RESUMO
Domesticated ungulate pluripotent embryonic stem (ES) cell lines would be useful for generating precise gene-modified animals. To date, many efforts have been made to establish domesticated ungulate pluripotent ES cells from early embryos without success. Here, we report the generation of porcine-induced pluripotent stem (iPS) cells using drug-inducible expression of defined factors. We showed that porcine iPS cells expressed alkaline phosphatase, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct3/4, Nanog, Sox2, Rex1 and CDH1. Pig iPS cells expressed high levels of telomerase activity and showed normal karyotypes. These cells could differentiate into cell types of all three germ layers in vitro and in teratomas. Our study reveals properties of porcine pluripotent stem cells that may facilitate the eventual establishment of porcine ES cells. Moreover, the porcine iPS cells produced may be directly useful for the generation of precise gene-modified pigs.
Assuntos
Técnicas de Cultura de Células/métodos , Doxiciclina/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Sus scrofa , Envelhecimento/fisiologia , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Análise por Conglomerados , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Vetores Genéticos/genética , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução GenéticaRESUMO
Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.
