Epigenetic Induction of Definitive and Pancreatic Endoderm Cell Fate in Human Fibroblasts

Figure 2

5-Azacytidine (5AZA) and Trichostatin A (TSA) treatment of primary adult fibroblast induces transient definitive endoderm and pancreatic endoderm markers. (a) qRT-PCR analysis demonstrated induction of endodermal genes (GATA4, MIXL1, EOMES, E-CADHERIN, SOX17, and CXCR4 but not SOX7, GSC, and FOXA2) in 5AZA + TSA-CRM treated primary human fibroblast cells. (b) qRT-PCR analysis demonstrated induction of pancreatic endoderm genes (HNF1B, PDX1, HLXB9, PAX6, and NKX6.1 but not SOX9, PTF1A, and NEUROD1) in 5AZA + TSA-CRM treated primary human fibroblast cells. (c) qRT-PCR analysis demonstrated induction of the pancreatic endocrine genes (ISL1, ARX, MAFB, and MAFA but not PAX4, NGN3, INS, SST, and GCG) in 5AZA + TSA-CRM treated primary human fibroblast cells. (d) qRT-PCR analysis demonstrated no induction of hepatocyte genes (ALB, AFP, and HNF4A) in 5AZA + TSA-CRM treated primary human fibroblast cells. (e) qRT-PCR analysis demonstrated induction of mesoderm lineage genes (MYOD1 and FLK1 but not TIE-2 and VE-CADHERIN) in 5AZA + TSA-CRM treated primary human fibroblast cells. (f) qRT-PCR analysis demonstrated no induction of pluripotency genes (OCT4, SOX2, and NANOG) in 5AZA + TSA-CRM treated primary human fibroblast cells. Black bar, CRM treated primary human fibroblast cells; light grey bar, DMSO-CRM treated primary human fibroblast cells; dark grey bar, 3 μM 5AZA-CRM followed by 100 μM TSA-CRM treated primary human fibroblast cells. Gene expression is shown relative to the housekeeping gene GAPDH. Data represent the mean ± SEM (standard error of mean) of three independent experiments. Statistical significance tests were performed between 5AZA + TSA treated versus untreated fibroblast and 5AZA + TSA treated versus DMSO treated fibroblast. , , and by unpaired 2-tailed Student’s -test. NS, not significant.

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