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

Abstract

The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%-90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a coculture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts

“…This strongly suggests a crosstalk between cFBs and hiPSC-CMs where the presence of cFBs influences the mechanoresponsiveness of hiPSC-CMs. This is in line with previous studies that showed that the number and activation state of cFBs modulated the electromechanical functioning of hiPSC-CMs [48][49][50][51]. Further studies should be directed to investigating the mechanisms that trigger the strain-induced reorientation response in the CM-cFB co-culture.…”

Label-free imaging for quality control of cardiomyocyte differentiation

“…At the end of our differentiation process, some cells were not positive for cTnT and therefore were not CMs. Previous studies have shown that these non-CMs at the end of the differentiation process are primary cardiac-like fibroblasts together with a small portion of non-differentiated hPSCs 41 . hPSC-derived CMs exhibit distinct metabolism from other hPSC-derived non-CMs 42 .…”

Label-free imaging for quality control of cardiomyocyte differentiation

“…At the end of our differentiation process, some cells were not positive for cTnT and therefore were not CMs. Previous studies have shown that these non-CMs at the end of the differentiation process are primary cardiac-like fibroblasts together with a small portion of non-differentiated hPSCs 35 . hPSC-derived CMs exhibit distinct metabolism from other hPSC-derived non-CMs 36 .…”

“…Co-culture of cardiac fibroblasts and CMs can induce fibroblast glycolytic activity and lactate secretion from fibroblasts 37 . Co-culture of cardiac fibroblasts and CMs also promotes a more mature phenotype in CMs along with increased reliance on oxidative phosphorylation 35 . Similarly, differences in NAD(P)H lifetime variables between CMs and non-CMs on day 8 (Figure 4h) are consistent with decreased glycolytic activity in the CMs (Figure S8).…”