Genetically Modified Human Bone Marrow Derived Mesenchymal Stem Cells for Improving the Outcome of Human Islet Transplantation

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.

本研究旨在探究人骨髓间充质干细胞（human bone marrow derived mesenchymal stem cells, hBMSCs）作为基因载体以提升人胰岛移植疗效的潜力。研究人员首先对hBMSCs的表型标记物表达情况进行鉴定，并通过Adv-hVEGF-hIL-1Ra转导hBMSCs，使其过表达人血管内皮生长因子（human vascular endothelial growth factor, hVEGF）与人白细胞介素1受体拮抗剂（human interleukin-1 receptor antagonist, hIL-1Ra）。随后将人胰岛与过表达hVEGF和hIL-1Ra的hBMSCs进行共培养，采用膜荧光法与葡萄糖刺激试验检测胰岛活性。将转导后的hBMSCs与人胰岛共同移植至糖尿病NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ（NSG）小鼠的肾被膜下，定期检测小鼠血糖水平随时间的变化，以验证经基因修饰的hBMSCs的治疗效果。实验终点时，对移植有胰岛的肾脏组织切片进行免疫荧光染色，检测胰岛素与血管性血友病因子（von Willebrand Factor, vWF）的表达。流式细胞术检测结果显示，hBMSCs阳性表达CD73、CD90、CD105、CD146及Stro-1表面标记物。通过干细胞标记物的mRNA表达水平以及转导后hBMSCs的成脂分化能力验证，腺病毒转染并未影响hBMSCs的干性及分化潜能。Adv-hVEGF-hIL-1Ra可高效转导hBMSCs并使其过表达hVEGF与hIL-1Ra。活死细胞染色与葡萄糖刺激试验结果表明，转导后的hBMSCs可提升胰岛对抗细胞因子混合物的存活率。将人胰岛与经基因修饰的hBMSCs共同移植后，糖尿病NSG小鼠的血糖控制情况得到改善，该结论通过平均血糖水平与腹腔葡萄糖耐量试验得以证实。肾脏组织切片的免疫荧光染色结果显示人胰岛素与vWF呈阳性表达。综上，本研究结果证实，hBMSCs可作为基因载体与滋养细胞，提升胰岛移植的治疗效果。