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|Title:||Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells|
|Source:||Yap, L.Y.W., Li, J., Phang, I.Y., Ong, L.T., Ow, J.Z.-E., Goh, J.C.H., Nurcombe, V., Hobley, J., Choo, A.B.H., Oh, S.K.W., Cool, S.M., Birch, W.R. (2011-02-01). Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells. Tissue Engineering - Part C: Methods 17 (2) : 193-207. ScholarBank@NUS Repository. https://doi.org/10.1089/ten.tec.2010.0328|
|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 5μg/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 250ng/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. © 2011 Mary Ann Liebert, Inc.|
|Source Title:||Tissue Engineering - Part C: Methods|
|Appears in Collections:||Staff Publications|
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