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1.
Stem Cell Reports ; 16(7): 1705-1717, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34171287

RESUMO

Hematopoietic stem cells (HSCs) are typically characterized by transplantation into irradiated hosts in a highly perturbed microenvironment. Here, we show that selective and temporally controlled depletion of resident HSCs through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. Strikingly, we achieved robust engraftment of donor HSCs even when delaying Gata2 deletion until 4 weeks after transplantation, allowing homing and early localization to occur in a completely non-perturbed environment. When HSCs from the congenic strains Ly5.1 and Ly5.2 were competitively transplanted, we found that the more proliferative state of Ly5.2 HSCs was associated with superior long-term engraftment when using conditioning by standard irradiation, while higher CXCR4 expression and a better homing ability of Ly5.1 HSCs strongly favored the outcome in our inducible HSC depletion model. Thus, the mode and timing of recipient conditioning challenges distinct functional features of transplanted HSCs.

2.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

3.
Cell Stem Cell ; 25(3): 407-418.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31303548

RESUMO

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.


Assuntos
Senilidade Prematura/patologia , Envelhecimento/fisiologia , Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Megacariócitos/fisiologia , Células Mieloides/fisiologia , Progéria/patologia , Agonistas Adrenérgicos/administração & dosagem , Envelhecimento/metabolismo , Senilidade Prematura/metabolismo , Animais , Diferenciação Celular , Encapsulamento de Células , Proliferação de Células , Modelos Animais de Doenças , Humanos , Interleucina-6/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismo , Progéria/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Nicho de Células-Tronco
5.
Blood ; 129(8): 950-958, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-27899358

RESUMO

Adhesion is a key component of hematopoietic stem cell regulation mediating homing and retention to the niche in the bone marrow. Here, using an RNA interference screen, we identify cytohesin 1 (CYTH1) as a critical mediator of adhesive properties in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs). Knockdown of CYTH1 disrupted adhesion of HSPCs to primary human mesenchymal stroma cells. Attachment to fibronectin and ICAM1, 2 integrin ligands, was severely impaired, and CYTH1-deficient cells showed a reduced integrin ß1 activation response, suggesting that CYTH1 mediates integrin-dependent functions. Transplantation of CYTH1-knockdown cells to immunodeficient mice resulted in significantly lower long-term engraftment levels, associated with a reduced capacity of the transplanted cells to home to the bone marrow. Intravital microscopy showed that CYTH1 deficiency profoundly affects HSPC mobility and localization within the marrow space and thereby impairs proper lodgment into the niche. Thus, CYTH1 is a novel major regulator of adhesion and engraftment in human HSPCs through mechanisms that, at least in part, involve the activation of integrins.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Fibronectinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos , Interferência de RNA
6.
Genes Dev ; 29(18): 1915-29, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26385962

RESUMO

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.


Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/fisiologia , Adesão Celular/genética , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Células Cultivadas , Deleção de Genes , Camundongos , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética , Regulador Transcricional ERG
7.
Curr Opin Hematol ; 21(4): 283-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24811164

RESUMO

PURPOSE OF REVIEW: The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. In this review, we will discuss how RNA interference (RNAi) screening has emerged as a new and powerful tool to molecularly dissect various functional aspects of both normal and malignant HSCs, and how this may ultimately enable the discovery of novel therapeutic targets for clinical applications. RECENT FINDINGS: Advances in RNAi technology and the creation of short hairpin RNA retroviral and lentiviral vector-libraries have provided tools to perform broad forward genetic screens in primary mammalian hematopoietic cells. Recent studies have identified novel fate determinants in murine HSCs as well as potential targets for ex-vivo expansion of human HSCs. RNAi screens have further unraveled tumor suppressor genes associated with hematopoietic neoplasms as well as candidate therapeutic targets in leukemic cells. SUMMARY: RNAi screening is a feasible tool to discover novel molecules that regulate both normal and malignant HSCs, thus increasing our general understanding of the physiology and pathophysiology of hematopoiesis. Moreover, RNAi technology holds great promise for the discovery of specific targets for therapeutic interventions.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Ensaios de Triagem em Larga Escala , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Animais , Diferenciação Celular/genética , Sobrevivência Celular/genética , Haploinsuficiência/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia/genética , Leucemia/terapia , Camundongos , Terapia de Alvo Molecular
8.
Chimerism ; 3(2): 48-50, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22854596

