Human pluripotent stem cells (hPSCs) have gained interest primarily because of their dual capabilities of self-renewal and differentiation into multiple cell types [1
]. Traditional culturing of hPSCs involves direct contact with a feeder cell layer, such as mouse embryonic fibroblasts (MEFs), to support their undifferentiated growth [5
]. These feeder cells not only produce extracellular matrix (ECM) components crucial for attachment of hPSCs, but also secrete factors that help maintain hPSCs in a pluripotent state. An important issue, however, is that direct contact of hPSCs with a feeder layer results in intermixing of cell types and may cause xenotypic contamination [6
]. Recent improvements in culture techniques include using human rather than mouse fibroblasts as feeder layers [7
] eliminating potential cross-species pathogen contamination; however separation of the feeder cells from the hPSCs, as is required for most differentiation protocols, still remains a technical challenge. For this reason, feeder-free systems have been developed, usually using purified substrates such as Matrigel™, fibronectin or laminin for attachment and replacing the other feeder cell-derived components with medium that is preconditioned by culture with feeder cells or defined medium containing high levels of synthetic growth factors [11
We report here an alternative method for culturing hPSCs that does not require mixing the cells with feeder cells, using pre-conditioned medium, or adding high levels of growth factors. We evaluated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture (MBIC) system that physically separates hPSCs from the feeder layer, while allowing for continuous conditioning of the medium by the feeder cells. In this study, we describe the first use of an MBIC system for hPSC propagation. We show that hPSCs cultured on membrane coated with human fibroblast-derived extracellular matrix and physically separated from human feeder cells (Supplementary Figure 1
) are phenotypically indistinguishable from those cultured in contact with feeder cells, having the same colony morphology, expression of pluripotency markers, in-vitro differentiation and global gene expression profiles. Use of a MBIC system for hPSC expansion allows for an economical alternative to synthetic media-based feeder-free system and is amenable to scaling up production of pure populations of hPSCs and their derivatives. The complete separation of the cell types will simplify the testing of multiple culture conditions for optimal hPSC growth, and will enable rapid changes in conditions, such as testing differentiation factors and/or other types of cells for co-culture, without the requirement for dissociating and replating the hPSCs.