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1.
PLoS Comput Biol ; 15(12): e1007543, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31815944

RESUMEN

Pluripotent stem cells retain the developmental timing of their species of origin in vitro, an observation that suggests the existence of a cell-intrinsic developmental clock, yet the nature and machinery of the clock remain a mystery. We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.


Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , RNA-Seq/estadística & datos numéricos , Animales , Línea Celular , Biología Computacional , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cinética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Medicina Regenerativa , Especificidad de la Especie
2.
Nat Methods ; 8(10): 821-7, 2011 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-21983960

RESUMEN

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.


Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteoma/análisis , Proteómica , Humanos , Proteoma/metabolismo
3.
Nat Methods ; 8(5): 424-9, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21478862

RESUMEN

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Animales , Biopsia , Bovinos , Proliferación Celular , Supervivencia Celular , Materiales Biocompatibles Revestidos , Medio de Cultivo Libre de Suero/química , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Expresión Génica , Sustancias de Crecimiento , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Albúmina Sérica Bovina , Piel/citología , Vitronectina
4.
PLoS Comput Biol ; 9(3): e1002936, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23505351

RESUMEN

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.


Asunto(s)
Ambystoma mexicanum/fisiología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Oncogenes , Análisis de Secuencia de ARN/métodos , Ambystoma mexicanum/genética , Amputación Quirúrgica , Animales , Análisis por Conglomerados , Extremidades/lesiones , Extremidades/fisiología , Regeneración/genética , Regeneración/fisiología , Regulación hacia Arriba , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología
5.
Cell Syst ; 9(2): 167-186.e12, 2019 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-31302154

RESUMEN

Neuroepithelial stem cells (NSC) from different anatomical regions of the embryonic neural tube's rostrocaudal axis can differentiate into diverse central nervous system tissues, but the transcriptional regulatory networks governing these processes are incompletely understood. Here, we measure region-specific NSC gene expression along the rostrocaudal axis in a human pluripotent stem cell model of early central nervous system development over a 72-h time course, spanning the hindbrain to cervical spinal cord. We introduce Escarole, a probabilistic clustering algorithm for non-stationary time series, and combine it with prior-based regulatory network inference to identify genes that are regulated dynamically and predict their upstream regulators. We identify known regulators of patterning and neural development, including the HOX genes, and predict a direct regulatory connection between the transcription factor POU3F2 and target gene STMN2. We demonstrate that POU3F2 is required for expression of STMN2, suggesting that this regulatory connection is important for region specificity of NSCs.


Asunto(s)
Células-Madre Neurales/metabolismo , Rombencéfalo/embriología , Médula Espinal/embriología , Diferenciación Celular/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células-Madre Neurales/fisiología , Células Neuroepiteliales , Neurogénesis , Neuronas/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Estatmina/genética , Estatmina/metabolismo , Transcriptoma/genética
6.
Stem Cell Reports ; 12(6): 1269-1281, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-31080110

RESUMEN

Contractile to synthetic phenotypic switching of smooth muscle cells (SMCs) contributes to stenosis in vascular disease and vascular transplants. To generate more contractile SMCs, we performed a high-throughput differentiation screen using a MYH11-NLuc-tdTomato human embryonic stem cell reporter cell line. We identified RepSox as a factor that promotes differentiation of MYH11-positive cells by promoting NOTCH signaling. RepSox induces SMCs to exhibit a more contractile phenotype than SMCs generated using PDGF-BB and TGF-ß1, two factors previously used for SMC differentiation but which also cause intimal hyperplasia. In addition, RepSox inhibited intimal hyperplasia caused by contractile to synthetic phenotypic switching of SMCs in a rat balloon injury model. Thus, in addition to providing more contractile SMCs that could prove useful for constructing artificial blood vessels, this study suggests a strategy for identifying drugs for inhibiting intimal hyperplasia that act by driving contractile differentiation rather than inhibiting proliferation non-specifically.


Asunto(s)
Diferenciación Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Células Madre Pluripotentes/metabolismo , Túnica Íntima/metabolismo , Animales , Becaplermina/metabolismo , Modelos Animales de Enfermedad , Humanos , Hiperplasia , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina/metabolismo , Células Madre Pluripotentes/patología , Ratas , Factor de Crecimiento Transformador beta1/metabolismo , Túnica Íntima/patología
7.
Exp Biol Med (Maywood) ; 242(17): 1679-1689, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28599598

RESUMEN

The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.


Asunto(s)
Encéfalo/citología , Modelos Biológicos , Células-Madre Neurales/citología , Células Madre Pluripotentes/citología , Ingeniería de Tejidos/métodos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Hidrogeles , Polietilenglicoles
8.
Cell Rep ; 2(3): 553-67, 2012 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-22981233

RESUMEN

Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFRα(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFRα(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.


Asunto(s)
Diferenciación Celular/fisiología , Hemangioblastos/metabolismo , Células Madre Pluripotentes/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Hemangioblastos/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes/citología
9.
Protein Expr Purif ; 55(1): 23-30, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17507238

RESUMEN

Shewanella oneidensis is of particular interest for research because of its unique ability to use a variety of metals as final respiratory electron acceptors and reduce them into insoluble oxides. A collection of monoclonal antibodies (mAbs) that were prepared towards Escherichia coli RNA polymerase (RNAP) was tested for reactivity with proteins extracted from S. oneidensis. Two polyol-responsive monoclonal antibodies (PR-mAbs) were used to purify RNA polymerase from S. oneidensis using immunoaffinity purification techniques. A collection of mAbs towards E. coli sigma subunits was also examined for cross-reactivity with S. oneidensis proteins. Reactions were identified with mAbs to E. coli sigma(70) and sigma(54). These mAbs will be useful tools for immunoaffinity purifying and studying the transcriptional machinery of S. oneidensis.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Shewanella/enzimología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Proteínas Bacterianas/química , Western Blotting , Cromatografía de Afinidad , ARN Polimerasas Dirigidas por ADN/química
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