RESUMO

Many sources of foreign or semi foreign cells, known as microchimerism (Mc), can be found in healthy individuals. We have recently shown in women with end stage renal disease (ESRD) that Mc frequencies and levels are exacerbated prior to kidney transplantation. Is Mc arising from pregnancy a protective factor for renal diseases explaining lower incidence in women? Is Mc helpful in slowing down disease progression? However, natural Mc is not the only actor as post blood transfusion Mc is also found at high levels in women with ESRD. The difficulty is therefore to distinguish the different types of Mc and this is made even more complicated when the recipient receives a potentially chimeric organ. What part does each source of chimerism play in disease and transplant fate, and can one decipher each role knowing that one chimerism may hide another?


Assuntos
Quimerismo , Falência Renal Crônica/imunologia , Transfusão de Sangue , Feminino , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/terapia , Transplante de Rim/imunologia , Gravidez , Doadores de Tecidos
9.
PLoS One ; 7(5): e36870, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22615829

RESUMO

Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc), none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, (67)FLEDR(71), shared by HLA-DRB susceptibility alleles, or (71)TRAELDT(77), shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient's clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ(2) = 28.4, p<10-6) and with anti-topoisomerase antibody (ATA) production (χ(2) = 43.9, p<10-9) whereas TRAELDT association is weaker in both subgroups (χ(2) = 7.2, p = 0.027 and χ(2) = 14.6, p = 0.0007 respectively). Moreover, FLEDR motif- association among patients with diffuse SSc remains significant only in ATA subgroup. The risk to develop ATA positive SSc is higher with double dose FLEDR than single dose with respectively, adjusted standardised residuals of 5.1 and 2.6. The increase in FLEDR motif is mostly due to the higher frequency of HLA-DRB1*11 and DRB1*15 haplotypes. Furthermore, FLEDR is always carried by the most abundantly expressed ß chain: ß1 in HLA DRB1*11 haplotypes and ß5 in HLA-DRB1*15 haplotypes.In French Caucasian patients with SSc, FLEDR is the main presenting motif influencing ATA production in dcSSc. These results open a new field of potential therapeutic applications to interact with the FLEDR peptide binding groove and prevent ATA production, a hallmark of severity in SSc.


Assuntos
Epitopos/genética , Grupo com Ancestrais do Continente Europeu/genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Alelos , Epitopos/imunologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Cadeias beta de HLA-DQ/genética , Cadeias beta de HLA-DQ/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Cadeias HLA-DRB5/genética , Cadeias HLA-DRB5/imunologia , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética
10.
PLoS One ; 7(3): e32248, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22403639

RESUMO

Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p<0.00001 and p = 0.0005 respectively). Male Mc was increased in women with ESRD whether they had or not a history of male pregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD.


Assuntos
Quimerismo/estatística & dados numéricos , Falência Renal Crônica/genética , Falência Renal Crônica/terapia , Transplante de Rim , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Idoso , Transfusão de Sangue , Estudos de Casos e Controles , Feminino , Humanos , Rim/patologia , Falência Renal Crônica/sangue , Falência Renal Crônica/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Gravidez , Fatores de Tempo , Adulto Jovem
11.
Chimerism ; 1(2): 56-60, 2010 10.
Artigo em Inglês | MEDLINE | ID: mdl-21327048

