The Experimental Study On Rat Bone Marrow-Derived Insulin-Producing Cells Autotransplantation In The Duodenal Wall For Treating Diabetes

Diabetes mellitus (DM) is currently one of major diseases threatening the health of humanity, particularly the elderly people, and has become the fourth-largest disease led to the death of the world's population. The current global diabetes has exceed 250 million people, and could reach to 380 million people in 20 years. All over the world every 10 seconds 2 people were diagnosed with diabetes, and one person died of diabetes-related diseases.DM is a chronic non-communicable diseases caused by lack of insulin or insulin resistance in vivo. It can be divided into type I and type II diabetes. The causes of type I diabetes mellitus are that, at the basis of genetic susceptibility genes and on the role of environmental factors, the body's own immune dysfunction are triggered which resulted in pancreaticβ-cell damage and destruction and the absolute lack of insulin secretion.Intensive insulin therapy is generally the best option for the treatment of diabetes mellitus. However, this therapy does not avoid serious microvascular complications. Therefore, pancreas and pancreatic islet transplantation are considered to be an effective approach for the treatment of diabetes. Currently, a limited supply of donor pancreatic islets and the risk of immunological rejection prevent widespread use of these approaches. In recent years, many researchers have tried to identify a substitute to pancreatic islet cell transplantation. Recently more and more attention has been paid to the study on stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types. According to their capacity of differentiation, stem cells have been divided into three major groups: totipotential stem cells, multipotential stem cells and unipotential stem cells. Unipotential stem cells, that is adult stem cells, exist in a variety of tissues and organs, have the self-replication capacity and can differentiate into cells of the host organizations to supplement the loss of physiological activity of cells or to repair damage in order to maintain the stability of the organization environment. It is believed that not only embryonic stem cells can differentiate into different organizations precursor cells, which develop into fuctional cells of the corresponding tissue, but also adult stem cells in specific conditions can trans-differentiate into other tissues. Because they can be easily harvested and, when autologously transplanted, there is no immunological rejection and ethical issues have broad application prospects.Researches have found that pancreatic ductal epithelial cells, hepatic oval cells and so on have characteristics of adult stem cells. However, the limited sources and self-renewal proliferation ability of these cells and a fewer of insulin-producing cells (IPCs) obtained after induction of these cells prevented it from becoming a seed cells for cell therapy of diabetes mellitus. Bone marrow contains hematopoietic stem cells and bone marrow-derived mesenchymal stem cells. The studies found that BMSCs in vitro and in vivo in different microenvironment could be induced to differentiate into multiple cells. Because of BMSCs with easily isolation, culture and in vitro amplification, and autologous transplantation without immune rejection characteristics, so the utilization of transdifferentiation potential of BMSCs, they can be induced to differentiate into specific tissue cells to repair damaged organization or organs.Many sites have been evaluated for engraftment of pancreatic islet cell; however, most of the experimental sites have no clinical applicability. The liver has been considered as the most adequate site. However, it is increasingly recognized that the liver has physiologic and anatomic characteristics that may contribute to the death of transplanted islets and technical difficulties limit the introduction of sufficient numbers of islets into the portal circulation. Bendayan and Park reported that typical islets of Langerhans are located in the connective tissue between the duodenal crypts and the muscle layer. They also found that islets of streptozotocin (STZ)-induced diabetic rats, mainly containing glucagon cells, were present in the duodenal mucosa and the insulin cells in the islets had disappeared. Moreover, the intestinal submucosa produces various growth factors, such as fibroblast growth factor-2, transforming growth factor and vascular endothelial cell growth factor, which are beneficial for cell growth. A recent study demonstrated that human islets maintain a higher level of function in vitro when cultured in processed submucosa In another study, improved islet function was observed when islets were transplant into a submucosal site. Therefore, we designed this study and the purpose of the study is to explore the potential of stem cells autotransplantation as a therapeutic strategy for diabetes mellitus. This study is a series which consists of three parts as follows: isolation, culture and induced differentiation of BMSCs in vitro; establishment of the rat model for experimental diabetes; aiutotransplantation of insulin-producing cells in the duodenal wall for treating diabetes. The paper was publicated in "The Anatomical Record".Part one: Isolation, culture and induced differentiation of BMSCsIsolation, culture and expanding of BMSCs in vitro are the premise of the total experiment. There are a few of BMSCs in bone marrow ( about 2-5 BMSCs per 10⁶ mononuclear cells), so it is important to expand and purify the BMSCs for the following experiments. BMSCs were isolated from adult rats using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for