| Objective: We isolated and proliferated Human umbilical cord mesenchymal stem cells (hUC-MSCS) and rat pancreatic islet cells, then the hUC-MSCS were induced towards pancreatic islet cells by co-culture system in rat pancreatic islet cells micro-environment, to investigate the differentiation potential and functional trends of hUC-MSCS in vitro to differentiate into islet-like cells in rat islet cells micro-environment.Methods:1 The umbilical cord was abtained under sterile conditions from full-term fetus of cesarean section (with informed consent of their families). Then the umbilical cord envelope was cut between the two umbilical arterial blood vessels along the long axis, and blood vessels were removed. The mesenchymal stem cells (MSCS) were isolated from human umbilical cord Wharton's Jelly and cultured by tissue adherent culture method. Cell morphology and growth pattern were observed under microscope. Cell-surface markers were all analyzed by flow cytometry using the following antibodies: CD34, CD45, CD14, CD 29, CD44 and CD 105.2 In order to isolate rat islet cells, we used collagenase in situ perfusion digestion method and filter filteing, then the cells were identified by specific dithizone(DTZ) staining.3 The 3rd or 4th passage of hUC-MSCS in good condition were inoculated into the bottom of 6-well Transwell plates(the co-culture group) and the ordinary 6-well plates(the simple culture group) at a density of 1×105cells/ml. After adheresion, we seeded the rat pancreatic islet cell clusters with at a density of 50clusters/well to the Transwell co-culture plate inserts. Co-culture method was adopted to induce hUC-MSCS to differentiate into pancreatic islet-like cells. Invert microscope was used to investigate the morphologic changes of the cells. The early-stage marker of pancreatic- Pancreatic Duodenal Homeobox-1 (PDX-1) was identified by immunocytochemistry staining. We also quantitatively measured the secretion levels of insulin and C-peptide by radioimmunoassay to observe the secretion function changing trend of cells and their activity in glucose-stimulated experiment in the co-culture group and the simple culture group.Results:1 The hUC-MSCS isolated from human umbilical cord Wharton's Jelly were observed under microscope and showed the following changes: on the 7th day, we could see spindle-like cells appeared around adherent tissue; on the 14th day, the cells became homogeneous long spindle-shaped; on the 20th day, the cells could be proliferated. After passaging, the cells arranged parallel or spiral-shaped. The 3rd passage of hUC-MSCS identified by flow cytometry strongly expressed the surface markers of mesenchymal cells CD 29, CD44 and CD105, did not express hematopoietic lineage markers CD 34, CD45 or CD14.We proliferated hUC-MSCS to the 10th passage, the flow cytometry results were conformed to the 3rd passage.2 The pancreatic islets, which were obtained from the normal SD rats weighing 250-300g, were separated by collagenase in situ perfusion digestion method and filter filteing and stained brownish red by DTZ.3 Under microscope, we saw the hUC-MSCS induced in the co-culture group showed the following changes in the induction process: on the 3rd day after induction, the cells gradually became elliptical; on the 7th days after induction, hUC-MSCS formed in cluster and stained brown by DTZ; on the 10th day after induction, the cells clusters became loose and irregular, and on the 14th day, the islet-like cell clusters decreased both in amound and volume. Cells in simple culture group still grew in a long spindle-shaped with no obvious changes.4 The expression of PDX-1 tested by immunocytochemistry was positive in the cells, which were induced in the co-culture group on the 3rd and 7th day after induction, while on the 10th day and the 14th day, the expression of PDX-1 was rare, and in the simple culture group, PDX-1 was negative.5 We detected insulin and C-peptide in the supernatant of co-culture group by radioimmunoassay. But in the simple culture group, we hardly detected any C-peptide, but a few of insulin. In the statistical analysis performed by SPSS13.0, we can see the insulin secretion levels were all have statistical differences between the co-culture group and simple group on the different day after induction (p<0.01). In the co-culture group, the secretion levels of insulin between the 3rd and 14th day after induction had no statistical differences (p>0.05), but compared with the 7th and 10th day, they all have statistical differences (p<0.01). The secretion level of insulin was the highest on the 7th day after induction. On the 10th day, the secretion level was justly inferior to the 7th day, but was obviously higher than the 3rd day and 14th day. The secretion trend of C-peptide was similar to the insulin. In the co-culture group, we saw the secretion levels of insulin and C-peptide were both incremental at first and then decreased along with the time.6 The cells cultured on the 7th day in the co-culture group and the simple culture group were both stimulated by different concentrations of glucose. In the co-culture group, the secretion level of insulin was 19.6025±5.9516μIU/ml in the serum-free culture supernatant of L-DMEM (1000mg /dl), and in the H-DMEM(4500mg/dl), the insulin secretion level was 27.6617±7.14825μIU/ml. In the simple culture group, the secretion level of insulin was 5.8000±1.006μIU/ml in supernatant of L-DMEM (1000mg /dl), and in the H-DMEM(4500mg/dl), the secretion level was 6.2100±1.1746μIU/ml. We saw the insulin secretion level increased obviously with the augmentation of glucose concentration in the co-culture group (p<0.01), the stimulation index of the islet-like cells was 1.4372±0.1390. But in the simple culture group, the insulin secretion level had no statistical differences (p>0.05).Conclusion:MSCS isolated from human umbilical cord Wharton's Jelly can be induced into islet-like cells in the micro-environment of pancreatic islet cells in vitro, and their insulin secretion function appeared to increase firstly and then decrease along with the time of culture in vitro.
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