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1.
PLoS Comput Biol ; 16(12): e1008491, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33362275

RESUMEN

Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness.


Asunto(s)
Redes Reguladoras de Genes , Células Madre Pluripotentes Inducidas/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Humanos
2.
Hum Genet ; 137(2): 183-193, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29417219

RESUMEN

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.


Asunto(s)
Secuenciación del Exoma , Variación Genética , Cardiopatías Congénitas/genética , Mosaicismo , Niño , Exoma/genética , Cardiopatías Congénitas/fisiopatología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Programas Informáticos
3.
Nature ; 465(7299): 808-12, 2010 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-20535210

RESUMEN

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.


Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Síndrome LEOPARD/patología , Modelos Biológicos , Medicina de Precisión , Adulto , Diferenciación Celular , Línea Celular , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias/metabolismo , Activación Enzimática , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome LEOPARD/tratamiento farmacológico , Síndrome LEOPARD/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Fosfoproteínas/análisis , Reacción en Cadena de la Polimerasa , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Factores de Transcripción SOXB1/genética
4.
Stem Cells ; 32(1): 191-203, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24022884

RESUMEN

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/metabolismo , Mesodermo/metabolismo , Células Madre Pluripotentes/metabolismo , Sialoglicoproteínas/biosíntesis , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Células Endoteliales/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Mesodermo/citología , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/citología , Sialoglicoproteínas/metabolismo , Análisis de Matrices Tisulares
5.
Blood ; 120(13): e35-44, 2012 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-22898598

RESUMEN

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cell yield by 6-fold. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34(high) cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34(+) cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34(high) cell therapies in a clinically relevant nonhuman primate model.


Asunto(s)
Antígenos CD34/metabolismo , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Animales , Western Blotting , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Linaje de la Célula , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Dinoprostona/genética , Dinoprostona/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Linfocitos/citología , Linfocitos/metabolismo , Macaca , Células Mieloides/citología , Células Mieloides/metabolismo , Purinas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Internalización del Virus , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
6.
Front Cell Dev Biol ; 11: 1148013, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37113767

RESUMEN

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

7.
Stem Cell Reports ; 16(12): 3036-3049, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34739849

RESUMEN

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.


Asunto(s)
Salud , Células Madre Pluripotentes Inducidas/citología , Adulto , Señalización del Calcio , Diferenciación Celular , Línea Celular , Células Clonales , Etnicidad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factores de Riesgo , Adulto Joven
8.
Stem Cell Res ; 45: 101837, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32413789

RESUMEN

Hemoglobin production during mammalian development is characterized by temporal switches of the genes coding for the α- and ß-globin chains. Defects in this controlled process can lead to hemoglobinapathies such as sickle cell disease and ß-thalassemia. The ability of human embryonic stem cells (hESC) to proceed through hematopoiesis could provide a clinically useful source of red blood cells. However, hESC-derived red cells exhibit an embryonic/fetal, but not adult, mode of hemoglobin expression. The resource described here is a hESC line engineered to express a reporter from its adult globin promoter, providing a screening platform for small molecules that lead to efficient induction of adult globin.


Asunto(s)
Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Adulto , Animales , Línea Celular , Células Madre Embrionarias , Hematopoyesis , Humanos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética
9.
Stem Cell Res Ther ; 10(1): 228, 2019 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-31358052

RESUMEN

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) show a great promise as a renewable source of cells with broad biomedical applications. Since insulin has been used in the maintenance of hiPSCs, in this study we explored the role of insulin in culture of these cells. METHODS: We report conditions for insulin starvation and stimulation of hiPSCs. Crystal violet staining was used to study the adhesion and proliferation of hiPSCs. Apoptosis and cell cycle assays were performed through flow cytometry. Protein arrays were used to confirm phosphorylation targets, and mRNA sequencing was used to evaluate the effect of transcriptome. RESULTS: Insulin improved the seeding and proliferation of hiPSCs. We also observed an altered cell cycle profile and increase in apoptosis in hiPSCs in the absence of insulin. Furthermore, we confirmed phosphorylation of key components of insulin signaling pathway in the presence of insulin and demonstrated the significant effect of insulin on regulation of the mRNA transcriptome of hiPSCs. CONCLUSION: Insulin is a major regulator of seeding, proliferation, phosphorylation and mRNA transcriptome in hiPSCs. Collectively, our work furthers our understanding of human pluripotency and paves the way for future studies that use hiPSCs for modeling genetic ailments affecting insulin signaling pathways.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Insulina/farmacología , Transcriptoma/efectos de los fármacos , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo
10.
Cell Stem Cell ; 20(4): 518-532.e9, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28017796

RESUMEN

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network, pathway, and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications.


Asunto(s)
Heterogeneidad Genética , Células Madre Pluripotentes Inducidas/metabolismo , Transcripción Genética , Alelos , Teorema de Bayes , Diferenciación Celular/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Humanos , Proteínas del Grupo Polycomb/metabolismo , Sitios de Carácter Cuantitativo/genética , Reproducibilidad de los Resultados
12.
Stem Cell Res ; 17(1): 93-96, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27558609

RESUMEN

We have generated a MIXL1-eGFP reporter human embryonic stem cell (hESC) line using TALEN-based genome engineering. This line accurately traces endogenous MIXL1 expression via an eGFP reporter to mesendodermal precursor cells. The utility of the MIXL1-eGFP reporter hESC line lies in the prospective isolation, lineage tracing, and developmental and mechanistic studies of MIXL1+ cell populations.

