Aislamiento y caracterización de células madre de membrana anmiótica murina

Zusammenfassung

Introduction: Human amniotic membrane has been previously described as a high throughput source for stem cells with capacity for proliferation and multipotent differentiation, reduced immunogenicity, and potent inmmunomodulatory properties. All of these characteristics make it a suitable cell source for further regenerative medicine approaches. Objectives: The main objective of this study was to isolate and characterize the murine amniotic stem cells in order to analyze their biological properties and specifically their pattern of expression of epithelial and mesenchymal markers, multipotent capacity and their inmunomodulatory properties in vitro, as well as their therapeutic efficacy in a murine model of haemophilia A. Methods: Amniotic membranes from pregnant females C57BL/6 mice (mAM) were carefully separated from the yolk sacs and collected. We carried out histological assays with whole mAMs as well as to study the expression of specific markers by PCR and inmunofluorescence. The different cell populations were isolated from the mAM and characterized by different techniques such as flow cytometry, inmunofluorescence and PCR. Furthermore, proliferation, differentiation capacity, cell cycle and clonogenic assays were performed. Moreover, the immunosuppressive capacity of the mAM cells was tested by using mixed lymphocyte cultures with allogeneic stimulator BALB/c and responder C57BL/6 splenocytes, cultured in presence of different ratios of mAM stem cells. Importantly, these cellular populations were used in a preclinical study of haemophilia A to analyze their therapeutic efficacy. In order to find out the more efficient route of administration of the cells, we performed intraportal and intraperitoneal transplants into the animals. Results: Two different types of cells were detected in freshly mAM, epithelial and mesenchymal stem cells. Both cell types expressed specific epithelial and mesenchymal markers, pluripotent and proliferation markers and also expressed the coagulation factors FVIII and von Willebrand factor. After setting up the most appropriate cell isolation protocol, we obtained two different types of cultures: one of epithelial-like cells and a CD44+ population cell culture. Epithelial-like cells showed signs of senescence at early passages in culture, negative expression for hematopoietic markers and positive for pluripotent and epithelial cell markers, although they also expressed mesenchymal markers in some culture passages. Furthermore, these cells were able to differentiate into lines of the mesoderm layer. On the other hand, CD44+ cells showed a high proliferation capacity and multipotent ability in all the studied culture passages. They expressed pluripotent, proliferative and other mesenchymal markers such as vimentine, CD90, CD105, and CD73. In addition, CD44+ cells displayed adequate clonogenic properties and induced a dose-dependent inhibition of the proliferation of activated C57BL/6 splenocytes in an allogeneic context. Finally, the mAM cell transplant into the haemophilic mice led to factor VIII expression in the hepatic tissue of the transplanted animals from 1 week up to 12 months post-transplant. Conclusions: mAM is composed by epithelial and mesenchymal stem cells that showed multipotent and proliferation properties in vitro. The population of epithelial-like cells displayed similar characteristics to epithelial stem cells, and at the same time, the population of CD44+ cells had similar characteristics to mesenchymal stem cells from other sources. Treatment with mAM stem cells in haemophilia A mice led to factor VIII gene expression, as well as to the production of the factor VIII protein in the liver of the transplanted haemophilic animals. Together, mAM may represent a rich source of pluripotent stem cells with immunomodulatory properties that could be effective for their therapeutical use in multiple murine preclinical studies in order to develop future human therapies.