Abstract
Cardiovascular diseases remain the leading causes of morbidity and mortality in the developed world. Cellular-based cardiac regenerative therapy serves as a potential approach to treating cardiovascular diseases. Although various cellular types have been tested, induced pluripotent stem cells (iPSCs) are regarded as a promising cell source for therapy. In this review, we will highlight some of the advances in generating iPSCs and differentiation to cardiac cells. We will also discuss the progress in modeling cardiovascular diseases using iPSCs-derived cardiac cells. As we continue to make progress in iPSC and cardiac differentiation technology, we will come closer to the application of cardiac regenerative medicine.
Introduction
Cardiovascular diseases remain a leading cause of morbidity and mortality in the developed world. Categorized by various ischemias and myopathies, cardiovascular diseases include heart failure and ultimately death. Many approaches have been taken to combat cardiovascular diseases such as preventative and palliative therapies. In the case of heart failure, currently the only strategy for providing a cure is heart transplantation. However, the use of this approach is limited due to the availability of donor hearts and immune rejection of the host.
As a potential treatment, cardiac regenerative cell therapy continues to serve as a hope for tackling cardiovascular diseases. Cell therapy for cardiac regeneration has evolved considerably during the last decade from skeletal myoblasts in the early stages to the use of mesenchymal and cardiac progenitor cells (CPCs). 1,2 One of the limitations of cell therapy is the generation of an unlimited number of cells capable of regenerating the damaged area or a whole organ. Embryonic stem cells (ESCs) were considered an important source of cells for organ repair due to their high proliferative capacity and the existence of established protocols to differentiate them into cell types from the 3 embryonic germ layers. The ethical problem of ESCs, primarily the destruction of embryos for their generation, was circumvented in 2006 with the appearance of induced pluripotent stem cells (iPSCs).3 Here, we will discuss the current state of utilizing iPSCs for cardiac regeneration and the potential avenues for personalized treatment of cardiovascular disease.
Discovery of iPSCs
In 2006, Takahashi and Yamanaka generated iPSCs that retained the pluripotent properties of ESCs, bypassing the ethical concerns associated with the use of ESCs in medical research. The investigators screened 24 candidate genes implicated in pluripotency and narrowed them down to 4 factors (Oct3/4, Klf4, Sox2, and c-Myc) sufficient to induce pluripotency in murine fibroblasts. 3 -5 A year later, several groups reprogrammed human somatic cells to generate human iPSCs using either Oct3/4, Klf4, Sox2, and c-Myc or Oct4, Sox2, Nanog, and Lin28, with 1 article suggesting that only Oct4 and Sox2 were essential for reprogramming human somatic cells. 6 -8 The authors note several limitations to their iPSCs, including the tumorigenic potential of c-Myc and Klf4 as well as subtle phenotypic differences between ESCs and iPSCs. 3,8 Also, microarray analyses showed that human iPSC global gene expression was similar but not identical to that of human ESCs. 6
Despite the perceived challenges, researchers became interested in potential medical applications of iPSCs, including disease modeling, drug screening, and autologous cell-based regenerative therapy. Importantly, the reports detailing generation of iPSCs from human somatic cells also demonstrated successful directed differentiation of human iPSCs toward neuronal and cardiac lineages using embryoid body (EB) culture, further suggesting that iPSC technology could provide a new source of cells for regenerative therapy. 6 -8
Advancements in iPSC Technology
The methods described involved viral integration of the transgenic factors into somatic cells. Researchers recognized a disadvantage of viral integration is the risk of tumorigenesis after reactivation of transgenes. Emphasis shifted toward generating transgene-free iPSCs that could potentially be used for cell-based regeneration (Table 1). Stadtfeld et al transiently infected mouse hepatocytes with adenoviruses expressing the 4 factors c-Myc, Klf4, Oct4, and Sox2 and obtained iPSCs with low efficiency (0.0001%-0.001%). 9 This method takes advantage of the low probability that adenoviruses will integrate into the host genome, and the authors report that their iPSCs retained pluripotency even after viral vectors have been diluted to undetectable levels in the cells. 9 Alternatively, plasmid vectors could be used to produce transgene-free iPSCs. Investigators describe an episomal-based strategy involving oriP/EBNA1 plasmid vectors derived from Epstein-Barr virus. 10 The episomal vectors expressed several combinations of reprogramming factors to induce iPSC generation in human foreskin fibroblasts. The authors showed through polymerase chain reaction (PCR) that plasmid vectors did not integrate into the host genome, also demonstrating through reverse transcriptase-PCR that iPSC lines did not express the transgenes. 10 Another group reported enhanced reprogramming efficiency (∼0.005%) in human adipose stem cells using a minicircle vector expressing a single reprogramming cassette containing Oct4, Sox2, Lin28, and Nanog. The authors obtained human iPSC colonies by days 14 to 16, and Southern blotting confirmed the lack of genomic integration of the minicircle vector in select colonies. 11
Advancements in iPSC Generation Technology.