RESUMO

We report the case of a 40-year-old man diagnosed with a scleroderma-like disease. Clinical similarities with graft versus host disease prompted initial testing for chimerism employing fluorescence in situ hybridization (FISH). Female cells were observed within peripheral blood mononuclear cells from the patient.Because maternal cells have been detected in healthy immunologically competent adults and patients with autoimmune conditions, we hypothesized that these cells were of maternal origin. Contrary to our expectations, HLA-specific quantitative PCR (QPCR) ruled out maternal microchimerism. However, HLA-specific QPCR testing was positive for the paternal HLA haplotype that the patient did not inherit. We reasoned that the most likely origin of chimerism with non-inherited paternal HLA alleles was from an unrecognized "vanished" twin. The patient had never received a blood transfusion.This report suggests that cells from a vanished twin are a possible source of chimerism. The frequency of chimerism from this source is not yet known and whether the scleroderma-like disease observed in the patient is anecdotal or implies a potential association with autoimmune disease remains to be elucidated.

12.
Chimerism ; 1(1): 23-5, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21327148

RESUMO

Rheumatoid arthritis, a chronic inflammatory joint disease, is strongly associated with HLA-DRB1*01 and *04 alleles that have in common similar 5-amino acid motifs in the third hypervariable region of DRB1 (QKRAA, QRRAA, RRRAA), the so called shared epitope (SE). Most patients with RA carry 1 or 2 doses of the SE, with particular genetic combinations at higher risk. In recent work we provided evidence that patients who lack HLA-DRB1*01 and/or *04 alleles can acquire RA susceptibility through fetal, maternal or iatrogenic microchimerism. We also discuss how Mc carrying HLA-DRB1*04 alleles is more likely to be present in the peripheral blood of RA patients compared to Mc carrying HLA-DRB1*01 alleles. We further analyze our results in light of the hierarchy for RA risk with different combinations of the SE. How Mc could contribute to RA susceptibility and whether it also contributes to the hierarchy of risk observed with particular combinations of SE-containing alleles is certainly the beginning of an intriguing story and may offer hope for future therapeutic and/or preventative interventions.


Assuntos
Quimerismo , Predisposição Genética para Doença , Alelos , Artrite Reumatoide/genética , Cadeias HLA-DRB1/genética , Humanos
13.
Rheumatology (Oxford) ; 48(4): 363-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19208687

RESUMO

OBJECTIVES: Male microchimerism (Mc) persisting from pregnancy has been found at greater frequencies and/or higher quantities in women with scleroderma (SSc) compared with controls, suggesting a possible role in disease development. Moreover, women with an HLA-compatible child have a higher risk to develop SSc. We tested the hypothesis, on our French SSc cohort, that women with lcSSc and dcSSc, two distinct clinical subsets, have a different profile in terms of Mc and HLA compatibility in families. METHODS: We studied 98 women (52 lcSSc and 46 dcSSc) for male Mc, by real-time PCR, in their whole blood and/or peripheral blood mononuclear cells (PBMC). Similarly, 91 matched healthy women were analysed. Complete HLA-DRB1 typing was obtained for 58 SSc and 68 control families (proband/children). RESULTS: Women with lcSSc (N = 50) had male Mc more often in their whole blood than women with dcSSc (N = 40, 20 vs 5%, P = 0.038), but not significantly more than controls. By contrast, women with dcSSc (N = 36) hold Mc more often in PBMC (25 vs 9%), but not significantly and have greater quantities than controls (N = 82, P = 0.048). This contrast is also visible in feto-maternal HLA-DRB1 compatibility, which was increased only among women with lcSSc (N = 33) compared with controls (N = 68, P = 0.003). CONCLUSION: For the first time, we showed that women with lcSSc and dcSSc hold male Mc in different blood compartments. Furthermore, a distinct pattern between the two SSc subtypes is observed for feto-maternal HLA-DRB1 compatibility. These results suggest a different mechanism behind each type of disease.


Assuntos
Quimerismo , Mães , Gravidez/genética , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Feminino , Antígenos HLA-DR , Cadeias HLA-DRB1 , Histocompatibilidade , Teste de Histocompatibilidade , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez/imunologia , Esclerodermia Difusa/genética , Esclerodermia Limitada/genética , Adulto Jovem
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