RESUMEN
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mosaicismo , Piel/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Células Clonales , Fibroblastos/citología , Perfilación de la Expresión Génica , Genoma Humano/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Neuronas/citología , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Piel/citologíaRESUMEN
The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-ß (TGF-ß), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-ß inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8(+) T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.
Asunto(s)
Antineoplásicos/administración & dosificación , Inmunoterapia/métodos , Interleucina-2/administración & dosificación , Nanoestructuras , Neoplasias Experimentales/terapia , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Inmunidad Adaptativa , Animales , Antineoplásicos/farmacología , Ciclodextrinas , Composición de Medicamentos , Geles , Inmunidad Innata , Interleucina-2/farmacología , Células Asesinas Naturales/metabolismo , Liposomas , Ratones , Ratones Endogámicos , Neoplasias Experimentales/inmunología , Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Cilia are present on nearly all cell types in mammals and perform remarkably diverse functions. However, the mechanisms underlying ciliogenesis are unclear. Here, we cloned a previously uncharacterized highly conserved gene, stumpy, located on mouse chromosome 7. Stumpy was ubiquitously expressed, and conditional loss in mouse resulted in complete penetrance of perinatal hydrocephalus (HC) and severe polycystic kidney disease (PKD). We found that cilia in stumpy mutant brain and kidney cells were absent or markedly deformed, resulting in defective flow of cerebrospinal fluid. Stumpy colocalized with ciliary basal bodies, physically interacted with gamma-tubulin, and was present along ciliary axonemes, suggesting that stumpy plays a role in ciliary axoneme extension. Therefore, stumpy is essential for ciliogenesis and may be involved in the pathogenesis of human congenital malformations such as HC and PKD.
Asunto(s)
Cilios/fisiología , Predisposición Genética a la Enfermedad , Hidrocefalia/genética , Enfermedades Renales Poliquísticas/genética , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/patología , Clonación Molecular , Biología Computacional , Perfilación de la Expresión Génica , Histocitoquímica , Hidrocefalia/metabolismo , Hibridación in Situ , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Enfermedades Renales Poliquísticas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tubulina (Proteína)/metabolismoRESUMEN
Gadd45beta (growth arrest and DNA damage-inducible, beta) is involved in cell cycle arrest, apoptosis, signal transduction and cell survival. In T cells, Gadd45b was rapidly induced by T cell receptor (TCR) and inflammatory signals. Deficiency of Gadd45beta in CD4+ T cells impaired their responses to TCR stimulation or inflammatory cytokines. ERK, p38 and JNK activation were all substantially suppressed in Gadd45beta-deficient CD4+ T cells. Cytokine production by Gadd45beta-deficient CD4+ T cells was also impaired. Furthermore, Gadd45beta mediated inflammatory cytokine production by dendritic cells, and Gadd45beta-deficient mice showed an impaired T helper type 1 response during Listeria monocytogenes infection. Gadd45beta is therefore a critical feedback regulator that perpetuates both cognate and inflammatory signals.