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1.
Blood ; 138(25): 2642-2654, 2021 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-34499717

RESUMO

Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages after transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. Although several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population and single-cell transcriptomes of unexpanded and ex vivo cultured cord blood-derived hematopoietic stem and progenitor cells as well as peripheral blood, adult bone marrow, and fetal liver. On the basis of these analyses, we propose the master transcription factor HLF (hepatic leukemia factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter. Most importantly, HLF-expressing cells comprise all stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human HSC state and outline a new approach to continuously mark these cells with high fidelity.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Transcriptoma , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Análise de Célula Única
2.
Blood ; 129(25): 3344-3351, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28408459

RESUMO

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.


Assuntos
Antígenos CD34/análise , Antígenos CD/análise , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Receptores de Superfície Celular/análise , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Receptor de Proteína C Endotelial , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID
3.
Blood ; 130(20): 2204-2214, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-28855157

RESUMO

Neomorphic missense mutations affecting crucial lysine residues in histone H3 genes significantly contribute to a variety of solid cancers. Despite the high prevalence of H3K27M mutations in pediatric glioblastoma and their well-established impact on global histone H3 lysine 27 di- and trimethylation (H3K27me2/3), the relevance of these mutations has not been studied in acute myeloid leukemia (AML). Here, we report the first identification of H3K27M and H3K27I mutations in patients with AML. We find that these lesions are major determinants of reduced H3K27me2/3 in these patients and that they are associated with common aberrations in the RUNX1 gene. We demonstrate that H3K27I/M mutations are strong disease accelerators in a RUNX1-RUNX1T1 AML mouse model, suggesting that H3K27me2/3 has an important and selective leukemia-suppressive activity in this genetic context.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Histonas/genética , Leucemia Mieloide Aguda/genética , Mutação de Sentido Incorreto , Transformação Genética , Adolescente , Idoso de 80 Anos ou mais , Animais , Metilação de DNA , Feminino , Humanos , Lisina/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Análise de Sequência de DNA
4.
Stem Cells ; 33(2): 342-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25286822

RESUMO

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75(NTR), the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75(NTR) axis plays a critical role in regulating breast CSC self-renewal and plasticity.


Assuntos
Comunicação Autócrina , Neoplasias da Mama/metabolismo , Proliferação de Células , Transição Epitelial-Mesenquimal , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Nicho de Células-Tronco , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo
5.
Cell Mol Life Sci ; 71(13): 2467-81, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24481864

RESUMO

p75(NTR), the common receptor for both neurotrophins and proneurotrophins, has been widely studied because of its role in many tissues, including the nervous system. More recently, a close relationship between p75(NTR) expression and pluripotency has been described. p75(NTR) was shown to be expressed in various types of stem cells and has been used to prospectively isolate stem cells with different degrees of potency. Here, we give an overview of the current knowledge on p75(NTR) in stem cells, ranging from embryonic to adult stem cells, and cancer stem cells. In an attempt to address its potential role in the control of stem cell biology, the molecular mechanisms underlying p75(NTR) signaling in different models are also highlighted. p75(NTR)-mediated functions include survival, apoptosis, migration, and differentiation, and depend on cell type, (pro)neurotrophin binding, interacting transmembrane co-receptors expression, intracellular adaptor molecule availability, and post-translational modifications, such as regulated proteolytic processing. It is therefore conceivable that p75(NTR) can modulate cell-fate decisions through its highly ramified signaling pathways. Thus, elucidating the potential implications of p75(NTR) activity as well as the underlying molecular mechanisms of p75(NTR) will shed new light on the biology of both normal and cancer stem cells.


Assuntos
Marcadores Genéticos , Proteínas do Tecido Nervoso/genética , Células-Tronco Pluripotentes/metabolismo , Receptores de Fator de Crescimento Neural/genética , Pesquisa com Células-Tronco , Apoptose/genética , Diferenciação Celular/genética , Movimento Celular/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais
6.
Cell Stem Cell ; 28(1): 48-62.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33417871

RESUMO

Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured ex vivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon ex vivo culture of human HSCs.


