Recent Progress Using Pluripotent Stem Cells for Cardiac Regenerative Therapy

Abstract

Pluripotent stem cells (PSCs) have gained interest for cell-based regenerative therapies because of their capacity to differentiate into most somatic cell types, including cardiomyocytes. Remarkable progress in the generation of PSC-derived cardiomyocytes has been made in this decade, and recent preclinical transplantation studies using various animal models have provided proof-of-principle for their use in heart regeneration. However, several obstacles preclude their effective and safe clinical application for cardiac repair, including the need for approaches that prevent tumorigenesis, arrhythmogenesis, and immune rejection. In this review, we focus on recent progress in the field of PSC-based cardiac regenerative therapy, including the remaining hurdles and potential approaches to circumventing them.

Chronic heart failure (CHF) is a leading cause of morbidity and mortality worldwide. Although pharmacological therapies can inhibit the progression of CHF, cardiac dysfunction never improves naturally. The prognosis for these patients is poor, with increased risk of death and reduced quality of life. Heart transplantation (Tx) is the only effective treatment for severe HF; however, donor shortage is a serious problem and many patients die while waiting for a transplant.^1,2 Therefore, novel treatment methods are needed and many studies on cardiac regenerative therapy have been performed.³

Pluripotent stem cells (PSCs) have gained interest as cell sources for cardiac regeneration because of their unlimited self-renewal and ability to differentiate into cardiomyocytes (CMs). Recently, Menasche et al initiated the first clinical trial for HF treatment using embryonic stem cell (ESC)-derived cardiac progenitors, and reported improved patient symptoms at 3 months post-operation.⁴ However, practical questions need to be addressed before large-scale clinical trials. Herein, we focus on the recent progress in cardiac regenerative therapy using PSCs, including the remaining hurdles and potential approaches to circumventing them.

Establishment of PSCs

Following the establishment of mouse ESCs in 1981,⁵ Thomson et al isolated similar cells from human blastocysts.⁶ Human ECSs have been widely utilized in various fields, including regenerative therapy and tissue engineering, and 378 cell lines have been registered in the National Institutes of Health Human Embryonic Stem Cell Registry (https://stemcells.nih.gov/research/registry.htm). However, ethical considerations remain a barrier to the clinical application of hESC-based therapies. Moreover, because ESC-derived cells or tissues are allogenic, immune rejection is a problem.

In 2006, Takahashi and Yamanaka developed a technique to reprogram adult mouse somatic cells into ESC-like pluripotent cells through the expression of 4 transcription factors (Oct3/4, Sox2, c-Myc, and Klf4), and named these “induced PSCs” (iPSCs).⁷ In 2007, They also generated human iPSCs (hiPSCs) using this method⁸ and Yu et al modified this method using a different set of transcription factors (Oct3/4, Sox2, Nanog, and Lin28).⁹ iPSC-based therapy is not associated with ethical issues because somatic cells are used; accordingly, autologous transplantation is theoretically feasible. Because of these characteristics, iPSCs are expected to be applied to regenerative therapy and tissue engineering.

Reprogramming methodology has been rapidly progressing during this decade. Retroviral and lentiviral vectors, which can cause mutagenesis and persistent transgene expression, were first used to deliver transcription factors. As such, Sendai virus-based vectors,¹⁰ which do not cause host genome mutations and are diluted out over several passages, or non-viral reprogramming methods including plasmid,¹¹ mini-circle based vector,¹² or episomal vector¹³ transfection, have been used. Reprogramming efficiency has increased with various methodologies such as PiggyBac transposon vectors^14,15 and transcription factor modification.¹⁶

Cardiac Differentiation of PSCs

Differentiation protocols of PSC-derived CMs (PSC-CMs) are summarized in the Figure. Doetschman et al reported that ESCs cultured in high concentrations of fetal bovine serum (FBS) without mouse embryonic fibroblasts form 3D structures, named embryoid bodies (EBs).¹⁷ Kehat et al confirmed the spontaneous generation of CMs after replacing EBs on gelatin-coated plates and culturing with DMEM containing 20% FBS.¹⁸ However, this protocol is inefficient, resulting in only 1% CMs.

