Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

Human Induced Pluripotent Stem Cell-Derived Non-Cardiomyocytes Modulate Cardiac Electrophysiological Maturation Through Connexin 43-Mediated Cell-Cell Interactions

“…This included greater AP amplitude and V max , although no difference in AP duration. The authors concluded that hiPSC-CMs cultured in those larger islands predominately mature by short-range interactions with neighboring CMs, although it was unclear whether those interactions were mechanical or biochemical in nature [41].…”

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

“…Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs, CDI) were grown on a substrate patterned with different sized islands of fibronectin [24] (see protocols for details). In brief, a polyacrylamide gel, which cells cannot adhere to, was deposited in the dish.…”

“…For instance, to study network dynamics in cultured neurons, one must record simultaneously from cells separated by millimeter distances, corresponding to the length of axonal projections in vitro. To study action potential propagation in cultured cardiomyocytes, one must image a sufficient area with high time resolution to map propagation of wavefronts at ~20 cm/s (corresponding to 200 μm/ms) [24]. Finally to probe the acute response of any cell type to addition of a drug, one can typically only sample a single FOV per well.…”

Geometry-dependent instabilities in electrically excitable tissues

“…6b). CheRiff was expressed using a lentiviral vector to allow patterned blue light stimulation 19 , and BeRST1 was used to image changes in membrane potential. We optogenetically paced 0D islands and 1D tracks simultaneously across a range of frequencies.…”

“…17,18 In complex cells such as cardiomyocytes, one typically cannot vary one parameter without affecting many others. For instance, growing hiPSC-CM on different size islands affects their patterns of gene expression 19 , which in turn can affect electrophysiology.…”