Isolation, Identification And Differentiation Of Human Endometrial Stem Cells

BackgroundStem cells are cells that have unlimited self-renewal and differentiation capacity under certain condition. According to the different period in life they can be divided into embryonic stem cells and adult stem cells. Adult stem cells are undifferentiated cells in various differentiated tissues. At present adult stem cells have been identified in diverse tissues (such as neural stem cells, hematopoietic stem cells and skin stem cells).The human endometrium shows remarkable regenerative capacity following menstruation, parturition and hormone-replacement therapy in post-menopausal women. Regeneration of endometrial glands and stroma form the new functionalis layer arises from their remnants in the basalis after menstruation, under the influence of rising oestrogen levels. Early in1978it was proposed that human endometrium may contain a population of stem cells responsible for its remarkable regenerative ability. Stem cells may exist in the basal layer of the endometrium,.including endometrial-myometrial junction.The subsequent clinical practice found women who had complete curettage of the endometrium for dysfunctional uterine bleeding had regenerated endometrium and got pregnant. These proved the existence of stem cells in the regenerated endometrium.Endometrial stem cell is a group cells with unlimited proliferative capacity, which can be identified functionally by monoclonal culture Monoclonal cell culture mainly includes soft agar culture method, limited dilution method, single cell micromanipulation method and flow cytometry separation method and so on. Limited dilution method is the most commonly used, whose principle is to continuously dilute the suspensed cells to extremely low density, then to inoculate them into a culture plate, culturing for several days. Cloning from single cell may occur. Compared with the soft agar method, single cell micromanipulation method and flow cytometry separation methods, it has the advantages of easy operation, short period and low cost.Endometrial stem cells are undifferentiated and immature cells, that can not be distinguished with the endometrial cells from the morphology but from the function. The best method is to use the specific histological and immunological markers to determine the stem cells. But specific markers has not yet been found. It has reported in the literature that CD90, CD29, CD73may be the alternative markers, but CD133, CD34, CD45may be not.Aldehyde dehydrogenase is a polymrophic enzyme responsible for the oxidation of retinol to retinoic acid in the early period of differentiation. ALDH1is an important enzyme for aldehyde oxidation.The ALDEFLUOR reagent system offers a non-immunological way to identify stem and progenitor cells on the basis of their ALDH (aldehyde dehydrogenase) activity. Uncharged ALDH-substrate, BAAA, is taken up by living cells through passive diffusion.BAAA is converted by intracellular ALDH into a negatively charged reaction product BAA-, which is retained inside cells expressing high levels of ALDH, causing the cells to become brightly fluorescent.The brightly fluorescent ALDH expressing cells (ALDHhigh) are detected in the green fluorescence channel of the standard flow cytometer.Since only cells with an intact cellular membrane can retain the ALDEFLUOR reaction product, only viable cells with the ALDHhigh are identified. ALDEFLOUR positive cells from not only nomal breast tissue but also breast carcinoma have self-renewal and multilineage differentiation capacity like stem cells while negative cells have not.There are high expression of ALDH1in multiple tissues such as hematopoietic cells, pancreatic and lung cancer. ALDH1is expected to become the common stem cell markers for separation and identification.It is not very clear that the differentiated mechanism and induced conditi on of stem cells. The differentiation of stem cells is closely related to the mi croenvironment which is called "niche" they grow in. It is probably because c ertain factors in the microenvironment are necessary for stem cells to different iate into target cell lineages.There was a research that MSCs have potential to differentiate into neurons, glials and retinal cells induced by supernatant of c ultured retinal cells. Another study was to identify the potentiality of induced MSCs into hair follicle stem cells by supernatant of cultured hair follicle.Endometrial stem cells are a particularly attractive source of cells for regenerative medicine because of their biological character.It has reported that endometrial stem cells promote angiogenesis in treatment of burn. Scientists have found that endometrial stem cells can generate dopamine for the treatment of Parkinson’s disease at the Parkinson model of mice. The endometrium is a hotbed