Figure.

Differentiation protocols of pluripotent stem cell-induced cardiomyocytes (PSC-CMs). AA, Activin A; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; CM, cardiomyocyte; EB, embryonic body; ESC, embryonic stem cell; hESC, human ESC; hiPSC, human induced pluripotent stem cell; mESC, mouse ESC; MG, Matrigel; VEGF, vascular endothelial growth factor.

The Mummery group was inspired by the role of the anterior endoderm in cardiogenesis, and developed the first directed differentiation method. They showed that P19 embryonal carcinoma cells differentiate into mesoderm-derived cells, including CMs, when grown in conditioned medium from END-2 cells, a visceral-endoderm-like derivative of P19s. Subsequently, they cocultured hESCs with END-2 cells and obtained 20–25% CMs.¹⁹

During fetal development, the heart originates from cardiac mesoderm, which is primarily regulated by 3 pathways, Nodal/Activin, bone morphogenetic protein-4 (BMP-4), and Wnt signaling.²⁰

Nodal/Activin

Nodal plays and essential role in gastrulation and germ layer formation.²⁰ In the PSC studies, Activin A was substituted by Nodal. When Activin A binds to the ALK4 receptor, activating Smad2/3 signaling, the primitive streak marker Brachyury is induced. With Brachyury expression, mesoderm formation is induced by low doses of Activin A, whereas endoderm is induced by high doses.²⁰

BMP-4

BMP-4 is also essential for gastrulation and germ layer establishment. In the presence of BMP-4, Brachyury and the mesodermal marker flk1 are induced and neuronal development is prevented.²⁰

Wnt Signaling

Wnt signaling plays a pivotal role in embryogenesis and patterning. When Wnt binds to a receptor complex consisting of a fizzled receptor and low-density lipoprotein receptor-related family (LRP) co-receptor, β-catenin is stabilized and translocates to the nucleus. Wnt3 can be detected before the onset of gastrulation, and if absent, the primitive streak, mesoderm, and node fail to develop.²¹ During cardiac differentiation, Wnt/β-catenin signaling has a biphasic effect; cardiac differentiation is enhanced before gastrulation and inhibited afterwards. The Wnt/β-catenin inhibitor Dkk1 is activated in the precardiac mesoderm and might promote cardiac differentiation by inhibiting Wnt/β-catenin signaling.²²

The first PSC-CM protocol using aforementioned factors was reported by Laflamme et al.²³ They used hESCs cultured on Matrigel-coated dishes. When cells reached almost 100% confluence (day 0), the medium was replaced with RPMI 1640 medium supplemented with B27 serum substitute (RPMI/B27) without insulin (RPMI/B27-ins) and Activin A was added. After 24 h, the medium was replaced with RPMI/B27-ins supplemented with BMP-4 for 4 days without medium change, which was followed by medium replacement with RPMI/B27 containing insulin every other day; >30% cardiac troponin T-positive hESC-CMs were obtained using this protocol.

Zhang et al modified this approach for their “matrix sandwich protocol”.²⁴ Extracellular matrix is critical for fetal development-associated processes such as cell adhesion and migration, coordinating growth factors and cytokines, and adjusting mechanical signaling. Matrigel was added to the medium on days −1 and 0, resulting in increased purity (40–90%) and maturation of hPSC-CMs 30 days after differentiation, with distinct subtypes of CMs including nodal, atrial, and ventricular.

Yang et al modulated Wnt signaling in addition to adding BMP-4 and Activin A.²⁵ Mouse and human ESC-derived EBs, formed via a feeder-depletion process, were cultured in serum-free defined medium (StemPro34, Invitrogen) supplemented with BMP-4 for 24 h. BMP-4, Activin A, and basic fibroblast growth factor (bFGF) were added to mimic primitive streak and mesodermal development. After 3 days, EBs were exposed to Dkk1 and vascular endothelial growth factor (VEGF) for 2 days, and bFGF was added for an additional 2 days; ultimately, >50% CM purity was obtained.