Other strategies to generate transgene-free iPSCs involve transient expression of reprogramming factors followed by faithful removal (excision) of the transgenes. One such method makes use of the Cre/loxP excision technology. Sommer et al reported using a single engineered lentiviral “stem cell cassette (STEMCCA)” vector expressing the 4 reprogramming genes (Oct4, Klf4, Sox2, and c-Myc) flanked by loxP sites to induce pluripotency in mouse tail-tip fibroblasts. 12 The authors selected clones with a single integration of STEMCCA using Southern blot, then used an adenoviral vector to transiently express Cre-recombinase in these clones to excise the STEMCCA, ultimately reporting 96% excision efficiency as verified by genomic PCR. The authors even report improved differentiation potential (both in vitro and in vivo) of the iPSCs postexcision compared to preexcision. 12
A different excision approach uses piggyBac (PB) transposons. This technique involves transfecting somatic fibroblasts with PB-TET transposon plasmids expressing c-Myc, Klf4, Oct4, and Sox2 driven by doxycycline inducible promoter. Culture in doxycycline-containing media induces the formation of iPSC clones that maintain expression of endogenous pluripotency markers after excision of transgenes by transposase. PiggyBac iPSCs could represent another source of xeno-free cells in clinical applications. 13
Although viral approaches for delivery of the reprogramming factors are the most widely used approaches for generating iPSCs, the integration of viral transgenes into the host genome raises the concern and risk of tumorigenicity. In order to circumvent the possible complications of viral integrations in the genome, Fusaki et al developed a transgene-free approach for generating iPSCs using a vector based on the Sendai virus, a nonintegrating RNA virus. 14 Generating iPSCs by using the nonintegrating Sendai virus vectors could be a more practical and safer solution for reprogramming. 19,20 The Sendai virus approach has also been used to generate iPSCs from circulating T cells collected from the peripheral blood, which could serve as an even more clinically relevant approach for practically generating patient-specific iPSCs. 19
Researchers have also developed protein-based, transgene-free methods to create iPSCs. Zhou et al used Escherichia coli to express recombinant forms of the 4 reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc), each with a polyarginine (11R) domain at the C terminus. 15 Proponents of protein-based reprogramming methods state that the lack of genetic manipulation and DNA transfection potentially enhances the safety of iPSCs for use in regenerative therapy. 15
New and recent work strive to produce iPSCs with high efficiency to supply the large number of cells needed for cell-based regenerative therapy. Warren et al devised a series of messenger RNA (mRNA) modifications, including treatment with phosphatase and substitution with altered nucelobases to decrease host interferon signaling in order to reduce the host cell’s immune response to foreign mRNA. 16 The authors created synthetic mRNA for Oct4, Sox2, Klf4, c-Myc, and LIN28 with modifications using in vitro transcription. The modified mRNAs of the reprogramming factors were repeatedly delivered to several human somatic cell types and iPSC colonies appeared as early as 2 weeks. Using this method, the authors report a high induction efficiency of 4.4% under low-oxygen conditions. 16 In another approach, researchers report the successful reprogramming of mouse and human fibroblasts with a lentiviral vector-expressing miRNA cluster miR302/367 known to be involved in Oct4 and Sox2 signaling. Importantly, the authors report that induction of pluripotency using the miRNA cluster was twice as efficient as using pluripotency factors in both mouse and human cells. 17
In a recent breakthrough in improving the iPSC reprogramming efficiency, Rais et al were able to reach near 100% efficiency in both mouse and human cells by depleting a single factor, Mbd3. 18 Mbd3 is a major factor in the NuRD complex, which is known as a nucleosome remodeling and deacetylation repressor complex that is ubiquitously expressed in all somatic cells. Genetic depletion of Mbd3 in mouse and human fibroblasts led to a >95% reprogramming efficiency of fully characterized iPSCs that could also form chimeras. 18 This drastic improvement in reprogramming efficiency could help overcome the challenges of scalability in regard to potential clinical application of generating patient-specific iPSCs.
