A Transition Research Of Human Early Embryo Mesenchymal Stem Cells To Epithelial Cells

The research of stem cells is the focus of life sciences and has great potential in fundamental medical research and clinical application. It has been discovered that stem cells exist in embryos and in adult tissues as well. These cells may function to maintain the normal function of the body and restore accidental injury. There are many types of adult stem cells such as neural stem cell, epithelial stem cell (ESC), and mesenchymal stem cell (MSC).MSC are easy to culture, expand rapidly, and are capable of multilineage differentiation.. MSC have many sources such as bone marrow, embryo bone marrow, synovium, the endothelium of umbilical cord, peripheral umbilical blood. The differentiation characteristic of MSC has been understood with deep research. Now it has been approved that MSC can differentiate into bone, cartilage, lipocyte, muscle, tendon, neuron and endothelium. The transplantation of MSC can relieve the symptom of Parkinson disease. The present technology permits in vitro expansion of hMSC without any apparent lost of phenotype or function. A number of cytokines are secreted by MSC, such as hematopoietic growth factors, nohematopoietic factors and chemokines, which modulate the bone marrow microenviroment and facilitate the proliferation and differentiation of MSC. MSC do not express major histocompatibility class II antigens or T-cell costimulatoy B7. Furthermore, human MSC are not substantially immunogenic and may actually inhibit both primary and secondary mixed lymhocyte reaction (MLR), which has a great capacity of immunomodulation. Animal models demonstrated the potential for clinical trials in the allogeneic setting. In addition, MSC could ameliorate or even prevent graft versushost disease(GVHD) reactions. The capacity of MSC to repair or replace stromal matrix damaged by genetic mutation or intensive chemotherapy, may improve the overall rate of engraftment. In acute renal failure, MSC can be used to improve the function and repair the kidney. People also find that MSC contribute to the repair of tendon, skin, brain musculoskeletal tissues and spinal cord. Through the implantation MSC into rat eyes, Kicic A and his colleagues provided a promising therapeutic strategy for some inherited retinal degenerations. MSC from early human embryo are more primordial than that from adule tissues. They could have a wider range of differentiated options and a shorter doubling time than MSC from adule tissue.The research of epidermal stem cells(ESC) can be traced back 30 years ago[62], recent work based on cell culture studies^63 -641 and a wide range of in vivo studies'65 ^ suggest a complex organization and distribution of stem cell in skin, with stem cells implicated at specific locations in the interfoUicular epidermis, in the upper regions of the outer root sheath of the hair follicle, and in the germinal matrix of growing hair follicles. ESC expand slowly in vitro, majorty of ESC are transit amplifying cellst6?1. ESC expand slowly in vitro, but in military medicine there is a big need of ESC. And the mechanism of epithelial development is studied. A lot of methods have been studied to induce hMSC into epithelial cells, one way is to use gene transfection, the other is to apply condition medium stimulated.During embryonic development, epithelial cells must escape the structural constraints imposed by tissue architecture and adopt a phenotype more amenable to cell movement, a process known as the epithelial-mesenchymal transition (EMT). Epithelium to mesenchyme (EMT) and mesenchyme to epithelium (MET) play a key role during embryonic development. Wnt-4 appears to act as an autoinducer of the mesenchyme to epithelial transition that underlies nephron development. Mesenchymal-epithelial transitions (MET) are crucial for vertebrate organogenesis. CDC42 and Racl play important and different roles in the MET that generates the vertebrate somites.Part one: Isolation, identification and in vivo expansion of humanearly embryo mesenchymal stem cellsMethods: hMSC were detected with specific antibody SH-2 by histological staining at different stages of human embryo from 4 to 7 weeks. We isolated and cultured the tissues in vitro. Culture medium is Dulbecco's Modified Eagles Medium High Glucose (DMEM-HG) supplemented with 10% fetal calf serum and 2mM Lglutamine, 100 U/ml penicillin, and 100ug/ml streptomycin (GIBCO). The cells were plated at a density of 105/ml in 90mm plate at 37°C with 5% CO2 in air. At the confluence of 85-90% cells were detached with 0.25% trypsin. Some were replated, the remainer were cryopreserved in liquid nitrogen with 10% dimethylsulfoxide, 40% fetal cattle serum and 50% DMEM-HG for 6 months. After thawing, the morphology, cell growth curve, cell cycle, phenotype, karotype and multi-potentiality of frozen stocks were detected in comparision with serial replating stocks in vitro. The expression of SH-2, CD44,OCT4,S-100,CD34, a - smooth muscle-actin in the cultured cells were detected by immunohistochemistry and Flow cytometry (FCM). The pathogens such as bacteria, fungi, chlamydia and mycoplasm in culture system and cells were detected. The safety of MSCs was evaluated in nude mice. Transplantation antigens, such as CD80, CD86, and MHC- II