Cardiac Cell Cycle Activation as a Strategy to Improve iPSC-derived Cardiomyocyte Therapy

Over the past 2 decades, scientists and clinicians have strived to leverage stem cell therapy as a treatment for heart failure. However, a myriad of clinical trials investigating the potential of adult stem cells to restore damaged myocardium showed inconsistent effectiveness.¹ A subsequent paradigm shift emerged with evidence that the benefits of such treatment, if any, were derived from cardioprotective paracrine factors released by stem cells as opposed to direct myocardial regeneration. As a result, researchers have attempted to regenerate the lost myocardium by delivering cardiac cells to the sites of myocardial damage.

With recent advances in induced pluripotent stem cell (iPSC) technology, cardiomyocytes can be efficiently and reproducibly generated from iPSCs.² The use of iPSCs in cell therapy is particularly attractive for the following reasons. First, iPSCs have extensive self-renewal and differentiation potential, which enables generation of a large number of iPSC-derived cardiomyocytes (iPSC-CMs) required for successful cell therapy. Second, this self-renewal property of iPSCs makes them amenable to desired genetic modification prior to differentiation.³ Third, iPSC-CMs exhibit properties of beating cardiomyocytes and have been shown to engraft in the host myocardium and electromechanically couple to neighboring native cardiomyocytes.⁴ Finally, as iPSCs are derived from patients’ own somatic cells, the use of autologous iPSC-CMs may help circumvent potential immunogenicity of the transplanted cells.⁵ Despite these beneficial characteristics of iPSCs, poor survival and engraftment of the transplanted cells remain major obstacles for efficient myocardial regeneration.¹

In this issue of Circulation Research,⁶ Zhu and colleagues sought to test whether the use of genetically engineered, cell cycle-activated iPSC-CMs could improve cardiac cell therapy. Recognizing that only a very small fraction of transplanted iPSC-CMs survive and engraft into the myocardium, they hypothesized that if iPSC-CMs could retain proliferative capacity, then even a small initial population of engrafted cells might proliferate and significantly restore myocardium. To investigate this hypothesis, Zhu et al. used a lentivirus to overexpress cyclin D2 (CCND2), an important cell cycle regulator of the G1-to-S phase transition, in iPSCs under the regulation of the α-myosin heavy chain promoter. Following differentiation, significantly more CCND2-overexpressing iPSC-CMs (iPSC-CCND2^OECMs) exhibited proliferative markers compared to wild-type CCND2 iPSC-CMs (iPSC-CCND2^WTCMs). In mouse myocardial infarction models, Zhu et al. observed significantly improved left ventricular systolic function and decreased scar size following the treatment of iPSC-CCND2^OECMs compared to iPSC-CCND2^WTCMs and sham injury groups. Consistent with this observation, bioluminescence imaging of the hearts showed increased engraftment of the cells in the iPSC-CCND2^OECMs treatment group. Immunostaining of the heart sections with human specific Troponin T and human nuclear antigen verified that the majority of these engrafted cells (~96%) were cardiomyocytes.

Cardiac Cell Cycle Activation

This work by Zhu et al. highlights the feasibility of resetting the regenerative potential of iPSC-CMs by inducing cell cycle activity to improve cell therapy.⁶ Human and animal studies have repeatedly shown that embryonic/fetal cardiomyocytes can proliferate. However, as they mature, they lose their ability to multiply and eventually undergo cell cycle arrest (post-mitosis).⁷ Similarly, differentiated iPSC-CMs also become post-mitotic and lose their ability to replicate as they mature. A potential solution for cardiac regeneration is to alter the cell cycle of cardiomyocytes to remain in the active replication cycle (Figure 1). To this end, extensive research has been conducted toward understanding how the cardiomyocyte cell cycle is regulated during cardiac development. Previous research found that while embryonic cardiomyocytes have a high expression of positive cell cycle regulators such as cyclins, cyclin dependent kinases, and proto-oncogenes, adult cardiomyocytes have a very low expression of these proteins.⁷ Thus, it has been postulated that direct overexpression of relevant cell cycle regulators may allow sustained myocardial proliferation. Among the regulators, D-type cyclins appear particularly promising in modulating myocardial proliferation. D-type cyclins have been shown to be highly expressed during embryogenesis, but their levels dramatically decrease after birth, reflecting corresponding cardiac cell cycle activity. While mice lacking D-type cyclins had progressive heart defects during embryogenesis that led to their premature death,⁸ CCND2 overexpression in mice resulted in significantly enhanced cell cycle activity with decreased infarct size following myocardial injury.⁹ Motivated by these studies, Zhu et al. created CCND2-overexpressing iPSC-CMs and demonstrated increased and sustained proliferative activity of the transplanted iPSC-CMs resulting in extensive myocardial regeneration in vivo.⁶

Figure 1. Strategies to activate cardiac cell cycle activity for myocardial regeneration.