In brief, the field of iPSCs has advanced significantly since the first reports of somatic cells reprogrammed to an embryonic like state, and recent methods to more efficiently generate transgene-free iPSCs suggest the possibility of using iPSCs as a source for autologous regenerative therapy. An exhaustive study of current iPSC technology is beyond the scope of this review but interested readers are directed to relevant reviews on this topic. 21 -23
Cardiomyocyte Differentiation of PSCs
Choosing a specific cell source that will provide a beneficial effect on regeneration of damaged heart tissue is important for cardiac cellular therapy. Although the early stages of cellular therapy started with skeletal myoblasts, the field has shifted its focus toward cardiac cells such as cardiomyocytes (CMs) and cardiac progenitors. The ability to obtain large, scalable quantities of cardiac cells was made possible by the development of technologies to differentiate cardiac cells from pluripotent stem cells (PSC; Figure 1). The primary method of human cardiac differentiation was pioneered by the work of Kehat 2001, where they showed differentiation of CMs from human ESCs (hESCs) using the EB method. 24 They were able to generate spontaneously contracting CMs that retained physiologically relevant structural and functional properties. Many other groups have also been able to induce the differentiation of hESCs to CMs and have also characterized their electrophysiological properties. 25 -27
Patient-specific induced pluripotent stem cell therapy for cardiovascular disease.
Due to the ethical challenges of potentially using hESCs clinically, generating CMs from iPSCs has become of great interest. Spontaneous cardiac differentiation can be achieved through forming EBs with iPSCs. However, due to challenges in low differentiation efficiency and purity of the derived CMs using the EB system, techniques have been developed to enhance CM differentiation, namely, directing the differentiation of CMs using various factors on monolayer cultures. Combinations of growth factors such as activin A, bone morphogenetic protein 4 (Bmp4), fibroblast growth factor (FGF) 2, wingless-type mouse mammary tumor virus integration site family (Wnt) member 3A, and vascular endothelial growth factor (VEGF) have been shown to induce CM differentiation with increased efficiencies (Figure 1). For example, with sequential treatment of activin A and Bmp4, Laflamme et al were able to achieve a high yield of differentiated CMs >30% using high-density monolayer cultures compared to <1% CMs using the EB method. 28 Upon further enrichment, this allowed them to obtain cultures with 71% to 95% CMs. With higher efficiency and purity of CM differentiation, this would also enable a more practically scalable application for the use of the differentiated cells.
Growth factors, however, can be expensive and it may be challenging to accurately control their concentrations as they can degrade quickly. More recently, small molecule-based approaches are indicating to be highly efficient in CM generation from iPSCs. 29,30 For example, Lian et al developed a robust system to generate CMs from human iPSCs by modulating the canonical Wnt signaling pathway. 30 Wnt signaling is a key regulator of cardiogenesis, and the temporal activation and inhibition of the pathway are important for cardiac development. Lian et al developed a chemically defined, growth factor-free, and serum-free monolayer-based method of differentiating highly purified CMs from iPSCs, which would also bypass the purification steps of the EB methods. 30 In this method, iPSCs are pretreated with a glycogen synthase kinase 3 inhibitor early in the differentiation process, and this is combined with Wnt inhibition to achieve a high yield (0.8-1.3 million CMs per cm2) of highly purified (80%-98%) CMs in 14 days without cell sorting. A defined system that can efficiently generate highly purified CMs, without the concern of pluripotent cell contamination, is likely to be a potential source for cardiac regenerative cell therapies (Figure 1).
