Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. effective therapies for the treatment of patients with severe diabetes1,2,3. However, the shortage of donor human pancreases limits such clinical therapy. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)4,5, have replication competence and the ability to differentiate into many cell types. Furthermore, they are reported to be a potential source of cells for cell therapy6,7. hPSCs may also be used as tools in drug discovery research, such as development of new drugs using disease model cells derived from disease-specific iPSCs8,9,10,11. For the realization of such therapies it is crucial to efficiently generate insulin-producing cells (IPCs) from PSCs. Several protocols have been reported to induce IPCs from hPSCs11,12,13,14,15,16,17,18,19,20,21,22, which mimic the differentiation process during pancreatic development. These methods are effective in IPCs induction. Pancreatic development is regulated Rabbit polyclonal to NPSR1 by transcriptional factors, including and and are expressed during this process. Subsequently, INS+ cells undergo maturation to fully functional islet cells that secrete insulin in response to glucose. These mature IPCs are thought to be characterized by their expression of maturation marker genes such as and and and and (Figs. 1A and S1A). The percentage of FOXA2+/SOX17+ cells was 91.6 0.3% of total cells (Fig. 1B). Wortmannin treatment for 4 days resulted in extensive cell death. Expression of FOXA2 and SOX17 proteins was examined by immunocytochemistry, revealing colocalization of these proteins (Fig. 1C). Treatment with other factors did not affect the expression of or as shown by quantitative PCR analysis (data not shown). These results showed that the combination of activin A, CHIR99021 and wortmannin synergistically induced DE cells from hPSCs. Differentiation of DE cells into pancreatic progenitor cells Because we established a highly efficient differentiation method that induced up to 90% of the total cell population into DE cells, we next tried buy Haloperidol (Haldol) to improve the differentiation efficiency of PDX1+ (pancreatic progenitor) cells from DE cells. It has been reported that treatment with Noggin, an inhibitor of bone morphogenetic protein (BMP) signalling, retinoic acid and fibroblast buy Haloperidol (Haldol) growth factor (FGF)7 or FGF10 induces PDX1+ cells from DE cells16,22,25,27,28. To evaluate these factors in differentiation of PDX1+ cells (Table S1), the expression of pancreatic progenitor markers and were analysed by quantitative PCR and the percentage of PDX1+ cells was examined by an immunochemical assay using an anti-PDX1 antibody. Treatment with Noggin or dorsomorphin, an inhibitor of BMP type I receptors ALK2, 3 and 6, increased the expression of and to similar levels (Fig. 2A), and the percentage of PDX1+ cells was also comparable (33.7 10.3% and 33.7 11.1%, respectively) (Fig. 2B). These results showed that both BMP signalling inhibitors acted with similar efficiencies, and the combination of these factors increased PDX1+ cells to 39.2 6.2% (Fig. 2B). Next, we used retinoic acid for differentiation of PDX1+ cells, and found that the percentage of PDX1+ cells was 18.4 6.5% (Fig. 2B). Because it was reported that activation of the ERK pathway antagonizes the effects of retinoic acid29, we examined the combination of “type”:”entrez-nucleotide”,”attrs”:”text”:”FR180204″,”term_id”:”258307209″,”term_text”:”FR180204″FR180204, an inhibitor of ERK1/2, and retinoic acid. As a result, the percentage of PDX1+ cells was increased to 58.2 6.2% (Fig. 2B). Next, we evaluated the combination of Noggin, dorsomorphin, {retinoic acid and “type”:”entrez-nucleotide”,FR180204, which increased the expression of and (Fig. 2A), and PDX1+ cells were 67.3 4.8% of total cells in the immunochemical assay (Fig. 2B). Moreover, we found that the combination of FGF10, retinoic acid, Noggin, dorsomorphin and “type”:”entrez-nucleotide”,”attrs”:”text”:”FR180204″,”term_id”:”258307209″,”term_text”:”FR180204″FR180204 increased the expression of and endocrine progenitor markers, including and (Figs. 2A and S2A).In addition, the percentage of PDX1+ cells was increased to 76.5 6.7% (Fig. 2B). FGF10 treatment was performed for 3 days because the expression of and the percentage of PDX1+ cells were not different at 3 and 6 days (Fig. S2B). The expression of was not significantly increased by treatment with other factors (data not shown). These results suggested that the combination of these five factors acted synergistically to efficiently induce PDX1+ cells from DE cells. Figure 2 Differentiation of DE cells into pancreatic progenitor cells. Next, we applied our differentiation method developed using KhES-3 cells to other hPSC lines, including H9, iPS (IMR90)-4 and iPS 253G1. All four cell lines showed differentiation into PDX1+ cells at an efficiency of 70C80%, suggesting our method is applicable to a wide variety of PSC lines (Fig. 2C). Induction of pancreatic progenitor cells into INS+ cells PDX1+ cells could be differentiated at a high efficiency of more than 70%. Next, we developed a differentiation method buy Haloperidol (Haldol) to induce INS+ cells. It has been reported that the factors.