The Study Of Getting Insulin Producing Cells Both Morphologically And Functionally From Human Bone Marrow MSCs Under Microenvironmental Manipulation In Vitro For Transplantation

BackgroundDiabetes mellitus (DM), which affects millions of people worldwide, is characterized by abnormally high levels of glucose in blood caused by either absolute (type 1 DM) or relative (type 2 DM) insulin deficiency due to the destruction of pancreaticβ-cells by T cells of the immune system or decreased insulin sensitivity respectively. In both types, an inadequate mass of functional islet cells is the major problem for the onset of hyperglycemia and the development of overt diabetes. Islet cell replacement is considered as the optimal treatment recently. However the deficiency of transplantable donor islets has historically hampered its further development.Lots of researchers have made great effort to expand pancreatic islets by stimulating endogenous regeneration of islets or generating islet-like cells from various sources of stem cells including embryonic stem cells (ESCs), multipotent stem cells within nonendocrine compartments of the pancreas, and various tissue precursor cells, such as hepatic oval cells, splenocytes. However even with these inspiring experimental advances, some obstacles, such as immune rejection, limited sources of stem cells from these organs, still remain.In the last decades, a new type of multipotent stem cells in adult bone marrow was reported. Bone marrow (BM) is a complex tissue containing two major stem cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Both of them are thought to be multipotent and can be induced to several cell types. Moreover BM-MSCs even can be induced to differentiate into ectodermal neural and epidermal-like cells and endodermal hepatic cells or maybe even other cell lineages from all three germ layers under appropriate experimental conditions. Comparing with other stem cells mentioned above, human BM-MSCs have several obvious advantages: it can be expanded in culture for many doublings, which provides a potentially unlimited source; it has a simple reliable and repeatable procedure to obtain, which makes autogenic transplantation come true and alleviates the major limitations of availability and allogenic rejection simultaneously. Most importantly, there is no need to consider the ethical problems, the major problem of ESCs. In the light of the actual worldwide diabetes epidemic, the generation of new insulin producing cells (IPCs) from human BM-MSCs would offer a particularly useful candidate for deficient donor islets.In the last 3 years, several studies have discussed the possibility of adult MSCs in BM to differentiate into IPCs with totally different induction protocols. However these articles mostly focus on murine or rat derived BM-MSCs, theβcell differentiation potential and characteristics of human BM-MSCs are poorly documented. Recently there have been three inspiring reports on human BM-MSCs expressing insulin under genetic modulation, and in the present study, we demonstrate that, under defined culture conditions, human BM-MSCs positive for the stem-cell marker Oct-4 express a series of pancreas developing markers including ISL-1, Beta2/NeuroD, Nkx6.1,Glut2, CK18 and CK19 without any induction, and can be induced to form islet-like aggregates morphologically. These aggregates can secrete insulin in a glucose related manner and express upregulated pancreatic developmental genes especially Nestin and PDX-1. Most importantly, after transplanted under the renal capsule of mice, the differentiated cells can decrease the blood glucose and rescue the diabetic mice.The 1st part: Isolation and cultivation of human BM-MSCs and biological features of the derived cellsObjective: To establish a method for isolation, cultivation and expansion of adult human BM-MSCs, and study their biological features and potential to form carcinomaMethod: 2-3 ml of heparinized adult bone marrow from nonleukemia persons were washed with PBS, and purified by Percoll gradient. Mononuclear cells at the Percoll interface were collected and seeded in expansion medium consisted of DMEM-LG supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The BM-MSCs were purified by their adhesiveness. The changes of cell's morphology were observed by phase contrast microscopy every day, and their expansion fashion at passage 3 was revealed by growth curve. Their expressions of stem cell marker Oct-4 were examined by RT-PCR, and cell surface markers were detected by Flow Cytometry. Their differentiation potential was examined by adipose induction, and their potential to form carcinoma were detected by soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.Result: Both primary and passage human BM-MSCs showed typically fibroblast- like morphology and had maintined highly proliferative capacity. The cells expressed mRNA of Oct-4, were negative for CD45, CD34, CD14, HLA-DR, and positive for D44, CD29, CD105. The cells showed adipose differentiation after 21 days' induction and no potential to form carcinoma both soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.The 2nd part: Expression of pancreas developing markers on undifferentiated human BM-MSCsObjective: To detect the expression of pancreas developing markers on undifferentiated human BM-MSCs in both mRNA and protein level. To reveal their potential to differentiate into insulin producing cells.Method: The mRNA expressions of pancreas developing markers including ISL-1,Beta2/NeuroD,Nkx6.1,Glut2,PDX-1,Nestin,CK18 and CK19 were detected by RT-PCR. The protein expressions of Glut2,ISL-1,CK18 and CK19 were further examined by Western Blot and immunocytochemistry.Result: Undifferentiated human BM-MSCs were positive for mRNA of ISL-1, Beta2/NeuroD, Nkx6.1, Glut2, CK18 and CK19. Western Blot showed that derived cells expressed CK18 and CK19, but did not express ISL-1 and Glut2. Immunocytochemistry confirmed most cells were positive for CK18 and CK19, and a small portion of cells were positive for ISL-1 and Glut2. The 3rd part: Differentiation of human BM-MSCs to insulin producing cells under microenvironmental manipulation in vitro.Objective: To found a three-stage induction protocol by microenvironmental manipulation to induce human BM-MSCs to differentiate into insulin producing cells in vitro. To reveal the possibility of human BM-MSCs to differentiate into islet-like cells without gene manipulation.Method: Human BM-MSCs at passage 3-5 were reseeded at a destiny of 2-3×10~4 cells per cm~2, and were treated sequentially by three-stage induction protocol composed of high glucose, bFGF, nicotinamide, EGF, exendin-4, B27, N2, HGF and activin-A et al. The changes of cell's morphology were observed by phase contrast microscopy every day. The mRNA and protein expression profiles of islet related markers were examined by RT-PCR and Western Blot respectively at the end of every induction stage. After three-stage induction, DTZ staining and immunocytochemistry were carried out to examine the insulin expression. Electronic microscopy was used to show the ultrastructure characteristics of differentiated cells and in vitro glucose stimulation was carried out for cells without induction, cells at the end of the second stage and cells after the entire induction.Results: Induced human BM-MSCs were found to form typical islet-like aggregates and ultrastructure characteristics of matureβcells were proved by electronic microscopy. They were also activated or upregulated to express multiple genes related to pancreaticβcell development and function (PDX-1, Nestin, pax-6, Glut2, ISL-1 and insulin). Insulin c-peptide and glucagon production were identified by immunocytochemistry. Insulin expression was further confirmed by DTZ staining and in vitro glucose stimulation which certified as a glucose related manner.The 4th part: Transplantation of insulin producing cells differentiated from human BM-MSCs can control diabetes of STZ induced diabetic nude mice.Objective: To investigate the in vivo effect of insulin producing cells differentiated from human BM-MSCs by microenvironmental manipulation on diabetes controls in nude mice Method: The diabetic nude mice were achieved by STZ (220 mg/kg) injection as blood glucose level was higher than 18mmol/l. Treated and nontreated human BM-MSCs were transplanted under the renal capsule of diabetic nude mice respectively. Blood glucose levels, weight and survival time were monitored for 30 days after transplantation. The graft status was examined by HE staining 2 days after transplantation and at the end of the observation period. Immunocytochemistry was carried out to detect the insulin expression.Result: Xenotransplantation of insulin producing cells from human BM-MSCs can decrease the hyperglycemia and prolong the survival time of STZ induced diabetic nude mice.Conclusion:1. Human BM-MSCs can be isolated and purified by Percoll gradient combined with adhesiveness selection. Derived cells can be cultured stably for 12 passages and expanded quickly.2. Expanded human BM-MSCs have the stem cell characteristics. They are positive for stem cell marker- Oct-4, and have the potential to differentiate to adipose cells.3. Expanded human BM-MSCs expresse several pancreas developing markers, indicating that they have the potential to differentiate into insulin producing cells.4. Human BM-MSCs can be induced into insulin producing cells in vitro by microenvironmental manipulation with a three-stage protocol. Differentiated cells have islet-like morphology and function.5. Differentiated cells transplantation can control diabetes of STZ induce nude mice, indicating human BM-MSCs can be another source of functionalβcells for diabetes treatment.