Análise comparativa do transcriptoma, capacidade de diferenciação e características imunofenotípicas de células estromais mesenquimais equinas obtidas de tecido adiposo e de tecido endometrial
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Data
2021-02-15
Autores
Bernardo, Ramona Bastos
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As células estromais mesenquimais (MSC’s) têm propriedades regenerativas e imunomoduladoras, tornando-as importantes na terapia veterinária. Nos equinos, estas células são frequentemente obtidas da medula óssea e do tecido adiposo, necessitando procedimento cirúrgico, com sedação e anestesia local. Assim, o tecido endometrial, obtido através de biópsia uterina, pode ser uma fonte mais acessível para o futuro uso terapêutico. MSC’s de origem endometrial (TE) e adiposa (TA) foram isoladas das mesmas éguas doadoras. As MSC’s foram avaliadas in vitro quanto ao potencial de diferenciação em tecido adipogênico e osteogênico. As MSC-TE e MSC-TA foram cultivadas in vitro e avaliadas quanto a expressão dos marcadores CD90, CD105, CD34 e MHC II, através de citometria de fluxo. Além disso, foi realizada análise transcriptômica comparativa destas células pela extração do RNA total seguido de Sequenciamento de Nova Geração (NGS). Foi analisada a expressão gênica diferencial de mRNA, assim como seus processos biológicos e vias metabólicas mais representadas em cada tipo celular. As MSC’s de ambos os tecidos apresentaram características específicas de células estromais mesenquimais, incluindo diferenciação nas linhagens adipogênicas e osteogênicas, além padrão fenotípico esperado. Os transcritos de todos os genes expressos se agruparam de acordo com a origem da MSC. Um total de 427 genes regulados positivamente e 587 genes regulados negativamente foram identificados nas MSC-TE. No entanto, estes valores representam somente 3,3% do total de transcritos identificados. As principais vias metabólicas expressas diferencialmente foram aquelas relacionadas ao alto padrão proliferativo do tecido endometrial. Portanto, as células estromais mesenquimais advindas do endométrio equino são capazes de ser isoladas através de biópsia uterina, podendo se tornar uma fonte usual de coleta de MSC’s, para fins terapêuticos, uma vez que apresentaram características imunofenotípica, de multipotencialidade e de produção de transcritos semelhantes ao tecido adiposo.
Mesenchymal stromal cells (MSC’s) have regenerative and immunomodulatory properties, making them important in veterinary therapy. In the equine species, MSC’s are frequently isolated from bone marrow and adipose tissue, requiring surgical procedure, with local anesthesia and sedation. Thus, the endometrium, through uterine biopsy, can be a more accessible source for future therapeutic use. Endometrial (TE) and adipose (TA) derived MSC’s were isolated from the same donors. MSC’s were evaluated in vitro for the potential for adipogenic and osteogenic differentiation. The MSC-TE and MSC-TA were cultured in vitro and the CD90, CD105, CD34 and MHC II markers were tested using flow cytometry. A comparative transcriptomic analysis was performed by extracting the total RNA followed by New Generation Sequencing (NGS). Differential gene expression of mRNA was analyzed, as well as its biological processes and metabolic pathways most represented in each cell type. The MSC’s of both tissues showed specific characteristics of mesenchymal stromal cells, including differentiation in adipogenic and osteogenic strains, in addition to the expected phenotypic pattern. The transcripts of all expressed genes were grouped according to the origin of the MSC. A total of 427 positively regulated genes and 587 negatively regulated genes were identified in the MSC-TE. However, these values represent only 3.3% of the total transcripts identified. The main metabolic pathways expressed differentially were those related to the high proliferative pattern of endometrial tissue. Therefore, mesenchymal stromal cells from the equine endometrium are able to be isolated through uterine biopsy, and can become a usual source of collection of MSC's, for therapeutic purposes, since they have immunophenotypic, multipotentiality and production of similar transcripts compared to adipose tissue.
Mesenchymal stromal cells (MSC’s) have regenerative and immunomodulatory properties, making them important in veterinary therapy. In the equine species, MSC’s are frequently isolated from bone marrow and adipose tissue, requiring surgical procedure, with local anesthesia and sedation. Thus, the endometrium, through uterine biopsy, can be a more accessible source for future therapeutic use. Endometrial (TE) and adipose (TA) derived MSC’s were isolated from the same donors. MSC’s were evaluated in vitro for the potential for adipogenic and osteogenic differentiation. The MSC-TE and MSC-TA were cultured in vitro and the CD90, CD105, CD34 and MHC II markers were tested using flow cytometry. A comparative transcriptomic analysis was performed by extracting the total RNA followed by New Generation Sequencing (NGS). Differential gene expression of mRNA was analyzed, as well as its biological processes and metabolic pathways most represented in each cell type. The MSC’s of both tissues showed specific characteristics of mesenchymal stromal cells, including differentiation in adipogenic and osteogenic strains, in addition to the expected phenotypic pattern. The transcripts of all expressed genes were grouped according to the origin of the MSC. A total of 427 positively regulated genes and 587 negatively regulated genes were identified in the MSC-TE. However, these values represent only 3.3% of the total transcripts identified. The main metabolic pathways expressed differentially were those related to the high proliferative pattern of endometrial tissue. Therefore, mesenchymal stromal cells from the equine endometrium are able to be isolated through uterine biopsy, and can become a usual source of collection of MSC's, for therapeutic purposes, since they have immunophenotypic, multipotentiality and production of similar transcripts compared to adipose tissue.
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Palavras-chave
Célula estromal mesenquimal, Endométrio, Equino, Expressão gênica, Mesenchymal stromal cells, Endometrium, Equine, Gene expression