I-31: Reconstruction of Human Endometrium from Somatic Stem Cells

s:3115:"Background: During reproductive life, the human endometrium undergoes around 500 cycles of growth, breakdown and regeneration. This outstanding regenerative capability is the basis for cyclic endometrial preparation and its dysfunction is involved in pathological disorders. Endometrial regeneration is mediated by the existence of a specialized endometrial stem cell (SC) population recently identified. Thispresentation will review the evidence available to date for the existence of endometrial SC in human endometrium detailing the functional approaches that have been used to identify their regenerative capacity and the translation to clinic in on-going clinical projects. Materials and Methods: Identification and localization of SSC is hard and laborious given the lack of clear differential morphological features and molecular markers. Two techniques based on general SSC properties, such as slow cycling and an ATP-binding cassette membrane, are available for this purpose. First, labelling with DNA marker 5-bromo-2-deoxyuridine (BrdU) has been used to identify the existence of SSC in animal models. BrdU signal progressively decreases in each division, and the retaining of the labelling either indicates no division or a very low division rate, one of the most important characteristics of SSC. Second, the Side Population (SP) method was initially created to identify SC in human hematopoietic tissue. In this technique, the cell-permeable DNA-binding dye Hoechst 33342 is loaded into the cell population of interest; SC have the ability to pump out this dye via an ATP-binding cassette membrane mechanism, resulting in a low-fluorescence side SP "tail" by flow cytometry. Results: We have proven the existence of endometrial SSC in mice endometrium by identifying the low cycling cell population using BrdU technique in combination with the co-localization of typical markers of undifferentiation1. In humans,we have demonstrated the existence of this endometrial SSC subset using the SP technique and further reconstructing the human endometrium from injecting isolated endometrial SP cells2 or SP cell lines3 into the NOD-SCID mice model. We have isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments identifyingits gene signature, characteristic immunophenotype and telomerase pattern. We analyzed their clonogenic activity under hypoxic conditions and the differentiation capability in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functionality of endometrial SP to develop human endometrium after subcutaneous or subcapsular kidney injection in NOD-SCID mice. We have also demonstrated the contribution of bone marrow to the reconstruction of the human endometrium and the SP subpopulation4. Conclusion: Based on the information gathered from these studies we have isolated the cell population responsible for the regeneration of the human endometrium and clinical trials using this technology in severe cases of Asherman syndrome and thin endometrium are in process.";