Search

Summary: The inability to quantify cardiomyocyte (CM) maturation remains a significant barrier to evaluating the effects of ongoing efforts to produce adult-like CMs from pluripotent stem cells (PSCs). Here, we present a protocol to quantify stem-cell-derived CM maturity using a single-cell RNA sequencing-based metric “entropy score.” We describe steps for generating an entropy score using customized R code. This tool can be used to quantify maturation levels of PSC-CMs and potentially other cell types.For complete details on the use and execution of this protocol, please refer to Kannan et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder responsible for nearly a quarter of sports-related sudden cardiac deaths. ACM cases caused by mutations in desmosome proteins lead to right ventricular enlargement, the loss of cardiomyocytes, and fibrofatty tissue replacement, disrupting electrical and mechanical stability. It is currently unknown how paracrine factors secreted by infiltrating fatty tissues affect ACM cardiomyocyte electrophysiology. Methods: A normal and a PKP2 mutant (c.971_972InsT) ACM hiPSC line were cultivated and differentiated into cardiomyocytes (CMs). Adipocytes were differentiated from human adipose stem cells, and adipocyte conditioned medium (AdCM) was collected. Optical mapping and phenotypic analyses were conducted on human iPSC-cardiomyocytes (hiPSC-CMs) cultured in cardiac maintenance medium (CMM) and either with AdCM or specific cytokines. Results: Significant differences were observed in voltage parameters such as the action potential duration (APD80, APD30), conduction velocity (CV), and CV heterogeneity. When cultured in AdCM relative to CMM, the APD80 increased and the CV decreased significantly in both groups; however, the magnitudes of changes often differed significantly between 1 and 7 days of cultivation. Cytokine exposure (IL-6, IL-8, MCP-1, CFD) affected the APD and CV in both the normal and PKP2 mutant hiPSC-CMs, with opposite effects. NF-kB signaling was also found to differ between the normal and PKP2 mutant hiPSC-CMs in response to AdCM and IL-6. Conclusions: Our study shows that hiPSC-CMs from normal and mPKP2 ACM lines exhibit distinct molecular and functional responses to paracrine factors, with differences in RNA expression and electrophysiology. These different responses to paracrine factors may contribute to arrhythmogenic propensity.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

Summary: A limitation in the application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. The mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, remain unclear. Here, we generate a single-cell RNA sequencing (scRNA-seq) reference of mouse in vivo CM maturation with extensive sampling of previously difficult-to-isolate perinatal time periods. We subsequently generate isogenic embryonic stem cells to create an in vitro scRNA-seq reference of PSC-CM-directed differentiation. Through trajectory reconstruction, we identify an endogenous perinatal maturation program that is poorly recapitulated in vitro. By comparison with published human datasets, we identify a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study can be leveraged toward improving the clinical viability of PSC-CMs.