Uso de matriz extracelular (Matrigel®) para estabelecimento de cultivo de células-tronco embrionárias de suínos e caracterização da expressão de moléculas associadas à pluripotência / Use of extracellular matrix (Matrigel®) for establishment of porcine embryonic stem cells and expression characterization of plurpotency related molecules
AUTOR(ES)
Marcelo Demarchi Goissis
DATA DE PUBLICAÇÃO
2008
RESUMO
Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers and differentiation in the three embryonic layers are necessary for validation of a pluripotent cell line. The objective of this study was to establish and characterize porcine ESC culture using Matrigel and compare the expression of pluripotency markers Oct-4, CD9 and α6-integrin with embryos. In vitro or in vivo porcine blastocysts were submitted to immunosurgery for culture of inner cell mass, fixation for immunocytochemistry or total RNA extraction for RT-PCR. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. Expression of Oct-4, CD9 and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. It was not possible to compare expression of pluripotency markers between porcine ESC and embryos. However, this study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts, which may not be related to pluripotent porcine embryonic cells.
ASSUNTO(S)
embryonic stem cells matrigel matrigel blastocyst blastocisto marcadores de pluripotência células-tronco embrionárias pluripotency markers porcine suínos
