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1.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299194

RESUMO

A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the RUNX1-JAK2 fusion into one endogenous RUNX1 allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets-confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Janus Quinase 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição STAT/metabolismo , Diferenciação Celular , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Janus Quinase 2/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais
2.
Sci Immunol ; 6(57)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664060

RESUMO

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.


Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19 , Epitopos de Linfócito T , Antígenos HLA-A/imunologia , Imunidade Celular , Mutação , SARS-CoV-2 , Linfócitos T CD8-Positivos/patologia , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Proliferação de Células , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferon gama/imunologia , Peptídeos/genética , Peptídeos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia
3.
Stem Cells Dev ; 27(19): 1376-1384, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30009677

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) derived from human induced pluripotent stem cells (hiPSCs) hold great promise for disease modeling, drug screens, and eventually cell therapy approaches. During in vitro differentiation of hiPSCs into hematoendothelial progenitors, the emergence of CD34-positive cells indicates a critical step of lineage specification. To facilitate the monitoring of hematopoietic differentiation of hiPSCs, we established fluorescent reporter cells for the stem and progenitor cell marker CD34. An IRES-GFP (internal ribosome entry site green fluorescent protein) construct was introduced by CRISPR/Cas9 into the 3' untranslated region of one endogenous CD34 allele. Single-cell clones were generated after excision of the floxed puromycin resistance cassette by Cre recombination and correct insertion was confirmed by genotyping polymerase chain reaction and Southern blot. To validate their functionality, the reporter hiPSCs were in vitro differentiated toward CD34+ cells using the STEMdiff Hematopoietic Kit combined with short-term inhibition of GSK3 (glycogen synthase kinase 3). All cells expressing nuclear GFP were positive for cell surface CD34, thus allowing the direct monitoring of the differentiation of hiPSCs into CD34+ cells either by flow cytometry or confocal microscopy. After fluorescence-activated cell sorting, cells displaying high GFP expression exhibited increased colony-forming potential in the MethoCult colony-forming unit assays as compared with CD34+ cells obtained by magnetic-activated cell sorting. In summary, we have generated functional CD34 GFP reporter hiPSCs, which not only permit label-free separation of HSPCs, but also tracing of the emergence and fate of CD34+ progenitors at the single-cell level.


Assuntos
Antígenos CD34/genética , Ensaio de Unidades Formadoras de Colônias/métodos , Proteínas de Fluorescência Verde/genética , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Nature ; 550(7674): 114-118, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28953874

RESUMO

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.


Assuntos
Bancos de Espécimes Biológicos , Genômica/métodos , Haploidia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Animais , Vasos Sanguíneos/citologia , Linhagem da Célula/genética , Resfriado Comum/genética , Resfriado Comum/virologia , Genes Essenciais/genética , Testes Genéticos , Células HEK293 , Homozigoto , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Neovascularização Fisiológica/genética , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Independentes de Cálcio/metabolismo , Rhinovirus/patogenicidade
5.
Oncotarget ; 7(16): 21362-80, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-26870995

RESUMO

The EPH and ephrins function as both receptor and ligands and the output on their complex signaling is currently investigated in cancer. Previous work shows that some EPH family members have clinical value in breast cancer, suggesting that this family could be a source of novel clinical targets. Here we quantified the mRNA expression levels of EPH receptors and their ligands, ephrins, in 65 node positive breast cancer samples by RT-PCR with TaqMan® Micro Fluidics Cards Microarray. Upon hierarchical clustering of the mRNA expression levels, we identified a subgroup of patients with high expression, and poor clinical outcome. EPHA2, EPHA4, EFNB1, EFNB2, EPHB2 and EPHB6 were significantly correlated with the cluster groups and particularly EPHB2 was an independent prognostic factor in multivariate analysis and in four public databases. The EPHB2 protein expression was also analyzed by immunohistochemistry in paraffin embedded material (cohort 2). EPHB2 was detected in the membrane and cytoplasmic cell compartments and there was an inverse correlation between membranous and cytoplasmic EPHB2. Membranous EPHB2 predicted longer breast cancer survival in both univariate and multivariate analysis while cytoplasmic EPHB2 indicated shorter breast cancer survival in univariate analysis. Concluding: the EPH/EFN cluster analysis revealed that high EPH/EFN mRNA expression is an independent prognostic factor for poor survival. Especially EPHB2 predicted poor breast cancer survival in several materials and EPHB2 protein expression has also prognostic value depending on cell localization.


Assuntos
Neoplasias da Mama/genética , Efrinas/genética , Regulação Neoplásica da Expressão Gênica , Receptor EphB2/genética , Receptores da Família Eph/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Estudos de Coortes , Efrinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Células HCT116 , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA , Receptor EphB2/metabolismo , Receptores da Família Eph/metabolismo
6.
J Tissue Eng Regen Med ; 9(2): 127-36, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23038666

RESUMO

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system.


Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Células Endoteliais/citologia , Fibrina/química , Células-Tronco/citologia , Técnicas de Cultura de Células , Técnicas de Cocultura/métodos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos Mononucleares/citologia , Metaloproteinase 14 da Matriz/metabolismo , Microscopia de Fluorescência , Neovascularização Patológica , Engenharia Tecidual/métodos
7.
Angiogenesis ; 17(4): 921-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25086616

RESUMO

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.


Assuntos
Tecido Adiposo/citologia , Células Endoteliais/citologia , Fibrina/química , Células-Tronco/citologia , Engenharia Tecidual/métodos , Diferenciação Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Morfogênese , Neovascularização Patológica , Neovascularização Fisiológica , Reação em Cadeia da Polimerase
8.
Cytotherapy ; 15(11): 1426-35, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24094492

RESUMO

BACKGROUND AIMS: Human endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge. METHODS: Fat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein. RESULTS: The expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel. CONCLUSIONS: Rare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.


Assuntos
Antígenos CD34/imunologia , Antígenos CD/imunologia , Células Endoteliais/citologia , Glicoproteínas/imunologia , Peptídeos/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Células-Tronco/citologia , 5'-Nucleotidase/biossíntese , Antígeno AC133 , Tecido Adiposo/citologia , Antígenos CD/biossíntese , Antígeno CD146/biossíntese , Caderinas/biossíntese , Diferenciação Celular/imunologia , Células Cultivadas , Endoglina , Células Endoteliais/imunologia , Proteínas Ligadas por GPI/biossíntese , Humanos , Lipectomia , Microesferas , Neovascularização Fisiológica/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Receptores de Superfície Celular/biossíntese , Células-Tronco/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
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