RESUMEN
Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.
Asunto(s)
Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Células-Madre Neurales/fisiología , Anilidas/farmacología , Animales , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Galectina 1/genética , Galectina 1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/genética , Humanos , Ratones , Neoplasias Experimentales , Receptor Patched-1/genética , Piridinas/farmacologíaRESUMEN
In this study, we take advantage of human induced pluripotent stem (iPS) cell-derived neural stem cells and brain organoids to study the role of p53 during human brain development. We knocked down (KD) p53 in human neuroepithelial stem (NES) cells derived from iPS cells. Upon p53KD, NES cells rapidly show centrosome amplification and genomic instability. Furthermore, a reduced proliferation rate, downregulation of genes involved in oxidative phosphorylation (OXPHOS), and an upregulation of glycolytic capacity was apparent upon loss of p53. In addition, p53KD neural stem cells display an increased pace of differentiating into neurons and exhibit a phenotype corresponding to more mature neurons compared to control neurons. Using brain organoids, we modeled more specifically cortical neurogenesis. Here we found that p53 loss resulted in brain organoids with disorganized stem cell layer and reduced cortical progenitor cells and neurons. Similar to NES cells, neural progenitors isolated from brain organoids also show a downregulation in several OXPHOS genes. Taken together, this demonstrates an important role for p53 in controlling genomic stability of neural stem cells and regulation of neuronal differentiation, as well as maintaining structural organization and proper metabolic gene profile of neural progenitors in human brain organoids.
Asunto(s)
Encéfalo/citología , Diferenciación Celular/genética , Inestabilidad Genómica , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Organoides/citología , Proteína p53 Supresora de Tumor/metabolismo , División Celular/genética , Línea Celular , Proliferación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Redes y Vías Metabólicas/genética , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Transcriptoma/genéticaRESUMEN
Neuroblastoma (NB) is an embryonic tumor of the peripheral nervous system and one of the most common solid cancers in infants. Mutations in the Anaplastic lymphoma tyrosine kinase (ALK) gene are common in NB. To study the contribution of ALK mutations in NB initiation and progression, we reprogrammed fibroblasts from two related NB patients carrying germline mutations in ALK (R1275Q) using non-integrating Sendai virus. The iPS cells are grown in a feeder- and xeno-free conditions, have normal karyotype, retain the ALK R1275Q mutation, have been characterized by expression of pluripotency markers and differentiation to all three germ layers.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Técnicas de Cultivo de Célula/métodos , Mutación de Línea Germinal/genética , Células Madre Pluripotentes Inducidas/patología , Neuroblastoma/genética , Neuroblastoma/patología , Secuencia de Bases , Línea Celular , Humanos , MasculinoRESUMEN
In vitro modeling of complex diseases is now a possibility with the use of patient-derived induced pluripotent stem (iPS) cells. Their stem cell properties, including self-renewal and their potential to virtually differentiate into any cell type, emphasize their importance as a translational tool for modeling disorders that so far have been limited by the unavailability of primary cell lines, animal models, or inaccessible human materials. Around 100 genes with germline mutations have been described to be responsible for cancer predisposition. Familial cancers are usually diagnosed earlier in life since these patients already carry the first transforming hit. Deriving iPS cells from patients suffering from familial cancers provides a valuable tool for understanding the mechanisms underlying pediatric cancer onset and progression since they require less mutation recurrence than adult cancers to develop. At the same time, some familial mutations are found in sporadic cases and are a valuable prognostic tool. Patient-derived iPS cells from germline malignancies can also create new tools in developing specific drugs with more personalized-therapy strategies.
RESUMEN
Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Cresta Neural/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Biomarcadores , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Expresión Génica , Humanos , Modelos Biológicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.