Bone Marrow Mesenchymal Stem Cells Induced To Differentiate Into Islet-like Cells In Vitro Experimental Study

Background and Objective In the bone marrow the self-renewal and pluripotent stem cells exist. Some studies showed that these stem cells could differentiate to tissues or cells of endoderm, mesoderm and ectoderm, such as liver cells, myocardial cells, muscle cells, nerve cells, adipose cells and insulin-producing cells et al. The aim of this study is to cultivate and isolate the rat bone marrow mesenchymal stem cells (BMSCs), and to investigate the biological characteristics of BMSCs by flow cytometer techniques, and to establish the basis for further study of differentiation of BMSCs to insulin-producing cells.Materials and methods After Wister rats were anesthetized to die, the rat bone marrow were douched out from shin bone and thigh bone by L-OMEM from one end under the asepsis condition. The bone marrow was blown lightly and softly to single cell suspension. After centrifuged and washed, the bone marrow cells were culture at a density of 1×10⁸/mL in 25cm² plastic culture flask under the condition of 37℃, 5% CO₂, and saturated humidity. When bone marrow cells were cultured for 48h, the culture fluid was changed for the first time and the non-adherent cells were discarded. After this, the culture medium was changed every two or three days and observe the cell morphologic change until the cells reached 80% confluence. Then the cells were detached from the culture flask walls by trypsin solution and replated. The expression of cell surface molecules CD29,CD90 and CD45 of the third generation of the cells were measured by flow cytometer techniques.Results In the first 48h, generous round cells were watched and were suspension growth. After cultured for 48h, some adherent cells could be watched. Along with the changed of medium the round and suspended cells decreased and adherent cells increased obviously at the fifth day. The adherent cells grew aggregately and exhibited a spindle and polygon shape. After about 10-14days the adherent cells bespreaded the flask wall and the shape of the adherent cells aggregate was like fish-clump or whirlpool. The morphology of the adherent cells showed a spindle-shape. The BMSCs were positive for CD29 (91.9%) and CD90 (51.3%) and negative for CD45 (6.9%).Conclusion In this study the rat bone marrow mesenchymal stem cells were successfully isolated and cultured by adhering to the culture plastic and isolated from the full bone marrow cells. Objective To observe the plasticity of human bone marrow mesenchymal stem cells(hBMSCs) differentiate into insulin-producing cells in medium contained high glucose, GLP-1, activin A, nicotinamide and BTC respectively in vitro.Materials and methods Human BMSCs were cultured in vitro and the cell morphologic change was observed until the cells reached 80% confluence, then the cells were detached from the culture flask walls by 0.1% trypsin solution and replated at a density of 2×10⁵/mL in 96-well plates. The hBMSCs were cultured in 23mmol/L of high glucose DMEM medium for 20 days, then the hBMSCs were divided into 7 groups: A group inducing with high glucose(23mmol/L); B group inducing with GLP-1(5nmol/L), C group inducing with activin A(10ng/ml); D group inducing with nicotinamide(10mmol/L); E group inducing with BTC(10ng/ml); F group inducing with all stimulating factors; G group inducing with common L-DMEM culture. All groups cultured for 16 days. The condition of the common L-DMEM was as control. The culture medium was changed every two or three days until the cells reached 80% confluence, then the cells were made to grow on cover glass. Insulin levels in the culture medium were measured by radioimmunoassay. Insulin mRNA of hBMSCs was examined by RT-PCR. The differentiated cells were examined for the expression of insulin, glucagons, somatostatin and nestin. To examine the insulin secretion on the condition of high concentration of glucose, the differentiated cells were plated in 24-well plates at 3×10⁵ cells per well and cultured ovemight. On the next day, the old culture medium was collected and the cells were washed by PBS for three times and then were cultured in L-DMEM for 1h. After that, all groups of the cells were cultured in 23mmol/L high glucose medium for 2h and the culture medium was collected at Omin, 30min, 1h and 2h for examining insulin concentration. The treated-cells were treated with acid alcohol overnight at -20℃and then were disrupted by the ultrasonic disrupter. The intra-cellular insulin was examined by radioimmunoassay and the total protein by Bradford technique.Results 1. Before induced hBMSCs were adherent, hBMSCs exhibited a spindle or polygon shape and transparent. The shape of the adherent cells aggregate was like fish-clump or whirlpool. The insulin level of before-induced hBMSCs (cell density 1.0×10⁶/ml ) was 2. 2867±0. 2665μU/ml (blank control). The insulin level of common L-DMEM culture was 2. 3650±0.4296μU/ml. There was no difference between the two groups (P=0.359＞0.05). 2. In this study we stimulated hBMSCs with high concentration of glucose (23mmol/L) for 20 days, and then were cultured with high glucose, GLP-1, activin A, nicotinamide, combined stimulating factors, BTC and common L-DMEM respectively for 16 days, at the end of the study, the insulin content of the differentiated cells increased 3.13, 3.00, 2.35, 2.94, 2.84, 3.22 and 2.32 fold respectively compared with before-induced hBMSCs ( Insulin content was 2.2867±0.2665μU/10⁶ cells) . Combined treatment with all stimulating factors did not result in the additive effects. 3. Insulin mRNA of hBMSCs was examined by RT-PCR and the results showed that insulin gene was expressed in GLP-1 and activin A group. The combined treatment group and BTC group could express two specific fragments, 900bp and 300bp.Notable exception was the 900bp fragment that was sequenced. Its sequence was different from human proinsulin gene with many mutations. So its character could not be confirmed now. Insulin mRNA exhibited obscurely in nicotinamide group. Insulin mRNA was absent in high glucose group and common L-DMEM group. 4. Immunocytochemistry confirmed that all stimulating groups were positive for insulin except of common L-DMEM group. Glucagon was negative in all stimulating groups. Except combined treatment group, somatostatin was negative in all stimulating groups. Only nicotinamide group and combined treatment group could express nestin weakly. 5. The results of high glucose-insulin secreting test showed that the insulin content increased to a certain extent in all stimulating groups. 6. Intra-cellular insulin content was highest in activin A, BTC and high glucose-common LDMEM group, and was lower in other groups.Conclusion In this study, human bone marrow mesenchymal stem cells were induced to differentiate into islet-like cells with high glucose, GLP-1, activin A, nicotinamide and BTC in vitro. High glucose, GLP-1, activin A, nicotinamide and BTC could induce hBMSCs into insulin producing cells, which was confirmed by radioimmunoassay, RT-PCR, immunocyto- chemistry. Combined treatment with all of the stimulating factors did not result in the additive effects.