Abstract
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl2 (n = 22). Patch-clamp techniques were used to record IKr and IK1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl2, at concentrations that selectively block IK1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible IK1 in 53% (20/38) of the cells studied, but presence of IKr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of IK1 in the majority of cells, but robust expression of IKr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on IKr due to the absence of IK1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.
Highlights
Like embryonic stem cells (ESCs), human induced pluripotent stem cells derived by reprogramming somatic cells can be cultivated in the pluripotency state or differentiated into somatic cell types including cardiomyocytes, neuronal cells and insulin producing beta cells of the islets of Langerhans [1,2]. hiPSCderived cardiomyocytes hold promise for use in a variety of applications including: 1) accelerated cost-effective drug development and safety pharmacology; 2) creation of in vitro models of genetic diseases to advance our knowledge of pathogenesis as well as to develop patient specific therapeutic modalities [2]; and 3) regenerative therapy
In an effort to assess the degree of homogeneity in the electrophysiologic profile of different batches of Embryoid bodies (EBs) derived from the same hiPSC line, we compared the spontaneous rate (BPM; beats per min) and action potential duration measured (APD) at 90% repolarization (APD90) as well as Bazett’s correction of APD90 [cAPD90-B]) in hiPSC-CM derived from all 103 beating clusters (BC) studied (Figure 2A and B; 17 batches of EBs) with those of a single batch in which we studied 27 BC (Figure 2C and D)
The results revealed no significant differences between the two groups suggesting that each batch of EBs-derived BC displays similar electrophysiologic characteristics
Summary
Like embryonic stem cells (ESCs), human induced pluripotent stem cells (hiPSCs) derived by reprogramming somatic cells can be cultivated in the pluripotency state or differentiated into somatic cell types including cardiomyocytes, neuronal cells and insulin producing beta cells of the islets of Langerhans [1,2]. hiPSCderived cardiomyocytes (hiPSC-CM) hold promise for use in a variety of applications including: 1) accelerated cost-effective drug development and safety pharmacology; 2) creation of in vitro models of genetic diseases to advance our knowledge of pathogenesis as well as to develop patient specific therapeutic modalities [2] (personalized medicine); and 3) regenerative therapy. HiPSCderived cardiomyocytes (hiPSC-CM) hold promise for use in a variety of applications including: 1) accelerated cost-effective drug development and safety pharmacology; 2) creation of in vitro models of genetic diseases to advance our knowledge of pathogenesis as well as to develop patient specific therapeutic modalities [2] (personalized medicine); and 3) regenerative therapy. To serve these functions, the hiPSC-CM must reasonably recapitulate the electrophysiological and pharmacological characteristics of adult native cardiomyocytes. We analyzed AP characteristics of 103 spontaneously contracting beating clusters (BC) at 11 to 119 days post-differentiation (dpd) and focused on their responses to IKr and IK1 block using E-4031 and BaCl2, respectively
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