Establishment of Clinically Compliant Human Embryonic Stem Cells in an Autologous Feeder-Free System

Abstract

Applications of human embryonic stem cells (hESCs) are limited by the use of mouse embryonic fibroblasts feeder and animal-derived components during culture. In this study, we demonstrated the potential use of extracellular matrix (ECM) derived from the autologous feeders to support long-term undifferentiated growth of hESCs in xeno-free, serum-free, and feeder-free conditions. Autologous H9 ebF (feeder cells derived from outgrowth of embryoid body [EB] predifferentiated from H9 hESCs) was derived from EBs predifferentiated from H9 hESCs through a direct-plating outgrowth system. The ECM comprising collagen VI, laminin, and fibronectin was extracted from H9 ebF through a freeze-thaw procedure. The autologous ECM together with animal component-free TeSR™2 medium was used to support long-term growth of H1 and H9 hESC lines for up to 20 passages. The maintenance of hESC undifferentiated state by autologous ECM was confirmed by the positive staining of hESC-specific markers (Oct4, SSEA-4, and Tra-1-60) and the expression of pluripotency marker genes (Oct4, Nanog, and Sox2). Flow cytometry further showed that more than 99% of hESCs retained the expression of SSEA-3/4 after long-term culture on autologous ECM. Pluripotency of hESCs on ECM was further proven by in vitro EB formation and in vivo teratoma assay. Overall, this study suggested a strategy for efficient propagation of clinically compliant hESCs in an autologous feeder-free culture system.