of embryonic development. The thin endometrium hinder zygotes from implantation and growth.which is the common cause for infertility and intrauterine adhesions. The most important strategy for the treatment is to promote endometrial regeneration and functional recovery. But current treatment fails to solve the endometrial regeneration for local severe or extensive injury in endometrium.In recent years, with the development of stem cell differentiation theory, stem cells gradually applied in clinical treatment research, which provides a new concept of the endometrial therapy.The number, function, regulation and positioning mechanism of Endometrial stem cell may also result in endometrial diseases.The law of endometrial growth, proliferation and evolution was found throuth the endometrial stem cell research which is a important guiding for prevention and treatment of enndometrial proliferation diseases, pregnancy, embryo implantation, postmenopausal hormone replacement therapy, cancer and so on.This paper to isolate, culture identify and induce endometrial stem cells intend to confirm endometrial stem cells existence, find the possible markers and explore the induction to endometrial cells.Methods1.Take the whole endometrium including5mm myometrium as sample.Human endometrial cells were isolated and dissociated mechanically and enzymatically from human endometrium. Stromal and epithelial cells were seperated by two series of filters(200mesh and400mesh). Stromal cells and epithelial cells was identified by Fluorescent staining for vimentin and keratin.2.Limiting dilution culture was performed to stromal cells and epithelial cells in some endometrial samples. The density of suspension cells was adjusted to respectively300stromal cells/cm2and500epithelial cells/cm2.After15days,cell morphology and growth characteristics were observed. Flow cytometry analysis was used to identify expression of CD133,CD34,CD45,CD90,CD73and CD29.3.ALDEFLUOR kit was used to label and sort stromal and epithelial cells in another endometrial samples by flow cytometry.The expressions of ALDHhigh cells were separated by fluorescence activated cell sorting(FACS).Limiting dilution culture was performed to these cells.Cell morphology and growth characteristics were observed. Flow cytometry analysis was used to identify expression of c-kit, CD90,CD73and CD29in cultured cells.4.The supernatants in cultured endometrial stromal and epithelial cells were collected as conditioned medium. The well-grown cells in endometrial stem cells that was cultured by the step2were chosen.Some were induced by sterile conditioned medium,which was the experimental group.The other were cultured by DMEM,which was the control group.Morphology and growth characteristics of induced cells were observed.Flow cytometry analysis was used to identify expression of CD13and CD9in induced stem cells that is respectively special for stromal cells and epithelial cellsResults1.The primary culture, observation and identification of endometrial stem cells(1)Epithelial cells were polygonal or oval,while stromal cells were shuttle-shaped.(2)Some proportion of attached epithelial cells formed small nests of5-10cells within5days of seeding.But by Days8-10in culture, most had become apoptotic and lifted off the plate. There was no colony in epithelial cells.(3)Stromal cells commenced proliferating after a period of3-4days. In15day culture period two types of colonies were distinguishable:colonies small in size, comprising large,loosely packed cells and large colonies, containing small, densely packed cells.The colony forming rate of the stromal cells that were not sorted by ALDH kit reached1.34±0.44%. The colony forming rate of large clone and small clone were respectively0.02%±0.06%and1.32%±0.44%.Cells were spiral-shaped and radial.(4)Results from flow cytometry showed that endometrial stem cells were positive for CD29,CD90and CD73but negative for CD34, CD45and CD133.2.Detection of endometrial stem cells by Aldehyde dehydrogenase activity(1)The proportion of ALDHhigh cells was1.88±0.17%in stromal cells, ALDHhigh cells were not detected in epithelial cells.(2)About15days later, the colony forming rate of the ALDHhigh cells reached4.50%±0.82%which were significandy higher than that of ALDHlow cells1.06%±0.34%(P<0.001). The colony forming rate of the ALDHhighcells in large clones reached0.11%±0.12%which were significantly higher than that of ALDHlow cells0.04%±0.08%(P<0.001). The colony forming rate of the ALDHhigh cells in small clones reached4.40%±0.81%which were significandy higher than that of ALDHlow cells1.02%±0.30%(P<0.001)(3)Results from flow cytometry showed that endometrial stem cells were positive for CD29,CD90,CD73and c-kit.3. The culture, observation and identification of endometrial stem cells after induction by the conditioned medium.