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1.
Nat Methods ; 19(6): 662-670, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35577954

RESUMEN

Spatial transcriptomics approaches have substantially advanced our capacity to detect the spatial distribution of RNA transcripts in tissues, yet it remains challenging to characterize whole-transcriptome-level data for single cells in space. Addressing this need, researchers have developed integration methods to combine spatial transcriptomic data with single-cell RNA-seq data to predict the spatial distribution of undetected transcripts and/or perform cell type deconvolution of spots in histological sections. However, to date, no independent studies have comparatively analyzed these integration methods to benchmark their performance. Here we present benchmarking of 16 integration methods using 45 paired datasets (comprising both spatial transcriptomics and scRNA-seq data) and 32 simulated datasets. We found that Tangram, gimVI, and SpaGE outperformed other integration methods for predicting the spatial distribution of RNA transcripts, whereas Cell2location, SpatialDWLS, and RCTD are the top-performing methods for the cell type deconvolution of spots. We provide a benchmark pipeline to help researchers select optimal integration methods to process their datasets.


Asunto(s)
Benchmarking , Transcriptoma , ARN , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos
2.
J Cell Physiol ; 238(1): 179-194, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36436185

RESUMEN

Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1+ cells overlapped with CD34+ CD144+ CD184+ CD73- arterial-type HE cells. Transcriptome analysis by RNA sequencing indicated that TAL1/SCL, GATA2, MYB, RUNX1 and other key transcription factors for hematopoietic development were mainly expressed in PEAR1+ cells, whereas the genes encoding for niche-related signals, such as fibronectin, vitronectin, bone morphogenetic proteins and jagged1, were highly expressed in PEAR1- cells. The isolated PEAR1+ cells exhibited significantly greater EHT capacity on endothelial niche, compared with the PEAR1- cells. Colony-forming unit (CFU) assays demonstrated the multilineage hematopoietic potential of PEAR1+ -derived hematopoietic cells. Furthermore, PEAR1 knockout in hPSCs by CRISPR/Cas9 technology revealed that the hematopoietic differentiation was impaired, resulting in decreased EHT capacity, decreased expression of hematopoietic-related transcription factors, and increased expression of niche-related signals. In summary, this study revealed a novel role of PEAR1 in balancing intrinsic and extrinsic signals for early hematopoietic fate decision.


Asunto(s)
Hemangioblastos , Hematopoyesis , Células Madre Hematopoyéticas , Células Madre Pluripotentes , Receptores de Superficie Celular , Humanos , Diferenciación Celular , Hemangioblastos/citología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/metabolismo
3.
Blood ; 137(7): 959-968, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33094331

RESUMEN

Genome-wide association studies have identified common variants associated with platelet-related phenotypes, but because these variants are largely intronic or intergenic, their link to platelet biology is unclear. In 290 normal subjects from the GeneSTAR Research Study (110 African Americans [AAs] and 180 European Americans [EAs]), we generated whole-genome sequence data from whole blood and RNA sequence data from extracted nonribosomal RNA from 185 induced pluripotent stem cell-derived megakaryocyte (MK) cell lines (platelet precursor cells) and 290 blood platelet samples from these subjects. Using eigenMT software to select the peak single-nucleotide polymorphism (SNP) for each expressed gene, and meta-analyzing the results of AAs and EAs, we identify (q-value < 0.05) 946 cis-expression quantitative trait loci (eQTLs) in derived MKs and 1830 cis-eQTLs in blood platelets. Among the 57 eQTLs shared between the 2 tissues, the estimated directions of effect are very consistent (98.2% concordance). A high proportion of detected cis-eQTLs (74.9% in MKs and 84.3% in platelets) are unique to MKs and platelets compared with peak-associated SNP-expressed gene pairs of 48 other tissue types that are reported in version V7 of the Genotype-Tissue Expression Project. The locations of our identified eQTLs are significantly enriched for overlap with several annotation tracks highlighting genomic regions with specific functionality in MKs, including MK-specific DNAse hotspots, H3K27-acetylation marks, H3K4-methylation marks, enhancers, and superenhancers. These results offer insights into the regulatory signature of MKs and platelets, with significant overlap in genes expressed, eQTLs detected, and enrichment within known superenhancers relevant to platelet biology.


Asunto(s)
Plaquetas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Megacariocitos/metabolismo , ARN/genética , Transcriptoma , Adulto , Población Negra/genética , Plaquetas/citología , Células Cultivadas , Femenino , Ontología de Genes , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Megacariocitos/citología , Especificidad de Órganos , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , ARN/biosíntesis , RNA-Seq , Población Blanca/genética , Secuenciación Completa del Genoma
4.
Am J Hematol ; 97(2): 194-202, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-34779029

RESUMEN

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.


