To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method
To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. is the generation of functional cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1C2 weeks aged mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2C3 weeks after birth, we hypothesized that this mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 g/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 g/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, LTX-315 and WAC proteins. Altogether, we exhibited the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic cells. The derivation Ocln of pancreatic cells from EC cells which are ES cell siblings would provide a useful experimental tool in study of pancreatic development and function as well as quick production of IPCs for transplantation. Introduction Diabetes mellitus is one of the most common chronic diseases which directly affects millions of people . LTX-315 Type 1 diabetes can be ameliorated by islet transplantation. The concept of transplanting pieces of pancreas in diabetic patients has over a century history. However, the major problem is the deficiency of transplantable cadaver islets. Many studies have been focused on how to develop renewable sources of islet-replacement tissue. Whereas some studies have shown the generation of insulin-producing cells (IPCs) from progenitor cells of the pancreas , liver , , pluripotent embryonic stem (ES) cells C, and skin-derived stem cells , the efficiency of in vitro generated IPCs is usually low. The existing protocols for generating IPCs from ES cells can be divided into spontaneous and induced differentiation . In the present work, using neonatal mouse pancreas extract (MPE) as a natural biological inducer, we developed a simple accessible way to generate functional IPCs from P19 embryonal carcinoma (EC) stem cell collection. Total removal of the pancreas in dogs produces severe and fatal diabetes . Daily injections of pancreatic extract prolonged life of a completely diabetic doggie. Subcutaneous administration of whole pancreas extract to the human subjects caused decrease in blood sugar and increased utilization of carbohydrate . Experimental studies exhibited that the supplementation of oral nutrition with pancreatic extract-enriched diet improved the nutritional status of the aged rats , . Reddy et al. showed that the oral administration of whole pancreas extract to young non-obese diabetic (NOD) mice, prevented autoimmune diabetes . Rat pancreatic extract (RPE) enhanced the expression of the required transcription factors for pancreas development . In vitro differentiation induction of rat mesenchymal cells into IPCs increased with the treatment of the cells by RPE , . Using RPE as a natural biological inducer, Zhang et al. differentiated human amniotic mesenchymal stem cells into insulin-secreting cells . Some studies show that RPE contains numerous growth factors and hormones related LTX-315 to pancreas regeneration , . The effects of RPE on IPCs differentiation of human adipose tissue-derived stem cells (hASCs) were evaluated. Genes involved in early pancreas development (such as Sox17 and IPF-1) were expressed in RPE-treated culture . Information regarding gene co-expression is useful to predict gene function , . Several databases have been developed for gene co-expression analysis based on a large amount of publicly available gene expression data measured by GeneChip platforms. DNA microarrays deposited in public databases provide.