Study On Mesenchymal Stem Cell And Insulin Producing Cell Therapy For Diabetes Mellitus

Backgrounds:Diabetes mellitus (DM) have higher morbidity rate and the complications, which seriously threat human health. The main treatments are oral hypoglycemic medications and/or insulin, but the patients need therapy for long-term, even lifetime, which has severe medication side effects. But the treatment can not fundamentally solve the problem. In recent years, many reports have confirmed that application of hematopoietic stem cell or islet transplantation for the treatment of type 1 diabetes was effective. Although the treatment has a certain effect, but we have taken notice of the high cost, high risk, and long-term use of immunosuppressive agents for the patients of islet transplant. Thus, to study a safe and long-acting method for the treatment of diabetes and its complications is an urgent need.Studies have shown that type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are closely related to the immune and inflammatory response or damage. Therefore, effectively inhibiting or blocking the immune injury can protect (3 cells from damage, maintain the efficacy of pancreas islet function and correct insulin resistance. Under certain conditions, Mesenchymal stem cells (MSC) can differentiate into pancreas islet β-like cells to secrete insulin. In addition, MSCs have other founctions for a tissue repairing, promoting angiogenesis, antioxidant effects, correcting metabolic disorders and insulin resistance. Thus, MSCs play an important role in the pathogenesis of multiple aspects of diabetes. The sources of MSC are wide. MSCs derived from bone marrow and adipose tissues have a high possibility of viral pollution, ability of differentiation decreased with age and possible shortcomings for promoting tumor growth. Umbilical cord derived mesenchymal stem cell (UMSC) has the same ability of multipotent differentiation, regulation of hematopoiesis and immune support as bone marrow and adipose derived MSC (BM-MSC and AD-MSC). Because of its wide variety of sources, easily obtained, without any ethics restrictions, strongly differentiation potential and proliferative capacity, low immunogenicity, rarely virus infection and no adverse effects on the donor, UMSC will become an important source of cell therapy instead of BM-MSC and AD-MSC.This study established the culture system of UMSC, and registered clinical trials (NCT01413035) of UMSC for treatment of diabetes mellitus in the NCBI Clinical Trial web. Based on the above framework, this study observed the safety, clinical effect and mechanisms of UMSC for the treatment of DM through a larger sample and in-depth study. The research result will guide the rational application of UMSC for the treatment of DM.Islet transplantation may be one of ways to cure diabetes mellitus. Because of limitation of donor source, survival period in vitro, as well as immune rejection after transplantation, its clinical application was greatly difficult. Therefore, looking for other sources, such as insulin producing cells (1PC), for replacing islet transplantation to treat DM becomes the research focus. The study was expected to provide a theoretical basis on the effective treatment for DM.The research advances of the stem cells provided the possibility for the differentiation of the stem cells into IPC in vitro. Embryonic stem cells (ESC) were totipotent stem cells that could be induced to differentiate into IPC. IPC derived from ESC played a hypoglycemic effect in vivo, but it may have some limitation of a tumorigenic and ethical problems. Induced pluripotent stem cells (IPS) can be induced to differentiate into IPC under certain conditions, but the generation of IPS needed help of virus vectors and there was also the risk of tumorigenicity. Human pancreatic stem cells can also differentiate into IPC, but the source and phenotype of pancreatic stem cell is not clear and its culture is very difficult. So, it is particularly important for looking for a safe and efficient stem cell to differentiate into IPC. UMSC can not only be induced into IPC, but has the effect of immunity suppression to reduce damage of islet β cells and insulin resistance. So UMSC become the ideal cells for IPC. Under the condition of current technology, IPC derived from UMSC have some shortcomings of the low differentiated degree and less insulin secretion which can not adapt the future need for large-scale clinical application. Thus, how to improve the differentiative efficiency from UMSC to IPC and increase insulin secretion of IPC are one of the key factors for clinical application.Laminin is a form of extracellular matrix proteins with a variety of biological functions. Laminin can promote expression of insulin gene and secretion of insulin on islet β cells. The mainly subtypes of laminin with this feature