The utilization of human-induced pluripotent stem cells (hiPSCs) in cell therapy has a tremendous potential but faces many practical challenges, including costs associated with cell culture media and growth factors. There is an immediate need to establish an optimized culture platform to direct the differentiation of hiPSCs into germ layers in a defined nutritional microenvironment to generate cost-effective and robust therapeutics. The aim of this study was to identify the optimal nutritional environment by mimicking the in vivo concentrations of three key factors (glucose, pyruvate, and oxygen) during the spontaneous differentiation of hiPSCs derived from cord blood, which greatly differ from the in vitro expansion and differentiation scenarios. Moreover, we hypothesized that the high glucose, pyruvate, and oxygen concentrations found in typical growth media could inhibit the differentiation of certain lineages. A design of experiments was used to investigate the interaction between these three variables during the spontaneous differentiation of hiPSCs. We found that lower oxygen and glucose concentrations enhance the expression of mesodermal (Brachyury, KIF1A) and ectodermal (Nestin, β-Tubulin) markers. Our findings present a novel approach for efficient directed differentiation of hiPSCs through the manipulation of media components while simultaneously avoiding the usage of growth factors thus reducing costs.
- design of experiments
- germ layer differentiation
- human-induced pluripotent stem cells