Phenotypic Variation Between Stromal Cells Differentially Impacts Engineered Cardiac Tissue Function

Throughout heart development, cardiomyocytes differentiate and mature in direct contact with nonparenchymal cell types, such as cardiac fibroblasts. Thus, when modeling myocardial tissue in vitro, tissue engineers include a supporting stromal cell population that is necessary for tissue formation, although the source of stromal cells has varied widely. This study systematically characterized the phenotype of commonly used stromal cell populations and analyzed the differential impacts of stromal phenotype on cardiac microtissue phenotype and function. Quantitative morphometric analysis, flow cytometry, unbiased morphological feature clustering, and RNA sequencing of the different stromal populations revealed variable cell morphologies, surface marker expression, and gene signatures, with primary adult stromal populations exhibiting more similar phenotypes to each other than to stem cell-derived and progenitor populations. The ability of self-assembled cardiac microtissues to consistently form tissues was highly dependent on the stromal population mixed with stem cell-derived cardiomyocytes, with cardiac fibroblasts and dermal fibroblasts (DFs) forming the most robust tissues compared with mesenchymal stromal cells and induced pluripotent stem cell-derived fibroblasts. Cardiac fibroblasts and DFs also resulted in cardiac microtissues displaying a more mature calcium handling profile, with increased amplitude and upstroke velocity. These results demonstrate the breadth of phenotypic variation across stromal populations owing to cell and tissue source, with certain primary populations, such as cardiac fibroblasts and DFs, supporting cardiac microtissue phenotype and improved calcium handling function. Impact Statement Understanding the relationship between parenchymal and supporting cell populations is paramount to recapitulate the multicellular complexity of native tissues. Incorporation of stromal cells is widely recognized to be necessary for the stable formation of stem cell-derived cardiac tissues; yet, the types of stromal cells used have varied widely. This study systematically characterized several stromal populations and found that stromal phenotype and morphology was highly variable depending on cell source and exerted differential impacts on cardiac tissue function and induced pluripotent stem cell-cardiomyocyte phenotype. Therefore, the choice of supporting stromal population can differentially impact the phenotypic or functional performance of engineered cardiac tissues.