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  • Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

Stem cells (Dayton, Ohio) (2015-02-03)
Miao Zhang, Jan Sebastian Schulte, Alexander Heinick, Ilaria Piccini, Jyoti Rao, Roberto Quaranta, Dagmar Zeuschner, Daniela Malan, Kee-Pyo Kim, Albrecht Röpke, Philipp Sasse, Marcos Araúzo-Bravo, Guiscard Seebohm, Hans Schöler, Larissa Fabritz, Paulus Kirchhof, Frank Ulrich Müller, Boris Greber
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 high-efficiency 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium selenite, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
Transferrin human, powder, BioReagent, suitable for cell culture
Millipore
Accutase cell detachment solution, A cell detachment solution of proteolytic & collagenolytic enzymes. The reagent is useful for creating single cell suspensions from clumped cell cultures for accurate cell counting, detachment of cells from primary tissue.