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1.
Dev Biol ; 439(1): 30-41, 2018 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-29678445

RESUMEN

During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak.


Asunto(s)
Gastrulación/genética , Línea Primitiva/embriología , Análisis de la Célula Individual/métodos , Animales , Embrión de Pollo , Pollos , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Línea Primitiva/fisiología , Análisis Espacio-Temporal , Transcriptoma/genética
2.
Proc Natl Acad Sci U S A ; 108(16): 6537-42, 2011 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-21464322

RESUMEN

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.


Asunto(s)
Atrofia Girata/enzimología , Atrofia Girata/genética , Ornitina-Oxo-Ácido Transaminasa/genética , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Células Madre Pluripotentes/enzimología , Células Cultivadas , Marcación de Gen/métodos , Estudio de Asociación del Genoma Completo , Inestabilidad Genómica/genética , Atrofia Girata/patología , Atrofia Girata/terapia , Humanos , Células Madre Pluripotentes/patología , Recombinación Genética
3.
Blood ; 117(6): 1977-85, 2011 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-21088132

RESUMEN

Epstein-Barr virus (EBV) encodes oncogenic information and, oftentimes concomitant with host immunosuppression, gives rise to malignancies in all major categories of lymphoma defined by the World Health Organization. Here, we conditionally evicted the viral extrachromosomal genome from tumor cells in vitro to examine the role of EBV in different lymphomas, including Burkitt lymphoma (BL) and posttransplant lymphoproliferative disorder. Cells derived from 2 canonical BLs were found to have the least dependence on the virus; some required EBV to prevent the inefficient induction of apoptosis. In contrast, cells derived from a subset of BL, Wp-restricted BL, required EBV to block a robust apoptotic program that involves the up-regulation of the proapoptotic protein Bim. Wp-restricted BL cells also relied on the virus to promote efficient proliferation, a distinction that highlights the multiple contributions EBV makes to affect proliferation of its host cells. Like Wp-BL cells, posttransplant lymphoproliferative disorder cells depended on the virus to inhibit apoptosis. They furthermore required the virus to drive them out of G(1)/G(0). Together, these results reveal a graded dependence on EBV among tumor cells that directly correlates with the number of viral genes expressed in the tumor cell.


Asunto(s)
Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Linfoma/genética , Linfoma/virología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Proliferación Celular , Infecciones por Virus de Epstein-Barr/patología , Genes myc , Genoma Viral , Humanos , Linfoma/patología , Trastornos Linfoproliferativos/etiología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/patología , Trastornos Linfoproliferativos/virología , Proteínas de la Membrana/genética , Modelos Biológicos , Oncogenes , Proteínas Proto-Oncogénicas/genética , Trasplantes/efectos adversos
4.
J Virol ; 83(7): 2930-40, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19129441

RESUMEN

We identified binding sites for Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in the human genome using chromatin immunoprecipitation and microarrays. The sequences for these newly identified sites were used to generate a position-weighted matrix (PWM) for EBNA1's DNA-binding sites. This PWM helped identify additional DNA-binding sites for EBNA1 in the genomes of EBV, Kaposi's sarcoma-associated herpesvirus, and cercopithecine herpesvirus 15 (CeHV-15) (also called herpesvirus papio 15). In particular, a homologue of the Rep* locus in EBV was predicted in the genome of CeHV-15, which is notable because Rep* of EBV was not predicted by the previously developed consensus sequence for EBNA1's binding DNA. The Rep* of CeHV-15 functions as an origin of DNA synthesis in the EBV-positive cell line Raji; this finding thus builds on a set of DNA-binding sites for EBNA1 predicted in silico.


Asunto(s)
ADN Viral/metabolismo , ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Replicación Viral , Animales , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , Genoma Humano , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , Análisis por Matrices de Proteínas , Unión Proteica
5.
Stem Cell Reports ; 10(1): 73-86, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29320761

RESUMEN

Arterial diseases continue to pose a major health concern but in vitro studies are limited because explanted cells can exhibit poor proliferative capacity and a loss of specificity. Here, we find that two transcription factors, MYCN and SOX17, induce and indefinitely expand in culture precursors of human arterial endothelial cells (expandable arterial endothelial precursors [eAEPs]). The eAEPs are derived from CD34+ cells found in umbilical cord blood or adult bone marrow. Independent eAEP lines differ in their proclivity to undergo an endothelial-to-mesenchymal transition (EndoMT), a hallmark event in a broad array of vascular diseases and disorders. Some cell lines spontaneously become mesenchymal over time in culture, an effect exacerbated by inhibition of the fibroblast growth factor receptor, while others do not readily convert. These distinctions were exploited to identify genes that correlate with resistance to an EndoMT and to elucidate transcriptional changes that underpin the transition.


Asunto(s)
Antígenos CD34/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Progenitoras Endoteliales/metabolismo , Sangre Fetal/metabolismo , Células de la Médula Ósea/citología , Células Progenitoras Endoteliales/citología , Sangre Fetal/citología , Humanos , Proteína Proto-Oncogénica N-Myc/metabolismo , Especificidad de Órganos , Factores de Transcripción SOXF/metabolismo
7.
Genome Biol ; 17(1): 173, 2016 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-27534536

RESUMEN

BACKGROUND: Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states. RESULTS: Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T) (+) mesendoderm toward a CXCR4 (+) DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation. CONCLUSIONS: We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.


Asunto(s)
Diferenciación Celular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/citología , ARN/genética , Técnicas de Cultivo de Célula , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/citología , Humanos , Células Madre Pluripotentes/citología , Análisis de la Célula Individual/métodos
8.
Stem Cell Reports ; 4(2): 171-80, 2015 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-25601207

RESUMEN

In this study, we demonstrate a newly derived mouse model that supports engraftment of human hematopoietic stem cells (HSCs) in the absence of irradiation. We cross the NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) strain with the C57BL/6J-Kit(W-41J)/J (C57BL/6.Kit(W41)) strain and engraft, without irradiation, the resulting NBSGW strain with human cord blood CD34+ cells. At 12-weeks postengraftment in NBSGW mice, we observe human cell chimerism in marrow (97% ± 0.4%), peripheral blood (61% ± 2%), and spleen (94% ± 2%) at levels observed with irradiation in NSG mice. We also detected a significant number of glycophorin-A-positive expressing cells in the developing NBSGW marrow. Further, the observed levels of human hematopoietic chimerism mimic those reported for both irradiated NSG and NSG-transgenic strains. This mouse model permits HSC engraftment while avoiding the complicating hematopoietic, gastrointestinal, and neurological side effects associated with irradiation and allows investigators without access to radiation to pursue engraftment studies with human HSCs.


Asunto(s)
Diferenciación Celular , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Subunidad gamma Común de Receptores de Interleucina/genética , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Linaje de la Célula , Genotipo , Xenoinjertos , Humanos , Inmunofenotipificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Fenotipo , Factores de Tiempo , Quimera por Trasplante
9.
Stem Cell Reports ; 3(6): 1043-57, 2014 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-25458896

RESUMEN

During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that "trap" murine cells in a proliferative state and endow them with a hemangioblast potential. These "expandable" hemangioblasts (eHBs) are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.


Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Hemangioblastos/citología , Hemangioblastos/metabolismo , Animales , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Perfilación de la Expresión Génica , Hemangioblastos/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunofenotipificación , Ratones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fenotipo , Transcriptoma
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