Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin-Producing Cells From Embryonic Stem Cells

Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin-Producing Cells From Embryonic Stem Cells Satsuki Miyazaki , Eiji Yamato and Jun-ichi Miyazaki From the Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Japan Address correspondence and reprint requests to Jun-ichi Miyazaki, MD, PhD, Division of Stem Cell Regulation Research (G6), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: jimiyaza{at}nutri.med.osaka-u.ac.jp Abstract Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. However, the differentiation is not efficient enough to produce insulin-secreting cells for future therapeutic use. Pdx-1, a homeodomain-containing transcription factor, is a crucial regulator for pancreatic development. We established an ES cell line in which exogenous pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus. Using this cell line, we examined the effect of pdx-1 expression during in vitro differentiation via embryoid body formation. The results showed that pdx-1 expression clearly enhanced the expression of the insulin 2, somatostatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2, and HNF6 genes in the resulting differentiated cells. Immunohistochemical examination also revealed that insulin was highly produced in most of the differentiated ES cells. Thus, exogenous expression of pdx-1 should provide a promising approach for efficiently producing insulin-secreting cells from human ES cells for future therapeutic use in diabetic patients. bFGF, basic fibroblast growth factor DMEM, Dulbecco’s modified Eagle’s medium Dox, doxytetracycline EB, embryoid body EGFB, enhanced green fluorescent protein ES, embryonic stem IRES, internal ribosome entry site LIF, leukemia inhibitory factor tTA, tetracycline-regulated transcriptional activator TUNEL, transferase-mediated dUTP nick-end labeling Footnotes Accepted January 16, 2004. Received July 31, 2003. DIABETES