(1) Endometrial stem cells in the control group were spindle-shaped with dim contour and abundant clear cytoplasm.The middle is a bit wide, while pointed at both ends, They were closely and radially arrayed.(2)After induction some endometrial stem cells underwent morphological changes, part of the cells were cluster arranged, polygonal, like epithelial cells, part of the cells were dispersed packed, flat, spindle shaped, like stromal cells.(3)Expression rate of CD13in experimental group was8.37%±0.51%, while in the control group that was1.88%±0.22%(P<0.001). Expression rate of CD9in experimental group was9.24%±0.90%, while in the control group that was1.68%±0.25%.(P<0.001).DiscussIn recent years, studies have shown that the presence of stem cells with high proliferation, self-renewal and differentiation potential in endometrium. Our results confirmed the presence of stem cells in endometrium.Two kinds of different sizes of clone were found in the stromal cells. Previous research discovered large clone is composed of endometrial precursor cells or stem cell, while small clone was originated from transit amplifying cells,(TA) that was differentiated by precursor cells. Proliferation ability of TA cell is weak than stem cells, and gradually differentiate into the end cells with different functions but without proliferation ability. The endometrial stem cells which were identified by flow cytometry were positive in CD29, CD90, CD73(bone marrow mesenchymal stem cell marker) but negative in CD133(epithelial stem cell factor) and hematopoietic cell surface markers such as hematopoietic precursor cell marker antigens CD34and CD45. The surface markers of endometrial stem cells by flow cytometry analysis were almost the same with that of bone marrow mesenchymal stem cell.They may be derived from mesenchymal stem cells.ALDH1may plays an important role in the early differentiation of stem cells.In a variety of tissue types, tumor stem cells have high expression of ALDH1, which have the characteristics of stem cells. At present, studies confirmed the stem cells were in the endometrium but they were very low, and the lack of specific markers.We also found the ALDHhigh cells from endometrial stem cells,which has the higher colony forming ability than ALDHlow cells,which will be a effective method to concentrate the endometrial stem cells. The endometrial stem cell express c-kit. C-kit is an oncogene, also known as stem cell factor receptor, which encodes a transmembrane tyrosine kinase receptor molecules. It is exist in hematopoietic stem cell membranes, the ligand of which are hematopoietic stem cell factor. Expression of surface antigen can be used as identification of endometrial stem cell.The supernatants in cultured endometrial cells induced endometrial stem cells to endometrial stromal and epithelial cells, which makes the formation of complete anatomical and functional endometrium possible. Endometrial stem cells have the potential of differentiation in vitro. With the development of the research in endometrial stem cells,stem cells would be induced into mature endometrial cells in order to treat endometrial injury.Endometrial reconstruction will become possible. So far,domestic and foreign scholars have been studying on the pathogenesis of intrauterine adhesions, have agreed that the disorder of endometrial repairment may be the main mechanism of intrauterine adhesions.In pathological conditions such as curettage after abortion or other uterine operation, endometrium can’t repair completely by endometrial cells instead of scar. Reduction or loss of Endometrial stem cells may be closely associated with endometrial repair disorder and intrauterine adhesions.This experiment by differentiation of endometrial stem cells in vitro, will explore transplantation to heal intrauterine adhesions, by the level of stem cells, and improve the cure rate and pregnancy rate of intrauterine adhesions.However, animal experiment have to be carried out to prove endometrial stem cells could successfully be induced to complete endometrium. At present, our research team is doing the animal experiment on treatment of intrauterine adhesions by stem cell.In addition, the molecular mechanism of the differentiation and the function of induced cells need to be further studied.ConclusionsResults have proved the presence of rare clonogenic stem cells with high proliferative potential and have suggested that endometiral stem cells may originate from BMSCs.Results have proved the ALDHhigh cells had a higher colony-forming efficiency in vitro than ALDHlow cells and have some markers of stem cells.Results have proved the supernatants from the culture of endometrial epithelial and stromal cells can partly induce the endometrial stem cells to endometrial stromal cells and epithelial cells. The exact mechanism remains to be further studied.