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1.
Cell ; 177(4): 910-924.e22, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-30982595

RESUMEN

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.


Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Análisis de la Célula Individual , Vía de Señalización Wnt
2.
Molecules ; 27(8)2022 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-35458632

RESUMEN

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC-OCT4-EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC-AAVS1-EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC-AAVS1-EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.


Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Línea Celular , Edición Génica/métodos , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos
3.
Biophys Physicobiol ; 21(Supplemental): e211015, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39175869

RESUMEN

During embryogenesis, human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT), in which the HE cells undergo drastic functional and morphological changes from flat, anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development, molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study, by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures, we achieved real-time observation of the human EHT in vitro. By continuous observation and single-cell tracking in the culture, it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably, HSC precursor emergence was not linked to asymmetric cell division, whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT, and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development.

4.
Sci Immunol ; 9(95): eade3814, 2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38787963

RESUMEN

Patients with heart failure (HF) often experience repeated acute decompensation and develop comorbidities such as chronic kidney disease and frailty syndrome. Although this suggests pathological interaction among comorbidities, the mechanisms linking them are poorly understood. Here, we identified alterations in hematopoietic stem cells (HSCs) as a critical driver of recurrent HF and associated comorbidities. Bone marrow transplantation from HF-experienced mice resulted in spontaneous cardiac dysfunction and fibrosis in recipient mice, as well as increased vulnerability to kidney and skeletal muscle insults. HF enhanced the capacity of HSCs to generate proinflammatory macrophages. In HF mice, global chromatin accessibility analysis and single-cell RNA-seq showed that transforming growth factor-ß (TGF-ß) signaling was suppressed in HSCs, which corresponded with repressed sympathetic nervous activity in bone marrow. Transplantation of bone marrow from mice in which TGF-ß signaling was inhibited similarly exacerbated cardiac dysfunction. Collectively, these results suggest that cardiac stress modulates the epigenome of HSCs, which in turn alters their capacity to generate cardiac macrophage subpopulations. This change in HSCs may be a common driver of repeated HF events and comorbidity by serving as a key carrier of "stress memory."


Asunto(s)
Insuficiencia Cardíaca , Inmunidad Innata , Memoria Inmunológica , Ratones Endogámicos C57BL , Animales , Insuficiencia Cardíaca/inmunología , Ratones , Masculino , Multimorbilidad , Factor de Crecimiento Transformador beta/metabolismo , Células Madre Hematopoyéticas/inmunología , Transducción de Señal/inmunología , Macrófagos/inmunología , Inmunidad Entrenada
5.
Proc Natl Acad Sci U S A ; 107(24): 10926-31, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20508149

RESUMEN

Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1beta (TIF1beta), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4-dependent transcription in a phosphorylation-dependent manner. TIF1beta is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1beta is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcd1 [corrected], Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1beta specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1beta regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1beta provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Sustitución de Aminoácidos , Animales , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Ratones , Mutagénesis Sitio-Dirigida , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Serina/química , Factores de Transcripción/metabolismo , Proteína 28 que Contiene Motivos Tripartito
6.
Cell Chem Biol ; 30(7): 780-794.e8, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37379846

RESUMEN

Overlapping principles of embryonic and tumor biology have been described, with recent multi-omics campaigns uncovering shared molecular profiles between human pluripotent stem cells (hPSCs) and adult tumors. Here, using a chemical genomic approach, we provide biological evidence that early germ layer fate decisions of hPSCs reveal targets of human cancers. Single-cell deconstruction of hPSCs-defined subsets that share transcriptional patterns with transformed adult tissues. Chemical screening using a unique germ layer specification assay for hPSCs identified drugs that enriched for compounds that selectively suppressed the growth of patient-derived tumors corresponding exclusively to their germ layer origin. Transcriptional response of hPSCs to germ layer inducing drugs could be used to identify targets capable of regulating hPSC specification as well as inhibiting adult tumors. Our study demonstrates properties of adult tumors converge with hPSCs drug induced differentiation in a germ layer specific manner, thereby expanding our understanding of cancer stemness and pluripotency.


Asunto(s)
Neoplasias , Células Madre Pluripotentes , Humanos , Diferenciación Celular/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Genómica
7.
J Biol Chem ; 286(23): 20345-53, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-21471226

RESUMEN

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.