expanding. Colonies of BMSCs were formed after 4 days at primary culture, after about 7 days, BMSCs got together and the number of division cells decreased. At a confluence of 90%, cells were resuspended with 0.05% trypsin-0.02%EDTA and seeded into fresh flasks. The BMSCs proliferated fast, and could be subcultured about every 5 days. The adherent, spindle-shaped BMSCs at passage 3 were harvested and analyzed by fluorescence-activated cell sorting. The result indicated that ralatively purified BMSCs were obtained after 3 generation.On the basis of the successful isolation and culture of BMSCs, we carried out an experiment on transdifferentiation of BMSCs into insulin-producing cells in vitro. After repeated experiments, we have successfully explored a best proposal of differentiation of BMSCs into insulin-producing cells: At passage 3, rat BMSCs were cultured in medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml epidermal growth factor (EGF) and 2% B₂₇ at a concentration of 1×10⁵ cells/ml. After 6 days, cells were cultured in a medium containing 10 ng/ml hepatocyte growth factor (HGF), 10 ng/mlβ-cellulin , 10 ng/ml activinA, 10 mmol/L nicotinamide and 2% B₂₇ for another 6 days. The medium was replaced every 3 days. Cells cultured in medium without an inducer were used as controls. After 6 days, there was a slight change in cell shape which are mostly round , a small number of cells were polygonal or long shuttle type, the refractive index significantly increased, and began to appear in small clusters of cells; 12 days after induction, the structures of cell clusters were similar to those of isolated islets. RT-PCR analysis revealed that these IPCs could express Ins1, Ins2, glucagon, glucose transporter-2 and pancreatic duodenal homeobox-1 (Pdx-1). Insulin production by the IPCs was confirmed by immunocytochemistry and Western blot analysis. The BMSCs in vitro are differentiated into functional isletβcells by compound inducer involved in present experiment which provide a cell source for cell autologous transplantation in the treatment of diabetes.Part two: Establishment of the rat model for experimental diabetesAccording to the typical pathophysiology of diabetes mellitus, diabetes mellitus was introduced by a single intraperitoneal dose (60 mg/kg) of streptozotocin dissolved in citrate buffer (pH 4.4) into 12 h-fasted rats, whereas control rats received only citrate buffer. After administration, polydipsia, weight loss and polyuria were obseved in STZ treatment group. The results of fasting blood glucose test showed that 3d after injection, the blood glucose levels in STZ treatment group were significantly higher than that before administration (P<0.01), while blood glucose levels in the control group before and after administration have no significant difference (P>0.05). 1w , 2w, 3w, and 4w after STZ treatment, the blood glucose levels bagan to elevate and remained to exceed 16.7mmol/L, whereas blood glucose levels in control group remained normal and there are significant difference in two groups (P<0.01 ). In the STZ-treated group, body weight decreased, whereas body weight in control group kept the increase, and there are significant difference in two groups (P<0.05). The animals in each group were humanely killed at 2 weeks and 4 weeks after STZ treatment. Pancreas were harvested for histologic examination. HE staining showed that pancreatic exocrine cells without abnormality could be seen and inflammatory cell infiltration was not found. Immunohistochemical staining for insulin demonstrated that normal pancreaticβcells existed in section of the control group, whereas there was a fewer of insulin-positive cells in the STZ-treated group. This animal model will provide the basis for further studies on pathophysiology and new strategy for stem cell transplantation for the treatment of the disease.Part three: Autotransplantation of insulin-producing cells in the duodenal wall for treating diabetes.After BMSCs were isolated from femur and tibia in diabetes rat model, the donor rats supplying the BMSCs were made diabetic. Insulin-producing cells differentiated from BMSCs by the utilization of a compound medium were autologously engrafted into the duodenal wall. Control group rats received only medium without cells. The blood glucose levels in cell-implanted group that received the cellular implantation gradually decreased and reverted to the physiological range after 1 week. However, blood glucose levels in control group which underwent sham surgery without cellular implantation remained elevated (P<0.01). Intraperitoneal glucose tolerance test-induced responses were observed in the cell-implanted group similar to that in the normal rats. 2, 4, and 8 weeks after transplantation, immunohistochemical staining for insulin indicated that insulin-positive cells were frequently observed in the cell-implanted group, and most transplanted cells had migrated into the submucosa of the duodenum and some had migrated into the mucosa. With time transplanted cells gradually gathered to form cell clusters similar to isolated islets. These results indicated that insulin-producing cells differentiated from BMSCs could survive in the duodenal wall, and reversed the body's hyperglycemia.ConclusionIn summary, we successfully set up an diabetes rat model, isolated, cultured, in vitro expanded and identified BMSCs, and induced BMSCs to differentiate into insulin-producing cells and then autotransplanted IPCs into the duodenal wall. It showed that the engrafted IPCs could survive and reversed hyperglycemia of rats. These results on autologous transplantation of IPCs derived from BMSCs into duodenal wall could offer a novel potential therapeutical protocol for diabetes mellitus.