Assuntos
Proteínas Correpressoras , Epigênese Genética , Células-Tronco Hematopoéticas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desacetilases/metabolismo , Humanos
7.
Lancet Haematol ; 7(2): e134-e145, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31704264

RESUMO

BACKGROUND: Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. METHODS: This single-arm, open-label, phase 1-2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3-64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged <18 years) were recruited. Patients aged younger than 50 years without comorbidities received a myeloablative conditioning regimen (cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, and 12 Gy total body irradiation) and patients aged older than 50 years and those with comorbidities received a less myeloablative conditioning regimen (cyclophosphamide 50 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, and 4 Gy total body irradiation). Patients were infused with the 7-day UM171-expanded CD34-positive cells and the lymphocyte-containing CD34-negative fraction. The primary endpoints were feasibility of UM171 expansion, safety of the transplant, kinetics of hematopoietic reconstitution (time to neutrophil and platelet engraftment) of UM171-expanded cord blood, and minimal pre-expansion cord blood unit cell dose that achieved prompt engraftment. We analysed feasibility in all enrolled patients and all other primary outcomes were analysed per protocol, in all patients who received single UM171-expanded cord blood transplantation. This trial has been completed and was registered with ClinicalTrials.gov, NCT02668315. FINDINGS: Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12-22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52 × 105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per µL was 9·5 days (IQR 8-12), median time to engraftment of 500 neutrophils per µL was 18 days (12·5-20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35-47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. INTERPRETATION: Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. FUNDING: Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Adolescente , Adulto , Autorrenovação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/transplante , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Intervalo Livre de Doença , Estudos de Viabilidade , Neutropenia Febril/etiologia , Feminino , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/etiologia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Resultado do Tratamento , Adulto Jovem
8.
Cell Rep ; 28(4): 1063-1073.e5, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340144

RESUMO

Transplantation of expanded hematopoietic stem cells (HSCs) and gene therapy based on HSC engineering have emerged as promising approaches for the treatment of hematological diseases. Nevertheless, the immunophenotype of cultured HSCs remains poorly defined. Here, we identify Integrin-α3 (ITGA3) as a marker of cultured human HSCs. Exploiting the pyrimidoindole derivative UM171 to expand cord blood (CB) cells, we show that ITGA3 expression is sufficient to separate the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population into two functionally distinct fractions presenting mostly short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells exhibit robust multilineage differentiation potential, serial reconstitution ability in immunocompromised mice, and an HSC-specific transcriptomic signature. Moreover, ITGA3 expression is functionally required for the long-term engraftment of CB cells. Altogether, our results indicate that ITGA3 is a reliable marker of cultured human long-term repopulating HSCs (LT-HSCs) and represents an important tool to improve the accuracy of prospective HSC identification in culture.


Assuntos
Biomarcadores/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Integrina alfa3/metabolismo , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Autorrenovação Celular , Regulação para Baixo , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Fenótipo , Proteínas Proto-Oncogênicas c-myc/metabolismo
9.
PLoS One ; 14(11): e0224900, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31703090

RESUMO

Elucidation of the molecular cues required to balance adult stem cell self-renewal and differentiation is critical for advancing cellular therapies. Herein, we report that the hematopoietic stem cell (HSC) self-renewal agonist UM171 triggers a balanced pro- and anti-inflammatory/detoxification network that relies on NFKB activation and protein C receptor-dependent ROS detoxification, respectively. We demonstrate that within this network, EPCR serves as a critical protective component as its deletion hypersensitizes primitive hematopoietic cells to pro-inflammatory signals and ROS accumulation resulting in compromised stem cell function. Conversely, abrogation of the pro-inflammatory activity of UM171 through treatment with dexamethasone, cAMP elevating agents or NFkB inhibitors abolishes EPCR upregulation and HSC expansion. Together, these results show that UM171 stimulates ex vivo HSC expansion by establishing a critical balance between key pro- and anti-inflammatory mediators of self-renewal.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Homeostase/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Biomarcadores , Diferenciação Celular , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Desintoxicação Metabólica Fase I , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Transcriptoma
11.
Nat Commun ; 7: 10399, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26822533

RESUMO

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.


Assuntos
Senescência Celular , Quebras de DNA de Cadeia Simples , Células Epiteliais/citologia , Neoplasias/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
12.
FEBS Lett ; 587(16): 2591-6, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23831066

RESUMO

The p75 neurotrophin receptor (p75NTR) undergoes sequential proteolytic cleavages leading to the generation of a carboxyl-terminal fragment (p75NTR-CTF) and an intracellular domain (p75NTR-ICD) in many cellular models. We have previously shown that p75NTR is involved in the survival of breast cancer cells. Here, we demonstrated that p75NTR cleavage occurs also in these cells. Surprisingly, p75NTR-CTF increased cell survival, whereas p75NTR-ICD had no effect. The pro-survival effect of p75NTR-CTF was associated with a decrease of TNF-related apoptosis-inducing ligand (TRAIL)-induced PARP and caspase 3 cleavages. Finally, our findings indicate that p75NTR could favor cell survival via its carboxyl-terminal fragment, independently of PI3-kinase, NF-κB, or MAP kinase signaling pathways.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor de Fator de Crescimento Neural/metabolismo , Caspase 3/metabolismo , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
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