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1.
Nature ; 630(8016): 412-420, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38839950

RESUMEN

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.


Asunto(s)
Autorrenovación de las Células , Células Madre Hematopoyéticas , Proteínas Nucleares , Animales , Femenino , Humanos , Masculino , Ratones , Células Cultivadas , Endocitosis , Endosomas/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Sangre Fetal/citología , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/embriología , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Análisis de Expresión Génica de una Sola Célula
2.
Nature ; 604(7906): 534-540, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35418685

RESUMEN

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.


Asunto(s)
Células Endoteliales , Células Madre Hematopoyéticas , Diferenciación Celular , Endotelio , Femenino , Hematopoyesis , Humanos , Mesonefro , Embarazo
3.
Blood ; 129(25): 3344-3351, 2017 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-28408459

RESUMEN

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.


Asunto(s)
Antígenos CD34/análisis , Antígenos CD/análisis , Sangre Fetal/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Receptores de Superficie Celular/análisis , Animales , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Receptor de Proteína C Endotelial , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones SCID
4.
Nat Methods ; 11(4): 436-42, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24562423

RESUMEN

Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.


Asunto(s)
Adenina/análogos & derivados , Técnicas de Cultivo de Célula/métodos , Indoles/farmacología , Leucemia/metabolismo , Células Madre Neoplásicas/fisiología , Pirimidinas/farmacología , Adenina/farmacología , Medio de Cultivo Libre de Suero , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mieloide Aguda , Estructura Molecular
5.
Curr Opin Hematol ; 22(4): 309-16, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26049751

RESUMEN

PURPOSE OF REVIEW: Hematopoietic stem and progenitor cell (HSPC) transplantation is frequently used in the treatment of hematological diseases. The outcome of the procedure is strongly influenced by the quantity of injected cells, especially if low cell numbers are infused as frequently encountered with cord blood transplants. Ex-vivo expansion of cord blood HSPCs would increase cell numbers, thus accelerating engraftment and reducing infectious complications and transplant-related mortality. In addition, expansion would maximize accessibility to better HLA-matched units, further improving patients' outcome. Similarly, in-vitro maintenance or expansion of leukemic stem cells (LSCs) would enable research into the much awaited targeted therapies that spare normal hematopoietic stem cells (HSCs). Here, we review recent findings on small molecules (excluding biologicals) regulating the activity of normal and leukemic stem cells and provide insights into basic science and clinical implications. RECENT FINDINGS: High-throughput screening of small molecules active on primary hematopoietic cells has led to the identification of two potent series of chemical compounds, best exemplified by StemRegenin1 and UM171, that both expand HSPCs. Current data suggest that the aryl hydrocarbon receptor antagonist StemRegenin1 is most active on primitive normal hematopoietic progenitors and LSCs and that UM171 expands long-term normal HSCs. SUMMARY: Small molecules are clinically useful and powerful tools for expanding HSPCs. They are also of potential value for dissecting the still elusive regulatory networks that govern self-renewal of human HSCs.


Asunto(s)
Sangre Fetal/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Purinas/farmacología , Pirimidinas/farmacología , Recuento de Células , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Sangre Fetal/citología , Sangre Fetal/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal
6.
Blood Cancer Discov ; 3(2): 116-135, 2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35015684

RESUMEN

Polycomb repressive epigenetic complexes are recurrently dysregulated in cancer. Unlike polycomb repressive complex 2 (PRC2), the role of PRC1 in oncogenesis and therapy resistance is not well-defined. Here, we demonstrate that highly recurrent mutations of the PRC1 subunits BCOR and BCORL1 in leukemia disrupt assembly of a noncanonical PRC1.1 complex, thereby selectively unlinking the RING-PCGF enzymatic core from the chromatin-targeting auxiliary subcomplex. As a result, BCOR-mutated PRC1.1 is localized to chromatin but lacks repressive activity, leading to epigenetic reprogramming and transcriptional activation at target loci. We define a set of functional targets that drive aberrant oncogenic signaling programs in PRC1.1-mutated cells and primary patient samples. Activation of these PRC1.1 targets in BCOR-mutated cells confers acquired resistance to treatment while sensitizing to targeted kinase inhibition. Our study thus reveals a novel epigenetic mechanism that explains PRC1.1 tumor-suppressive activity and identifies a therapeutic strategy in PRC1.1-mutated cancer. SIGNIFICANCE: We demonstrate that BCOR and BCORL1 mutations in leukemia unlink PRC1.1 repressive function from target genes, resulting in epigenetic reprogramming and activation of aberrant cell signaling programs that mediate treatment resistance. Our study provides mechanistic insights into the pathogenesis of PRC1.1-mutated leukemia that inform novel therapeutic approaches. This article is highlighted in the In This Issue feature, p. 85.


Asunto(s)
Carcinogénesis , Leucemia , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Cromatina , Epigénesis Genética/genética , Humanos , Leucemia/genética , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Transducción de Señal/genética
7.
Cell Rep ; 28(4): 1063-1073.e5, 2019 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-31340144

RESUMEN

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.


Asunto(s)
Biomarcadores/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Integrina alfa3/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células , Regulación hacia Abajo , Sangre Fetal/citología , Perfilación de la Expresión Génica , Humanos , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismo
8.
PLoS One ; 14(11): e0224900, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31703090

RESUMEN

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.


Asunto(s)
Autorrenovación de las Células/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Homeostasis/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Biomarcadores , Diferenciación Celular , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Fase I de la Desintoxicación Metabólica , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacos , Transcriptoma
9.
Mol Ther Methods Clin Dev ; 10: 156-164, 2018 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-30101153

RESUMEN

Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34+ and CD34+CD45RA- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the ex vivo production of gene-modified cells and for reducing requirements of virus for a broad range of applications.

10.
J Clin Invest ; 126(12): 4569-4584, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27797342

RESUMEN

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.


Asunto(s)
Estradiol/análogos & derivados , Células Madre Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Microambiente Tumoral/efectos de los fármacos , 2-Metoxiestradiol , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Estradiol/farmacología , Humanos , Células Jurkat , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Ratones , Células Madre Neoplásicas/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Microambiente Tumoral/genética , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de Xenoinjerto
11.
Science ; 345(6203): 1509-12, 2014 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-25237102

RESUMEN

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.


Asunto(s)
Sangre Fetal/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Sangre Fetal/citología , Sangre Fetal/fisiología , Terapia Genética/métodos , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/fisiología , Humanos , Huésped Inmunocomprometido , Indoles/química , Ratones , Pirimidinas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
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