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1.
STAR Protoc ; 5(4): 103408, 2024 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-39436772

RESUMEN

A combination of hematopoietic stem cell (HSC)- and endothelial cell (EC)-lineage-tracing mouse models enables us to determine blood cell origins. We present a protocol to induce cell labeling in vivo and to trace labeled hematopoietic cells to segregate their origins. We describe the steps for harvesting various hematopoietic tissues, antibody staining, and analyzing the Tomato+ percentages within each immune cell population. We also show how to estimate HSC- and EC-derived percentages of the target cell populations. For complete details on the use and execution of this protocol, please refer to Kobayashi et al.1.

2.
Mol Cancer Res ; 22(1): 94-103, 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-37756563

RESUMEN

Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.


Asunto(s)
Leucemia Mieloide Aguda , Monoéster Fosfórico Hidrolasas , Animales , Ratones , Leucemia Mieloide Aguda/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/genética
3.
Dev Comp Immunol ; 147: 104897, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37516328

RESUMEN

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.


Asunto(s)
Bombyx , Nucleopoliedrovirus , Humanos , Animales , Nucleopoliedrovirus/fisiología , Baculoviridae , ADN Viral/genética , Receptores de Reconocimiento de Patrones/genética , Antivirales , Proteínas de Neoplasias/genética , ARN Helicasas DEAD-box/genética
4.
Cell Rep ; 42(3): 112239, 2023 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-36906851

RESUMEN

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.


Asunto(s)
Células Endoteliales , Células Madre Hematopoyéticas , Animales , Ratones , Linaje de la Célula , Médula Ósea , Hematopoyesis
5.
Immunol Rev ; 315(1): 11-30, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36929134

RESUMEN

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.


Asunto(s)
Sistema Inmunológico , Sistema Inmunológico/citología , Sistema Inmunológico/crecimiento & desarrollo , Humanos , Animales , Hematopoyesis , Embrión de Mamíferos/citología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Linaje de la Célula
6.
Blood ; 141(3): 244-259, 2023 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-36206490

RESUMEN

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.


Asunto(s)
Leucemia Mieloide Aguda , Ubiquitina-Proteína Ligasas , Humanos , Animales , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Transducción de Señal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Mutación
7.
Front Cardiovasc Med ; 9: 988713, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36426217

RESUMEN

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated ß-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

8.
Front Immunol ; 13: 896396, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35898504

RESUMEN

Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained via an HSC-independent pathway, the earliest HSCs to emerge in the AGM and seed the early FL can produce MCs, but only during a minimal time window. Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.


Asunto(s)
Mastocitos , Mesonefro , Animales , Médula Ósea , Gónadas , Células Madre Hematopoyéticas/metabolismo , Ratones
9.
Artículo en Inglés | MEDLINE | ID: mdl-35801078

RESUMEN

Numerous studies have revealed the critical role of premature senescence induced by various cancer treatment modalities in the pathogenesis of aging-related diseases. Senescence-associated secretory phenotype (SASP) can be induced by telomere dysfunction. Telomeric DNA damage response induced by some cancer treatments can persist for months, possibly accounting for long-term sequelae of cancer treatments. Telomeric DNA damage-induced mitochondrial dysfunction and increased reactive oxygen species production are hallmarks of premature senescence. Recently, we reported that the nucleus-mitochondria positive feedback loop formed by p90 ribosomal S6 kinase (p90RSK) and phosphorylation of S496 on ERK5 (a unique member of the mitogen-activated protein kinase family that is not only a kinase but also a transcriptional co-activator) were vital signaling events that played crucial roles in linking mitochondrial dysfunction, nuclear telomere dysfunction, persistent SASP induction, and atherosclerosis. In this review, we will discuss the role of NAD+ depletion in instigating SASP and its downstream signaling and regulatory mechanisms that lead to the premature onset of atherosclerotic cardiovascular diseases in cancer survivors.

