Future Treatment of Heart Failure Using Human iPSC-Derived Cardiomyocytes
Heart transplantation can drastically improve survival in patients with a failing heart; however, the shortage of donor hearts remains a serious problem with this treatment strategy and the successful clinical application of regenerative medicine is eagerly awaited. To this end, we developed a novel method to generate human induced pluripotent stem cells (iPSCs) from circulating human T lymphocytes using Sendai virus containing Yamanaka factors. To establish an efficient cardiac differentiation protocol, we then screened factors expressed in the future heart site of early mouse embryos and identified several growth factors and cytokines that can induce cardiomyocyte differentiation and proliferation. Subsequent transcriptome and metabolome analysis on undifferentiated stem cells and cardiomyocytes to devise a specific metabolic environment for cardiomyocyte selection revealed completely different mechanisms of glucose and lactate metabolism. Based on these findings, we succeeded in metabolically selecting cardiomyocytes using glucose-free and lactate-supplemented medium, with up to 99 % purity and no teratoma formation. Using our aggregation technique, we also showed that >90 % of the transplanted cardiomyocytes survived in the heart and showed physiological growth after transplantation. We expect that combining these techniques will achieve future heart regeneration.
KeywordsInduced pluripotent stem cell Purification Cardiomyocyte Transplantation Human
Heart disease remains a leading cause of death despite recent medical advances, and heart transplantation remains the ultimate treatment for severe heart failure. However, limited donor numbers remain an unsolved problem for transplantation therapy, and both patients and clinicians hold great hope for the future success of heart regenerative cell therapies as an alternative strategy . Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-renew infinitely and are potential mass production sources for therapeutic cardiomyocytes. In particular, human iPSCs have the huge advantage of avoiding immunological rejection after cell transplantation.
Human iPSCs were first generated from dermal fibroblasts by Takanashi and Yamanaka in 2007 , using a retroviral transduction system. Subsequently, the methodology for generating iPSCs has dramatically improved. We previously reported that integration-free iPSCs could be easily and rapidly generated from terminally differentiated circulating T lymphocytes in peripheral blood using Sendai virus . Our method makes it possible to generate iPSCs from any patients including children, girls, and the very elderly by blood sampling alone.
4.2 Cardiac Differentiation from Human iPSCs
Many approaches using ESCs have been investigated to induce cardiac differentiation. In general, the differentiation of ESCs into any cell lineage is based on the mechanism of normal early development . The visceral endoderm is known to play a key role in the differentiation of cardiac precursors that are present in the adjacent mesoderm during development, and Mummery et al.  previously reported that human ESCs effectively differentiate into cardiomyocytes when cocultured with mouse visceral endoderm-like (END-2) cells. In an attempt to improve cardiac differentiation efficiencies inexpensively and easily, Takahashi et al.  screened a chemical compound library approved by the United States Food and Drug Administration (FDA) and found that ascorbic acid efficiently induced cardiac differentiation. However, this chemical alone might be not enough to induce cardiac differentiation from iPSCs.
Several studies have shown that various combinations of heart development-related proteins including BMP, activin, Wnt, BMP inhibitor, and Wnt inhibitor induce cardiomyocytes from ESCs [7, 8, 9, 10]. We reported that the context-dependent differential action of BMPs in cardiomyocyte induction is explained by the local action of Noggin and other BMP inhibitors and, accordingly, developed a protocol to induce cardiac differentiation of mouse ESCs through transient administration of Noggin . However, to obtain hundreds of millions of cardiomyocytes, it is necessary to establish a cardiac differentiation method that is both efficient and cost-effective due to the many expensive recombinant protein factors used. To address this problem, Minami et al.  screened small-molecule compounds to identify those that significantly increase cardiac differentiation induction, and they revealed some inhibitors of canonical Wnt signaling as candidates. In addition, recently novel efficient protocols using small molecules and/or chemically defined media have been reported (Lian et al. Nat Protocols 2013;8:162–175, Burridge et al. Nat Methods. 2014;11:855–60).
