RESUMO
The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-ß inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/- HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/- HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/- NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro.
Assuntos
Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Disostose Mandibulofacial/genética , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Benzamidas/farmacologia , Morte Celular , Movimento Celular , Embrião de Galinha , Dioxóis/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Disostose Mandibulofacial/patologia , Crista Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in FBN1, which encodes the extracellular matrix protein fibrillin-1. To investigate the pathogenesis of aortic aneurysms in MFS, we generated a vascular model derived from human induced pluripotent stem cells (MFS-hiPSCs). Our MFS-hiPSC-derived smooth muscle cells (SMCs) recapitulated the pathology seen in Marfan aortas, including defects in fibrillin-1 accumulation, extracellular matrix degradation, transforming growth factor-ß (TGF-ß) signaling, contraction and apoptosis; abnormalities were corrected by CRISPR-based editing of the FBN1 mutation. TGF-ß inhibition rescued abnormalities in fibrillin-1 accumulation and matrix metalloproteinase expression. However, only the noncanonical p38 pathway regulated SMC apoptosis, a pathological mechanism also governed by Krüppel-like factor 4 (KLF4). This model has enabled us to dissect the molecular mechanisms of MFS, identify novel targets for treatment (such as p38 and KLF4) and provided an innovative human platform for the testing of new drugs.