Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings

Abstract

Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications.

Fig. 1. Evolution of human pluripotent stem cell (hPSC) culture

The in vitro culture of hPSCs has evolved to achieve xenogeneic-free and defined conditions. The illustration depicts this progression (left to right) from co-culture with feeder-cells and serum-containing medium, to feeder-independent cultures in chemical-defined medium. Feeder-free conditions have progressed from the use of complex combinations of extracellular matrix (ECM) proteins like Matrigel™ as a substrate, to individual ECM molecules such as laminin-511, vitronectin and fibronectin. The third generation substrates for hPSC culture is defined by the use of synthetic components in combination with biologic motifs. Advanced materials now provide a fully synthetic substrate that support clonal growth, derivation and long-term culture of genomically stable hPSCs. The gradient transition from dark to light red color (left to right) indicates the complexity of the culture medium, from undefined to defined components. hPSCs are illustrated as the rectangular cells with a prominent red nuclei, while fibroblasts are shown as elongated cells with blue nuclei.