are laminin 111, laminin 411 and laminin 511, which are expressed on endothelial cells of capillaries around islets. The study found that there was a distinct bilayer structure of basement membrane in human islets, suggesting that laminin 111, laminin 411 and laminin 511 may have a regulatory function of differentiation from MSC to IPC. Based on above study, we optimized a differentiation system according to the traditional method. During differentiation, we added laminin 411 to observe differentiation efficiency and insulin secretion of IPC. The study will provide a theoretical basis on further enhancing differentiation efficiency from UMSC to IPC and insulin secretion of IPC.This study consisted of three parts:the first part was a clinical study on the UMSC therapy for T2DM and its complications; the second part discussed the differentiation and regulation from UMSC into IPC; the third part was next work plan. As follows:The first part:Clinical study on umbilical cord mesenchymal stem cell therapy for type 2 diabetes mellitusObjective:The aim of this study is to evaluate safety and potential therapeutic effects of UMSC on T2DM.Materials and methods:Acquired the ethical approval from the Ethics Committee of the second hospital of Shandong University, after signed informed consent in donors, we collected umbilical cord tissue of healthy puerpera with cesarean delivery. According to the standard operation procedure, UMSC was isolated, cultured and amplificated in the GMP Lab. Then UMSC was successfully confirmed by morphology, flow cytometryanalysis and identification of multi-directional differentiation potential according to the criterion of International Society of Cytotherapy in 2006. From July 2009 to October 2012,18 patients with T2DM were enrolled according to the inclusion and exclusion criteria. UMSC was intravenously transfused 2-3 times for interval 2 weeks. All patients were followed up in the first, third and sixth month after UMSC transfusion. Age, gender, diabetes duration and medications as well as weight, height and BMI were recorded. Fasting plasmaglucose (FPG), postprandial blood glucose (PBG), HbA1c, C-peptide andsubsets of T cells were measured. All adverse reactions were carefully documented. An effective criterion was made and data was analyzed using SPSS 19.0 software.Result:UMSC was successful obtained. UMSC has following characteristics:(1) fusiform fibroblast-like cells, adherent growth and stable passage; (2) high expression surface markers of CD73, CD90, CD105 and CD166, low or no expression surface markers of HLA-DR, CD34, CD45 and CDllb; (3) multipotent differentiation potential, which can be differentiated into osteoblasts, chondrocytes and adipocytes under appropriate conditions. According to the evaluation standard of curative effect, patients were divided into effective group and ineffective group. There was no statistical significance (p>0.05) in the clinical baseline characteristics between the effective group and ineffective group. FBG and PBG of the patients in effective group were significantly reduced (p<0.05) after UMSC transfusion. Plasma C-peptide levels and regulatory T cell (Treg) in the effective group were numerically higher after UMSC transfusion. However, the difference of both parametersdid not reach significance (p>0.05). Only 4 out of 18 patients (22.2%) had slight transient fever. Up to 6 months after UMSC transfusion, all patients continued to have a feeling of well-being and were physically more active.Conclusion:UMSC transfusion is safe and well tolerated, effectively alleviates blood glucose, and increases the generation of C-peptide levels and Tregs in a subgroup of T2DM patients. This pilot study provides fundamental for further study of UMSC transfusion on control of blood glucose as well as morbidity of T2DM in a larger cohort.The second part:Differentiation and regulation of umbilical cord mesenchymal stem cells into insulin producing cellObjective:To observe laminin411 affect efficiency of differentiation of UMSC into IPC, and insulin secretion of IPC sources from UMSC, which provide a clinical basis for IPC.Materials and methods:The protocol of isolation, cultivation, expansion and identification of UMSC is the same as the first part. After four stages, UMSC has been differentiated into IPC (UMSC-IPC), and the expression of cell surface molecules, mRNA, protein levels, islet cell morphology, insulin secretion and other aspects of the induced cells were identified by flow cytometry, RT-PCR, Western blot, DTZ staining, immunofluorescence detection methods. During the induction process, laminin 411 was added and laminin 411-IPC was induced and differentiated from UMSC. Firstly, efficiency of differentiation and insulin secretion of laminin 411-IPC were observed in vitro and mechanisms were further studied. Secondly,60mg/kg streptozotocin (STZ) was given through wistar rats intraperitoneal injection for destruction of pancreatic β cell, which can established animal models of T1DM. Rats were intravenously infused UMSC, UMSC-IPC and laminin 411-IPC. The clinical symptoms, blood glucose levels, C peptide changes, weight changes, survival analysis were observed to assess therapeutic effect of UMSC, UMSC-IPC and laminin 411-IPC on diabetic rats.Result:Under the premise of successful acquired UMSC according to GMP standard, UMSC was successfully induced to differentiate into IPC through four stages in the role of insulin-transferrin (ITS), nicotinamide and other factors on 2 weeks. The IPC has the morphology of islet β cells, insulin secretion (189.8 ± 93.05) μIU/ml, high expression of islet P cell markers (such as Pdxl, Insulin, etc.). In the process of induction, laminin 411 was added. The generation capacity and insulin secretion of laminin 411-IPC were increased significantly and insulin secretion of laminin 411-IPC increased more than three-fold to (612.1±40.78) μIU/ml. As the same time, laminin 411 can significantly increase the expression on mRNA and proteins of Pdxl, Ngn3, Pax4, insulin on IPC. The mRNA expressions of Soxl7 and Foxa2, which were the decisive endothelial marker genes, on Laminin 411-IPC group were significantly higher than UMSC-IPC group (p<0.05). The correlation analysis showed that the mRNA expressions of insulin, Pdxl and Foxa2 were positively related to secretion (p<0.05). At the animal level, the rat model of T1DM was successfully constructed through intraperitoneal injection of STZ. The fasting blood glucose in diabetic rats after Laminin 411-IPC infusion for 3 days was significantly lower than that of UMSC-IPC infusion (p<0.05). Laminin 411-IPC group could significantly reduce the level of HbAlc compared with UMSC-IPC group (p<0.05). Compared with the UMSC group, laminin 411-IPC group could significantly decrease the level of HbAlc (p<0.01). Laminin 411-IPC and UMSC-IPC groups could obviously improve the survival rate of diabetic rats and increase the weight, but there were no significant difference between the two groups. In addition, obviously adverse reactions were not been found.Conclusion:UMSC can differentiate into IPC under suitable conditions, and laminin 411 can effectively improve the degree of differentiation and increase the secretion of insulin of generated IPC. Laminin411-IPC can obviously reduce blood sugar in diabetic rats, improve the clinical symptoms and prolong survival. Most important of all, IPC infusion is convenient, safe and reliable. The study lays the foundation on IPC replace pancreatic β cells for future large-scale clinical applications in the treatment of diabetes mellitus.The third part:next work1. Clinical ResearchOur early work is to evaluate safety and potential therapeutic effects of UMSC on T2DM. UMSC transfusion is safe and well tolerated, effectively alleviates blood glucose, and increases the generation of C-peptide levels and Tregs in a subgroup of T2DM patients. The therapeutic effect was partly effective in the short-term (within six months). To further evaluate the long-term effect, we will continue to observe the current cases by follow-up. For currently less clinical cases, the next step we will actively apply for the national stem cell clinical research base. And the therapeutic effect will be verified by large-scale clinical trials. Then we will further explore the mechanism of UMSC effective treatment of diabetes and complications. The aim is to provide a theoretical basis for the clinical application of UMSC.2. Fundamental Research(1) To search for new drugs improve conversion efficiency from UMSC to IPC and enhanced the insulin secretory function of IPC in vitro, which can provide a new and efficient ways to satisfy future large-scale clinical application.(2) How to reduce the immunogenicity of IPC and avoid implanted IPC from attack by immune system is the focus of future research. We will solve these problems in the following ways:1) Using UMSC derived IPC:Since UMSC has a low immunogenicity, we presume that UMSC-IPC has also a low immunogenicity to reduce the damage by body’s immune system.2) Whether UMSC and IPC combined transplantation can further reduce the immune system attack to IPC.3) In order to completely solve the problem of immune rejection, autologous UMSC was used to different into IPC and improve the conversion efficiency and secrete insulin function of IPC.(3) Consistent with the problems with islet transplantation, IPC transplant also need to select the ideal transplant way. The next step, we will compare the impact of different methods, such as intravenously transfusion, subcutaneousu injection and intramuscular injction, etc, on blood glucose and survival period. At last, we will find the best transplant way to treat effectively diabetes mellius.