Abstract
Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.
Highlights
Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem cells
We recently showed that IaI activates the Yes/Yes-associated protein (YAP)/TEAD transcription factor pathway and induces expression of Oct[4] and Nanog in mouse ES cells[24]
Even though the use of the separate IaI domains showed HC2 to be responsible for the human pluripotent stem (hPS) cell attachment, all three antibodies inhibited IaI-induced attachment regardless of which domain was being targeted, suggesting that antibody binding hinders IaI-mediated attachment when used in solution (Fig. 1c)
Summary
Scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. IaI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. We describe a new, simplified and time-efficient cell culture method based on the E8 medium supplemented with soluble IaI ( called E8:IaI) This method requires no surface coating and supports long-term propagation of hPS cells, clonal expansion and single-cell passaging even in the absence of ROCKi
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