RESUMEN
Whole genome duplication is frequently observed in cancer, and its prevalence in our prior analysis of end-stage, homologous recombination deficient high grade serous ovarian cancer (almost 80% of samples) supports the notion that whole genome duplication provides a fitness advantage under the selection pressure of therapy. Here, we therefore aim to identify potential therapeutic vulnerabilities in primary high grade serous ovarian cancer with whole genome duplication by assessing differentially expressed genes and pathways in 79 samples. We observe that MHC-II expression is lowest in tumors which have acquired whole genome duplication early in tumor evolution, and further demonstrate that reduced MHC-II expression occurs in subsets of tumor cells rather than in canonical antigen-presenting cells. Early whole genome duplication is also associated with worse patient survival outcomes. Our results suggest an association between the timing of whole genome duplication, MHC-II expression and clinical outcome in high grade serous ovarian cancer that warrants further investigation for therapeutic targeting.
Asunto(s)
Cistadenocarcinoma Seroso , Neoplasias Ováricas , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Humanos , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Regulación Neoplásica de la Expresión Génica , Duplicación de Gen , Genoma Humano , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismoRESUMEN
Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions.
Asunto(s)
Reprogramación Celular , Cromatina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/metabolismo , Animales , Reprogramación Celular/genética , Cromatina/genética , Femenino , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Factores de Transcripción/genéticaRESUMEN
Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.
Asunto(s)
Fibroblastos/citología , Neutrófilos/citología , Animales , Reprogramación Celular/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Fibroblastos/fisiología , Citometría de Flujo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Queratinocitos/citología , Queratinocitos/fisiología , Neutrófilos/fisiología , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.
Asunto(s)
Linaje de la Célula/fisiología , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Proteínas de Homeodominio/metabolismo , Mioblastos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Línea Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Proteínas de Homeodominio/genética , Ratones , Mioblastos/citología , Factores de Transcripción , Proteínas de Pez Cebra/genéticaRESUMEN
Mature cells of the body can be reprogrammed towards a pluripotent state by forced expression of the transcription factors Oct-4, Klf-4, Sox2, and C-Myc (OKSM) at very low efficiency. To study the reprogramming process in detail the rare intermediates of the reaction need to be separated from the bulk population. Using a genetically engineered reprogrammable mouse strain we describe how to isolate intermediates from reprogramming cultures of mouse embryonic fibroblasts via antibody labeling of cell surface markers and fluorescence-activated cell sorting (FACS).
Asunto(s)
Separación Celular , Reprogramación Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular/métodos , Citometría de Flujo/métodos , RatonesRESUMEN
Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).
Asunto(s)
Antígenos de Superficie/análisis , Citometría de Flujo/métodos , Células Madre Pluripotentes Inducidas/citología , Animales , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/biosíntesis , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/metabolismo , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/biosíntesis , Embrión de Mamíferos/citología , Molécula de Adhesión Celular Epitelial , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Antígeno Lewis X/análisis , Antígeno Lewis X/metabolismo , Masculino , Ratones , Ratones Transgénicos , Embarazo , Antígenos Thy-1/análisis , Antígenos Thy-1/metabolismo , Factores de Transcripción/biosíntesisRESUMEN
Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.
Asunto(s)
Células Endoteliales/citología , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/metabolismo , Somitos/citología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Aorta/citología , Aorta/embriología , Biomarcadores/análisis , Movimiento Celular , Quimiocina CXCL12/análisis , Quimiocina CXCL12/metabolismo , Embrión de Pollo , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Humanos , Ratones , Músculos/citología , Músculos/metabolismo , Mutación/genética , Somitos/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/genética , Proteínas Wnt/análisis , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/análisis , Proteínas de Pez Cebra/genéticaRESUMEN
Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
Asunto(s)
Reprogramación Celular , Técnicas Citológicas/métodos , Células Madre Pluripotentes Inducidas/citología , Animales , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Factores de Transcripción/metabolismoRESUMEN
Human induced pluripotent stem cells (hiPSCs) have led to an important revolution in stem cell research and regenerative medicine. To create patient-specific neural progenitors (NPs), we have established a homogenous, expandable, and self-renewable population of multipotent NPs from hiPSCs, using an adherent system and defined medium supplemented with a combination of factors. The established hiPSC-NPs highly expressed Nestin and Sox1. These NPs were continuously propagated for ~1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Voltage clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and developmental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.