Volchkov PY1*, Lagarkova MA1,2*, Prokhorovich MA3, Kiselev SL2
(1 - Institute of Gene Biology, Russian Academy of Science, Moscow; 2 - Vavilov Institute of General Genetics RAS, Moscow ; 3 - Institute of Cytology and Genetics SB RAS, Novosibirsk; maryalag@yahoo.com)
Human embryonic stem cells (hESCs) are a valuable resource in regenerative medicine because of their capacity to differentiate into all cell types. We have established a culture system for the efficient differentiation of endothelial cells from hESCs without embryoid bodies formation step. Using defined culture medium and growth factors cocktail we obtained endothelial cells primary culture with up to 50% purity.
We applied immunomagnetic cell sorting using CD 31 antibody to purify the primary culture of hESC-derived endothelium. Cells sorted with this technique expressed endothelial-specific genes (vWF, CD105, eNOS) and formed cord-like structures in Matrigel assay and on collagen matrix. We compared status of GATA-2, GATA-3 and eNOS gene’s promoters methylation in hESCs, hESCs-derived purified endothelial cells and human umbilical vein endothelial cells (HUVEC)
Hypermethylation of promoter regions of genes studied in hESCs correlates with gene silencing whereas hypomethylation in hESCs-derived endothelial cells and HUVECs correlated with high level of expression. Overall our data indicate the importance of epigenetic regulation for tissue-specific differentiation and provide the evidence that in the course of in vitro differentiation ESCs undergo the same epigenetic program as differentiating cells of embryo in vivo.
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