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1.
Blood ; 141(16): 1990-2002, 2023 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-36652668

RESUMO

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.


Assuntos
Células-Tronco Hematopoéticas , Mitógenos , Adulto , Humanos , Animais , Camundongos , Integrina alfa6/metabolismo , Mitógenos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Antígenos CD34/metabolismo , Proliferação de Células , Pontos de Checagem do Ciclo Celular , Ciclo Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
2.
Blood ; 136(24): 2764-2773, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33301029

RESUMO

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.


Assuntos
Regulação Leucêmica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transplante de Neoplasias
3.
Blood ; 133(9): 927-939, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30622121

RESUMO

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.


Assuntos
Antígenos CD34/metabolismo , Linhagem da Célula , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteoma/análise , Análise de Célula Única/métodos , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteoma/metabolismo
4.
Blood ; 129(3): 307-318, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-27827829

RESUMO

Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and ß-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and ß-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.


Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Fatores de Transcrição
5.
J Vis Exp ; (197)2023 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-37590518

RESUMO

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.


Assuntos
Células-Tronco Pluripotentes Induzidas , Transplantes , Humanos , Neurônios , Procedimentos Neurocirúrgicos , Transplante de Células
6.
Exp Hematol ; 109: 18-26, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35288232

RESUMO

Previous studies of aging have revealed intrinsically determined alterations in the properties of the hematopoietic stem cell (HSC) and progenitor compartments in mice, with variable evidence of an extension of these findings to humans. To examine more closely the surface phenotypes within the CD34+ compartment of human blood and bone marrow from birth to old age, we undertook a 13-parameter phenotypic profile analysis of samples from healthy human donors aged 0-76 years. The results indicate a conserved stability of canonically defined phenotype frequencies within the CD34+ compartment across this age spectrum, in contrast to previously reported losses of historically defined progenitor phenotypes associated with lymphoid-restricted outputs with advancing age. Interestingly, multidimensionality reduction of the data also produced an unexpected age-independent landscape that, nevertheless, revealed conserved phenotypic differences between cells isolated from blood or bone marrow samples. These source-specific differences were most notable in the HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ fraction, where they were driven largely by differences in cell surface expression of CD34, CD45, CD90, and CD38. Coordinated changes in the expression of several surface markers were also observed during downstream transitions within the CD34+ compartment, suggesting potential new strategies for isolating cell types with more narrowly defined functional properties. Overall, these findings indicate a general conservation during human aging of the phenotypic changes that segregate the major historically defined stages of differentiation within the human CD34+ compartment and underscore the selection processes that govern those that enter the circulation or alter their phenotypes therein.


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Fenótipo , Antígenos Thy-1
7.
Cancer Discov ; 11(11): 2884-2903, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34021002

RESUMO

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.


Assuntos
Anoikis , Proteína Acessória do Receptor de Interleucina-1 , Sarcoma de Ewing , Adulto , Linhagem Celular Tumoral , Criança , Humanos , Proteômica , Receptores de Interleucina-1 , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia
8.
Blood Adv ; 3(21): 3201-3213, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698451

RESUMO

Human B-cell precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed that BCP-ALL cells display unique and clonally heritable, stable DNA replication timing (RT) programs (ie, programs describing the variable order of replication and subnuclear 3D architecture of megabase-scale chromosomal units of DNA in different cell types). To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients with varied genetic subtypes and outcomes. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that seem to be associated with relapse. These results suggest that the genesis of BCP-ALL involves alterations in RT that reflect biologically significant and potentially clinically relevant leukemia-specific epigenetic changes.


Assuntos
Cromossomos/genética , Período de Replicação do DNA , Leucemia/genética , Leucemia/patologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores , Neoplasias do Sistema Nervoso Central/secundário , Biologia Computacional/métodos , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Variação Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos , Humanos , Imunofenotipagem , Leucemia/mortalidade , Masculino , Camundongos , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia
9.
Stem Cell Reports ; 11(2): 578-592, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30078558

RESUMO

Increasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.


Assuntos
Metilação de DNA , Epigênese Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Biologia Computacional/métodos , Ilhas de CpG , Perfilação da Expressão Gênica , Genômica/métodos , Camundongos , Análise de Célula Única
10.
Nat Cell Biol ; 20(6): 710-720, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29802403

RESUMO

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.


Assuntos
Proliferação de Células , Separação Celular/métodos , Sangue Fetal/citologia , Mitose , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Metilação de DNA , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Masculino , Camundongos Transgênicos , Fenótipo , Fatores de Tempo , Transcriptoma
11.
Stem Cell Reports ; 8(1): 152-162, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076756

RESUMO

The role of growth factors (GFs) in controlling the biology of human hematopoietic stem cells (HSCs) remains limited by a lack of information concerning the individual and combined effects of GFs directly on the survival, Mitogenesis, and regenerative activity of highly purified human HSCs. We show that the initial input HSC activity of such a purified starting population of human cord blood cells can be fully maintained over a 21-day period in serum-free medium containing five GFs alone. HSC survival was partially supported by any one of these GFs, but none were essential, and different combinations of GFs variably stimulated HSC proliferation. However, serial transplantability was not detectably compromised by many conditions that reduced human HSC proliferation and/or survival. These results demonstrate the dissociated control of these three human HSC bio-responses, and set the stage for future improvements in strategies to modify and expand human HSCs ex vivo.


Assuntos
Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Integrina alfa6/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Fenótipo
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