Células tronco embrionárias e induzidas ao estado de pluripotência em bovinos: características moleculares e aplicação em quimeras embrionárias
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Data
2021-05-05
Autores
Botigelli, Ramon César
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Universidade Estadual Paulista (Unesp)
Resumo
Células tronco pluripotentes são uma ferramenta fundamental para os estudos da embriologia, para o desenvolvimento de estudos clínicos in vitro, para a medicina regenerativa e mais recentemente, para a produção de gametas in vitro. Em 2006, Takahashi e colaboradores demonstraram ser possível a obtenção de células-tronco pluripotentes por indução gênica (induced pluripotent stem cells ou iPSCs). No entanto, condições ambientais e de cultura in vitro, como a utilização de diferentes suplementos para a manutenção da pluripotência podem diminuir e modular a eficiência da reprogramação à pluripotência. Dessa forma, a presente tese teve como objetivo estudar os efeitos do oxigênio e de diferentes suplementos para a manutenção da pluripotência no cultivo e reprogramação de células iPSCs de bovinos (biPSCs). No primeiro estudo, fibroblastos fetais de bovinos foram reprogramados em diferentes tensões de oxigênio (baixo ou alto oxigênio) e diferentes suplementos para a manutenção de pluripotência, bFGF ou bFGF+LIF+CHIR+PD (bFL2i). Em nossas condições experimentais, baixo oxigênio não aumentou a eficiência de reprogramação quando comparada a reprogramação em alto oxigênio. Por outro lado, a suplementação bFL2i aumentou a eficiência de reprogramação das biPSCs. As biPSCs derivadas com bFL2i em baixo oxigênio indicaram ausência de habilidade de auto renovação. As linhagens caracterizadas foram positivas para fosfatase alcalina, positivas para a expressão de genes marcadores de pluripotência (SOX2, OCT4 e STELLA), no entanto, negativas para NANOG. Ainda, baixo oxigênio aumentou a expressão de GLUT1 e GLUT3. As biPSCs também foram positivas na imunofluorescência para OCT4 e SOX2, mas negativas para NANOG. As biPSCs foram competentes em formar corpos embrióides e estes expressão marcadores de mesoderme e ectoderme, ainda, uma linhagem apresentou expressão de endoderme. No segundo estudo, fibroblastos fetais de bovinos foram reprogramados com diferentes suplementos para a manutenção da pluripotência (fatorial 2x2; bFGF, bFGF2i, LIF, LIF2i), ainda, linhagens de células tronco embrionárias foram isoladas para serem comparadas com as biPSCs geradas. A reprogramação de biPSCs foi mais eficiente com bFGF. As linhagens derivadas com LIF2i indicaram ausência de habilidade de auto renovação. As linhagens biPSCs e bESCs foram positivas para fosfatase alcalina. As biPSCs e bESCs foram positivas para a expressão de genes marcadores de pluripotência (SOX2, OCT4, STELLA, LIFr e OTX2) e somente as biPSCs expressaram NANOG e ESRRb. Ainda, biPSCs e bESCs foram positivas para SOX2, OCT4 e H3K27me3, negativas para NANOG na imunofluorescência. As biPSCs e bESCs foram competentes em formar corpos embrióides, estes expressaram marcadores de mesoderme, ectoderme e endoderme. Ainda, nossos resultados indicaram que em nossas condições, as biPSCs foram mais competentes comparadas as bESCs para a contribuição de blastocistos quiméricos quimeras. Por fim, nossos resultados demonstram que a reprogramação de biPSCs é possível em baixo e alto oxigênio. No entanto, a suplementação LIF2i e bFL2i (baixo oxigênio) não promoveram a auto renovação das biPSCs. Também, as biPSCs e bESCs geradas e isoladas apresentaram características de pluripotência, onde, biPSCs foram mais competentes em contribuir em quimeras embrionárias
Pluripotent stem cells are a crucial tool to embryology studies, to development in vitro clinical studies, to regenerative medicine and more recently, to gametes in vitro production. In 2006, Takahashi and collaborators demonstrated can be possible generate pluripotent stem cells by gene expression induction (induced pluripotent stem cells or iPSCs). However, in vitro culture and environmental conditions can modulate the reprogramming efficiency to pluripotency. Thus, the present thesis aimed to study the effects of oxygen and different sets of supplements for maintenance the pluripotency state during the bovine iPSC reprogramming window (biPSCs). In the first study, bovine fetal fibroblasts were reprogrammed in low or high oxygen and in different sets supplements for maintenance of pluripotency, bFGF or bFGF+LIF+CHIR+PD (bFL2i). In our conditions, low oxygen did not increase the reprogramming efficiency when compared to normoxia. On the other hand, bFL2i supplementation increased the reprogramming efficiency of biPSCs. The biPSCs derived in bFL2i low oxygen indicated the absence of self-renewal ability. The characterized biPSCs were positive for alkaline