Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells.
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.
Yap
Lynn Y W
LY
Stem Cells and Tissue Repair Group, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Li
Jian
J
Phang
In Yee
IY
Ong
Lay Ting
LT
Ow
Jo'an Zhu-En
JZ
Goh
James C H
JC
Nurcombe
Victor
V
Hobley
Jonathan
J
Choo
Andre B H
AB
Oh
Steve K W
SK
Cool
Simon M
SM
Birch
William R
WR
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*
D015415:Biomarkers; D019096:Vitronectin