Similar to Lian et al, Minami et al were also able to use chemically defined conditions using small molecules to modulate cardiac differentiation to achieve high efficiency of differentiation. 31 Screening for small molecules, they discovered a potent small molecule KY0211, which leads to consistent and highly efficient cardiac differentiation in multiple human pluripotent stem cell (hPSC) lines. KY0211 is thought to promote cardiac differentiation by inhibiting Wnt signaling. In addition, temporal modulation with Wnt activators such as BIO and CHIR in the early phase of cardiac differentiation can produce up to 98% CMs. Compared to Lian et al’s differentiation method which primarily produces immature myosin light chain (MLC) 2v/MLC2a ventricular CMs (VCMs), Minami et al’s method mainly produces MLC2v-positive/MLC2a-negative more mature VCM and pacemaker cells. 31 This suggests that although both small molecules target Wnt signaling, the slight differences in differentiation protocols using these small molecules could lead to controlling the maturity and production of specific populations of CMs.
In addition to developing protocols for the efficient generation of CMs from iPSCs, it may also become important to assess the identity of the differentiated cells. Chamber-specific CMs would need to be derived efficiently because pacemaker, atrial, and ventricular myocytes have distinct functional properties that may contribute to arrhythmias when delivered into the infarcted area. 32 In the case of myocardial infarction, which commonly affects the left ventricle, generating a homogenous population of VCMs may help to regenerate the cardiac muscle. However, current differentiation methods yield heterogeneous populations of CMs with varying levels of VCM. In order to better understand the derivation of VCMs, Xu et al compared the derivation of VCMs from the standard fibroblast-derived iPSCs with VCM-derived iPSCs (ViPSCs). 32 Their study showed that ViPSCs lead to a significant increase in the differentiation of functional VCMs compared to fibroblast iPSCs. 32 Furthermore, it appears that the ViPSCs retain unique transcriptional and epigenetic signatures that serve as a “memory” of ViPSCs in their original ventricular identity. Further understanding how transcriptional and epigenetic modifications regulate the identity of the VCMs could help in improving the derivation of large numbers of chamber-specific VCMs for cellular replacement therapies. 33
Differentiation of iPSCs to CPCs
Differentiated CMs have been previously used as cell therapy for myocardial infarction in animal models. However, these CMs have poor cell engraftment and survival in the infarcted area. The harsh ischemic environment may not be suitable for the long-term survival and maintenance of the CMs due to lack of nutrients in the ischemic area. An alternative source of cells that could be more advantageous is the use of cardiac progenitor cells (CPCs). Cardiac progenitor cells are precursors to the terminally differentiated CMs and are marked by expression of various genes including Flk1, Isl1, Gata4, Tbx20, Nkx2.5, etc. It is thought that CPCs have the multipotent ability to differentiate into various cardiovascular cell types including CMs, smooth muscle cells (SMCs), and endothelial cells (ECs). The formation of SMCs and ECs would ideally help with angiogenesis and revascularization of the ischemic area, which would provide perfusion of blood to deliver nutrients, improving the survival of CPCs and host CMs.
Several types of cardiac progenitors have been derived from iPSCs and assessed for their therapeutic potential. One population of cardiac progenitors that expresses the surface marker fetal liver kinase (Flk) 1 was derived from mouse iPSCs by Mauritz et al. 34 Mouse iPSCs were differentiated using the EB method and subsequently sorted for Flk1-positive populations. Compared to the Flk1-negative and unsorted populations, the Flk1-positive progenitors were reported to show multipotent differentiation potential in vitro and in vivo. Furthermore, the Flk1-positive progenitors appeared to show modest improvements in cardiac function in the case of a mouse model of myocardial infarction. Although these results are promising, the authors were not able to see structural organization and alignment of the donor cells, neither the cross-striation pattern of the donor CMs, suggesting poor cell engraftment and donor–host coupling. In addition, the study assessed the donor cells for a short term of 2 weeks and long-term studies would be needed to determine the therapeutic potential.