Several studies then showed that induction techniques in ESCs could also be applied to iPSCs, although differentiation efficiencies were suggested to be inferior compared to ESCs. However, as such differentiation efficiencies vary greatly with different cell lines , further investigation is needed in the future. Furthermore, to efficiently obtain large quantities of cardiomyocytes inexpensively, it is necessary to continue refining efficient cardiac differentiation systems combined with the use of small-molecule compounds. However, despite improved cardiac differentiation efficiencies, it is inevitable that human PSC derivatives will contain not only cardiomyocytes but also undifferentiated stem cells and/or noncardiac cells because all PSCs cannot differentiate into cardiomyocytes. Therefore, to confirm safety after transplantation, it is necessary to remove noncardiac cells and undifferentiated stem cells that could cause tumors.
4.3 Nongenetic Methods for Purifying Cardiomyocytes
One of the biggest risks with in vitro-generated cardiomyocytes for clinical use is teratoma formation due to residual PSC contamination . Current procedures for eliminating such contamination and boosting cardiomyocyte enrichment involve genetic modification [14, 15] and nongenetic methods using a mitochondrial dye  or antibodies to specific cell-surface markers . However, none of these methods are ideal for the therapeutic application of PSC-derived cardiomyocytes due to insufficient stability, genotoxicity, and the use of fluorescence-activated cell sorting (FACS). To address this issue, we sought to purify cardiomyocytes efficiently and inexpensively, based on differences among cell-specific nutrition sources.
4.4 Transplantation of Human PSC-Derived Cardiomyocytes
Many studies have been conducted regarding cell transplantation therapies in animal models using human PSC-derived cardiomyocytes. The survival of dispersed transplanted cardiomyocytes is reportedly very low , and some ingenuity is required in the method of transplantation. Our group previously reported that transplanted cell survival was dramatically improved by transplantation after formation of cardiomyocyte aggregates , while Laflamme et al.  showed a similar effect using a prosurvival cocktail that inhibited apoptosis.
With regard to the effectiveness of cell transplantation, Laflamme et al.  also demonstrated that transplanted human ESC-derived cardiomyocytes could improve cardiac function after 4 weeks in a rat myocardial infarction model. In addition, Shiba et al.  demonstrated that transplanted human ESC-derived cardiomyocytes electrically coupled to the host cardiomyocytes and suppressed arrhythmias in a guinea pig myocardial infarction model. Furthermore, with respect to cell transplantation in large animals, Kawamura et al.  recently reported that cardiac cell sheets comprising purified human iPSC-derived cardiomyocytes generated using our method  improved cardiac function in a pig myocardial infarction model. Thus, efficacies have been achieved in cell transplantation therapies using human iPSC-derived cardiomyocytes, although many such studies showed only short-term effectiveness. Thus, careful evaluation of the efficacy and safety of human iPSC-derived cardiomyocytes in cell transplantation over the longer term must be ongoing.
4.5 Future Directions
The discovery and refinement of human iPSCs generation is expected to advance not only regenerative medicine but also drug discovery and analyses of genetic disorders using patient-specific iPSCs . The major and common problem remaining in this quest is securing sufficient numbers of mature and functional cardiomyocytes with high purity. To solve this problem, it is essential to develop a stable and efficient mass culture system and to establish a simple system to analyze the electrophysiological function of the generated cardiomyocytes (Fig. 4.1). Furthermore, the future realization of clinical applications using human iPSCs will necessitate a better understanding of the cell biology and techniques involved in tissue engineering generally. Only then will we be able to achieve long-term safety and efficacy in the heart failure models of large animals and finally realize human heart regenerative therapies.
Open Access This chapter is distributed under the terms of the Creative Commons Attribution-Noncommercial 2.5 License (http://creativecommons.org/licenses/by-nc/2.5/), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. The images or other third party material in this chapter are included in the work's Creative Commons license, unless indicated otherwise in the credit line; if such material is not included in the work's Creative Commons license and the respective action is not permitted by statutory regulation, users will need to obtain permission from the license holder to duplicate, adapt or reproduce the material.