Synergistic Effect of Medium, Matrix, and Exogenous Factors on the Adhesion and Growth of Human Pluripotent Stem Cells Under Defined, Xeno-Free Conditions

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), share the properties of unlimited self-renewal and the capacity to become any cell type in the body, making them well suited for regenerative medicine and cell therapy. So far, almost all hPSC lines have been directly or indirectly exposed to animal-derived products, which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation, propagation, and cryopreservation of hPSCs. Moreover, the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined, xeno-free conditions, 2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium, matrix, and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor, Rho-associated kinase inhibitor (ROCK), and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system, and not at all in another. In this study, we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined, xeno-free conditions.