With studies during mouse embryonic development and mouse ESC differentiation which suggest CMs, SMCs, and ECs arise from a common Flk1+ (kinase insert domain protein receptor [KDR]) cardiovascular progenitor, Yang et al 35 were able to determine that a common progenitor was also present during human cardiogenesis. The authors used an EB differentiation protocol and various factors including activin A, Bmp4, basic FGF, VEGF, and Dickkopf 1. Using this method, they were able to sort out a progenitor population using KDR and C-Kit as markers. A KDRlow/C-Kitneg population was defined as a distinct population with high expression of cardiac markers such as islet 1 (Isl1), Nkx2.5, and Tbx5 and was found to have the potential to differentiate into CMs, ECs, and SMC both in vitro and in vivo. This suggests that the KDRlow/C-Kitneg population could be representative of a multipotent cardiac progenitor population during human cardiac development. Several other groups have also used various methods to derive cardiac progenitors from iPSCs. 36 -38
During cardiac development, a multipotent cardiovascular progenitor marked by the expression of the transcription factor Isl1 is known to be involved in developing more than two-thirds of the heart. These Isl1+ CPCs can differentiate into CMs, SMCs, and ECs both in vitro and in vivo. 39 -41 Because of their known developmental origin in developing the heart and their multipotent differentiation potential, the Isl1+ progenitors can provide an advantageous source for cell therapy. Moretti et al were able to generate Isl1+ CPCs from both mouse and human iPSCs. 42 Using the EB method, the authors differentiated CPCs from Isl1-Cre/R26R-LacZ (Cre-recombinase expression driven by Isl 1 promoter) iPS clones, which allowed them to sort for the Isl1+ cells. The authors reported successful generation of iPSCs-derived Isl1+ CPCs and that they were able to differentiate them into CMs, SMCs, and ECs both in vitro and in vivo. However, the use of the Isl1 promoter in this strategy would isolate not only Isl1+ CPCs but also other cell types that express Isl1 transcription factor. Moreover, the potential of the Isl1+ CPCs as a source for cell therapy in the context of a myocardial infarction or disease has not yet been assessed.
Application of iPSC Technology for Cardiac Repair
As iPSC technology continues to improve, the potential for iPSCs in cardiac regenerative therapy continues to become highly attractive. Cardiac cells derived from iPSCs could provide a powerful tool for cellular therapy by either deriving patient-specific iPSCs or generating an expandable bank of iPSCs that can be used at any time. If the clinical safety challenges of iPSC technology can be overcome, iPSC-derived cardiac cells would be advantageous compared to current strategies because of (1) the less invasive nature of generating iPSCs from skin and blood cells and (2) the capability to generate large quantities of cardiac cells from iPSCs, which would be more scalable than isolating primary cells from the heart. Having an expandable and scalable population of cells is especially beneficial for cellular therapy in areas such as myocardial infarctions where the heart experiences a significant loss of cells and would require a replacement of the working myocardium. The iPSCs-derived cardiac cells would be a valuable and unlimited source of CMs for patient-specific cardiac repair.
Already, researchers have demonstrated the ability to derive patient-specific iPSCs capable of CM differentiation. Zwi-Dantsis et al created iPSC lines from the dermal fibroblasts of 2 male (51 and 61 years old) patients with ischemic cardiomyopathy having advanced heart failure. 43 The authors found that these heart failure iPSC lines differentiated into EBs to form CMs that coupled electrically and mechanically with rat ventricular myocytes in vitro and in vivo. Importantly, the authors report no significant difference between iPSCs derived from these patients with heart failure and control iPSCs derived from a healthy individual’s foreskin fibroblasts. The authors reason that patient-specific iPSCs could serve as a source of CMs for cell-replacement therapy because the heart failure in patients was acquired and does not adversely affect the genotypic qualities of the iPSCs. 43 It is important to note, however, that higher propensities for heart disease associated with family history will still translate to these patient-specific iPSCs.
With the advancements in iPSC generation and cardiac differentiation, assessing the therapeutic potential of iPSC-derived cells is important for translating cardiac regenerative therapies to the clinic. Several initial reports have tested the delivery of iPSC-derived CMs through direct injection into infarcted myocardium. Such as in Carpenter et al, the authors injected human cardiac progenitors to the peri-infarct region of a rat heart after coronary artery ligation and reperfusion. 44 Although the cardiac cells engrafted and differentiated into CMs and SMCs, the authors observed a modest trend in cardiac functional improvement.