Applying defined factors such as Activin A, BMP-4, and Dkk1 resulted in enhanced PSC to CM differentiation, when compared with non-directed EB-based methods.¹⁹ However, these protocols are associated with limitations, namely, large variations in differentiation efficiency between cell lines and high costs.

Paige et al showed that Wnt/β-catenin signaling modulates Activin A/BMP-4-mediated cardiac differentiation and has a biphasic effect on hESCs.²⁶ Lian et al addressed this by using 2 small molecules, CHIR99021, a glycogen synthase kinase 3 inhibitor that activates Wnt signaling and promoting mesoderm induction, and IWP-2/IWP-4, a Wnt inhibitor.²⁷ hiPSCs, in monolayer culture on Matrigel- or Synthemax-coated dishes, were treated with RPMI/B27-ins containing CHIR99021 for 24 h. On days 3–5, IWP-2 or IWP-4 was added, and >82% hiPSC-CM purity was obtained. Burridge et al used a similar method and reported >90% purity.²⁸

Rodriguez et al combined these methods and added to RPMI/B27-ins medium CHIR99021 before differentiation, Activin A and BMP-4 from days 1–3, and XAV939, a Wnt inhibitor, after the BMP-4 phase.²⁹

hPSC-CMs generated using these methods are generally a mixture of atrial-, ventricular-, and nodal-like cells.³⁰ Obtaining specific CM subtypes is important for regenerative therapy and drug screening. Regarding specific differentiation, substantial work has been performed. For instance, retinoic acid signaling has been shown to mediate the direct differentiation of atrial and ventricular CMs,^31–33 and nodal CMs seemed to result from Activin/nodal/TGF and FGF inhibition in addition to retinoic acid activation.³⁴

Preclinical Transplantation of PSC-Derived CMs

Preclinical studies using PSC-CMs for cardiac repair are summarized in the Table. The first preclinical trial of mESC-CM-based therapy was performed in 1996.³⁵ mESC-CMs were injected into uninjured mouse ventricular myocardia, and grafts were monitored for up to 7 weeks. The Xiao group extended these data, demonstrating that mESC-CMs implanted into infarcted rat hearts survived for 32 weeks and improved cardiac function.^36,37

Table. Preclinical Studies of Pluripotent Stem Cell-Induced Cardiomyocyte (hPSC-CM) Transplantation

Author	Year	Animal	Cell type (cell no.)	MI (induction method)	Delivery (timing)	Duration post Tx (weeks)	Results
							Cardiac function	Tumor	Other
Min36	2002	Rat	Beating mESCs (1×104)	+ (ligation)	Injection (immediately after MI)	6	Improved	–
Laflamme38	2005	Rat	hESC-CMs (5×106)	−	Injection	4	ND	–	Graft size improved by heat-shock pretreatment
van Laake41	2007	Mouse	hESC-CMs (1×106)	+ (ligation)	Injection (immediately after MI)	12	No change	–	Cardiac function improved at 4 weeks after Tx
Dai40	2007	Rat	hESC-CMs (2×106)	+ (reperfusion)	Injection (immediately after MI)	4	ND	–
Caspi39	2007	Rat	hESC-CMs (1.5×106)	+ (ligation)	Injection (7–10 days after MI)	4	Improved	–
Laflamme23	2007	Rat	hESC-CMs (5×106)	+ (reperfusion)	Injection (4 days after MI)	4	Improved	–	Graft survival improved by using pro-survival cocktail
Mauritz42	2011	Mouse	miPSC-CPCs (5×105)	+ (ligation)	Injection (immediately after MI)	2	Improved	–	Flk-1-positive CPCs were implanted
Kawamura83	2012	Pig	hiPSC-CMs (2.5×107)	+ (ligation)	Scaffold free patch (28 days after MI)	8	Improved	–	Almost no graft survived at last Paracrine effect improved cardiac function
Xiong55	2013	Pig	hiPSC-ECs, vSMCs (2×106 each)	+ (reperfusion)	Cell loaded patch (immediately after MI)	4	Improved	–	No arrhythmia
Shiba50	2014	Guinea pig	hESC-CMs (8×107)	+ (cryoinjury)	Injection (28 days after MI)	4	No change	–	Arrhythmia was observed after Tx Host-graft electrical coupling was proved
Chong51	2014	Monkey	hESC-CMs (1×109)	+ (reperfusion)	Injection (14 days after MI)	12	No change	–	Arrhythmia was observed after Tx Host-graft electrical coupling was proved
Ye54	2014	Pig	hiPSC-CMs, ECs, vSMCs (3.5–4×105 each)	+ (ligation)	Injection with IGF patch (7 days after MI)	4	Improved	–	No arrhythmia
Shiba52	2016	Monkey	Monkey iPSC-CMs (4×108)	+ (reperfusion)	Injection (28 days after MI)	12	Improved	–	Arrhythmia was observed after Tx Host-graft electrical coupling was proved

