Maintenance Of Rat Pancreatic Islet Functions For Transplantation By Co-culture With Mesenchymal Stem Cells

Background:Diabetes mellitus is a complex metabolic disease with an estimated worldwide prevalence of 171 million cases among the adult population and will up to 300 million cases in 2025 by World Health Organization (WHO). It becomes the third non-contagious disease after cardiovascular disease and cancer. It is associated with severe long-term micro-and macro-vascular complications, and carries high rates of morbidity and mortality. China’s population prevalence rate of diabetes mellitus has reached 5% now, similar to developed countries. Although it represents only 10% of the diabetic population worldwide, the ongoing increasing incidence of type 1 diabetes (up to 41/100,000 population/year in Europe and up to 25/100,000 population/year in North America) represents a major global health issue.Type 1A diabetes mellitus is a T cell-mediated, organ-specific autoimmune disorder leading toβ-cell distruction and lowered insulin production, characterized by the presence of anti-islet cell antibodies (currently the best criterion for a diagnosis of type 1A diabetes), severe insulitis and evidence of autoimmune destruction of pancreaticβ-cell.Standard treatment strategies for type 1 diabetes are based on different regimes of insulin injections with careful blood glucose monitoring. However, insulin therapy is based on severe hypoglycaemic episodes as it can not exactly mimic the physiology of insulin secretion. At the same time, it increases the risk of severe hypoglycemia also.Currently, the only way to achieve and maintain normal blood sugar level, while avoiding the risk of hypoglycaemia, is islet replacement therapy: pancreas transplantation and islet transplantation. Now, however,β-cell replacement therapies including pancreatic or islet transplantation have demonstrated partially successful results in reversing long-term renal and neural complications in selected type 1 diabetic patients. In the last 20 years, pancreas transplantation in the treatment of type 1 diabetes achieved good results. Meanwhile, research on islet transplantation has achieved remarkable results. First, the human islet transplantation for the treatment of diabetes has been reported successful:Shapiro et al. reported insulin independence in 21 out of 36(58%) islet-transplant recipients after 1 year of follow-up, of whom five maintained insulin independence for 2 years. In a 5-year follow-up study of 65 islet-transplant recipients, Ryan et al. reported a median duration of insulin independence of 15 months. In fact, approximately 10% of all patients maintain long-term insulin independence. Second, with the improvements in pancreas preservation, isolation rate of islet and reasonable treatments of anti-inflammatory and immunosuppressive, islet transplantation has achieved similar results with pancreatic transplantation; Third, the studies show that islet cells differentiated from stem cells can reverse diabetes, which suggested that islet transplantation has a potentially unlimited source of tissue.Islet transplantation has been shown to reduce blood sugar effective, to make the insulin-dependent diabetic patients become non-dependent, or to reduce the amount of insulin, and to prevent the patients who can not rely on insulin therapy from chronic complications of diabetes greatly. In addition islet transplantation also has advantages such as transplantation security, basically non-invasive, fewer complications and easily repeated. Islet transplantation is considered to be the final plan which can cure type 1 diabetes completely and prevent the progress of the complications.In order to maintain normal blood glucose and glycosylated hemoglobin levels, the amount of islet for transplantation should amount to at least 10% to 20% of the whole pancreas. Pancreatic islets of Langerhans comprise only 2% of the pancreatic cell mass, and they are difficult to isolate and purify from the massive exocrine pancreas tissue. Islet’s death and apoptosis were caused by damage during islet isolation and purification, longer time of isolation, no microvascular regeneration at the early stage of transplantation. Under normal circumstances, sufficient numbers of islets could be harvested from only half the donor pancreases in order to meet the quantity requirement of one clinical islet transplantation therapy. In its 2006 annual report, the Collaborative Islet Transplant Registry presented data from 225 islet-transplant recipients between 1999 and 2005. Nearly two thirds of the recipients achieved insulin independent (defined as no need for insulin administration for at least 14 days) during the year following transplantation, which dropped by one third over the second year.The major goal of future diabetes therapy is to promoteβcell regeneration, which could be accomplished byβcell selfreplication differentiation from progenitor cells with the use of stem cell therapy to over come autoimmunity and to improve endogenous insulin secretion.Researchers over the world are study the following aspects actively:①Obtain a sufficient quantity and quality of islets by improving the technology of islet isolation.②Immunosuppressive solutions without islet-toxic effection.