Abstract
The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF). hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet transplantation.
Highlights
Type I diabetes is an autoimmune disease characterized by the destruction of islets
The primary antibodies for human insulin, human von Willebrand Factor, and the Dylight 488-conjugated secondary antibody were purchased from Abcam (Cambridge, MA)
Real Time RT-PCR data have shown that expression of these markers was not affected by the adenovirus transduction suggesting that human bone marrow derived mesenchymal stem cells (hBMSCs) can be used as gene delivery vehicles without affecting their stemness (Figure 2A)
Summary
Type I diabetes is an autoimmune disease characterized by the destruction of islets. Transplantation of human islets has been clinically used to achieve insulin independence. In addition to long-term graft rejection, clinical success of islet transplantation is limited by primary non function (PNF) of islets [1]. PNF is characterized by poor glycemic response due to b-cell death in the first two weeks after transplantation [2]. There is marked b-cell death immediately after transplantation due to hypoxia and exposure to inflammatory cytokines released by infiltrating immune cells [3]. Human islets upon exposure to inflammatory cytokines show increased expression of apoptosis markers [4]. Interleukin-1 beta (IL-1b) produced by islets and macrophages plays a key role in the apoptosis of islets after transplantation [5]. Interleukin-1 beta (IL-1b) produced by islets and macrophages plays a key role in the apoptosis of islets after transplantation [5]. hIL-1Ra competitively blocks IL-1b binding and has been shown to minimize the detrimental effect of IL-1b on islet viability [5,6]
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