Abstract
Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.
Highlights
Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate in vivo and in vitro into all cell types of the embryo proper
Generation of embryonic stem cells ES cells were generated from C57BL/6 mouse 3.5 day post coitum blastocyst stage embryos by plating the embryos into a 96 well dish with irradiated feeder cells and ES cell media consisting of high glucose containing Dulbecco’s modified Eagle media (DMEM) supplemented with 15% fetal bovine serum (FBS), 100 U/ml+100 mg/ ml penicillin (100 U/ml)-streptomycin (100 mg/ml), 100 mM 2mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10 ng/ml Leukemia Inhibitory Factor [4] in the presence of 25 mg of PD98059 as described by Buehr and Smith [25]
C57BL/6 mouse ES cells were cultured on a feeder layer of gamma irradiated PEFs and incubated at 37uC with 95% O2/5% CO2 in an ES cell maintenance medium comprised of 15% FBS (Gemini Bio-Products, Woodland, CA), 1 mM sodium pyruvate (Stem Cell Technologies, Vancouver, Canada), 2 mM glutamine (Stem Cell Technologies), 0.1 mM non-essential amino acids (Stem Cell Technologies), 10 ng/ml leukemia inhibitory factor (Chemicon International, Temecula, CA), 100 mM 1-thioglyercol (MTG)
Summary
Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate in vivo and in vitro into all cell types of the embryo proper. These cells represent a potentially unlimited source of differentiated cells or tissue for transplantation for common diseases such as type 1 diabetes mellitus. D’amour et al, by using activin A, exendin, cyclopamine and retinoic acid, showed that human embryonic stem cells can differentiate into endocrine cells capable of synthesizing pancreatic hormones [7]. Other scientists have differentiated human ES cells into insulin producing cells [9]
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