Asunto(s)
Transfusión de Eritrocitos , Eritrocitos/citología , Eritropoyesis , Células Madre Pluripotentes Inducidas/citología , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID
5.
Mol Ther ; 29(5): 1918-1932, 2021 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-33484967

RESUMEN

Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.


Asunto(s)
Eritroblastos/citología , Transfusión de Eritrocitos/métodos , Eritrocitos/citología , Leucocitos Mononucleares/citología , Complejo Represivo Polycomb 1/genética , Adulto , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Sangre Fetal , Humanos , Ratones , Modelos Animales
6.
Blood ; 131(2): 191-201, 2018 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-29101237

RESUMEN

Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Megacariocitos/citología , Receptor Notch4/genética , Trombopoyesis , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Mutación Puntual , Receptor Notch4/metabolismo , Transducción de Señal
7.
Stem Cells ; 37(6): 779-790, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30811771

RESUMEN

Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communications and exert various biological activities via delivering unique cargos of functional molecules such as RNAs and proteins to recipient cells. Previous studies showed that EVs produced and secreted by human mesenchymal stem cells (MSCs) can substitute intact MSCs for tissue repair and regeneration. In this study, we examined properties and functions of EVs from human induced pluripotent stem cells (iPSCs) that can be cultured infinitely under a chemically defined medium free of any exogenous EVs. We collected and purified EVs secreted by human iPSCs and MSCs. Purified EVs produced by both stem cell types have similar sizes (∼150 nm in diameter), but human iPSCs produced 16-fold more EVs than MSCs. When highly purified iPSC-EVs were applied in culture to senescent MSCs that have elevated reactive oxygen species (ROS), human iPSC-EVs reduced cellular ROS levels and alleviated aging phenotypes of senescent MSCs. Our discovery reveals that EVs from human stem cells can alleviate cellular aging in culture, at least in part by delivering intracellular peroxiredoxin antioxidant enzymes. Stem Cells 2019;37:779-790.


Asunto(s)
Senescencia Celular/genética , Vesículas Extracelulares/química , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Peroxirredoxinas/genética , Antioxidantes/metabolismo , Transporte Biológico , Comunicación Celular , Vesículas Extracelulares/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Células Madre Mesenquimatosas/citología , Peroxirredoxinas/metabolismo , Fenotipo , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Transducción Genética , Transgenes
8.
Nature ; 515(7527): 414-8, 2014 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-25132547

RESUMEN

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.


Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Trastornos Mentales/patología , Sinapsis/patología , Animales , Diferenciación Celular , Fibroblastos , Glutamina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Trastornos Mentales/genética , Trastornos Mentales/metabolismo , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Linaje , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , Prosencéfalo/metabolismo , Prosencéfalo/patología , Unión Proteica , Sinapsis/metabolismo , Transcriptoma
9.
J Cell Physiol ; 231(5): 1065-76, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26395760

RESUMEN

Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34(+) CD31(+) CD144(+) population derived from hPSCs contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial-to-hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34(+) CD31(+) CD144(+) HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation.


Asunto(s)
Células Endoteliales/citología , Hematopoyesis , Células Madre Multipotentes/citología , Células Madre Pluripotentes/citología , Antígenos CD/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos
10.
Blood ; 124(12): 1926-30, 2014 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-25114263

RESUMEN

Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.


Asunto(s)
Trastornos de la Coagulación Sanguínea Heredados/genética , Trastornos de la Coagulación Sanguínea Heredados/terapia , Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/terapia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutación Missense , Reparación del Gen Blanco/métodos , Animales , Trastornos de la Coagulación Sanguínea Heredados/patología , Trastornos de las Plaquetas Sanguíneas/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/trasplante , Leucemia Mieloide Aguda/patología , Ratones , Trombopoyesis/genética
11.
Stem Cells ; 33(5): 1470-9, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25702619

RESUMEN

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult ß-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa ß-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.


Asunto(s)
Anemia de Células Falciformes/genética , Diferenciación Celular , Eritrocitos/metabolismo , Células Madre Pluripotentes Inducidas/patología , Mutación Puntual/genética , Edición de ARN/genética , Globinas beta/genética , Adulto , Anemia de Células Falciformes/patología , Línea Celular , Células Eritroides/citología , Células Nutrientes/citología , Marcación de Gen , Sitios Genéticos , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo
12.
Mol Ther ; 23(3): 570-7, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25418680

RESUMEN

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.


