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| Author(s): |
Flavia Regina Oliveira de Barros
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2009-01-22 |
| Examining board members: |
José Antonio Visintin;
Mayra Elena Ortiz D\'Avila Assumpção;
Fabíola Freitas de Paula Lopes
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| Advisor: | José Antonio Visintin |
| Abstract | |
Embryonic stem cells (ESC) represent a useful tool to study embryonic development, cell differentiation and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral and religious considerations by scientists and laymen alike. Therefore, an animal model like the pig (Sus scrofa) is valuable by overcoming such hurdles, since this species holds physiologic parameters similar to humans. In spite of the high biomedical potential of ESC, many difficulties have been faced to maintain these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro and in vitro produced swine blastocysts, (2) to compare two in vitro culture conditions to maintain isolated inner cell masses (ICM), MEF or Matrigel and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2 and FoxD3 at ESC and in vitro and in vitro produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vitro produced blastocysts were obtained by superovulation followed by artificial insemination of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing five days after ovulation. in vitro and in vitroproduced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. The resultant ICM was cultured in stem cells media (GMEM added by 15% SFB, 0.1 mM ß-mercaptoethanol, 1% non essential amino acids and 4 ng/mL of bFGF) over monolayer of irradiated murine fetal fibroblasts (MEF) or Matrigel. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro and in vitro produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis of in vitro and in vitro produced blastocysts showed that Nanog and Sox2 are less expressed in in vitro produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Since no ESC lineage was obtained in swine until now, we believe this species have different requirements compared to murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC. (AU) | |