16. PMID: 30217728; PMCID: PMC6224789 Zhang J, Wilson GF, Soerens AG et al (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104(3):e30–e41

2

Cardiac Reprogramming with Stem Cells: An Advanced Therapeutic Strategy in Advanced Heart Failure Alexander E. Berezin and Alexander A. Berezin

Abstract

Heart failure (HF) remains the most prominent cause of cardiovascular morbidity and mortality. The underlying molecular mechanisms of the natural evolution of HF affect cardiac myocytes’ loss and extracellular matrix rearrangement, resulting from the persistence of several etiological factors, including coronary artery disease, viral infections, epigenetic alterations, genetic mutations, adverse immune reactions, and primary cardiac toxicity. Although there are welldeveloped approaches to pharmacological therapy and device care, i.e., bridgeto-destination, bridge-to-bridge, and bridge-to-transplantation therapies, the mortality from advanced HF is still unacceptably high. Current efforts to improve clinical outcomes for these patients are attributed to translational medical care, which mainly directs to cardiac regeneration and reprogramming with stem cells. Recent studies in patients with ischemia and non-ischemia severe HF have shown conflicting survival results after implantation of bone marrow mesenchymal stem cells and human induced pluripotent stem cells. Still, cardiac function was substantially improved in the majority of the investigations. It has been disputed that improving stem-derived cardiac cell maturation before implantation and using alternative endogenous sources for stem cells, i.e., endogenous skeletal myoblasts, may accelerate morphofunctional recovery after implantation. Indeed, direct cardiac reprogramming has been considered a promising therapeutic A. E. Berezin (✉) Internal Medicine Department, Zaporozhye State Medical University, Zaporozhye, Ukraine Department of Internal Medicine II, Division of Cardiology, Paracelsus Medical University, Salzburg, Austria A. A. Berezin Internal Medicine Department, Zaporozhye Medical Academy of Post-Graduating Education, Zaporozhye, Ukraine # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 K. H. Haider (ed.), Cardiovascular Applications of Stem Cells, https://doi.org/10.1007/978-981-99-0722-9_2

23

24

A. E. Berezin and A. A. Berezin

approach to rejuvenate damaged myocardium by previously transforming endogenous skeletal myoblasts into cardiac myocyte-like cells. This approach being occurred to be substantially progressive is now under scientific discussion. The chapter depicts the challenging clinical perspectives for implanting cardiac reprogrammed stem cells in severe HF. Keywords

Advanced heart failure · Cardiomyocytes · Stem cells · Cardiac reprogramming · Cardiac regeneration

Abbreviations 6-MWTD ACC AHA AHF AMPK Arrdc1 CMP CSCs CVD ECM EPCs ERK ESC GDMT HF HFA HFmrEF HFpEF HFrEF HFSA HIF HSPs INTERMACS iPSCs JAK JNK″ LVEF MAPK MI MSC

6-Minute walk test distance American College of Cardiology American Heart Association Advanced heart failure AMP-activated protein kinase Arrestin-domain containing protein 1 Cardiomyopathy Cardiac stem cells Cardiovascular disease Extracellular matrix Endothelial progenitor cells Extracellular signal-regulated kinase European Society of Cardiology Guideline-directed medical therapy Heart failure Heart Failure Association Heart failure with a mildly reduced ejection fraction Heart failure with a preserved ejection fraction Heart failure with a reduced ejection fraction Heart Failure Society of America Hypoxia-inducible factor Heat shock proteins Interagency Registry for Mechanically Assisted Circulatory Support Inducible pluripotent stem cells Janus kinase c-Jun N-terminal kinase Left ventricular ejection fraction Mitogen-activated protein kinase Myocardial infarction Adipose tissue-derived stem cells and bone marrow-derived MSCs

2

Cardiac Reprogramming with Stem Cells: An Advanced Therapeutic Strategy. . .

Nkx2.5 NPs NT-proBNP NYHA SDF-1 STEMI TGF VEGF Yap

2.1

25

NK2 Homeobox 5 protein Natriuretic peptides N-terminal brain natriuretic pro-peptide New York Heart Association Stromal cell-derived factor-1 ST segment elevation myocardial infarction Transforming growth factor Vascular endothelial growth factor Yes-associated protein

Introduction

Heart failure (HF) remains a serious problem of public health that is strongly associated with increased all-cause and cardiovascular death, urgent hospital admission, and poor quality of life among patients with established cardiovascular disease (CVD) worldwide (Virani et al. 2021; Groenewegen et al. 2020). Albeit there is a general trend of reducing new cases of HF with reduced ejection fraction (HFrEF) in developed countries (Chan et al. 2021), global growth of HF prevalence relates to a srelative increase in HF with preserved ejection fraction (HFpEF) mainly in developing countries (GBD 2017 Disease and Injury Incidence and Prevalence Collaborators 2018). Most patients with advanced HF seem to have an ischemic etiology of the condition, whereas non-ischemic HF has been detected as sufficiently rarer than ischemic. However, the exposure to effective chemotherapy technologies in managing malignancy, infective myocarditis, peripartum cardiomyopathy, primary cardiomyopathies, and autoimmune disorders led to a dramatically prolonged life span and a significant increase in the number of survivors (Leczycki et al. 2022; Chuda et al. 2022). Consequently, the risk of transforming any phenotype of HF to advanced HF in these patient cohorts is regarded as an undeniable fact (Ávila et al. 2014; Stolfo et al. 2021). Indeed, the disproportion in a presence of primary etiology causes, comorbidity signatures, important clinical and organizational factors, and affordability of healthcare systems among HF patients from different countries ensure a sharp difference in HF outcomes, including those that tackle transitions to advanced HF from its phenotypes (Savarese et al. 2019; Vedin et al. 2017). Although the development of advanced HF seemed to have been disputed due to the predominantly natural progression of HFrEF, a recent observational study revealed that more than 50% of patients with advanced HF had HFrEF or HFpEF (Dunlay et al. 2021). Moreover, all-cause mortality and risk of urgent hospitalization in advanced HF patients were not related to phenotypes of HF and exact LVEF value (Dunlay et al. 2021; Liang et al. 2022). These data result in the hypothesis that a transition of any phenotype of HF to advanced HF can be an attributable factor to an impact of comorbidities and etiologies on adverse cardiac remodeling directly related to progressive myocyte loss and consistent extracellular matrix rearrangement (Bhatt

26

A. E. Berezin and A. A. Berezin

et al. 2017). Despite varying HF treatment approaches to prevent either progression or reversal of adverse cardiac remodeling with guideline-directed medical therapy (GDMT), revascularization, device-based management, and rehabilitation programs, several modalities of impaired myocardium in advanced HF patients cannot be consistently repaired otherwise as by cell therapy to improve prognosis (Zhao et al. 2020; Yang et al. 2020; Venugopal et al. 2022; Steffens et al. 2020; Hoeeg et al. 2020). Preclinical and clinical studies have yielded numerous optimistic results of the therapeutic potency of the cardiac regenerative approach with implanted stem cells, but these encouraging results necessitate large confirmatory trials, which seemed to have been controversial enough (Berezin and Berezin 2021). The chapter aims to elucidate the challenging clinical perspectives for implanting cardiac reprogrammed stem cells in advanced HF.

2.2

Advanced Heart Failure: Definition and Contemporary Strategy

The definition of advanced HF has been subject to significant changes since the strict criteria of the one that first appeared (Fig. 2.1). It includes initially symptomatic HF patients with serious limitation of physical exercise, sustainable resting left ventricular ejection fraction (LVEF)