Efficient long-term engraftment and survival of cells upon direct cell transplantation to the heart still remains one of the major challenges regarding cellular therapy. Many reports have shown that survival of transplanted stem cells remains low, often with >90% of cells disappearing within the first few days and <2% remaining 4 weeks after transplantation. 45 In order to improve the survival and engraftment of the transplanted cells, various bioengineering techniques such as scaffold-based and scaffold-free approaches and injectable matrices are also being tested as cell delivery methods for iPSC-derived cardiac cells.
Scaffold materials have been tested for use in cell delivery because it is thought to provide an adequate matrix that would sufficiently stabilize and anchor the cells in a particular region of the heart. The supporting scaffold matrix is thought to provide chemical and biological cues that would mimic the native microenvironment, thus supporting the engraftment of cardiac cells to a matrix and preventing programmed cell death due to loss of matrix attachment, also known as anoikis. Furthermore, the ideal goal of scaffold-based approaches would be to generate functional cardiac tissue of clinically relevant thickness (∼1 cm) that can provide a replacement for the damaged myocardium.
46
Various cardiac cells have been seeded on synthetic (polyglycolic acid, poly-
Although scaffolds can provide a controlled structural support for the cells, it is thought that scaffolds may limit the force that can be generated by the cells. Therefore, scaffold-free and scaffold-based approaches that allow the self-assembly of cells without scaffolds has been developed. One method is the use of cell sheets, which are monolayers of cells that potentiate cell–cell contacts and extracellular matrix secretion. Upon differentiation of iPSCs to cardiac cells using combinations of growth factors, cells are seeded upon the surface of temperature sensitive dishes. 47,48 Cell culture surfaces are covalently grafted with the temperature responsive polymer poly (N-isopropylacrylamide). To form cell sheets in temperature-sensitive plates, cells are typically cultured at 37°C until they are 100% confluent and then are detached from the plate as a cell sheet when they are cultured at 20°C. Several cell sheets can be layered one on top of each other to generate a multilayered cell sheet. Cell sheets are advantageous because they reinforce the cell–cell contact of donor cells before implantation and aid in the formation of a basal lamina that prevent anoikis.
Transplantation of cell sheets developed from iPSC-derived CMs has been tested in various models including a rat model for myocardial infarction. 48,49 Upon delivery of the cell sheets, the rats showed significant improvement in cardiac function and attenuation of ventricular modeling. They also observed neovascularization of the cell sheet graft. However, cells were not able to engraft in the long term and disappeared after 4 weeks upon transplantation.
Kawamura et al also developed cell sheets from human iPSC-derived CMs using the temperature-sensitive dishes and transplanted the sheets into a porcine model for myocardial infarction. 50 They observed enhancement in cardiac performance and attenuation of left ventricular modeling. Although the hiPSC-CM sheets were detectable after 8 weeks, there were few that survived for a longer term. Although different delivery systems have been tested for the therapeutic efficacy of iPSC-derived cardiac cells, many challenges remain, including long-term engraftment and survival of the transplanted cells.
More recently, Kawamura et al developed another strategy for improved engraftment and survival using hiPSC-CM cell sheets by transplanting the cell sheets with an omentum flap. 51 The omentum is part of the abdominal peritoneum and has historically been used in surgical revascularization due to its rich vasculature. The authors were able to surgically transplant the cell sheets with the omentum flap onto the myocardium. By tracking the cells using superparamagnetic iron oxide labeling and magnetic resonance imaging (MRI), they found that cell sheets with the omentum were able to survive for 8 weeks after transplantation into minipigs than those without the omentum flap. 78% of the cells survived with the omentum compared to 42% without. In addition to the improvement in long-term cellular survival, they also reported increased angiogenesis due to the omentum. Combining various bioengineering methods to improve the engraftment and vascularization of the transplanted cells may improve the survival and function of cells in cardiac repair.
