Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium

Abstract

The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for β cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-β. The expression of genes related to pancreatic β cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic β cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd.

Induction of pancreatic β cell gene expression in mesenchymal stem cells

“…Furthermore, immunoreactivity for proinsulin along with C-peptide in the current study exhibited the presence of endogenously produced insulin. De novo synthesis and processing of insulin as a feature of normal b cells was reported previously (Brol en et al, 2005;Zulewski, 2006;Ebrahimie et al, 2014;Mansouri et al, 2014). In the current study, insulin production by IPCs was confirmed by immunoflourescence, ELISA, and real-time PCR analysis.…”

“…The imported genes in constructed network were CREB1 , EP300 , and PDX‐1 , which are the major regulators in differentiation of MSCs to IPCs. Interestingly, we observed the upregulation of these transcription factors at the following two experiments: (1) Production of IPCs from P19 EC stem cells under the treatment of PCM (Mansouri et al, ), and (2) Production of IPCs from P19 EC stem cells using mouse neonate pancreas extract (Ebrahimie et al, ). Upregulation of CREB1 , EP300 , and PDX‐1 transcription factors in production of IPCs from different types of stem cells, such as P19 EC stem cells and MSCs, and under different inducers, such as PCM and mouse neonate pancreas extract, confirms the key role of CREB1, EP300 , and PDX‐1 in generation of IPCs.…”

“…Based on our recent reports, INS2 and PDX‐1 are the central proteins for induction of IPCs. Furthermore, several transcription factors, including EP300 and CREB1, have positive regulatory effects on crosstalk and activation of PDX ‐1 and INS2 (Ebrahimie et al, ; Mansouri et al, ).…”

“…Some studies have reported that MSCs isolated from various tissues are able to differentiate into functional IPCs (Oh et al, 2004;Chang et al, 2007;Sun et al, 2007;Chao et al, 2008;Liu and Han, 2008;Zhang et al, 2009;Xie et al, 2013). We recently introduced two successful induction systems using neonate MPE or PCM (Mansouri et al, 2014) as natural native inducers to produce IPCs from P19 EC cells. Our observations documented that both MPE and PCM Figure 6 Detection of secreted (A) and intracellular versus secreted (B) insulin in undifferentiated mesenchymal stem cells (MSCs) and differentiated cells (IPCs).…”

“…We recently introduced two successful induction systems using neonate mouse pancreas extract (MPE) (Ebrahimie et al, ) and pancreas‐conditioned medium (PCM) (Mansouri et al, ) as natural native inducers to produce IPCs from P19 embryonal carcinoma (EC) cells. Based on our observations, both MPE and PCM are able to efficiently induce IPC differentiation.…”