ABSTRACT
Aims: Mesenchymal stem cells (MSCs) can differentiate into multiple cell types including insulinproducing cells. However, these cells usually cannot be directed to efficiently differentiate into β cells in vitro. The present study aimed to explore whether the pancreatic microenvironment could induce bone marrow-derived (BM)-MSCs to differentiate into β cells to compensate for insufficient β-cells. Methodology: We directly transplanted male enhanced green fluorescence protein (EGFP)- expressing BM-MSCs into the pancreas of female diabetic Sprague-Dawley rats by multi-point injection. Results: BM-MSCs could restore serum insulin and C-peptide levels and reverse hyperglycemia by intra-pancreatic transplantation. BM-MSCs from male donors could differentiate into pancreatic stem/progenitor cells and β cells under female pancreas micro-environment. Neogenesis islets derived from BM-MSCs were verified in pancreatic tissue by histology and the expression of genes related to β cell gene biomarker was determined by RT-PCR and quantitative real time-PCR. Ychromosome SRY and PDX-1 mRNA have expressed simultaneously in neogenesis β cells. Polyploidy and aneuploid DNA were not observed. Conclusion: This study showed that transplanted BM-MSCs did not fuse with pancreatic cells and could contribute to repair, paracrine and differentiation into new islet β cells in the pancreatic microenvironment.
ABSTRACT
The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes (Pdx-l,Pax4,MafA,and Nkx6.1,etc) were investigated.The promoter methylation status of islet differentiation-associated genes (Pdx-1,Pax4,MafA and Nkx6.1),Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR (MeDIP-qPCR) techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification (DNA methylation,H3 acetylation,H3K4m3 and H3K9m3) of these genes and their expression was analyzed.The results showed that:(1) the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; (2) NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells (P＜0.05); (3) NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells (P＜0.05),with significantly increased level of gene expression; (4) NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell (P＜0.05),with no detectable mRNA expression of these genes.It was concluded that histone modification (H3K4m3 and H3K9m3) and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.