RESUMO
Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models. For RC rat-only weight regression, both cells had the same accordance (50%), while for RC rat-only millimole regression, the accordance was 50% for ADSC and 42% for 3T3s. Thus, ADSC have similar capability for GHS class prediction as the 3T3 cell line for the evaluated reference substances. Therefore, ADSCs showed the potential to be considered a novel model for use in evaluating cytotoxicity in drug development and industry as well as for regulatory purposes to reduce or replace the use of laboratory animals with acceptable sensitivity for toxicity prediction in humans. These cells can be used to complete the results from other models, mainly because of its human origin. Moreover, it is less expensive in comparison with other existing models.
Assuntos
Tecido Adiposo/citologia , Bioensaio , Células-Tronco/efeitos dos fármacos , Testes de Toxicidade Aguda/métodos , Animais , Células 3T3 BALB , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Camundongos , Reprodutibilidade dos Testes , Medição de Risco , Células-Tronco/patologiaRESUMO
Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that â¼ 1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/fisiologia , Fibroblastos/fisiologia , Polirribossomos/metabolismo , Células Estromais/fisiologia , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Polirribossomos/genética , RNA Mensageiro/genética , Células Estromais/metabolismoRESUMO
Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life.