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1.
Cell ; 153(2): 281-3, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23582317

RESUMEN

A core network of genes maintaining pluripotency has been at least partially defined. How the genetic switch is flipped to differentiation is the subject of a new study that reveals some unexpected players.

2.
J Cell Sci ; 135(21)2022 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-36217793

RESUMEN

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.


Asunto(s)
Centrosoma , Centro Organizador de los Microtúbulos , Humanos , Centro Organizador de los Microtúbulos/metabolismo , Centrosoma/metabolismo , Intestinos , Diferenciación Celular , Proteínas/metabolismo , Mucosa Intestinal/metabolismo
3.
Development ; 147(21)2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33033118

RESUMEN

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.


Asunto(s)
Diferenciación Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Endodermo/embriología , Enfermedades de la Vesícula Biliar/genética , Enfermedades de la Vesícula Biliar/patología , Células Madre Pluripotentes Inducidas/patología , Atresia Intestinal/genética , Atresia Intestinal/patología , Mutación/genética , Páncreas/embriología , Factores de Transcripción del Factor Regulador X/genética , Alelos , Secuencia de Bases , Diferenciación Celular/genética , Cromatina/metabolismo , Consanguinidad , Diabetes Mellitus/diagnóstico por imagen , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Familia , Femenino , Enfermedades de la Vesícula Biliar/diagnóstico por imagen , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Atresia Intestinal/diagnóstico por imagen , Masculino , Linaje , Transcripción Genética , Transcriptoma/genética , Microtomografía por Rayos X
4.
BMC Biol ; 20(1): 47, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35164755

RESUMEN

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.


Asunto(s)
Hialuronoglucosaminidasa , Degeneración Macular , Anciano , Coroides/irrigación sanguínea , Coroides/patología , Células Endoteliales , Angiografía con Fluoresceína , Glicocálix/patología , Humanos , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/uso terapéutico , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/patología
5.
Mol Psychiatry ; 26(10): 5766-5788, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-32647257

RESUMEN

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.


Asunto(s)
Enfermedad de Alzheimer , Síndrome de Down , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Síndrome de Down/genética , Genes Supresores , Humanos , Organoides/metabolismo , Trisomía
6.
Development ; 145(17)2018 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-30115640

RESUMEN

The historic town of Taos, New Mexico, with its rich multicultural history of art and craft, was the site of the second Keystone Symposium on 'Endoderm Development and Disease', which was held in February 2018. The theme of the meeting was 'Cross-Organ Comparison and Interplay', emphasizing an integrative and multisystem approach to the broad topics of organ physiology, homeostasis, repair, regeneration and disease. As we review here, participants shared their recent discoveries and discussed how new technologies developed in one organ system might be applied to answer crucial questions in another. Other integrative themes were how agents such as parasites, microbes, immune cells, physical forces and innervation can affect tissue organization and progenitor cell dynamics, and how defects in the development of an organ can impact its adult function. Participants came away with a broader vision of their field and a renewed sense of collective energy empowered by novel tools and fresh ideas.


Asunto(s)
Endodermo , Animales , Congresos como Asunto , Humanos , New Mexico
7.
Genes Dev ; 26(22): 2471-6, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23154981

RESUMEN

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.


Asunto(s)
Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Diferenciación Celular/genética , Línea Celular , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Mesodermo/metabolismo , Transducción de Señal , Proteína Smad2/genética , Proteína smad3/genética
8.
Differentiation ; 99: 62-69, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29239730

RESUMEN

Corneal tissue is the most transplanted of all body tissues. Currently, cadaveric donor tissues are used for transplantation. However, a global shortage of transplant grade material has prompted development of alternative, cell-based therapies for corneal diseases. Pluripotent stem cells are attractive sources of cells for regenerative medicine, because large numbers of therapeutically useful cells can be generated. However, a detailed understanding of how to differentiate clinically relevant cell types from stem cells is fundamentally required. Periocular mesenchyme (POM), a subtype of cranial neural crest, is vital for development of multiple cell types in the cornea, including clinically relevant cells such as corneal endothelium and stromal keratocytes. Herein, we describe protocols for differentiation of POM from pluripotent stem cells. Using defined media containing inhibitors of TGFß and WNT signalling, we generated neural crest cells that express high levels of the POM transcription factors PITX2 and FOXC1. Furthermore, we identified cells resembling POM in the adult cornea, located in a niche between the trabecular meshwork and peripheral endothelium. The generation and expansion of POM is an important step in the generation of a number of cells types that could prove to be clinically useful for a number of diseases of the cornea.


Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias Humanas/citología , Cresta Neural/citología , Células Madre Pluripotentes/citología , Células Cultivadas , Córnea/citología , Humanos , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
9.
Genes Dev ; 25(3): 238-50, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21245162

RESUMEN

Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective toward the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells and mouse epiblast stem cells to study specification of definitive endoderm in vitro. Using a combination of whole-genome expression and chromatin immunoprecipitation (ChIP) deep sequencing (ChIP-seq) analyses, we established an hierarchy of transcription factors regulating endoderm specification. Importantly, the pluripotency factors NANOG, OCT4, and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMESODERMIN (EOMES), which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.


Asunto(s)
Diferenciación Celular , Endodermo , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes , Proteínas de Dominio T Box/metabolismo , Activinas/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular , Endodermo/citología , Endodermo/metabolismo , Redes Reguladoras de Genes/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Proteína Homeótica Nanog , Proteína Nodal/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas de Dominio T Box/genética
10.
Development ; 141(18): 3505-16, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25183869

RESUMEN

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.


Asunto(s)
Gastrulación/fisiología , Genes MCC/genética , Morfogénesis/fisiología , Vía de Señalización Wnt/fisiología , Pez Cebra/embriología , Animales , Western Blotting , Polaridad Celular/fisiología , Inmunoprecipitación , Hibridación in Situ , Luciferasas , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Dominios PDZ/genética , Reacción en Cadena de la Polimerasa , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteínas de Pez Cebra/metabolismo
12.
Biol Open ; 13(10)2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39387302

RESUMEN

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types.


Asunto(s)
Diferenciación Celular , Células Epiteliales , Pulmón , Células Madre Pluripotentes , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Pulmón/citología , Pulmón/metabolismo , Pulmón/embriología , Células Epiteliales/metabolismo , Células Epiteliales/citología , Biomarcadores , Proteínas Fetales/metabolismo , Proteínas Fetales/genética , Antígenos CD/metabolismo , Técnicas de Cultivo de Célula , Molécula de Adhesión Celular del Leucocito Activado
13.
Stem Cells ; 30(4): 631-42, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22893457

RESUMEN

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic ß cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.


Asunto(s)
Activinas/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Animales , Diferenciación Celular/fisiología , Endodermo/citología , Humanos , Ratones , Transducción de Señal
14.
Stem Cells ; 29(8): 1176-85, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21630377

RESUMEN

Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions.


Asunto(s)
Activinas/metabolismo , Endodermo/citología , Redes Reguladoras de Genes , Proteína Nodal/metabolismo , Células Madre/metabolismo , Secuencia de Bases , Diferenciación Celular , Línea Celular , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Humanos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Proteína Homeótica Nanog , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Células Madre/citología
15.
Dev Dyn ; 240(9): 2166-74, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22016184

RESUMEN

Mutated in Colorectal Cancer (MCC) encodes a multiple PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein implicated, as its name suggests, in the pathogenesis of human colon cancer. To date, however, what role, if any, MCC plays in normal tissue homeostasis and development remains unclear. In an effort to expand our understanding of MCC function and distribution, we examined the expression of the evolutionarily conserved mouse Mcc homolog between embryonic days (E) 6.5 and 12.5 using conventional whole-mount in situ hybridization and two independent Mcc reporter alleles. Mcc is expressed in the posterior primitive streak during gastrulation and in diverse tissues of both mesodermal and endodermal origin. In addition, Mcc transcripts localize to the posterior neural tube and identify discrete neuronal subtypes and ganglia within the developing central nervous system. Genetically, however, Mcc is entirely dispensable, as mice homozygous for the Mcc(Gt(D062B07)) gene trap allele, which generates a loss-of-function mutation, are viable and fertile, with no ostensible phenotype.