Asunto(s)
Glicómica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Lectinas/química , Polisacáridos/metabolismo , Análisis por Matrices de Proteínas/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Polisacáridos/genética
8.
Bioinformatics ; 26(19): 2486-7, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20709690

RESUMEN

UNLABELLED: Text mining from the biomedical literature is of increasing importance, yet it is not easy for the bioinformatics community to create and run text mining workflows due to the lack of accessibility and interoperability of the text mining resources. The U-Compare system provides a wide range of bio text mining resources in a highly interoperable workflow environment where workflows can very easily be created, executed, evaluated and visualized without coding. We have linked U-Compare to Taverna, a generic workflow system, to expose text mining functionality to the bioinformatics community. AVAILABILITY: http://u-compare.org/taverna.html, http://u-compare.org.


Asunto(s)
Minería de Datos/métodos , Biología Computacional , Bases de Datos Factuales , Interfaz Usuario-Computador , Flujo de Trabajo
9.
Cell Rep ; 34(10): 108818, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33691101

RESUMEN

Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.


Asunto(s)
Autorrenovación de las Células/fisiología , Histonas/metabolismo , Células Madre Neoplásicas/citología , Células Madre Pluripotentes/citología , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Linaje de la Célula , Ectodermo/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/deficiencia , Histonas/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Mesodermo/metabolismo , Células Madre Neoplásicas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Nucleosomas/metabolismo , Fosforilación , Células Madre Pluripotentes/metabolismo
10.
Cell Rep Med ; 2(2): 100202, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33665638

RESUMEN

The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.


Asunto(s)
Hematopoyesis/fisiología , Leucemia Mieloide Aguda/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Receptores Dopaminérgicos/metabolismo , Tioridazina/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal/fisiología
11.
Biochem Biophys Res Commun ; 401(1): 1-6, 2010 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-20807502

RESUMEN

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Epéndimo/citología , Proteína smad6/fisiología , Proteína smad7/fisiología , Animales , Línea Celular , Cilios/fisiología , Epéndimo/fisiología , Ratones , Proteína smad6/genética , Proteína smad7/genética
12.
FASEB J ; 23(1): 114-22, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18809738

RESUMEN

Formation of the primitive streak (PS) is the initial specification step that generates all the mesodermal and endodermal tissue lineages during early differentiation. Thus, a therapeutically compatible and efficient method for differentiation of the PS is crucial for regenerative medicine. In this study, we developed chemically defined serum-free culture conditions for the differentiation of embryonic stem (ES) cells into the PS-like cells. Cultures supplemented with Wnt showed induction of expression of a PS marker, the brachyury gene, followed by induction of the anterior PS markers goosecoid and foxa2, a posterior PS marker, evx1, and the endoderm marker sox17. Similar differentiation of PS by Wnt was also observed in human ES cells. Moreover, we revealed that the activation of the Wnt canonical pathway is essential for PS differentiation in mouse ES cells. These results demonstrated that Wnt is an essential and sufficient factor for the induction of the PS-like cells in vitro. These conditions of induction could constitute the initial step in generating therapeutically useful cells of the definitive endoderm lineage, such as hepatocytes and pancreatic endocrine cells, under chemically defined conditions.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Línea Primitiva/efectos de los fármacos , Línea Primitiva/fisiología , Proteínas Wnt/farmacología , Activinas/farmacología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Transducción de Señal , Proteínas Wnt/administración & dosificación , Proteína Wnt3
13.
Nat Cell Biol ; 19(11): 1336-1347, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29035359

RESUMEN

Acute myeloid leukaemia (AML) is distinguished by the generation of dysfunctional leukaemic blasts, and patients characteristically suffer from fatal infections and anaemia due to insufficient normal myelo-erythropoiesis. Direct physical crowding of bone marrow (BM) by accumulating leukaemic cells does not fully account for this haematopoietic failure. Here, analyses from AML patients were applied to both in vitro co-culture platforms and in vivo xenograft modelling, revealing that human AML disease specifically disrupts the adipocytic niche in BM. Leukaemic suppression of BM adipocytes led to imbalanced regulation of endogenous haematopoietic stem and progenitor cells, resulting in impaired myelo-erythroid maturation. In vivo administration of PPARγ agonists induced BM adipogenesis, which rescued healthy haematopoietic maturation while repressing leukaemic growth. Our study identifies a previously unappreciated axis between BM adipogenesis and normal myelo-erythroid maturation that is therapeutically accessible to improve symptoms of BM failure in AML via non-cell autonomous targeting of the niche.


