TY - JOUR
T1 - Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells
AU - Yap, Lynn Y.W.
AU - Li, Jian
AU - Phang, In Yee
AU - Ong, Lay Ting
AU - Ow, Jo'An Zhu En
AU - Goh, James C.H.
AU - Nurcombe, Victor
AU - Hobley, Jonathan
AU - Choo, Andre B.H.
AU - Oh, Steve K.W.
AU - Cool, Simon M.
AU - Birch, William R.
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 2011/2/1
Y1 - 2011/2/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=79551514298&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79551514298&partnerID=8YFLogxK
U2 - 10.1089/ten.tec.2010.0328
DO - 10.1089/ten.tec.2010.0328
M3 - Article
C2 - 20726687
AN - SCOPUS:79551514298
VL - 17
SP - 193
EP - 207
JO - Tissue Engineering - Part C: Methods
JF - Tissue Engineering - Part C: Methods
SN - 1937-3384
IS - 2
ER -