phosphatase, positive for expression of pluripotency marker genes (SOX2, OCT4 and STELLA), however, negative to NANOG. In addition, low oxygen increased the expression of GLUT1 and GLUT3. biPSCs were positive for OCT4 and SOX2, but negative for NANOG in immunofluorescence. The biPSCs were competent in forming embryoid bodies (EBs), also, EBs expressed markers of mesoderm and ectoderm, also, one biPSCs line expressed endoderm marker. In the second study, fetal bovine fibroblasts were reprogrammed with different supplements to maintain pluripotency (factorial 2x2; bFGF, bFGF2i, LIF, LIF2i), and embryonic stem cell lines were isolated to be compared with the generated biPSCs. The bFGF supplementation increased the reprogramming efficiency of biPSCs. The biPSCs derived with LIF2i indicated the absence of self-renewal ability. The biPSCs and bESCs were positive for alkaline phosphatase. The biPSCs and bESCs were positive for the expression of pluripotency marker genes (SOX2, OCT4, STELLA, LIFr and OTX2) and only the biPSCs expressed NANOG and ESRRb. In addition, biPSCs and bESCs were positive for SOX2, OCT4 and H3K27me3, negative for NANOG in immunofluorescence. The biPSCs and bESCs were competent to form embryoid bodies, which expressed markers of mesoderm, ectoderm and endoderm. Still, our results indicate biPSCs were more competent compared to bESCs for the chimeric blastocyst contribution. Finally, our results demonstrate the reprogramming of biPSCs is possible in low and high oxygen. However, supplementation LIF2i and bFL2i (low oxygen) did not promote self-renewal ability on biPSCs. Also, biPSCs and bESCs were generated and isolated and showed pluripotency characteristics, where biPSCs were more competent in contributing to embryonic chimeras.
Pluripotent stem cells are a crucial tool to embryology studies, to development in vitro clinical studies, to regenerative medicine and more recently, to gametes in vitro production. In 2006, Takahashi and collaborators demonstrated can be possible generate pluripotent stem cells by gene expression induction (induced pluripotent stem cells or iPSCs). However, in vitro culture and environmental conditions can modulate the reprogramming efficiency to pluripotency. Thus, the present thesis aimed to study the effects of oxygen and different sets of supplements for maintenance the pluripotency state during the bovine iPSC reprogramming window (biPSCs). In the first study, bovine fetal fibroblasts were reprogrammed in low or high oxygen and in different sets supplements for maintenance of pluripotency, bFGF or bFGF+LIF+CHIR+PD (bFL2i). In our conditions, low oxygen did not increase the reprogramming efficiency when compared to normoxia. On the other hand, bFL2i supplementation increased the reprogramming efficiency of biPSCs. The biPSCs derived in bFL2i low oxygen indicated the absence of self-renewal ability. The characterized biPSCs were positive for alkaline phosphatase, positive for expression of pluripotency marker genes (SOX2, OCT4 and STELLA), however, negative to NANOG. In addition, low oxygen increased the expression of GLUT1 and GLUT3. biPSCs were positive for OCT4 and SOX2, but negative for NANOG in immunofluorescence. The biPSCs were competent in forming embryoid bodies (EBs), also, EBs expressed markers of mesoderm and ectoderm, also, one biPSCs line expressed endoderm marker. In the second study, fetal bovine fibroblasts were reprogrammed with different supplements to maintain pluripotency (factorial 2x2; bFGF, bFGF2i, LIF, LIF2i), and embryonic stem cell lines were isolated to be compared with the generated biPSCs. The bFGF supplementation increased the reprogramming efficiency of biPSCs. The biPSCs derived with LIF2i indicated the absence of self-renewal ability. The biPSCs and bESCs were positive for alkaline phosphatase. The biPSCs and bESCs were positive for the expression of pluripotency marker genes (SOX2, OCT4, STELLA, LIFr and OTX2) and only the biPSCs expressed NANOG and ESRRb. In addition, biPSCs and bESCs were positive for SOX2, OCT4 and H3K27me3, negative for NANOG in immunofluorescence. The biPSCs and bESCs were competent to form embryoid bodies, which expressed markers of mesoderm, ectoderm and endoderm. Still, our results indicate biPSCs were more competent compared to bESCs for the chimeric blastocyst contribution. Finally, our results demonstrate the reprogramming of biPSCs is possible in low and high oxygen. However, supplementation LIF2i and bFL2i (low oxygen) did not promote self-renewal ability on biPSCs. Also, biPSCs and bESCs were generated and isolated and showed pluripotency characteristics, where biPSCs were more competent in contributing to embryonic chimeras.
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Palavras-chave
Células pluripotentes, Fatores de crescimento, Reprogramação celular