Research On Induction Of Insulin Producing Cells From Mesenchymal Stem Cells

BackgroundDiabetes mellitus (DM) is one group of metabolic diseases characterized by high blood glucose level resulting mainly from hyposecretion of and tissue resistance to insulin. With improvement in economics and changes in life style, there will be approx.114million patients with diabetes mellitus in China by2014. DM requires constant and daily based medical treatment in order to control hyperglycaemia and related complications. At present, commonly used-insulin sensitization agent and exogenous insulin administration can only temporarily alleviate hyperglycemia condition, increase sensitivity of the target organs to insulin, but could not cure diabetes. Rencently, Islet transplantation has been tried in clinic and showed some clinical benefits. However, limited treatment efficacy due to short survival rate of transplanted islets and host immune rejection, and limited source of donors hinders developing an islet transplantation-based treatment approach for DM.Application of the insulin producing cells instead of islets seems to be an attractive approach for DM therapy in the future. In recent years, previous studies demonstrated that embryonic stem cells (ESC) could be in vitro induced to differentiate into insulin producing cells (IPC) in a culture system containing multiple cytokines and/or genetic modification, and transfusion of these IPCs reduced blood glucose level in diabetes animal models. However, to apply ESC-derived IPCs in clinic is still a long way to go because of its ethical issues and risk to develop a teratoma. Therefore to identify new sources of stem cells for IPC are urgently required.Mesenchymal stem cells (MSCs) are one type of adult stem cells having characteristics of self-renew and multi-directional differentiation capacity. Except from their differentiation activity, MSCs are endowed with potent immunosuppressive capacity, by which MSCs may excert therapeutic effects on auto-immune diseases and diabetes mellitus. Moreover, studies have demonstrated that bone marrow derived-MSC (BM-MSC) and adipose derived-MSC (AD-MSC) are able to differentiate into IPCs under suitable in vitro culture conditions. Howerve, their differentiation capacity are relative limited and also there were age-related issues in these kinds of MSCs. Furthermore, in vitro studies have also demonstrated that adiposed-derived MSCs support cancer cell growth while umbilical cord tissues derived MSCs not. In contrast, umbilical cord tissues are "wastes" of normal delivery, therefore ethical issues can be avoided. Moreover there was no risk of teratoma. Apparently, umbilical cord tissue is a good source of MSC for induction of IPC differentiation.Laminin is a heterotrimer glycoprotein that contains alpha, beta, and gamma chains. At least19laminin isoforms have been identified nowadays. Laminin is named according to its subchains (e.g., laminin411comprises the a4, β1, and γ1chains). Laminin is a key component of the basement membrane, and is involved in the structural scaffold, cell proliferation, and differentiation. Jiang, et al found that laminin111promoted the differentiation of fetal mouse pancreatic beta cells, whereas Leite et al reported that laminin could induce the expression of islet cell markers in the hepatic oval cells in vitro. Laminin was recently shown to promote the differentiation of hTERT-over-expressed human BM-MSCs into IPCs by activating Akt and Erk signals.In this study, we sought to induce human umbilical cord Wharton’s jelly derived MSCs (UC-MSC) into IPCs using laminin without any gene manipulation. In our culture system, insulin release increased significantly after a high concentration of glucose stimulation, and the yield of IPCs was much higher compared to that using published differentiation methods. In the animal tests, fasting blood glucose (FBG) levels declined rapidly following the administration of laminin-induced IPCs and glycosylated haemoglobin (HbAlc) level was significantly reduced70days after transfusion of IPC with laminin than IPC and MSC groups.Materials and MethodsThe culture of the UC-MSC and identificationAcquired the ethical approval from the Ethics Committee of Second Hospital of Shandong University, after signed informed consent in donors, we collect maternal health cesarean delivery of umbilical cord tissue, treated with collagenase and trypsin digestion, to obtain mesenchymal stem cells (MSC), and then made in a large number of amplification in vitro. MSCs were successfully confirmed by morphology, flow cytometry analysis and identification of multi-directional differentiation potential according to minima cretiaria of International Society of Cytotherapy in2006. MSCs can be applied to clinical treatment in accordance with GMP conditions.The IPC differentiation and improvement of published protocolsThen refer to the published literature, the differentiation of IPC was induced in presence of insulin transferrin (ITS), nicotinamide, with a four stages of differentiation. The IPC differentiation was confirmed by cell morphology, DTZ staining, RT-PCR, immunofluorescence, and Western blot. And then we explored adding laminin for further improvement of IPC differentiation. The mRNA and the protein level of IPC was confirmed again using RT-PCR, Western blot, immunofluorescence chemistry, and flow cytometry to determine the effect of adding laminin. And insulin release assay was also used to test the maturation and effecacy of adding laminin in the process of differentiation.Animal experimentIn the in vivo experimental section, after approved by the Ethics Committee of Second Hospital of Shandong University, we injected STZ intraperitoneal directly destroy the β cells of Wistar rats to make animal model of type1diabetes