Human Fetal Bone Marrow Mesenchymal Stem Cells And Treatment Of Mouse Diabetes

Diabetes mellitus has already been the third human illness following cardiovascular disease and tumor. At present, the treatment method of diabetes is mainly injection of insulin. However, exotic insulin can not keep blood glucose level constant and can not prevent from occurrence and development of the complications, such as diabetes-nephropathy, diabetes-coronary heart disease and diabetes-eye disease, etc. The one ideal procedure of clinical treatments for diabetic patient with serious complications was the transplantation of pancreas islets. Unfortunately, islet transplantation has historically been hampered by immune rejection and the scarcity of donor islets. Bone marrow mesenchymal stem cells (BMMSCs) may derive from a patient with diabetes. If the BMMSCs from a patient with diabetes are induced to differentiate into pancreatic islet-like cells and transplanted into patient, the diabetes may be treated without limitation from immune rejection and scarcity of islet donor. This study is to isolate BMMSCs of human abortuses at age of 2～3 months, in vitro induce the BMMSCs to differentiate into pancreatic islet-like cells with extract of fetal pig's pancreas and other factors, and transplant the pancreatic islet-like cells into STZ-induced hyperglycemic nude mice model for treatment of diabetes. This study provides support for continuing efforts aimed at utilizing BMMSCs as a steady and renewable source of autologous insulin-producing cells for transplantation in patients with diabetes.1 Study on biological properties of human fetal bone marrow mesenchymal stem cellsFetal BMMSCs between embryonic stem cells and adult stem cells, especially BMMSCs from fetus at age of 2～3 months, closer to embryonic stem cells, may be more ideal seed cells for human tissue engineering and regeneration medicine than their adult counterparts.This study was to isolate BMMSCs from human abortuses at age of 2～3 months by scissoring their long bones lengthwise, followed by rinsing and culturing whole marrow cells. Basic medium and serum concentration for BMMSCs culture were optimized and growth curves made both with MTT reduction assay. Isolated cells were identified with flow-cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and tumor forming experiment. The differentiating ability of isolated cells was demonstrated by inducting to neurocytes and cardiomyocytes. The results showed that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful to recover the BMMSCs from human abortuses at the age of 2～3 months, in the present experiment,α-MEM+20%FCS was the best culture system for the BMMSCs in vitro, the third passage BMMSCs expressed Oct4, SSEA3 and SSEA4 beside the surface markers of their adult counterparts, the population doubling time of the BMMSCs of passage 6, 12 and 24 were 34,36 and 40h respectively, the cells of passage 6, 12 and 24 showed diploid karyotype and formed no tumor in the nude mice, and the BMMSCs possessed very strong ability of differentiation into neurocytes and cardiomyocytes. The BMMSCs of human abortuses at the age of 2～3 months proved by all of above results to have a part of biological properties of embryonic stem cells and be biologically safe and ideal seed cells for researches on human tissue engineering and regeneration medicine.2 In vitro transdifferentiation of marrow mesenchymal stem cells into pancreatic islet-like cells(1)The BMMSCs of human abortuses at the age of 2～3 months passed through domestication culture with high glucose medium and inducing with a 4-step inducement system. Half of them gradually formed ball-like cell clusters with 148.6±63.4(n=986) cells each and 125.84±53.45μm (70～180μm) diameter, which resembled very closely islets of Langerhans. Islet-like clusters and a part of adherent cells were stained into crimson with DTZ. The induced cells were demonstrated by RT-PCR and immunocytochemistry to express multiple genes related to pancreatic endocrine cell function (proinsulin, Glut-2, glucagon, somatostatin and pancreatic polypeptide), and demonstrated by radioimmunoassay(RIA) to secrete insulin and C-peptide upon glucose challenge. Islet-like clusters secreted more insulin than adherent cells. Under stimulation of 5, 10 and 20mmol/L glucose for 30min, the induced cells secreted 25.99±11.07, 89.95±21.65 and 320.66±30.73μIU/ml insulin respectively,which were remarkably higher than control group (P<0.01) and remarkably different each other (P<0.01).