| Objective At the present stage, the main materials used in the study of stem cells derived from embryonic stem cells and bone marrow mesenchymal stem cells. But there are some limitations those cells, For example:traumatic, ethical, etc. Therefore, it is urgent to find a new type of stem cells in order to avoid the above-mentioned problems such as ethics controversy, the lack of sources and the potential of tumor formation and so on. The main purpose of this research is to study the acquisition and culture, proliferation and identification the of menstrual blood-derived mesenchymal stem cells in vitro. The morphology and growth of cells were observed. And in vitro induction of osteogenic and adipogenic differentiation were also observed. To investigate whether the differentiation of menstrual blood-derived mesenchymal stem cells (MenSCs) into endometrial epithelial and stromal cells is feasible following the exposure to various concentrations of estradiol under in vitro conditions.It is to lay a foundation for its application in clinical practice.Methods In this experiment, MenSCs were isolated using Ficoll density gradient method, which from menstrual blood of the second day of the menstrual cycle in healthy females. The classical MTT method was used to describe the growth curve of the second generation cells in vitro. The morphology and the changes of the dynamic proliferation of cells were observed under the inverted optical microscope. The expression of the immune phenotype was detected by flow cytometry in second passages.MenSCs of the second generations were divided into two groups:the induction group and the control group. Respectively, cells were inoculated by 5* 104/mL into 6 well plates by gelatin coated.After 24 hours,Cells adherent to the wall were replaced by osteogenic induction medium and FBS, penicillin-streptomycin,200 umol/L ascorbic acid,10 mmol/L p-glycerophosphate and 0.1 mol/L dexamethasone.In the control group, the culture medium was changed by L-DMEM and 10% FBS. Cells were cultured in 37℃ and 5% CO2 cell culture box. The culture medium was changed every two days. The cells were cultured for three weeks. Respectively, At the end of 1,2 and 3 weeks, By detecting the expression of ALP, collagen and calcium nodules, the effect of bone induction was observed. The grouping method is same as above. Cells were inoculated by 5* 104/mL into 6 well plates by gelatin coated.After 24 hours, Cells adherent to the wall were replaced by adipogenic induction medium and FBS,1* 10-6 mol/L dexamethasone,10 mol/L insulin,0.5 mmol/L IBMX,0.2 mmol/L indomethacin. In the control group, the culture medium was changed by L-DMEM and 10% FBS. Cells were cultured in 37℃ and 5% CO2 cell culture box. The culture medium was changed every two days. The cells were cultured for four weeks.At the end of 2,3and 4weeks.Oil red O stain was carried out and the results of oil red staining were observed under microscope. By detecting the induction of MenSCs into osteogenic differentiation and adipogenic differentiation.To explore the potential of osteogenic and adipogenic differentiation in vitro.MenSCs from healthy women’s menstrual blood were cultured. The culture media contained growth factors [TGF-β (10 ng/ml); EGF (10 ng/ml); PDGF-BB (10 ng/ml)]. Cultured MenScs were divided into four groups based on the specific concentrations of 17 β-estradiol, they were exposed for five days; 1 X 10-9 mol/L (group 1),1 X 10-8 mol/L (group 2),1×10-7 mol/L (group 3) and 1 X 10-6 mol/L (group 4). A control group was maintained in parallel where MenSCs were cultured in a medium without growth factors and estradiol. Immunocytochemistry was used to characterize the incubated cells by testing the expression of specific endometrial epithelial and stromal cell markers; cytokeratin (CK) and Vimentin (VIM). Furthermore, Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed to detect the mRNA and protein expression levels of CK and VIM, respectively, to further characterize the differentiated MenSCs.Results by using Ficoll density gradient centrifugation, The results of this study showed that the stem cells could be isolated and cultured from menstrual blood of healthy female. This primary cell is approximately 1-2weeks up to 85%-90% fusion. The cells were spindle shaped, whirlpool, and radiation. By MTT method, the cell doubling time was about 30-32h.After passage, the cells could grow stably, and the morphology of the cells was dominated by fiber like cell morphology. By passages, cells were seeded 1 to 2 days, the cells proliferation slowly, to 3-4 days, the cells grow rapidly after medium was changed. To 5-6 days, which entered the logarithmic growth phase, The results showed by flow cytometry, cells showed that it is positive of CD29, CD44, CD90 and CD105.CD34, CD45, CD14, HLA-DR were negative, low expression of HLA-ABC. This indicates that the cultured cells are stem cells. That cultured cells into stem cells. Induced differentiation experiment showed that the expression of collagen in the early induction group was higher than that in the later stage of MenSCs induction group. MenSCs after two weeks of induction by alizarin red staining showed obvious calcium nodules. In induced group, ALP activity - osteoblast differentiation and maturation of the important signs along with the induction time was increased, strong positive. It is negative in the control group. After 4 weeks, there were obvious lipid droplets, and the oil red O staining was positive, and the control group was negative.Menstrual blood-derived cells showed clonal growth, in which more than 90% of cultured cells were MenSCs identified by flow cytometry. After culture for 5 days, most cells differentiated from fibroblast-like to elliptical or fusiform shaped cells. The expressions of CK and VIM in experimental groups (scores between 4 and 6) were significantly higher than that of the control group (scores between 1 and 2) in during immunocytochemical analysis as well as at mRNA and protein levels. Highest CK mRNA levels were observed in group 2,while VIM mRNA levels increased paralleled with the increased estradiol concentration.The quantification of protein CK and VIM also showed the same pattern during Western BlotConclusion By Ficoll density gradient centrifugation, which could obtain menses. The detection of specific cell surface markers also confirmed stem cells. Further experiments also showed that it could induce the differentiation of osteoblasts and adipocytes in vitro. They had advantages of a wide range of sources, high proliferation ability, and lower immune original and multi-directional differentiation potential. Those stem cells provide certain theoretical basis for the clinical application, which were expected to become the ideal tissue engineering seed cells. MenSCs possess a potential of differentiation into endometrial epithelial and stromal cells in an appropriate environment in vitro. Both growth factors and 17 β-estradiol play a key role in stimulating the differentiation of MenSCs towards epithelial and stromal cells, and estradiol presents a dose-related effect. |
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