CMs, cardiomyocytes; ECs, endothelial cells; ESC, embryonic stem cell; mESC, mouse ESC; hESC, human ESC; iPSC, induced pluripotent stem cell; hiPSC, human iPSC; MI, myocardial infarction; Tx, tranplantation; vSMCs, vascular smooth muscle cells.

Early transplantation studies using human ESC-CMs (hESC-CMs) used uninjured heart models; Laflamme et al injected “heat shock” pretreated hESC-CMs, which sufficiently formed intracardiac grafts.³⁸ Next, myocardial infarction (MI) was induced in mice and rats by various methods, including ligation or occlusion of the coronary artery with/without reperfusion and cryoinjury; implantation of hESC-CMs was performed 5 min to 4 weeks post-MI. Although most reports showed engraftment and improved cardiac function, the observation periods were within 4 weeks.^23,39,40 van Laake et al showed significant improvement in cardiac function at 4 weeks post-transplantation, but at 12 weeks there was no difference in cardiac function and small graft sizes were observed.⁴¹

Preclinical studies using iPS cell-derived CMs (hiPSC-CMs) have also been performed.^42–48 Most studies using ES- and iPS cell-derived hPSC-CMs have provided proof-of-concept, including graft survival and mechanical improvement of injured hearts. However, until recently, it was unknown if the mechanical benefits of transplantation were directly attributable to the contractile properties of transplanted PSC-CMs. A fundamental question was whether hPSC-CMs could couple electrically with host CMs. Our group created a hESC line expressing the fluorescent Ca indicator GCaMP3 for CM differentiation. These cells synchronously flashed green with each contraction, allowing for the identification of graft contraction in vivo. When we transplanted GCaMP3-expressing hESC-CMs into injured guinea pig hearts, most grafts coupled electromechanically with the host myocardium,^49,50 but some contracted independently. Chong et al transplanted GCaMP-expressing hESC-CMs into infarcted monkey hearts and found that all grafts coupled and contracted synchronously with the host myocardium.⁵¹ Further, we transplanted monkey iPS cell-derived CMs expressing a brighter Ca indicator, G-CaMP7.09, into infarcted monkey hearts and found electrical coupling; however, electrical conduction was delayed when the heart rate was increased by electrical stimulation.⁵² This slower propagation likely reflects insufficient gap junction development in the graft.

Another issue is whether PSC-CM transplantation affects ventricular arrhythmia incidence.^49–55 Roell et al showed that the expression of the gap-junction protein, connexin 43, in either embryonic CMs or skeletal myoblasts suppressed ventricular arrhythmia in mice.⁵³ Similarly, we showed that ventricular arrhythmia is suppressed by hESC-CM transplantation in a guinea pig model of acute heart injury,⁴⁹ whereas the incidence was unchanged in chronically injured hearts.⁵⁰ In contrast, Chong et al observed ventricular tachycardia after hESC-CM transplantation.⁵¹ Subsequently, our group showed that iPSC-CM transplantation significantly increased ventricular tachycardia incidence in a monkey model of acute MI.^51,52 Inconsistent results are most likely because of differences in heart sizes and beating rates. In small animals, electrical reentry is less likely to occur because of the small heart size.⁵⁶ In addition, small animals have faster heart rates than humans, which could mask ventricular arrhythmia.