(2)In 4-step inducement system, all of steps and factors were useful to differentiation of BMMSCs into islet-like cells. The first step was to induce BMMSCs for 1h with H-DMEM containing 4mmol/Lβ-mercaptoethanol, which promoted differentiation of BMMSCs into Nestin+- cells. If the first step lacked, the insulin secretion of induced cells decreased 35%.The second step was to induce BMMSCs for 4～6 d with H-DMEM containing 10ng/mL EGF, bFGF and HGF,2%B27 and 4% extract of fetal pig's pancreas, which promoted proliferation, aggregation, clustering and differentiation of Nestin+- cells into islet-like cells. If the second step lacked, the insulin secretion of induced cells decreased 61%. The third step was to induce BMMSCs for 4 d with H-DMEM containing 10ng/mL EGF and HGF, 10mmol/L nicotinamide, 25μmol/L Zinc acetate, 2%FCS and 4% extract of fetal pig's pancreas, which promoted further differentiation of Nestin+-cells into islet-like cells, and tightening, becoming round and mature of islet-like clusters. If the third step lacked, the insulin secretion of induced cells decreased 63%. The fourth step was to induce BMMSCs for 2 d with L-DMEM containing 10nmol/L exendin-4 and GLP-1, 25μmol/L zinc acetate, 2%FCS and 4%extract of fetal pig's pancreas, which promoted maturation of pancreatic incretion cells and synthesization of proinsulin, and enhance sensitivity ofβ-cells to glucose. If the fourth step lacked, the insulin secretion of induced cells decreased 47%. If anyone among HGF, extract of fetal pig's pancreas, bFGF, EGF, zinc acetate dehydrate, nicotinamide, exendin-4 and GLP-1 lacked, insulin secretion of induced cells remarkably decreased (P<0.01).3 Transplantation of pancreatic islet-like cells for treatment of diabetes mellitus Pancreatic islet-like cells labeled with CM-DiI were transplanted into the right testis of STZ-induced diabetic mice model, and right testis of a part of mice were removed after one month to observe change of blood glucose level and examine whether transplanted cells existed and secreted insulin. The result was as followed:(1)Molding 20 Balb/c male nude mice received intraperitoneal injections of 50mg STZ/kg once a day for five consecutive days. Beginning from 72h following the last STZ injection, fasting blood glucose level was determined using a standard blood glucose meter (Sure StepTM Plus, LIFESCAN) once a day for three consecutive days. If three fasting blood glucose levels were all higher than 18mmol/L, the mouse would be used to experiment. After STZ injection, three fasting blood glucose levels of 17 of 20 nude mice were all higher than 18mmol/L and successful rate of molding was 85%(17/20).(2)Cell labeling BMMSCs and induced pancreatic islet-like cells were incubated in the working solution of CM-DiI for 5 minutes at 37℃, and then for an additional 15 minutes at 4℃. After labeling, the cells were washed with phosphate-buffered saline(PBS),resuspended in fresh medium,and observed with a microscope.The labeling rate of the cells was 100%.(3)Treatment of diabetes 17 hyperglycemic mice were randomly divided into 3 groups, test group(7 mice), control group(5 mice) and blank group(5 mice). Labeled pancreatic islet-like cells (1×105), BMMSCs(1×105) and PBS(same dosage) were transplanted into the right testis of test, control and blank groups respectively. After transplantation, hyperglycemia of the test group(7 mice) lowered to normal level within 2 weeks and their weight slightly increased. After one month of transplantation, the testis of 3 mice in the test group were removed, blood glucose levels of the mice went up to former level(>18mmol/L), their weight decreased in line, and all of them died within 30d after operation. The blood glucose of the remainder 4 mice in the test group maintained normal levels. Hyperglycemia of control and blank group did not lower, their weight gradually decreased, and all of them died within 60d after transplantation.(4) Examination of grafts Dewaxed sections of nude mice testis in test group were observed with Confocal Microscope. Red fluorescent light mainly distributed to area surrounding seminiferous tubules. The area surrounding seminiferous tubules appeared green fluorescent light after the dewaxed sections were stained with anti-insulin monoclonal antibody and FITC Goat anti-mouse IgG, indicating that transplanted pancreatic islet-like cells distributed to area surrounding seminiferous tubules and produced insulin.