Asunto(s)
Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Western Blotting , Ectodermo/citología , Ectodermo/metabolismo , Embrión de Mamíferos/citología , Desarrollo Embrionario/genética , Endodermo/citología , Endodermo/metabolismo , Femenino , Masculino , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Xenopus laevis
16.
Sci Rep ; 11(1): 4712, 2021 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-33633173

RESUMEN

Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.


Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Pulmón/citología , Células Madre Pluripotentes/citología , Factor Nuclear Tiroideo 1/análisis , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Línea Celular , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Luminiscentes/genética , Pulmón/metabolismo , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Factor Nuclear Tiroideo 1/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteína Fluorescente Roja
17.
Sci Rep ; 10(1): 11801, 2020 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-32678226

RESUMEN

Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related protein superfamily. MFAP4 is produced by vascular smooth muscle cells and is highly enriched in the blood vessels of the heart and lung, where it is thought to contribute to the structure and function of elastic fibers. Genetic studies in humans have implicated MFAP4 in the pathogenesis of Smith-Magenis syndrome, in which patients present with multiple congenital abnormalities and mental retardation, as well as in the severe cardiac malformation left-sided congenital heart disease. Comprehensive genetic analysis of the role of MFAP4 orthologues in model organisms during development and tissue homeostasis is however lacking. Here, we demonstrate that zebrafish mfap4 transcripts are detected embryonically, resolving to the macrophage lineage by 24 h post fertilization. mfap4 null mutant zebrafish are unexpectedly viable and fertile, without ostensible phenotypes. However, tail fin amputation assays reveal that mfap4 mutants have reduced numbers of macrophages, with a concomitant increase in neutrophilic granulocytes, although recruitment of both cell types to the site of injury was unaffected. Molecular analyses suggest that loss of Mfap4 alters the balance between myeloid and lymphoid lineages during both primitive and definitive haematopoiesis, which could significantly impact the downstream function of the immune system.


Asunto(s)
Proteínas de la Matriz Extracelular/genética , Hematopoyesis/genética , Pez Cebra/genética , Animales , Proteínas Portadoras , Desarrollo Embrionario/genética , Proteínas de la Matriz Extracelular/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas , Humanos , Recuento de Leucocitos , Microfibrillas/metabolismo , Fenotipo , Pez Cebra/embriología , Pez Cebra/metabolismo
18.
Sci Adv ; 6(2): eaax9852, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31950080

RESUMEN

Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease-causing mutations. We have identified a new mutation in the PRDM15 gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine Prdm15 causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.


Asunto(s)
Polaridad Celular , Proteínas de Unión al ADN/genética , Holoprosencefalia/genética , Mutación con Pérdida de Función/genética , Receptores Notch/metabolismo , Factores de Transcripción/genética , Vía de Señalización Wnt , Animales , Tipificación del Cuerpo/genética , Encéfalo/anomalías , Encéfalo/embriología , Polaridad Celular/genética , Estudios de Cohortes , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Placa Neural/metabolismo , Embarazo , Transcripción Genética , Dedos de Zinc
19.
Stem Cell Reports ; 12(1): 57-70, 2019 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-30629940

RESUMEN

Heterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward ß-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ ß-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny.


Asunto(s)
Diferenciación Celular , Endodermo/metabolismo , Factor de Transcripción GATA6/genética , Redes Reguladoras de Genes , Células Secretoras de Insulina/metabolismo , Páncreas/anomalías , Enfermedades Pancreáticas/congénito , Células Madre Pluripotentes/metabolismo , Linaje de la Célula , Células Cultivadas , Endodermo/citología , Factor de Transcripción GATA6/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/citología , Páncreas/metabolismo , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/metabolismo , Células Madre Pluripotentes/citología , Unión Proteica , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
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