Although scaffold-based and scaffold-free approaches would help in the structural support of transplanted cells, these methods would require surgery to implant the cardiac patches to the heart. As a more noninvasive approach, injectable materials have also been tested to serve as matrices to support the engraftment and survival of transplanted cells. Tested biomaterials include fibrin, collagen, Matrigel, alginate, self-assembling peptides, chitosan, and hydrogel. 52 Combinations of these materials are still being investigated for their use with iPSCs.
iPSC Technology for Cardiovascular Disease Modeling
In addition to cellular therapies for cardiac regeneration, iPSC technology can be utilized for modeling cardiovascular diseases. The advantage of using iPSCs is that it would enable modeling the disease as closely to the patient’s physiology and genetics as possible by isolating patient-specific fibroblasts. This could provide more insights into understanding various diseases since current animal models may not accurately encapsulate the comprehensive human disease phenotype. The first diseases to successfully be modeled using patient-specific iPSCs were the long-QT syndromes which are a series of disorders marked by a prolonged QT interval and a high susceptibility to sudden cardiac arrest due to ventricular tachyarrhythmia. 53 It is likely that long-QT syndrome was the earliest genetic cardiovascular disease to be modeled using iPSCs due to their clinical importance, identifiable genetic mutations, cell-autonomous pathology, and assayable electrophysiological properties. 53 Moretti et al were the first to model long-QT syndrome type 1 using CMs derived from patient-specific iPSCs and identified an autosomal dominant mutation in the KCNQ1 gene. 54 They were able to recapitulate the electrophysiological properties of the disease including the prolonged action potential in ventricular and atrial cells. Using patient-specific iPSCs serves as a more representative model of the pathogenesis of the disease compared to mouse models, which do not recapitulate the human disease phenotype. In addition, disease modeling using iPSCs would serve as a good platform for testing alternative approaches to developing candidate drugs as was seen by Moretti et al where treatment with β-blocker propranolol showed protective effects from induced tachyarrhythmia. Other long-QT syndromes have also been modeled using iPSCs, such as type 2 and type 8. 54 Already, several cardiovascular disease models have been generated using iPSCs. Diseases such as LEOPARD syndrome, Timothy syndrome, familial dilated cardiomyopathy, and other inherited arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia, have been modeled using in vitro iPSC-derived CM from patients. 54 -57 Using iPSC-derived CMs to model cardiovascular disease will help understand the mechanisms underlying the patient-specific characteristics of the disease.
Vascular diseases such as supravalvular aortic stenosis (SVAS) and Williams-Beuren Syndrome (WBS) have also been modeled using patient-specific iPSC-derived SMC; SVAS is an autosomal dominant disease marked by haploinsufficient mutations in the elastin (ELN) gene, leading to abnormal proliferation of vascular SMCs. 58 Supravalvular aortic stenosis can lead to blockage of arterial vessels and sudden cardiac death. Similarly, WBS, a neurodevelopmental disorder characterized by a distinctive “elfish” facial appearance, also exhibits SVAS with ELN haploinsufficiency, leading to vascular SMC proliferation and stenoses. In Ge et al, our group was able to generate patient-specific, iPSC-derived SMCs of 2 different ELN mutations. We were able to recapitulate the pathologies of the SVAS disease including, disorganization of smooth muscle α-actin filament bundles, increased rate of proliferation, and chemokine induced migration. Furthermore, we were able to rescue SM α-actin bundle formation with recombinant ELN or RhoA signaling and inhibition of hyperproliferation with attenuation of extracellular signal-regulated kinase 1/2 activity. 58 Induced pluripotent stem cell-SMCs could provide a useful platform for understanding the pathogenesis of SVAS and other vascular diseases and development of novel strategies for treatment.
In addition to using patient-specific iPSCs for modeling of cardiovascular diseases, differentiated patient-specific iPSCs can also be used for drug screening purposes. By using patient specific-derived CMs, this would allow for screening of the efficacy and toxicity of various drugs or combinations of drugs on the particular patient’s cells, thus enabling an even more personalized version of therapy to treat the disease. Patient-specific iPSC-derived CMs would serve as a closer model for simulating human conditions, compared to mouse models of large animals, which would be more informative, especially in preclinical testing of drugs.
