Universal Cardiac Induction of Human Pluripotent Stem Cells in Two and Three-Dimensional Formats: Implications for In Vitro Maturation

Abstract

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal highefficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation. STEM

Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs

“…Cardiac induction was performed under serum and albumin-free conditions in a monolayer format, as described (Zhang et al, 2015). Signaling factors in short-term growth factor stimulation experiments were BMP4 (1-20 ng/ml, R&D), CHIR99021 (1-3 mM, Axon Medchem), Dorsomorphin (250nM, Santa Cruz), and IWP-2 (2mM, Santa Cruz).…”

“…We have recently developed a directed differentiation protocol for cardiac induction of hESCs that shares the basic principle of two-step signaling pathway perturbation with previous reports (Burridge et al, 2012;Zhang et al, 2015). Here, we analyze the signaling framework driving this procedure by mimicking signaling gradients in vitro and systematically investigating downstream transcriptional programs.…”

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

“…Given that after 4 weeks of differentiation, the cells reach a rather stable transcriptomic state in 2D and 3D cultures, it is hypothesized that additional time is necessary for functional and ultrastructural maturation. 158 Differentiation can be further enhanced in 3D cultures by culturing cells in bovine (type I) collagen/Matrigel hydrogels yielding cells (2-3 weeks) expressing cardiac genes at comparable levels to the adult left ventricle. 159 Fibrin-based hydrogels, biodegradable scaffolds, and scaffold-free constructs have also been used for promoting maturation of hPSC-heart muscle cells.…”

WNT and BMP regulate roadblocks toward cardiomyocyte differentiation: lessons learned from embryos inform human stem cell differentiation

“…Several signaling pathways synergize with Wnt/β-catenin signaling to promote more efficient cardiomyocyte differentiation (11,12). Greber and colleagues found that particularly BMP signaling cooperates with Wnt/β-catenin signaling in the early stages of the differentiation protocol.…”