الفهرس 
Introduction & Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Diabetes mellitus is a widespread endocrine devastating disease affecting millions of people worldwide every year. Maintaining normal glycemic control with exogenous insulin imposes a burden on patients. Transplantation of an intact pancreas or isolated pancreatic islets are alternative methods for diabetic patients treatment. However, the shortage of cadaveric organs and the necessity of immunosuppressive drugs are limiting factors. SSEA-3 is an embryonic surface marker and it was suggested that SSEA-3 have a role in stem cell fate and is responsible for the differentiation capacity of stem cells. Recent reports reported that SSEA-3 expressed on a rare subpopulation within mesenchymal adult stem cells (MSCs), and these cells called Muse cells. The present study aims to isolate and differentiate a rare subpopulation among MSCs with pluripotent characteristics into IPCs. This SSEA-3 embryonic-like stem cells sub-population was characterized after FACS separation by different methods to confirm its pluripotency and the capacity of insulin production after in vitro differentiation. MSCs were obtained from fresh human fat liposuctions. characterization of the isolated adherent ASCs was performed by the expression of several surface antigens, plastic adherence, and multilineage differentiation capacity. Muse cells were isolated from ASCs by BD FACSAria III. To quantify the purity of isolated Muse cells, staining with anti-SSEA-3 and observation under the confocal microscope were performed and qRT-PCR was performed for endocrine precursor hormone Nestin and pluripotent genes and PDX-1. Isolated Muse and non-Muse cells were differentiated into insulin-producing cells through a two stages protocol: Differentiated Muse and non-Muse cells were exposed to different glucose concentrations, cells were incubated 1 hour in three different concentrations of Krebs-Ringer bicarbonate buffer (KRB) [5.5, 12, 25mM]. KRB samples were collected and measured for insulin and c-peptide by enzyme-linked immunosorbent assay. Moreover, verification of insulin produced by the differentiated Muse and non-Muse cells was revealed by flow cytometeric analysis. Gene expression levels were determined by RT-PCR by using the most characteristic genes of islets. To investigate the expression of pancreatic hormones, immunofluoresence analysis was performed for insulin and c-peptide in differentiated cells. Flow cytometeric analysis of the isolated adherent ASCs revealed that these cells expressed low levels of hematopoietic surface markers CD14, CD45, and CD34 and expressed high levels of mesenchymal surface markers CD90, CD105, and CD73. These results indicated that relatively purified ASCs was successfully isolated. The results of insulin release and c-peptide content which represents the mean of 6 donors can be summarized as following:- 1- The mean of insulin release was 0.008±0.0008 ng/μg/hr in Muse cells and 0.005±0.001 ng/μg/hr in non-Muse cells in response to 5.5 mM glucose concentration, 0.015±0.002 ng/μg/hr in Muse cells and 0.009±0.002 ng/μg/hr in non-Muse cells in response to 12 mM glucose concentration and 0.025±0.002 ng/μg/hr in Muse cells and 0.014±0.004 ng/μg/hr in non-Muse cells in response to 25 mM glucose concentration. 2- The mean of c-peptide release in the differentiated cells was 0.009±0.004 ng/μg/hr in Muse cells and 0.0045±0.0018 ng/μg/hr in non-Muse cells in response to 5.5 mM glucose concentration, 0.016±0.0027 ng/μg/hr in Muse cells and 0.008±0.0029 ng/μg/hr in non-Muse cells to 12 mM glucose concentration and 0.027±0.003 ng/μg/hr in Muse cells and 0.016±0.003 ng/μg/hr in non-Muse cells in response to 25 mM glucose concentration. These data indicated that there was a stepwise increase of the insulin content release by differentiated cells in response to increasing glucose concentrations. Flow cytometeric analysis revealed that 10.2±1.7% of Muse cells and 4.3±2.1% of non-Muse cells were positive for insulin expression. RT-qPCR of pluripotent markers was performed to confirm the pluripotency of Muse cells. The expression of pluripotent genes was multiplicated in Muse cells compared to non-Muse cells by 6-fold in nestin, 28-fold in nanog, 12-fold in oct3/4 and 60-fold in sox2 and 10.8-fold in PDX-1. At the end of differentiation protocol, the relevant endocrine genes, insulin, glucagon, PDX1 and somatostatin were expressed in Muse and non-Muse cells, the expression was multiplicated in Muse cells compared to non-Muse cells by, by 15-fold in insulin, 6-fold in PDX-1, 4-fold in glucagon and 4-fold in somatostatin, relative to human islet gene expression. The immunofluoresence analysis and immunocytochemical study for the differentiated Muse and non-Muse cells showed that 5.8±1.2% of Muse cells were positive for insulin, but the insulin was undetected in non-Muse cells. The co-expression of insulin and c-peptide was observed within the same cells by electronic merging. In conclusion, our findings present an evidence that adult stem cells contain a pluripotent subpopulation which is able to differentiate into IPCs .Immunofluoresence and Immunohistochemical studies showed a high positive staining percentage of insulin in differentiated Muse cells, also flow cytometeric analysis and expression of endocrine genes proved these findings. Also, we reported the down-regulation of SSEA-3 and pluripotent genes during in vitro passaging.