Although the significant advances in cardiac differentiation of PSCs will lead to further improvements in cellular therapy, disease modeling, and drug screening for cardiovascular diseases, the maturation status of differentiated cardiac cells remains a challenge and may have important implications regarding the clinical applicability of the derived cells. Previous studies on CM differentiation of PSCs have consistently shown that although PSCs can differentiate into functional beating CMs, they adopt a more embryonic and fetal-like phenotype rather than adult-like CMs. 24,59 For example, PSC-derived CMs will exhibit spontaneous contractile activity, express disorganized sarcomeres, and have small, circular morphology compared with adult-like CMs that do not have spontaneous contractile activity and express organized sarcomere formation, with large, rectangular cell morphology. 60 In addition, differentiated CMs exhibit reduced strength of electrophysiological properties and calcium handling properties. 59,60
Because of the maturation state of iPSC-derived CMs using current differentiation protocols, there may be challenges in the immediate use of iPSC-CMs for clinical applications. Regarding cellular therapy, it is unknown whether iPSC-CMs can electrically couple with the host CMs and produce adequate contractile strength to match that of adult CMs. In regard to disease modeling, being able to control the maturation stages of CMs would be important for testing diseases of congenital nature versus later-adult stage diseases because drug efficacy and cytotoxicity may be different in early stage CMs compared to late-stage CMs.
Although there are challenges regarding the maturation state of iPSC-CMs, further understanding of the maturation process and development of novel differentiation methods may improve the maturity of iPSC-CMs for clinical use. Recently, Lundy et al did a comparative study on the maturation state of iPSC-CMs at an earlier stage of differentiation (20-40 days) compared to late-stage-derived CMs (80-120 days) to determine whether the iPSC-CMs could develop a more adult-like phenotype. 61 Late-stage iPSC-CMs were able to exhibit morphology, increased structural organization, and electrophysiological properties that were more similar to adult-CMs compared to early iPSC-CMs. This suggests that iPSC-CMs are able to adopt a highly mature phenotype under certain differentiation conditions. Developing methods on modulating the differentiation conditions, either through induction of growth factors or altering the 3D environment of the cells, may lead to derivation of even more adult-like CMs, which would enhance the use of PSC-CMs in cardiac regenerative strategies.
Recently, Nunes et al were able to develop a platform to alter the 3-dimensional (3D) culture microenvironment, leading to maturation of hPSC differentiated CMs. 62 They created a platform that would generate a microenvironment with architectural and electrical cues that would be conducive to maturation of both hESC and hiPSC-derived CMs. The novel platform, termed “biowires,” consists of seeding hPSC-derived CMs into a microfabricated polydimethylsiloxane (PDMS) channel around a sterile surgical suture in type I collagen gels. Seeded cells were able to remodel and contract the collagen gel matrix after the first week of culture. After a week of preculture, the authors initiated electrical stimulation of the biowires at varying frequencies. Compared to nonstimulated controls, the electrically stimulated hPSC-CMs on biowires showed an increase in rod-like CMs, enhanced sarcomeric organization, improved conduction and calcium handling properties, and functional electrophysiological properties indicative of mature CMs. 62 Further understanding how to manipulate the structural and electrical components of the 3D microenvironment will lead to improved maturation of hPSC differentiated CMs, enabling the use of hPSC-CMs for clinical applications.
Conclusions
As cardiovascular diseases continue to be a leading cause of morbidity and mortality in the developed world, it is essential to develop strategies to combat these diseases. iPSC technology has been heralded as the key for therapeutic regenerative strategies due to its potential of differentiating into any cell type of interest. Many advances have been made since the first establishment of iPSC technology although many challenges remain before application to the clinic. Induced pluripotent stem cells can be applied for cardiac regeneration as well for cellular therapies and disease modeling. Already, a significant progress has been made in assessing the therapeutic potential of cardiac cells derived from iPSCs. Furthermore, various diseases have been modeled using patient-specific iPSCs, which can give a more comprehensive and accurate understanding of cardiovascular diseases. Continuing to improve iPSC generation, cardiac differentiation, and clinically applicable delivery strategies will lead to the development of technologies for cardiac regeneration.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received the following financial support for the research, authorship, and/or publication of this article: NIH 1K02HL101990-01 and 1R01HL116705-01, Connecticut Stem Cell Research Program 12-SCB-YALE-06, 11SCA33 and 11SCB18, AHA 09SDG2080420, and Cardiovascular Medical Research and Education Fund (Y.Q.); Connecticut Stem Cell Research Program 13-SCA-Yale-32 (O.B); National Science Foundation Graduate Research Fellowship DGE-1122492 (C.S).