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1.
BMC Bioinformatics ; 23(1): 276, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35831796

RESUMO

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) allows the detection of rare cell types in complex tissues. The detection of markers for rare cell types is useful for further biological analysis of, for example, flow cytometry and imaging data sets for either physical isolation or spatial characterization of these cells. However, only a few computational approaches consider the problem of selecting specific marker genes from scRNA-seq data. RESULTS: Here, we propose sc2marker, which is based on the maximum margin index and a database of proteins with antibodies, to select markers for flow cytometry or imaging. We evaluated the performances of sc2marker and competing methods in ranking known markers in scRNA-seq data of immune and stromal cells. The results showed that sc2marker performed better than the competing methods in accuracy, while having a competitive running time.


Assuntos
Análise de Célula Única , Software , Perfilação da Expressão Gênica/métodos , RNA-Seq , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Sequenciamento do Exoma
2.
Blood ; 136(18): 2051-2064, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32726410

RESUMO

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.


Assuntos
Medula Óssea/patologia , Fibrose/patologia , Inflamação/patologia , Transtornos Mieloproliferativos/complicações , Fator Plaquetário 4/metabolismo , Mielofibrose Primária/patologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Proliferação de Células , Progressão da Doença , Fibrose/etiologia , Fibrose/imunologia , Fibrose/metabolismo , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos , Camundongos , Camundongos Knockout , Mutação , Fator Plaquetário 4/genética , Mielofibrose Primária/etiologia , Mielofibrose Primária/imunologia , Mielofibrose Primária/metabolismo
3.
Nat Cardiovasc Res ; 3(8): 951-969, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39155965

RESUMO

Myelofibrosis and osteosclerosis are fibrotic diseases disrupting bone marrow function that occur in various leukemias but also in response to non-malignant alterations in hematopoietic cells. Here we show that endothelial cell-specific inactivation of the Lats2 gene, encoding Hippo kinase large tumor suppressor kinase 2, or overexpression of the downstream effector YAP1 induce myofibroblast formation and lead to extensive fibrosis and osteosclerosis, which impair bone marrow function and cause extramedullary hematopoiesis in the spleen. Mechanistically, loss of LATS2 induces endothelial-to-mesenchymal transition, resulting in increased expression of extracellular matrix and secreted signaling molecules. Changes in endothelial cells involve increased expression of serum response factor target genes, and, strikingly, major aspects of the LATS2 mutant phenotype are rescued by inactivation of the Srf gene. These findings identify the endothelium as a driver of bone marrow fibrosis, which improves understanding of myelofibrotic and osteosclerotic diseases, for which drug therapies are currently lacking.

4.
Blood Adv ; 8(3): 766-779, 2024 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-38147624

RESUMO

ABSTRACT: It is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1-/+, Egr1-/+, Csnk1a1/Egr1-/+ mice. A transplantable acute leukemia only developed in the Csnk1a1-/+Trp53-edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The "Myc signature" closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Humanos , Camundongos , Medula Óssea/metabolismo , Deleção Cromossômica , Haploinsuficiência , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Cell Rep ; 43(1): 113608, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38117649

RESUMO

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.


Assuntos
Antineoplásicos , Transtornos Mieloproliferativos , Neoplasias , Humanos , Camundongos , Animais , Proteínas Hedgehog/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Leucócitos Mononucleares/metabolismo , Hematopoese
6.
Nat Nanotechnol ; 18(4): 336-342, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37037895

RESUMO

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.


Assuntos
Aumento da Imagem , Rim , Animais , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Microscopia Confocal/métodos
8.
Exp Hematol ; 110: 28-33, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35341805

RESUMO

Within the heterogenous pool of bone marrow stromal cells, mesenchymal stromal cells (MSCs) are of particular interest because of their hematopoiesis-supporting capacities, contribution to disease progression, therapy resistance, and leukemic initiation. Cultured bone marrow-derived stromal cells (cBMSCs) are used for in vitro modeling of hematopoiesis-stroma interactions, validation of disease mechanisms, and screening for therapeutic targets. Here, we place cBMSCs (mouse and human) in a bone marrow tissue context by systematically comparing the transcriptome of plastic-adherent cells on a single-cell level with in vivo counterparts. Cultured BMSCs encompass a rather homogenous cell population, independent of the isolation method used and, although still possessing hematopoiesis-supporting capacity, are distinct from freshly isolated MSCs and more akin to in vivo fibroblast populations. Informed by combined cell trajectories and pathway analyses, we illustrate that TGFb inhibition in vitro can preserve a more "MSC"-like phenotype.


Assuntos
Células da Medula Óssea , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Fibroblastos , Hematopoese/fisiologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Análise de Célula Única
9.
Blood Adv ; 6(6): 1780-1796, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35016204

RESUMO

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.


Assuntos
Deleção Cromossômica , Síndromes Mielodisplásicas , Haploinsuficiência , Células-Tronco Hematopoéticas/patologia , Humanos , Síndromes Mielodisplásicas/patologia
10.
Cell Stem Cell ; 28(4): 637-652.e8, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33301706

RESUMO

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.


Assuntos
Células-Tronco Mesenquimais , Transtornos Mieloproliferativos , Mielofibrose Primária , Alarminas , Animais , Medula Óssea , Humanos , Camundongos
11.
Front Genome Ed ; 2: 618308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-34713241

RESUMO

The contribution of bone marrow stromal cells to the pathogenesis and therapy response of myeloid malignancies has gained significant attention over the last decade. Evidence suggests that the bone marrow stroma should not be neglected in the design of novel, targeted-therapies. In terms of gene-editing, the focus of gene therapies has mainly been on correcting mutations in hematopoietic cells. Here, we outline why alterations in the stroma should also be taken into consideration in the design of novel therapeutic strategies but also outline the challenges in specifically targeting mesenchymal stromal cells in myeloid malignancies caused by somatic and germline mutations.

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