title: 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.
authors.affiliation: Stem Cell Laboratory, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, National University of Singapore, Singapore, Singapore.
authors.forename: Xin
authors.initials: X
authors.lastname: Fu
authors.forename: Wei Seong
authors.initials: WS
authors.lastname: Toh
authors.forename: Hua
authors.initials: H
authors.lastname: Liu
authors.forename: Kai
authors.initials: K
authors.lastname: Lu
authors.forename: Mingming
authors.initials: M
authors.lastname: Li
authors.forename: Tong
authors.initials: T
authors.lastname: Cao
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007621:Karyotyping; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction