Inducing Differentiation Of Human Placenta Mesenchymal Stem Cells Into Islet β-like Cells By Four Protocols

Objectives:To isolate mesenchymal stem cells from human placenta and induce them to differentiate into isletβ-like cells by four protocols. Methods:1. Isolation, culture and identification of human placenta mesenchymal stem cells.Mesenchymal stem cells were isolated from human placenta by digestion of collagenaseⅡand adhesive screening method. Cell cycle and the expressions of cell surface antigens were detected by flow cytometry. RT-PCR detected the expression of SSEA-4. The cells were cultured in an adipogenic medium or in an osteogenic medium. After induction, the cells were observed by oil red O staining, alkaline phosphatase staining and Von Kossa staining.2. Differentiation of placenta mesenchymal stem cells into islet P-like cells.Protocol 1: MSCs from placenta were cultured in IMDM medium supplemented with p-mercaptoethanol (β-Me) and 5%FBS for 2 days. After digestion, cells were cultured in IMDM medium supplemented with Activin A, ATRA and 2%B27 for 2 days. Then cells were switch to IMDM medium supplemented with EGF, bFGF, FN and 3%FBS for 5 days. Finally, cells were cultured in DMEM/F12 medium supplemented with BTC,GLP-1, NIC and 3%-5%FBS for 7-9 days.Protocol 2: The procedures were similar to Protocol 1 .But Protocol 2 reduced the dose of EGF, and without FN and digestion.Protocol 3: The procedures were similar to Protocol 1.But Protocol 3 applied IMDM medium not DMEM/F12 medium in the final procedure.Protocol 4: Recombinant adenovirus vector carrying pdx-1 gene was constructed and transfected into 293 cell line to generate recombinant adenovirus (pAdxsi-CMV-Pdx1). Then hPL-MSCs were infected with pAdxsi-CMV-Pdx1.After digestion, cells were induced by EGF, bFGF, hepatocyte growth factor (HGF), BTC, GLP and NIC.After induction, the levels of insulin secretion, C peptide secretion and glucose-simulated insulin release test were examined by chemiluminescence immunoassay. The genes' expression related to isletβcells were detected by RT-PCR. PDX-1, insulin and C peptide in the treated cells were examined by immunocytochemistry and immumofluorescence method. The expressions of PDX-1 and insulin were examined by Western blot. 3. The effect of induced cells in type 1 diabetic mice.Type 1 diabetic mice were made by intraperitoneal injection of streptozocin. The induced cells were implanted into the right renal subcapsular space of diabetic mice. Blood glucose levels were monitored every 3 days. The right kidneys were removed at the 24th day after implantation and blood glucose levels were still monitored. At last, the pancreata and the grafts in the right kidneys were detected for immunohi stochemi stry. Results:1. Isolation, culture and identification of human placenta mesenchymal stem cells.Human placenta mesenchymal stem cells expressed CD44 and CD29, but didn't express CD34, CD45, CD106, CD14, CD40 and HLA-DR. 92.6% of cells was in G0/G1 phase. After induction, the cells were positive for oil red O staining, alkaline phosphatase staining and Von Kossa staining. And hPL-MSCs could express SSEA-4.2. Identification of islet (3-like cells.After induction, islet-like cell clusters formed. The genes' expression related to isletβcells were found by RT-PCR. The differentiated cells expressed PDX-1, insulin and C peptide, which were confirmed by immunocytochemistry, immumofluorescence method and Western blot.The chemiluminescence immunoassay demonstrated that the cells induced by Protocol 1 secreted much more insulin than that of Protocol 2 group (222.00±84.97mU/L vs. 72.00±27.75mU/L, P<0.05) , pAdxsi-CMV-EGFP group (222.00±84.97mU/L vs. 1.40±0.57mU/L, ,P<0.05) and MSC group (222.00±84.97mU/L vs. 0.08±3.03mU/L, P<0.05) . And protocol 3 secreted much more insulin than that of MSC group (226.00±73.35mU/L vs. 0.08±3.03mU/L, P<0.05) . The clusters induced by Protocol 4 secreted much more insulin than that of Protocol 1 group (722.00±179.36mU/L vs. 222.00±84.97mU/L, P<0.05) . The isletβ-like cells aquired by the four protocols stimulated index under 5.5mmol/L and 25mmol/L glucose for one hour was 4.69±2.18, 2.40±1.04, 3.49±1.70, 4.91±1.80, respectively.The clusters induced by Protocol 4 secreted much more C peptide than that of Protocol 1 group ( 3.380±2.310ng/mL vs. 0.447±0.381ng/mL, P < 0.05 ) , pAdxsi-CMV-EGFP group (0ng/mL, P<0.05) and MSC group (Ong/mL, P<0.05). 3. The effect of induced cells in type 1 diabetic mice.After differentiated cells transplantation, the blood glucose levels began to decrease. At the end of the experiment, the blood glucose levels reached to 6-8 mmol/L (Protocol 4 group) or 10mmol/L (Protocol 1 group). But the blood glucose levels of some mice emerged undulation during the declining course. The control groups remained hyperglycemia. When the kidneys that contain the differentiated cells were removed, the hyperglycemia reappeared. Immunohistochemistry showed the differentiated cells were positive for insulin. Conclusions:1. Mesenchymal stem cells can be isolated from human placenta. Human placenta MSCs express CD29 and CD44, but no CD34, CD45, CD106, CD14, CD40 and HLA-DR. Moreover, hPL-MSCs express SSEA-4, which is one of the specific markers of embryonic stem cells.2. They have the ability to differentiate into adipocyte and osteoblast in vitro.3. Four protocols all can induce hPL-MSCs to differentiate into isletβ-like cells. The protocol that pAdxsi-CMV-Pdxl combined with cytokines is more effective than other protocols. The differentiated cells induced by this protocol express insulin stably, and normalize the blood levels of diabetic mice.