TGF-β2 allows pluripotent human embryonic stem cell proliferation on E6/E7 immortalized mouse embryonic fibroblasts

Abstract In this study we report observations that mouse embryonic fibroblasts (MEF) capable of supporting expansion of pluripotent, human embryonic stem cells (hESC) fail to support after immortalization using E6/E7 oncogenes in serum conditions; however this can be reversed following addition of exogenous TGF-β2. Microarray analysis of immortalized and non-immortalized MEF revealed differential gene expression of several TGF-β related genes. By supplementing TGF-β2 into E6/E7 immortalized MEF cultures, this enabled proliferation of undifferentiated, pluripotent hESC as demonstrated by marker expression (Oct-4, SSEA-4, alkaline phosphatase) and teratoma formation representing three germ layers following hESC injection into immuno-deficient mice. Subsequent investigation using quantitative real-time PCR highlighted differential gene expression of several extracellular matrix related transcripts in primary and immortal (±TGF-β2) feeder cells including the induction of osteopontin following addition of TGF-β2. Our results demonstrate that TGF-β2 and its related genes in MEF play a role in the support of pluripotent hESC expansion.

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