③Using stem cell technology and islet xenotransplantation to overcome the shortage of the donor’s islet source. The amount of required islets for transplantation will be greatly reduced if the time of pancreatic islets in vitro can be extended and the good features kept. The treatment of diabetes will access to new development by overcoming the shortage of human islet donors.Mesenchymal stem cells (MSCs) represent an interesting therapeutic option due to their immunomodulatory properties and their potential for in vitro differentiation into insulin-secreting cells, therefore achieving the major therapeutic goals for type 1 diabetes.Friedenstein et al. first identified mesenchymal stem cells (MSCs) as a fibroblast-like cell population capable of generating osteogenic precursors, the mesenchymal stromal cells derived from the bone marrow are heterogeneous, stromal population of multipotent non-haematopoietic progenitor cells with the capacity to differentiate into multiple mesenchymal lineages, including bone, fat and cartilage. After more than 10 passages amplification in vitro MSCs can still maintain their pluripotency, and MSCs can be induced amplification in the context of injury, participating in tissue repair or regeneration reaction. MSCs can secrete a variety of hematopoietic and non hematopoietic growth factor, interleukin and chemokine, such as M-CSF, SCF, FIT-3 ligand, TPO, IL-6,7,8,11,12,14,15 and LIF et al. These cytokines play an important role in the hematopoietic stem cell proliferation and differentiation. At present, we can identify MSCs according to their morphology, antigenic phenotype and differentiation potential. MSCs express several cell surface antigens, such as CD73, CD90, CD29, CD44, CD106, CD120a, CD124. As MSCs are a non-haematopoietic cell line, they do not express haematopoietic markers such as CD34, CD 14, CD45, CD117 and And the surface markers closely related to graft rejection such as HLA-DR, B7-1(CD80), B7-2(CD86), CD40 and Fas apoptosis receptor. MHCs express intermediate level of major histocompatibility complex (MHC) classⅠmolecules on their cell surface, but not MHC classⅡ, properties that allow their transplantation across MHC barriers.This research (partⅠ) is focusing on the method to establish a method of isolation, subculture and amplified of Wistar rat marrow mesenchymal stem cells (BMSCs) fitting the requirement of ex-perimentation for further study.MSCs can induce immune tolerance. Research has shown that bone marrow mesenchymal stem cells have the same organizational identity which can suppress T cell activation to formate microchimerism between the receptor and the islet when United islet transplantation, so that recipients need not have long-term use of anti-rejection drugs to achieve long-term survival of transplanted islets.The death of islets, including the loss ofβ-cells during isolation and culture, is still a major obstacle for successful islet transplantation. The death of islet cells (including apoptosis and inactivation ofβ-cells) is mostly due to the hypoxia/nutritional deficiency-induced injury at the process of islet isolation, purification and culture. Therefore, in order to prevent the death and enhance the function of transplanted islet, many means to improve the cell culture conditions have been used.Recent researches indicate that MSCs can promote the repair of injured tissue or cells and maintain the normal function of tissue through cytokines, cell-cell contact or cell confluence. The potential nutritional effects of MSCs on the transplanted islets is not yet entirely clear currently. Although studies have confirmed that factors secreted by the MSCs, such as interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), can improve the quality of transplanted islet after transplantation. However, MSCs as feeder cells to produce trophic effects and mechanism of related molecular signal transduction cascade is still poorly understood.The effects about MSCs to extend survival time and protect function of islet for transplantation by being co-cultured with them were observed in this research (partⅡ) in order to do some exploration for preserving the function of transplanted islets effectively before islet transplantation.PartⅠIsolation, subculture and identity of Wistar Rat mesenchymal stem cellsObjectiveTo establish a method of isolation, subculture and amplified of Wistar rat marrow mesenchymal stem cells (BMSCs) fitting the requirement of ex-perimentation for further study.MethodsTo separate, subculture and amplified in vitro MSCs from bone marrow of Wistar rats by density gradient centrifugation combined with adherence culture. All MSCs were tested for their phenotypes by using FITC-conjugated mouse anti-rat CD90, CD45 monoclonal antibodies with flow cytometry. ResultsMSCs obtained from passages 3 used for this study showed CD90+(95%), CD45-(94%) by flow cytometry and their fluorescence intensity exhibition have significantly statistical difference.Conclusion and SignificanceIt is a simple and practical method to separate MSCs from the bone marrow of adult Wistar rats by means of density gradient centrifugation and adherence. MSCs can greatly proliferate in vitro and offer seed cells for further study.Part IIMaintenance of rat pancreatic islet functions for transplantation by co-culture with mesenchymal stem cellsObjectiveResearches indicate that mesenchymal stem cells (MSC) can not only differentiation cross over embryonic germ layers division under specified conditions but promote the repair of injured tissue or cells and maintain the normal function of tissue through cytokines, cell-cell contact or cell confluence. Our study is to observe the effect of MSC on prolonging the survival time and maintaining the function of islet in vitro by co-culture with rat’s islet.MethodsIslets of Wistar rats were isolated and purified by one-step method using Histopage-1077; Four groups were divided randomly:islet cultured alone (basic cultivation; high glucose cultivation), islet cocultured with MSC (basic cultivation; high glucose cultivation), with 15 holes in each group; The status and survival rate of islets in four groups were observed, The amount of insulin secretion was measured by enzyme-labeled immunosorbent assay (ELISA) and insulin stimulation index was calculated in the insulin release test.ResultsThe survival rate of islets in group coculture was significantly higher than that in group culture alone both in 7th and 14th day (P<0.01); The levels of insulin secretion and the insulin stimulation index in group coculture were also higher than in group culture alone in 7th day (P<0.01).ConclusionMSC can significantly prolong the survival time and maintain the viability of islets when coculturing islets with MSC.SignificanceMaintenance of rat pancreatic islet functions for transplantation by co-culture with mesenchymal stem cells was proved. It provides a new method to save the number and maintain the normal function of islet for clinical islet transplantation.