Asunto(s)
Adipocitos/patología , Médula Ósea/patología , Eritropoyesis/fisiología , Leucemia Mieloide Aguda/patología , Adipogénesis/fisiología , Adulto , Anciano , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Técnicas de Cocultivo/métodos , Femenino , Células Madre Hematopoyéticas , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , PPAR gamma/metabolismo , Células Madre/patología , Adulto Joven
14.
Elife ; 3: e01846, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24714494

RESUMEN

Direct lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin(+) ß-cells, glucagon(+) α-cells, and somatostatin(+) δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to ß-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and ß-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001.


Asunto(s)
Reprogramación Celular , Células Secretoras de Glucagón/fisiología , Células Secretoras de Insulina/fisiología , Páncreas Exocrino/fisiología , Células Secretoras de Somatostatina/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula , Transdiferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Células Secretoras de Glucagón/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Páncreas Exocrino/citología , Páncreas Exocrino/metabolismo , Células Secretoras de Somatostatina/metabolismo , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Proteína Fluorescente Roja
15.
Nat Biotechnol ; 32(12): 1223-30, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25402613

RESUMEN

Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage-reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.


Asunto(s)
Células Acinares/citología , Linaje de la Célula , Diabetes Mellitus Experimental/terapia , Células Secretoras de Insulina/patología , Animales , Glucemia , Diferenciación Celular/genética , Metilación de ADN/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Humanos , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos NOD
16.
Stem Cells Transl Med ; 2(4): 265-73, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23526252

RESUMEN

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 10(4) M(-1)) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells.


Asunto(s)
Lectinas/metabolismo , Sondas Moleculares/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Anticuerpos/metabolismo , Biomarcadores/metabolismo , Análisis por Conglomerados , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ligandos , Ratones , Peso Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Células Madre Pluripotentes/citología , Polisacáridos/metabolismo , Unión Proteica
17.
Int J Dev Biol ; 54(8-9): 1287-94, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20711997

RESUMEN

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.


Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Células Madre Embrionarias/citología , Cresta Neural/citología , Animales , Apoptosis/efectos de los fármacos , Blastocisto/citología , Blastocisto/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Nestina , Cresta Neural/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Tiempo , Factores de Transcripción/genética
19.
Dev Dyn ; 238(6): 1309-20, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19441056

RESUMEN

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309-1320, 2009. (c) 2009 Wiley-Liss, Inc.


Asunto(s)
Tipificación del Cuerpo/fisiología , Inducción Embrionaria , Xenopus laevis/embriología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Endoteliales/fisiología , Corazón/embriología , Corazón/fisiología , Riñón/anatomía & histología , Riñón/embriología , Riñón/fisiología , Tejido Nervioso/anatomía & histología , Tejido Nervioso/embriología , Páncreas/anatomía & histología , Páncreas/embriología , Ingeniería de Tejidos , Xenopus laevis/anatomía & histología
20.
Differentiation ; 75(1): 1-11, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17244017

RESUMEN

The in vitro formation of organs and/or tissues is a major goal for regenerative medicine that would also provide a powerful tool for analyzing both the mechanisms of development and disease processes for each target organ. Here, we present a method whereby pancreatic tissues can be formed in vitro from mouse embryonic stem (ES) cells. Embryoid body-like spheres (EBSs) induced from ES cell colonies were treated with retinoic acid (RA) and activin, which are candidate regulators of pancreatic development in vivo. These induced tissues had decreased expression of the sonic hedgehog (shh) gene and expressed several pancreatic marker genes. ES cell-derived pancreatic tissue was composed of exocrine cells, endocrine cells, and pancreatic duct-like structures. In addition, the ratio of exocrine to endocrine cells in the induced tissue was found to be sensitive to the concentrations of RA and activin in the present experiment.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Páncreas/crecimiento & desarrollo , Técnicas de Cultivo de Tejidos , Activinas/farmacología , Animales , Biomarcadores , Diferenciación Celular/genética , Células Madre Embrionarias/química , Células Madre Embrionarias/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Glucagón/genética , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Insulina/genética , Ratones , Páncreas/química , Páncreas/citología , Polipéptido Pancreático/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Transactivadores/genética , Tretinoina/farmacología , alfa-Amilasas/genética
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