Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

Abstract

A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.

Bioreactor Suspension Culture: Differentiation and Production of Cardiomyocyte Spheroids From Human Induced Pluripotent Stem Cells

“…The experimental manipulation of stem/progenitor cells has led to continuous improvements in cell viability, differentiation efficiency, and functional activity (Laco et al, 2018;Le et al, 2018;Biermann et al, 2019;Cai et al, 2019;Leitolis et al, 2019;Valls-Margarit et al, 2019;Yang et al, 2019;Zhu et al, 2020). Contemporary protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into cardiomyocytes (hiPSC-CMs) are often based on the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to alternately activate and then suppress the Wnt signaling pathway (Mazzola and Pasquale, 2020).…”

Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

“…The KOSM4 hiPSC line, established using a SeVdp-iPS vector 14 , 15 , was used for all experiments unless otherwise stated. The cells were plated on a fibronectin-coated dish with serum-free medium and were maintained in an ESF9a-based medium with daily medium changes 14 .…”

“…In this study, we tested our hypothesis that auto/paracrine factors and Wnt-modulating signals are the primary effects of high cell density during cardiomyocyte differentiation from hiPSCs. To clarify the effects of cell density, we have previously established a method to induce cardiomyocytes from hiPSCs at an initial low cell density (1% confluence) 14 . However, low differentiation efficiency was obtained with only 4% cardiac troponin T (cTnT) expression.…”

Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes

“…Once the differentiation procedure is optimized, it enables the control of differentiation process without relying on cell density. That leads higher productivity even differentiation induction is commenced at 1% cell confluency [88].…”

“…Regarding the homogeneity in cell population of the 2D monolayer culture, several groups noticed that more beating cells can be obtained in the peripheral side of the culture dishes, so-called the rim effect [88,96,97]. Laco et al demonstrated that the peripheral cells tend to have higher level of cell proliferation, thus promoting cell contraction earlier and stronger than the center cells [98].…”