title: Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer.
abstract: An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional β 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 1-2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the 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 µg/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 β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/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, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation.
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran.
authors.forename: Marzieh
authors.initials: M
authors.lastname: Ebrahimie
authors.affiliation: Department of Biology, Faculty of Basic Sciences, University of Isfahan, Isfahan, Iran; Research Institute of Biotechnology, Shahrekord University, Shahrekord, Iran.
authors.forename: Fariba
authors.initials: F
authors.lastname: Esmaeili
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Azad Islamic University of Shahrekord, Shahrekord, Iran.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Cheraghi
authors.affiliation: Department of Physiology, Faculty of Medical Sciences, Shahrekord University of Medical Sciences, Shahrekord, Iran.
authors.forename: Fariba
authors.initials: F
authors.lastname: Houshmand
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran; Research Institute of Biotechnology, Shahrekord University, Shahrekord, Iran.
authors.forename: Leila
authors.initials: L
authors.lastname: Shabani
authors.affiliation: Institute of Biotechnology, Shiraz University, Shiraz, Iran; School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia.
authors.forename: Esmaeil
authors.initials: E
authors.lastname: Ebrahimie
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects; D054278:Embryonal Carcinoma Stem Cells / Q000473:pathology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D053263:Gene Regulatory Networks / Q000187:drug effects; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013194:Staining and Labeling; D014020:Tissue Extracts / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D007328:Insulin; D014020:Tissue Extracts; D014157:Transcription Factors