title: Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells.
abstract: Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™), which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives, expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for > 20 passages on tissue culture-treated polystyrene plates, coated from 5 μg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-, endo-, and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy, atomic force microscopy, and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density, via the concentration of depositing solution, revealed a threshold surface density of 250 ng/cm², which is required for hESCs attachment, proliferation, and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable.
authors: Stem Cells and Tissue Repair Group, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors: Lynn Y W
authors: LY
authors: Yap
authors: Jian
authors: J
authors: Li
authors: In Yee
authors: IY
authors: Phang
authors: Lay Ting
authors: LT
authors: Ong
authors: Jo'an Zhu-En
authors: JZ
authors: Ow
authors: James C H
authors: JC
authors: Goh
authors: Victor
authors: V
authors: Nurcombe
authors: Jonathan
authors: J
authors: Hobley
authors: Andre B H
authors: AB
authors: Choo
authors: Steve K W
authors: SK
authors: Oh
authors: Simon M
authors: SM
authors: Cool
authors: William R
authors: WR
authors: Birch
mesh_terms: D000327:Adsorption / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D013057:Spectrum Analysis; D013499:Surface Properties / Q000187:drug effects; D013997:Time Factors; D019096:Vitronectin / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D019096:Vitronectin