were detected by FCM.Results: hMSC were localized in the limb bud under the neurocoele (not including primitive gut and hepatic bud). After isolation, we got anchorage dependent fibroblast-like cells. The hMSC took no significant biological changes after passage for many times. They were spindle shaped and had a high proliferation capacity in vitro. Analysis of cell surface molecules was performed using FCM as the following procedures. No hematopoietic markers were found on each passage, such as CD34, CD45. In addition, no CD80, CD86 and MHC-II were expressed in this population, which are correlated with immune rejection. Cells of all passages were positive for CD29, CD44, CD90 and the homogeneity can reach 99%. With the induction of reagents in vitro, MSC can differentiate into osteoblast cells, adipocytes, neurons and chondrocytes. No tumors were formed after they were inoculated subcutaneously in nude mice 4 weeks later. Through detection bacteria, fungi, chlamydia andmycoplasm were negative in the supernatant of culture medium and cells. Conculsion A large number of hMSC are in the limb bud under the neurotube. After in vitro isolation and culture we can get hMSC. Their biological properties make no change after passage for many times. It demonstrated that hMSC through serial passage and those frozen stocks were homogenous in biological characteristics. Cryopreservatiom did not significantly alter the properties of hMSC. In vitro expanded hMSC maintained its biological characteristics so as to be used for clincial trials.Part two: differentiation ability of human early embryomesenchymal stem cellsMethods: HMSC were seeded at the concentration of 2x104 /cm2 per well in 6-well plate. Each culture plate was kept in stimulated medium until cell confluence. The suitable reagents were used for the differentiation of cells. Under phase-contract microscope, the shape of cells changed. By immunohistochemical staining we detected the expression of special antigens of osteoblast cells, adipocytes, neurons and chondrocytes. To investigate the differentiation ability of hMSC in vivo, we located the cells with green fluorescence protein (GFP). The cells were injected into the subcutis of mice back withlO6 cells per mouse for each week at same location and totally three times. Two weeks after the last injection, the mice were put to dead and the tissue samples were detected with the goat polyclonal antibody GFP. By H.E staining we compared the difference between the subcutaneous microstructure of positive experimental mice and that of negative control mice.Results: HMSC were grown in standard medium until the cell reached 100% confluence. Then they were stimulated to differentiate. Under different conditions, we could see that nearly 100%, about 90%, over 80%, and approximately 80% of the cells can differentiate into osteoblasts after 14 days, chondrocytes after 10 days, adipocytes 20 days later and neurons 14 days later, respectively. During the induction, the negative control cells had no change and the expression of collagen I, collagen II, neurofilament and lipid droplet were not detected. Our data showed a hierarchyexisting in the hMSC differentiation pathway. Since the cells spent different time to complete their differentiation, maybe there was a hierarchy of differentiation. In fact, the cells were easy to finish the differentiation into osteobalsts and chondrocytes. There were some difficulties to differentiate into adipocytes and neurons.The number of hair follicles of positive group was much larger than that of the negative group. In the experimental mice, some cell masses could be found below the skin. The engraftment of hMSC can be find in the follicles and cell masses, even in sweat gland subcutaneously. By H.E staining the subcutaneous microstructure of positive experimental mice and negative control mice showed evident difference. Conclusion: Human early embryo mesenchymal stem cells have the ability to differentiate into many kinds of lineages in vivo and in vitro. Subcutaneous microstructure of the experiment mice have changed distinctly and follicles and cell mass could be observed.Part three: transition of human early embryo mesenchymal stemcells to epithelial cells.Methods: The cells were transfected with pcDNA3.0 EGF or pcDNA3.0 VEGF by FuGENE 6 at the confluence of 70%, then the positive clones were selected with G418. The condition medium included different Ga2+ density. Under phase control microscope we observed the change of cells' shape. By immunohistochemical staining we detected the expression of special antigens of epithelial cells. The change of CDC42 and Smad3 was detected by RT-PCR. Western-blot was used to detect the change of P-FAK and E-cadherin protein. The induced cells were put to clinical application to estimate their clinical effect..Results: The functional DNA flag made a great effect on the differentiation of hMSC in transfection. After transfection of VEGF into hMSC, the cells changed as epithelial cells and expressed vWF and VEGF. After EGF transfection keratin 19 was expressed in about 90% cells, the expression of EGF and E-cadherin could also be detected. Within the limited Ga2+ density, E-cadherin expression increased with the increment of the Ga2+ density. The quantities of CDC42 and Smad3, P-FAK were changed...