13.
Nat Protoc ; 10(3): 413-25, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25654758

RESUMEN

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-ß (TGF-ß) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.


Asunto(s)
Diferenciación Celular/fisiología , Células Epiteliales/citología , Células Madre Pluripotentes/citología , Sistema Respiratorio/citología , Células Madre/citología , Ingeniería de Tejidos/métodos , Humanos , Técnicas In Vitro/métodos
14.
Cell Rep ; 13(3): 504-515, 2015 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-26456833

RESUMEN

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Leucemia Mielomonocítica Juvenil/metabolismo , Células Mieloides/citología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/patología , MicroARNs/genética , Mutación , Células Mieloides/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Regulación hacia Arriba
15.
PLoS One ; 9(7): e101316, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25010565

RESUMEN

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.


Asunto(s)
Citometría de Flujo/métodos , Atrios Cardíacos/citología , Células Madre Pluripotentes Inducidas/citología , Proteínas Musculares/genética , Miocitos Cardíacos/citología , Proteolípidos/genética , Calcio/metabolismo , Diferenciación Celular , Cromosomas Artificiales Bacterianos/genética , Fenómenos Electrofisiológicos , Expresión Génica , Genes Reporteros/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo
16.
Stem Cells Dev ; 21(3): 404-10, 2012 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-21631388

RESUMEN

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings, however, requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study, we show that once specified to neuro-epithelial lineage, hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However, our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes, namely, migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes, a new potential source for therapeutic use for brain tumors and other neurological diseases.


Asunto(s)
Astrocitos/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Astrocitos/metabolismo , Línea Celular , Linaje de la Célula , Ensayos de Migración Celular , Movimiento Celular , Humanos , Inmunofenotipificación , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Tiempo
17.
Nat Biotechnol ; 29(3): 267-72, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21358635

RESUMEN

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-ß and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.


Asunto(s)
Células Madre Embrionarias/citología , Endodermo/citología , Intestinos/citología , Células Madre Pluripotentes/citología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Células Cultivadas , Humanos , Intestinos/embriología
18.
Blood ; 109(7): 2679-87, 2007 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-17148580

RESUMEN

The onset of hematopoiesis in the mouse embryo and in the embryonic stem (ES) cell differentiation model is defined by the emergence of the hemangioblast, a progenitor with both hematopoietic and vascular potential. While there is evidence for the existence of a hemangioblast in the mouse, it is unclear if this progenitor develops during the establishment of the human hematopoietic system. In this report, we have mapped hematopoietic development in human ES cell (hESC) differentiation cultures and demonstrated that a comparable hemangioblast population exists. The human hemangioblasts were identified by their capacity to generate blast colonies that display both hematopoietic and vascular potential. These colony-forming cells express the receptor tyrosine kinase KDR (VEGF receptor 2) and represent a transient population that develops in BMP-4-stimulated embryoid bodies (EBs) between 72 and 96 hours of differentiation, prior to the onset of the primitive erythroid program. Two distinct types of hemangioblasts were identified, those that give rise to primitive erythroid cells, macrophages, and endothelial cells and those that generate only the primitive erythroid population and endothelial cells. These findings demonstrate for the first time the existence of the human hemangioblast and in doing so identify the earliest stage of hematopoietic commitment.


Asunto(s)
Células Madre Embrionarias/citología , Hematopoyesis/fisiología , Secuencia de Bases , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Citocinas/fisiología , Cartilla de ADN/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Eritropoyesis/efectos de los fármacos , Eritropoyesis/genética , Eritropoyesis/fisiología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
19.
Blood ; 105(10): 3862-70, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15677567

RESUMEN

In this report, we have defined the stage at which Scl functions in the establishment of the hematopoietic system and provide evidence that its primary role is in the generation of the hematopoietic lineages from a progenitor called the blast colony-forming cell (BL-CFC), a cell considered to be the in vitro equivalent of the hemangioblast. Using an embryonic stem (ES) cell line in which lacZ cDNA has been targeted to the Scl locus, we show that most of the BL-CFCs are detected in the SCL/lacZ- population, indicating that this progenitor does not express Scl. In the blast colony assay, Scl-/- cells initiate colony growth but are unable to generate endothelial and hematopoietic progeny and thus form colonies consisting of vascular smooth muscle cells only. The capacity to give rise to blast colonies can be rescued by retroviral transduction of a wild-type Scl gene into Scl-/- FLK-1+ cells, suggesting that the BL-CFC is generated in this population. Finally, we show that Scl-/- endothelial cells display a growth deficiency in monolayer cultures that can be partially overcome by maintaining this population as 3-dimensional aggregates indicating that specific cellular interactions are required for maintenance of the Scl-/- endothelial lineage in vitro.


Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Hematopoyesis , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Proteínas de Unión al ADN/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
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