Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes
Introduction
Heart disease is the leading cause of death in developed countries, accounting for over 36% of all deaths in the United States [1], yet the mechanistic study of human heart disease beginning at the cellular level has been limited by the lack of suitable human cardiomyocyte models. However, thanks to recent revolutionary advances in cellular reprogramming, and directed differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocytes (iPSC-CMs), a number of in vitro models of healthy and diseased human cardiac tissues have been developed [2], [3]. Despite these advances, an important concern regarding the use of iPSC-CMs is their functional immaturity relative to primary cardiomyocytes. For instance, early studies indicated that cardiomyocytes generated in vitro from pluripotent stem cells exhibit fetal phenotypes with respect to their physiological performance [4], [5]. Several recent studies have addressed maturation of iPSC-CMs or embryonic stem cell-derived cardiomyocytes (ESC-CMs) [6], [7], [8], [9], [10], but the majority were limited to calcium handling and electrophysiological evaluation. Thus, considerable unmet needs remain for the adequate study of in vitro maturation of iPSC-CMs, particularly at the cell and molecular levels, and factors that modulate it. One such factor influencing cell maturation, including cardiomyocytes, is the tissue microenvironment. In particular, cell-substratum interaction is essential for proper development and maintenance of tissue architecture and function. In many complex organisms, the extracellular matrix (ECM) plays a critical role in cardiomyocyte development, but the full mechanism of its impact remains unknown due to the ECM's heterogeneity in both composition and structural orientation. Yet, despite considerable progress being made to engineer niches that control cellular responses through purpose-specific biomaterial designs (e.g., surface patterning, biomolecule addition) that would encompass some of the native ECM properties, the direct effects of characteristic biochemical and biophysical properties of unmodified materials alone have largely been underexplored. To address the need, we employed a library of copolymer scaffolds with varying physicochemical properties as culture substrates [11]. The copolymer library contained different mole percentages of three components: hydrophilic poly(ethylene glycol) (PEG), hydrophobic poly(ε-caprolacton) (PCL), and negatively-charged carboxylated-PCL (cPCL). Each copolymer subunit was selected for the specific properties it contributed to the resulting copolymer: PCL is a semi-crystalline, biodegradable, and hydrophobic, as well as being FDA-approved in medical devices [12]; PEG is a biocompatible, hydrophilic, and repellent polymer that reduces protein adsorption and cell attachment through steric exclusion [13], [14]; and cPCL facilitates cell attachment to the scaffold surface by providing a negative charge, effectively counteracting the PEG's repellant effects [14]. These combinatorial polymers were electrospun to make fiber mesh scaffolds that mimic ECM fiber structure and orientation, and subsequently used as test culture substrates.
Human iPSCs were differentiated into human iPSC-CMs through a directed differentiation protocol [15]. After 15–30 days of culture on each copolymer scaffold, we examined the effects of the copolymer composition on iPSC-CM phenotype by evaluating beating behavior, mitochondrial function and gene expression profiles. Our results indicate that certain combinatorial polymer scaffolds, especially a 4%PEG-96%PCL copolymer, promote the acquisition of several phenotypic features of mature ventricular myocytes including organized sarcomeres, abundant mitochondria, increased contractility and higher expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7, each of which have been associated with cardiac/ventricular maturation [16], [17], [18]. Moreover, 4%PEG-96%PCL was associated with enhanced expression of intermediate filament-associated proteins involved in transducing integrin-mediated mechanical signals to the myofilaments. These results suggest the synthetic biomaterial promoted cardiac maturation by mimicking some features of basement membrane-integrin/sarcolemma interactions seen in normal development. In summary, our study suggests that specific chemical compositions of synthetic extracellular substrates can exert profound influence on in vitro maturation of iPSC-CMs.
Section snippets
Reprogramming of human dermal fibroblasts and maintenance of human iPSCs
A human iPSC line (CC2) was generated from a healthy control subject using an episomal approach and validated, as we have previously described, following the work of Dr. Shinya Yamanaka [19], [20], [21]. Maintenance and culture of human iPSCs followed our established methods [19], [20], [21], [22]. Pluripotency was validated by PluriTest, a bioinformatics assay [23], using a teratoma-validated line as a positive control, and normal chromosomal karyotype was confirmed (Genetic Associates,
Generation of human iPSC-CMs
Validated human iPSCs from a healthy control subject, generated via an episomal-based reprogramming method developed by Shinya Yamanaka and colleagues, were used for all studies (Fig. S2a) [19], [20], [21]. Direct differentiation of these iPSCs cultured on Matrigel, outlined in Fig. S2b, successfully yielded iPSC-CMs. Visual inspection of the iPSC-CMs 7–8 days after differentiation showed individual beating cells, and 10–13 days after differentiation the iPSC-CMs formed beating sheets of cells (
Discussion
New breakthroughs in cellular reprogramming to generate human iPSCs, which are capable of differentiating into cardiomyocytes, offer extraordinary opportunities to study human heart diseases at the cellular level. However, despite their vast potential, iPSC-CMs generated with existing methods appear to be immature, resembling embryonic or fetal myocytes; thus, iPSC-CMs do not yet fully recapitulate characteristics of adult human myocytes. In this study, we succeeded in enhancing the maturation
Conclusion
Synthetic combinatorial polymer substrates were found to have distinct effects on in vitro maturation of human iPSC-CMs. Specifically, the 4%PEG-96%PCL scaffold enhanced iPSC-CM contractility and mitochondrial activity. This functional improvement was accompanied by the increased expression of MLC-2v, a marker of mature ventricular myocytes, and cTnI, a cardiac adult marker. While the precise mechanism is unknown, our results suggest that the chemical composition of a synthetic substrate
Acknowledgments
Authors thank Dr. Bjorn Knollmann's laboratory for serving healthy rabbit adult myocytes to us for mitochondrial functional assay. This research work was funded and supported by NIH R01HL104040, NIH UH2 TR000491, NIH R01ES010563, NIH R01ES016931, 1R01 NS078289, VA Merit 101BX000771, NSF CAREER CBET 1056046 and Clinical Translational Science Award No. UL1TR000445 from the National Center for Advancing Translational Sciences. Polymer characterization was conducted through the use of the core
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