Role of Egr1 on Pancreatic Endoderm Differentiation.

The low efficiency of in vitro differentiation of human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (iPSCs) into insulin-producing cells is a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). Our previous investigation into the key factors for the differentiation of PSCs into insulin-producing cells suggested that the expression of GATA binding protein 6 (GATA6) and Gremlin 1 (GREM1) and inhibition of early growth response protein 1 (Egr1) may be important factors. In this study, we investigated the role of Egr1 in pancreas development. The transfection of small interfering RNA (siRNA) of Egr1 in the early phase induced the differentiation of iPSCs derived from fibroblasts (FiPSCs) into pancreatic endoderm and insulin-producing cells. In contrast, the downregulation of Egr1 in the late phase suppressed the differentiation of FiPSCs into pancreatic endoderm and insulin-producing cells. In addition, the overexpression of Egr1 suppressed the differentiation of iPSCs derived from pancreatic cells into pancreatic endoderm and insulin-producing cells. These data suggest that the downregulation of Egr1 in the early phase can efficiently induce the differentiation of iPSCs into insulin-producing cells.

Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.

The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells.