TGF-beta2 allows pluripotent human embryonic stem cell proliferation on E6/E7 immortalized mouse embryonic fibroblasts.
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-beta2. Microarray analysis of immortalized and non-immortalized MEF revealed differential gene expression of several TGF-beta related genes. By supplementing TGF-beta2 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-beta2) feeder cells including the induction of osteopontin following addition of TGF-beta2. Our results demonstrate that TGF-beta2 and its related genes in MEF play a role in the support of pluripotent hESC expansion.
Stem Cell Group, Bioprocessing Technology Institute, Centros, 20 Biopolis Way, #06-01, Singapore 138668, Singapore.
Stephen
S
Chen
Andre
A
Choo
Angela
A
Chin
Steve K W
SK
Oh
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology*; D012795:Sialoglycoproteins / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2
C118860:E6 protein, Human papillomavirus type 6; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D053495:Osteopontin