Asunto(s)
Alelos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma Humano , Células Madre Pluripotentes Inducidas/metabolismo , Sitios de Carácter Cuantitativo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Línea Celular , Reparación del ADN por Unión de Extremidades , Endonucleasas/genética , Endonucleasas/metabolismo , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Datos de Secuencia Molecular , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Reparación del ADN por Recombinación , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
13.
Proc Natl Acad Sci U S A ; 110(31): 12601-6, 2013 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-23858432

RESUMEN

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.


Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Matriz Extracelular/química , Neovascularización Fisiológica , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos/métodos , Línea Celular , Células Endoteliales/citología , Endotelio Vascular/citología , Humanos , Células Madre Pluripotentes/citología , Medicina Regenerativa/métodos
14.
Nat Genet ; 39(2): 237-42, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17211412

RESUMEN

Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.


Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Genes Supresores de Tumor , Células Madre/metabolismo , Adulto , Proliferación Celular , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Células Tumorales Cultivadas
15.
Am J Hum Genet ; 90(2): 295-300, 2012 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-22305531

RESUMEN

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.


Asunto(s)
Genes Ligados a X , Mutación de Línea Germinal , Hemoglobinuria Paroxística/genética , Proteínas de la Membrana/genética , Adulto , Animales , Cromosomas Humanos X/genética , Exoma/genética , Salud de la Familia , Femenino , Genotipo , Heterocigoto , Humanos , Masculino , Ratones , Linaje , Fenotipo , Embarazo , Transfección/métodos
16.
Blood ; 121(15): 2882-90, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23372166

RESUMEN

Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with ß-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Western Blotting , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Proliferación Celular , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/citología , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Microscopía Confocal , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
17.
Stem Cells ; 32(2): 349-63, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23765875

RESUMEN

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) shares much similarity to the cancer initiation process, and the molecular mechanisms underlying both processes remain to be elucidated. Here, we report that a tumor- or embryonic stem cell-specific Ras gene ERas, which encodes a constitutively active form of GTPase, and its downstream Phosphoinositide-3 kinase/Akt signaling pathway are important facilitators for the somatic reprogramming process. We found that overexpression of ERas retrovirally enhanced mouse iPSC induction while ERas knockdown repressed it. Modulation of Akt signaling by genetic or chemical means greatly impacted the reprogramming efficiency. Forced expression of a constitutively active Akt1 gene could rescue the reduced efficiency resulting from ERas knockdown, and point-mutation analyses further revealed that ERas is tightly coupled with Akt signaling to enhance reprogramming. Mechanistically, the forkhead transcription factor FoxO1 can function as a barrier to the iPSC induction, and the inactivation of FoxO1 by Akt-dependent phosphorylation largely accounts for the enhancing effect of ERas-Akt signaling on reprogramming. Collectively, these results unravel the significance of the ERas-Akt-FoxO1 signaling axis in iPSC generation, suggesting a possibly shared molecular basis for both somatic reprogramming and cancer initiation.


Asunto(s)
Células Madre Embrionarias/metabolismo , Factores de Transcripción Forkhead/genética , Proteína Oncogénica p21(ras)/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Diferenciación Celular/genética , Reprogramación Celular/genética , Proteína Forkhead Box O1 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neoplasias/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética
18.
Stem Cells ; 32(1): 269-78, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24105986

RESUMEN

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.


Asunto(s)
Eritroblastos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Diferenciación Celular/efectos de los fármacos , Eritroblastos/enzimología , Eritropoyesis/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/enzimología , Janus Quinasa 2/genética
20.
Cancer Cell ; 12(3): 230-8, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17785204

RESUMEN

The antitumorigenic activity of antioxidants has been presumed to arise from their ability to squelch DNA damage and genomic instability mediated by reactive oxygen species (ROS). Here, we report that antioxidants inhibited three tumorigenic models in vivo. Inhibition of a MYC-dependent human B lymphoma model was unassociated with genomic instability but was linked to diminished hypoxia-inducible factor (HIF)-1 levels in a prolyl hydroxylase 2 and von Hippel-Lindau protein-dependent manner. Ectopic expression of an oxygen-independent, stabilized HIF-1 mutant rescued lymphoma xenografts from inhibition by two antioxidants: N-acetylcysteine and vitamin C. These findings challenge the paradigm that antioxidants diminish tumorigenesis primarily through decreasing DNA damage and mutations and provide significant support for a key antitumorigenic effect of diminishing HIF levels.


Asunto(s)
Acetilcisteína/farmacología , Antineoplásicos/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Factor 1 Inducible por Hipoxia/fisiología , Animales , Línea Celular , Inestabilidad Genómica , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Transgénicos , Mutación
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