University of Khartoum

In-Vitro Differentiation of Mesenchymal Stem Cells to Pancreatic Islet Β- Like Cells with In Vivo Insulin Secretion

In-Vitro Differentiation of Mesenchymal Stem Cells to Pancreatic Islet Β- Like Cells with In Vivo Insulin Secretion

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Title: In-Vitro Differentiation of Mesenchymal Stem Cells to Pancreatic Islet Β- Like Cells with In Vivo Insulin Secretion
Author: Ismail, Nihad Elsadig Babiker
Abstract: Background: Regenerative medicine has recent advances in stem cell research. The last couple of decades has shown glimpses of the exciting potential of stem cells in tissue regeneration as well as in the restoration of diseased organs and systems but the stem cell therapy is not yet considered as a routine form of clinical treatment. The ideal stem cell population should be present and accessible in abundant numbers, harvestable by a relatively non invasive procedure, able to differentiate into a variety of cell lineages and easy to transplant to an autologous or allogeneic host. Research studies on stem cells in Sudan have not yet being well established. The aim of the study is to establish stem cells research in Sudan by transferring the technology for isolation and differentiation of human mesenchymal stem cells (MSCs) from cord blood and adipose tissue. Further the isolated MSCs were differentiated in vitro into islet β- like cells, and then the therapeutic effect of islet β-like cells was determined in vivo using experimental animals. Material and Methods: This is an experimental study conducted at the National Center for Neurological Sciences (NCNS), Khartoum, Sudan. Cord blood and adipose tissue samples were processed for MSCs isolation and differentiation.For adipose tissue, enzymatic and density centrifugation was used to isolate MSCs. Ficoll-Hypaque gradient density protocol was used to isolate MSC from the Cord blood. An inverted microscope was used for identification of isolated MSCs and then followed for 32 weeks. After the process of isolation, 3-step protocol was used to differentiate MSCs into islet β- like cells. Eight albino Wister rats were injected intraperitoneally by sterptozotocin (STZ) solution to induce type 1diabetes mellitus. The diabetic rats were divided into two groups 4each. One group for determination of the therapeutic effect and the other was control.The test group was injected intraperitoneally with the differentiated islet β- like cells and the blood glucose monitored every day for 7 days. Results: Small size growing cells with plastic adherent characteristic appeared in the first week. The characteristic shape of MSCs was detected three weeks after culturing. The phenotype of these cells showed positivity of CD13 in cord blood while CD34, CD45 and HLADR were negative. In the adipose sample CD34 and CD13 were positive while CD45 and HLADR were negative findings that supported real MSCs. Isolated MSC differentiated into islet β- like cells showed no changes of MSCs morphology in step1, while in step 2 decreasing of proliferation rate was noted and the MSCs shape changed into short and round. In step 3 clusters of islet -like cell were formed. The differentiated cells were positive for diathizone stain and displayed positive imuno-reactivity to antihuman insulin antibody. ELIZA results of insulin secretion by islet β- like cells in high glucose concentration medium showed positive result with >3.5 u/ml scale reading. The in vivo result of diabetic rats, the response in the test group occurred after 24h, one week later the glucose level decreased from 400mg/dl to 96mg/dl (P=0.021), while in the control group the glucose raised up to 600mg/dl. Conclusion: In this studywe successfully transferred the technology for isolation and differentiation of MSCs from CB and ADSCs and open the door for establishing stem cells research in Sudan. Also these MSCs can differentiate into islet β- like cells in vitro and function as insulin producing cells both in vitro and in vivo, however these cells are promising source of stem cells for β- cells regeneration. Finally we recommend more researches with a large number of samples using different sources of stem cells and further works is required to examine the curative effects of islet β- like cells on the human.
Description: 72page
URI: http://khartoumspace.uofk.edu/123456789/26480


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