There have been several approaches to delivering PSC-CMs to infarcted myocardium. The most straightforward approach is direct intramyocardial injection. When cells are injected into the infarcted myocardium, most of them disappear immediately. To improve hESC-CM engraftment efficacy, Laflamme et al developed a new cocktail of pro-survival factors, named the “pro-survival cocktail”.²³ This consisted of Matrigel to prevent anoikis, pinacidil to induce ischemic conditions, IGF-1 to activate Akt, the caspase inhibitors ZVAD-fmk and Bcl-XL, and cyclosporine A to block mitochondria-related death. The pro-survival cocktail significantly improved graft survival at 4 weeks post-transplantation, whereas individual factors had no effect. Subsequently, we and others have reported successful engraftment of transplanted cells using various animal models.^49,51,52,57 Of note, the pro-survival cocktail contains Matrigel, an extracellular matrix extracted from EHS mouse sarcoma cells, and a possible safety concern. As a substitute, Cheng et al⁵⁸ injected human cardiosphere-derived cells suspended in hydrogel-based hyaluronic acid into the infarct border zone of injured mouse hearts. Implanted cells survived for 3 weeks and improved cardiac function.

Hypoxia at the injection site could also prevent implanted cell survival and engraftment.^59–61 Activated heme oxygenase-1 (HO-1) reduces infarct size, myocyte apoptosis, and myocardium remodeling.⁶² Luo et al pretreated hESC-CMs with cobalt protoporphyrin (CoPP), a pharmacologic inducer of HO-1, which resulted in larger grafts and improved cardiac function at 8 weeks post-implantation.^63,64 Further studies are required to find xeno-free, chemically defined factors that promote graft survival.

Another approach to deliver PSC-CMs is epicardial transplantation.^65–68 Engelmayr et al showed that honeycomb scaffolds promote the alignment of grafts to neonatal rat CMs and mechanical and electrical properties in vitro.⁶⁵ Miyagawa et al demonstrated that CM sheets, which were constructed using neonatal rat cardiac cells on poly(N-isopropylacrylamide)-grafted polystyrene dishes, integrated with injured rat hearts to improve cardiac function.⁶⁶ Caspi et al reported a 3D vascularized cardiac tissue using hESC-derived CMs, ECs, and embryonic fibroblasts; the 3D-tissue construct developed early cardiac tissue-like structures and functions.⁶⁷ Bioartificial whole rat hearts that were decellularized and loaded with rat neonatal CMs were reported by Ott and colleagues.⁶⁸ These hearts had macroscopic structure and generated pump functions after electrical stimulation.

Remaining Hurdles

In 2015, the first clinical trial of cardiac regenerative therapy using hESC-derived cardiac progenitor cells (hESC-CPC) was reported.⁴ A patient with Class III HF caused by MI, refractory to conventional treatment, underwent coronary bypass surgery with hESC-CPC-infiltrated fibrin patch implantation. After 3 months, symptoms and cardiac function improved, and contraction at the implantation site was evident. Although an implantable cardioverter defibrillator was used, there were no subsequent arrhythmias or adverse events. Accordingly, cardiac regenerative therapy using PSC-derived cells is associated with high expectations. However, some important hurdles (as follows) must be addressed.

