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Obtainment, cultivation and expansion of stem cells from the yolk sac of domestic pigs (Sus scrofa)

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Author(s):
Bruno Machado Bertassoli
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
Defense date:
Examining board members:
Antônio Chaves de Assís Neto; Paula Fratini; Ana Claudia Oliveira Carreira Nishiyama
Advisor: Antônio Chaves de Assís Neto
Abstract

Fetal attachments as umbilical cord, amnion and amniotic fluid have recently been suggested as ideal sources of different strains of stem cells, due to the noninvasive nature of the isolation procedure, the large mass of tissue to harvest cells with high efficiency and potential differentiation. Therefore, this study aimed to analyze morphologically membranes extraembryonic by light microscopy and immunohistochemistry and analyze the cells from the yolk sac to characterize this cells as to potential as a possible source of pluripotent stem cells for future use in regenerative therapy. For immunohistochemistry, the membranes were processed by routine histological techniques, and then the blocks were cut sequentially and placed on silanized slides previously, the protocol was the immunohistochemistry conventional using the following antibodies: Cytokeratin and VEGF. Trials were made for concentration and cell viability, cell growth evaluation, and the characterization by flow cytometry using specific antibodies (CD105, NANOG, CD45 and Oct-3/4) and characterization by immunohistochemistry using the following antibody: CD90, CD105, CD117, vimentin, Stro-1, Oct-4, VEGF, beta tubulin, Cytokeratin and PCNA. The yolk sac stood in the ventral portion of the embryo near the umbilical cord, proving how a structure small, few vessels and with yellowed, this also presented as a trilaminar structure (endoderm, mesenchyme and mesothelium), where the endoderm had a simple cubic epithelium, the mesenchyme with a large amount of connective tissue and the mesothelium with flattened cells. The amnio shown to be deeply linked to the embryo, being the first membrane fetal considering the order embryo/uterus, and had oval-shaped transparent coloring, histologically this membrane is composed of two distinct layers, a simple squamous epithelium and mesenchyme containing an amorphous substance with a small amount of cells. The chorionic membrane showed little wrinkles and alantoic membrane structure as a crescent-shaped, having an intense vascularization, and the evolution of the pregnancy is almost impossible the perception of the two, forming the membrane chorioallantoid. The corium is constituted by a layer of round cells, arranged in linear format, as the allantois is characterized by its mesenchyme amorphous with a small amount of cells. Relative to immunohistochemical all membranes expressing VEGF and cytokeratin markers. The yolk sac cells showed adherence to plastic and a spindle-like morphology, similar to fibroblasts, they reached confluence of 70% in about 20 days, these cells were maintained until passage 4, where later cell death occurred. These cells had similar imunofenoipagem the mesenchymal stem cells and hematopoietic cells expressing markers such as CD105, CD90, CD117, vimentin, Stro-1, Oct-4, VEGF, Beta Tubulin, Cytokeratin, Nanog and PCNA. From the study it can be concluded that: there are specific cells in each extraembrionária membrane, and that the cells derived from the yolk sac have characteristics similar to mesenchymal stem cells and embryonic, and being considered an important tool for future experimental studies of cell therapy in medicine veterinary regenerative. (AU)

FAPESP's process: 11/03656-9 - Obtainment, cultivation and expansion of stem cells from the yolk sac of pigs (Sus scrofa)
Grantee:Bruno Machado Bertassoli
Support Opportunities: Scholarships in Brazil - Master