Positive cell cycle regulators such as cyclin-CDK complexes facilitate cell cycle entry and progression. Overexpression (OE*) of cyclins have been shown to promote cell cycle activity. Treatments with various hormones, growth factors, microRNAs, or small molecules have also been used to 1) induce reentry into the cell cycle or 2) modulate activities of direct cell cycle regulators and their molecular correlates (e.g., tumor suppressor RB) along the various points of the cell cycle. BMP, bone morphogenetic protein; CDK, cyclin-dependent kinase; FGF, fibroblast growth factors; G-CSF, granulocyte colony stimulating factor; GSK3, glycogen synthase kinase 3; HASF, hypoxia-regulated Akt-mediated stem cell factor; IGF, insulin-like growth factor; miR, microRNA; RB, retinoblastoma; T3, triiodothyronine.

Another approach to promote cell cycle activity is by treating cardiac cells with hormones, growth factors, microRNAs, or small molecules to enhance cell cycle signaling or to induce reentry of differentiated myocytes (Figure 1).¹⁰ For example, treatment of embryonic stem cell (ESC)-derived CMs with insulin-like growth factor was shown to enhance their proliferation by activating the phosphoinositide 3- kinase/Akt signaling pathway.¹¹ Via a different pathway of JAK/STAT, treatment of iPSC-CMs and ESC-CMs with granulocyte colony-stimulating factor (GCSF) resulted in significant proliferation of the derived cardiomyocytes.¹² A number of microRNAs were also reported to enhance cardiac cell cycle activity when they were overexpressed or inhibited/knocked down in cardiomyocytes in vivo.¹⁰ More recently, using a high-throughput combinatorial screening approach, several small molecules enhancing iPSC-CM proliferation have also been identified.¹³

Significant research efforts have been made to discover genetically modifiable or druggable targets to enhance cardiac cell cycle activity. While promising, we still know relatively little about the mechanism underlying cardiac cell cycle regulation. Due to the complex molecular circuits involved in this process, it is difficult to fully understand how the treatment of the “pro-proliferative” molecules affect overall cell cycle activity and function. Rather than targeting upstream signaling pathways, overexpression of direct cell cycle regulators may provide a more reliable and consistent method to activate the cell cycle. In particular, with advances in genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, genetic engineering of iPSCs has become highly efficient, precise, and convenient.¹⁴ This ability to genetically modify iPSCs is particularly attractive because it does not require temporal and spatial regulation of drug treatments. The continued myocardial regeneration of iPSC-CMs observed by Zhu et al.⁶ also supports the durable effectiveness of genetically engineered cell cycle activation. While more research is needed to understand the long-term effects of CCND2 overexpression in iPSC-CMs, the approach by Zhu et al. to genetically reset cardiac cell cycle activity appears to be an attractive strategy for efficient myocardial regeneration.

Potential Hurdles for Safe and Effective Clinical Application

Prior to the use of cell cycle-activated iPSC-CMs for safe and effective clinical translation, multiple hurdles need to be addressed. First, the generation of iPSCs takes time (6-12 weeks) and remains costly, especially if any genetic modifications are needed. Second, the iPSC-CM therapy may increase the risk of developing potentially life-threatening arrhythmias. Shiba et al. recently reported allogeneic transplantation of iPSC-CMs in a non-human primate model using cynomolgus monkey (Macaca fascicularis).⁴ While they observed successful engraftment of the transplanted iPSC-CMs, all five recipient monkeys developed transient episodes of ventricular tachycardia. Currently, it is unknown whether the proliferation of cell cycle-activated iPSC-CMs increases the risk of arrhythmia, as small animal models do not accurately recapitulate the underlying arrhythmia risk. Thus, further investigation of their arrhythmogenicity is essential before commencing safe clinical translation. Lastly, transplantation of iPSC-CMs may carry a risk of tumorigenicity.¹⁵ This risk increases with the extent of incompletely differentiated cells remaining in culture prior to transplantation.¹⁵ With advances in our differentiation and maturation strategies, we are now able to generate >90% pure, functional iPSC-CMs.² Yet, whether cell cycle-activated iPSC-CMs have increased oncogenic potential remains unknown and requires further studies. Additionally, tumorigenicity may be increased in the presence of culture-acquired mutations,¹⁶ especially when iPSCs undergo genetic modification such as with lentivirus-mediated transduction. While it is encouraging that Zhu et al. did not observe tumor formation at 4 weeks following transplantation, this does not rule out potential long-term tumorigenic risk in humans. Thus, extended monitoring in pre-clinical stage will be crucial to demonstrate safety.

Conclusion

The study by Zhu et al.⁶ is a valuable addition to the growing body of literature investigating cell cycle activation as a strategy to improve iPSC-CM regeneration. By genetically engineering iPSCs to overexpress an important cell cycle regulator CCND2, Zhu et al. showed significantly increased and sustained proliferation of the differentiated iPSC-CMs. Transplantation of these cell cycle-activated iPSC-CMs resulted in extensive myocardial regeneration and subsequent improved cardiac function following myocardial injury in vivo. While more studies are warranted to address potential risks of arrhythmogenicity and tumorigenicity, CCND2-overexpressing iPSC-CMs appear to be a promising solution to improve our current state of cardiac cell therapy.