Tumor Formation

Because cardiac regeneration requires many PSC-CMs, tumorigenesis is a concern. There have been several reports of teratoma formation after undifferentiated PSC transplantation in animal models. Ahmed et al injected miPSCs into injured mouse hearts, and found a myocardial intramural teratoma rate of 37.5%.⁶⁹ Currently, there is no evidence that hPSC-CM transplantation results in teratoma formation, based on observations using several animal models (using 10⁶ CMs in mice, 10⁷ in rats, 10⁸ in guinea pigs, and 10⁹ in monkeys). However, in humans, with longer follow-up periods and the requirement for more CMs, further attention is required. It is important to establish large-scale cardiac differentiation protocols with consistently high purity. Effective purification methods will facilitate this. An initial purification method was demonstrated by Klug et al,³⁵ wherein mESCs were transfected with a neomycin resistance transgene activated by α-myosin heavy chain. CMs derived from these ESCs were selected, resulting in >99.6% CMs. Huber et al⁷⁰ used another transgenic hESC line expressing a reporter gene (eGFP) under the control of the human myosin light chain-2v (MLC2v) promoter. They identified >95% eGFP-positive cells during differentiation. Human CM-specific cell surface markers are also useful for purification. Antibodies against SIRPA (signal regulatory protein α)⁷¹ and VCAM1 (vascular cell adhesion molecule 1)⁷² resulted in >95% PSC-CM purity. However, these cell selection methods have disadvantages for clinical application, including insufficient purity, genotoxicity, and the requirement of FACS and/or antibodies. To overcome this, new purification methods utilizing the unique metabolic properties of PSCs and CMs have been developed.^73,74 When PSC-derived cells were cultured with glucose-depleted culture medium containing abundant lactate, only PSC-CMs survived and CM purity was almost 99%.⁷³ Recently, this method was improved by removing glutamine from the culture medium.⁷⁴

Arrhythmia

There have been conflicting reports as to whether PSC-CM transplantation alters the incidence of ventricular arrhythmias.⁷⁵ Cardiac grafts could possibly contribute to all 3 fundamental arrhythmia mechanisms, namely automaticity,¹⁸ reentry,³⁹ and triggered activity.⁷⁶ As mentioned before, conflicting results have been obtained regarding whether these cardiac grafts cause arrhythmias.^49–53 However, it remains a potential issue to be further explored. To control post-transplantation arrhythmia, more studies are required using large-animal models, and ideally with allogeneic transplantation, in order to match the CM beating rate between the host and graft animals.

Immunogenicity

The immune response against implanted PSC-derived cells or organs is a critical obstacle. Pharmacological immunosuppression is one solution to control immune reactions after implantation, but this strategy increases the risk of infection and malignancy. Human leukocyte antigen (HLA; the major histocompatibility complex (MHC) in humans) is known to play an essential role in graft rejection, and theoretically, acute graft rejection could be inhibited by HLA-matched transplantation. Lu et al utilized the HLA class I-knockdown method through β 2-microglobulin disruption and confirmed the hypoimmunogenicity of HLA class I-deficient hESCs.⁷⁷ Although this approach could provide a “universal donor” human PSC line, HLA class I also inhibits the activation of natural killer (NK) cells,^78,79 suggesting that this approach might induce NK cell-related graft rejection. Our group transplanted MHC-matched monkey PSC-CMs into injured monkey hearts with mild immunosuppression and observed long-term graft survival without acute rejection.⁵² Currently, several groups are generating iPS cells from individuals with homozygous HLA alleles to provide off-the-shelf iPS cells for which HLA will be matched with multiple recipients.^80,81 In Japan, 50 donor iPSC lines are needed to match >90% of the Japanese population, requiring 24,000 donors for screening. HLA-matched iPSC banking will also reduce the cost of PSC-based clinical therapies.⁸²

The ultimate solution to control graft rejection is autologous transplantation; however, this is associated with substantial challenges. First, the production of many quality-controlled, patient-specific CMs would be challenging. Clinical-grade CMs should be pure, karyotypically normal, genetically intact, and produced in a GMP-grade facility. Second, many high-quality CMs need to be affordably generated; it is not clear if this is commercially viable. Finally, although proof-of-concept has been established for PSC-CMs using acute or subacute models of MI, it would not be feasible to prepare patient-specific PSC-CMs in this situation. Thus, new methodologies using chronic MI models are required.

Conclusions

PSCs have prompted novel cell-based heart regenerative therapies. Improvements in the differentiation and purification of PSC-CMs will warrant larger-scale production of clinical-grade CMs. Preclinical studies, initially in small animals and recently in swine and non-human primate models, have suggested the effectiveness of PSC-based therapy. Certainly, some obstacles preclude safe clinical use, such as tumor formation, arrhythmias and immune rejection. Advanced preclinical studies will expedite the clinical application of PSC-based heart regeneration.

Acknowledgments

This work was partially supported by JSPS KAKENHI Grant Number JP17K16589.

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