Wistar rats. After successfully established animal model of type1diabetes, infusion cells for cytotherapy group (normal saline control group, the group treated with infusion of MSC infusion treatment group, IPC and add laminin differentiation of IPC in treatment group, etc.), and observed the different processing of IPC effect on the treatment of diabetic rats, including clinical symptoms, animal weights, levels of fasting blood glucose level, glycosylated hemoglobin, survival analysis, etc.Clinical trialsTo evaluate safty and treatment effects of UC-MSC on type2DM, we have carried on one clinical trial (NCT01413035). From July2009to October2012,18patients with type2DM were enrolled the clinical trial from the Department of Endocrine and Metabolic diseases in the Second Hospital of Shandong University. Diagnoses were made based on the diagnostic criteria of World Health Organization (WHO). The study protocol was approved by the Ethical Committee of the Second Hospital of Shandong University. Informed consent according to the Declaration of Helsinki was obtained from every participated patient. The average dose of cell infusion was1.8X106MSC/kg for each patient.ResultsUC-MSCs are adherence to plastic flasks in accordance to the standards of The International Society for Cellular Therapy (ISTC). UC-MSCs were high expression CD73, CD90, CD105, CD166and low expression of HLA-DR, CD34, CD45. UC MSC has a multi-directional differentiation potential under the appropriate differentiation condition in vitro. MSCs could differentiate into adipocytes, osteoblasts and chondrocytes.UC-MSC can also be differentiated to IPCs under appropriate conditions, have been verified by multiple methods, it has insulin secretion function in vitro. Insulin secretion in vitro can reach189.8±93.05μIU/ml. And in the mRNA and protein levels, high expression of islet β cells associated genes (such as Pdxl, Insulin, etc.). Adding appropriate concentration of reconstituted human laminin, can significantly promote the generation and mature of IPCs, compared with no added group. In the mRNA and protein level, adding laminin could significantly improve the gene expression of Pdxl, Ngn3, Pax4and insulin. In the insulin releasing test in vitro, under the environment of high glucose stimulation, IPCs with laminin could secrete large amounts of insulin, insulin secretion in vitro can reach612.1±40.78μIU/ml in adding laminin411group, while laminin511adding group insulin secretion in vitro can achieve:561.6±31.81μIU/ml, add laminin group were more than three times higher than not.Laminin411and laminin511effectively induced IPC differentiation from MSCs, and upregulated both mRNA insulin expression and protein levels. The expression of genes known to govern insulin expression such as Pdxl and Ngn3were robustly induced by laminin411and laminin511, which suggested that Pdxl and Ngn3functioned as signaling pathways in the regulation of insulin expression and IPC differentiation induced by laminin. Administration of laminin induced-IPCs (laminin-IPCs) rapidly and significantly down-regulated fasting blood glucose level, and markedly improved the symptoms and survival of T1DM rats compared with controls.In animal experiments, adding laminin compared to MSC infusion group, the survival time, blood glucose levels and levels of weight are obviously improved. Add laminin411IPC infusion set, can significantly reduce the blood glucose level rapidly at0.5week after transfusion. Add laminin511IPC infusion treatment group, could effective control of blood glucose for longer periods of time.In the clinical trials, after six months of follow-up, found no obvious side effects, the overall treatment efficiency is as high as44.4%.Of clinical trial results, we found that after MSC infusion treatment, the fasting blood glucose and postprandial blood glucose levels can alleviate effectively.ConclusionsUC-MSC can be differentiated into mature insulin producing cells in vitro under the appropriate condition in the presence of laminin. IPC significantly induced IPC maturity and large amount of insulin secretion in vitro. In animal experiments, adding laminin differentiation of IPC, animals can successfully control blood glucose, ameliorate the clinical symptoms, and improve survival. Our results revealed that laminin411and511could induce Pdxl and Ngn3expression both at mRNA and protein levels, which provides a clue of how laminin increases the maturation of IPCs. Mature pancreatic (3-cells are also Pdxl positive. Our current protocols of IPCs differentiation are to form Pdxl+expressing pancreatic progenitors, indicating that laminin may play essential roles in affecting the yield of IPCs.Laminin acted as a potent differentiation inducer of IPCs from MSCs derived from umbilical cord tissue. Laminin induced differentiation through the Pdxl and Ngn3signaling pathways. Laminin-IPCs significantly reduced blood glucose level and improved the survival of T1DM rats. Our novel finding suggested the potential clinical use of laminin-IPCs in the treatment of T1DM. However, further studies on the biosafety of these cells in animal models are warranted before a clinical trial can be launched. In addition, the safety and efficacy of laminin-IPCs in human diabetes patients should be investigated.To conclude, laminin411and511significantly and efficiently induced differentiation of IPCs from MSCs derived from human cord tissues, and these IPC produced large amount of insulin through Pdxl and Ngn3signalling pathways. Moreover, IPC with laminin transfusion resulted in a long-term therapeutic effects on T1DM. Our findings here highlight a crucial role of laminin in the induction of IPCs differentiation from human MSC and a potential clinical use of IPCs with laminin in the treatment of T1DM as well as type2DM in the future.