RESUMO
UNLABELLED: Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. OBJECTIVE: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. MATERIAL AND METHODS: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. RESULTS: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). CONCLUSIONS: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Modelos Animais , Porco Miniatura , Engenharia Tecidual/métodos , Animais , Antígenos CD/análise , Brasil , Técnicas de Cultura de Células , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Masculino , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. .
Assuntos
Animais , Masculino , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Modelos Animais , Porco Miniatura , Engenharia Tecidual/métodos , Antígenos CD/análise , Brasil , Técnicas de Cultura de Células , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Over the last years an expanding family of small RNAs (i.e. microRNAs, siRNAs and piRNAs) was recognized as key players in diverse forms of gene silencing and chromatin organization. Effectors functions of these small RNAs are achieved through ribonucleoprotein (RNP) complexes containing at their center an Argonaute/Piwi protein. Although these proteins and their small RNA-associated machinery can be traced back to the common ancestor of eukaryotes, this machinery seems to be entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, which are unable to trigger RNAi related phenomena. Speculating about the presence of alternate small RNA-mediated pathways in these organisms, we constructed and analyzed a size-fractionated cDNA library (20-35 nt) from epimastigotes forms of T. cruzi. Our results showed the production of an abundant class of tRNA-derived small RNAs preferentially restricted to specific isoacceptors and whose production was more accentuated under nutritional stress. These small tRNAs derived preferentially from the 5' halves of mature tRNAs and were recruited to distinctive cytoplasmic granules. Our data favor the idea that tRNA cleavage is unlikely to be the consequence of non-specific degradation but a controlled process, whose biological significance remains to be elucidated.