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Universidad de Murcia

1. García Guillén, Ana Isabel. Aislamiento y caracterización de células madre de membrana anmiótica murina.

Degree: Departamento de Medicina Interna, 2017, Universidad de Murcia

ABSTRACT 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… Advisors/Committee Members: [email protected] (authoremail), false (authoremailshow), García Bernal, David (director), Marín Atucha, Noemí Teresa (director), true (authorsendemail).

Subjects/Keywords: Células madre; Oftalmología; Ciencias de la salud; 5; 57; 576; 6; 61; 617

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APA (6th Edition):

García Guillén, A. I. (2017). Aislamiento y caracterización de células madre de membrana anmiótica murina. (Thesis). Universidad de Murcia. Retrieved from http://hdl.handle.net/10803/463041

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

García Guillén, Ana Isabel. “Aislamiento y caracterización de células madre de membrana anmiótica murina.” 2017. Thesis, Universidad de Murcia. Accessed November 20, 2018. http://hdl.handle.net/10803/463041.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

García Guillén, Ana Isabel. “Aislamiento y caracterización de células madre de membrana anmiótica murina.” 2017. Web. 20 Nov 2018.

Vancouver:

García Guillén AI. Aislamiento y caracterización de células madre de membrana anmiótica murina. [Internet] [Thesis]. Universidad de Murcia; 2017. [cited 2018 Nov 20]. Available from: http://hdl.handle.net/10803/463041.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

García Guillén AI. Aislamiento y caracterización de células madre de membrana anmiótica murina. [Thesis]. Universidad de Murcia; 2017. Available from: http://hdl.handle.net/10803/463041

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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