Polygene-modified mouse induced differentiation of pluripotent stem cells into insulin-producing cells
10.3724/SP.J.1008.2013.00700
- Author:
Xiang-Jun FAN
1
Author Information
1. Department of General Surgery, The First Affiliated Hospital of Soochow University
- Publication Type:Journal Article
- Keywords:
Cell differention;
Induced pluripotent stem cells;
Insulin-secreting cells;
Transcription factors
- From:
Academic Journal of Second Military Medical University
2013;34(7):700-707
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the effects of key msulm gene transcsipiion regulators (PDX-1, NeuroD1 and MafA) on the differentiation of nduced pluripotent stem cells (iPSCs) nnto insulin-producing cells. Methods Mouse embryonic fibroblasts (MEFs) were nnfected with lentivirus (LV-ef1a-Hygromic'rn-TRE-Oct4/Sxx2/Kl/4/cMyc) at a muitiplicity of infection, and iPSCswere selected and identified. Then the iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad-mNeuroD1-IRES -GFP andAd-mMa/A-IRES -GFP) to nnduce differentiation nnto insulin-producing cells cc vitro. RT-PCR was applied to detect expression of functional genes nn pancreatic islet B cells; immunofluorescence was used to examine the expresson and location of rnsuHn protenn; and ELISA was used to deterrmne the volumes of secreted insulin at different concentrations of glucose (0, 5, 10, 20, 30, and 40 mmoVL). Results The iPSCs derived from MEFs could form intensive llones with smooth boundary, express embryonic stem cell-specific cell surface markers, including Nacog, SSEA-1 and Rex-1, and differentiate nnto three embryonic iayers, which indicating that MEFs were successfully reprogrammed nto iPSCs. Mouse iPSCs nnfected with Ad-PDX-1-IRES-GFP, Ad-mNeuroD-IRES-GFP, and Ad-mMa/A-IRES-GFP could d i fferentiate nto pancreatic illet B cells. RT-PCR resuits showed that polygene-modffied iPSCs and pancreatic illet B-cell lnne MIN6 had simlar gene expresson profile. Immunofluorescence analyses confirmed insulin expresson nn the d ifferentiated cells. Resuits of ELISA showed that polygene-modified iPSCs had a satisfactory response to d ifferent concentrations of glucose. Conclusion Key insulin gene transcription reguiatorsPDX-1, NeuroD! and MafA can work synerglstically to nnduce mouse iPSCs differentiation into pancreatic islet B cells capable of insulin biosynthesis and secretion.