10.
Stem Cell Rev Rep ; 17(6): 2304-2313, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34561772

RESUMEN

Polycomb group protein Bmi1 is essential for hematopoietic stem cell (HSC) self-renewal and terminal differentiation. However, its target genes in hematopoietic stem and progenitor cells are largely unknown. We performed gene expression profiling assays and found that genes of the Wnt signaling pathway are significantly elevated in Bmi1 null hematopoietic stem and progenitor cells (HSPCs). Bmi1 is associated with several genes of the Wnt signaling pathway in hematopoietic cells. Further, we found that Bmi1 represses Wnt gene expression in HSPCs. Importantly, loss of ß-catenin, which reduces Wnt activation, partially rescues the HSC self-renewal and differentiation defects seen in the Bmi1 null mice. Thus, we have identified Bmi1 as a novel regulator of Wnt signaling pathway in HSPCs. Given that Wnt signaling pathway plays an important role in hematopoiesis, our studies suggest that modulating Wnt signaling may hold potential for enhancing HSC self-renewal, thereby improving the outcomes of HSC transplantation.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Vía de Señalización Wnt , Animales , Hematopoyesis/genética , Células Madre Hematopoyéticas , Ratones , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Vía de Señalización Wnt/genética
11.
Cytotechnology ; 73(4): 643-655, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34349353

RESUMEN

The cell line NISES-AnPe-428 (AnPe), derived from the Chinese oak silkworm Antheraea pernyi, was characterized for its permissiveness and productivity for six different nucleopolyhedrovirus (NPV) species. These NPVs included homologous Antheraea pernyi NPV (AnpeNPV) and heterologous Autographa californica multiple NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Spodoptera exigua MNPV (SeMNPV), and Lymantria dispar MNPV (LdMNPV), representing viruses that had been isolated from insect species belonging to five different families (Saturniidae, Noctuidae, Bombycidae, Arctiidae, and Lymantriidae). We found that AnPe cells supported productive replication of AnpeNPV, AcMNPV, BmNPV, HycuMNPV, and SeMNPV to varying degrees. Upon infection with SeMNPV, a subset of AnPe cell population in the culture underwent apoptosis, while remaining cells produced limited amounts of progeny virions and polyhedra. AnPe cells were refractory to LdMNPV infection and failed to support replication of viral DNA, indicating that viral replication was restricted at or prior to the step of viral DNA replication. These results indicated that AnPe cells have the potential to provide excellent systems for studying the molecular mechanisms underlying cellular permissiveness for NPV replication and host-range determination of NPVs.

12.
Development ; 148(15)2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-34370006

RESUMEN

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Desarrollo Embrionario/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Animales , Diferenciación Celular/inmunología , Células Endoteliales/inmunología , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones Endogámicos C57BL
13.
Vis Comput ; 37(12): 2931-2949, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34177023

RESUMEN

Image processing and computer vision on mobile devices have a wide range of applications such as digital image enhancement and augmented reality. While images acquired by cameras on mobile devices can be processed with generic image processing algorithms, there are numerous constraints and external issues that call for customized algorithms for such devices. In this paper, we survey mobile image processing and computer vision applications while highlighting these constraints and explaining how the algorithms have been modified/adapted to meet accuracy and performance demands. We hope that this paper will be a useful resource for researchers who intend to apply image processing and computer vision algorithms to real-world scenarios and applications that involve mobile devices.

14.
Front Cell Dev Biol ; 9: 634151, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33996794

RESUMEN

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10-15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.

16.
J Immunol ; 204(12): 3262-3272, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32332108

RESUMEN

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1 Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1 -/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.


Asunto(s)
Subgrupos de Linfocitos B/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Subgrupos de Linfocitos B/fisiología , Médula Ósea/metabolismo , Linaje de la Célula/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Microambiente Celular/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Linfocitos/metabolismo , Linfocitos/fisiología , Linfopoyesis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID
17.
Virus Res ; 276: 197832, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31794797

RESUMEN

Bombyx mori cells induce antiviral responses including global protein synthesis shutdown, rRNA degradation, and apoptosis upon infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We previously demonstrated that five and six amino acid residues located at positions between 514 and 599 of AcMNPV P143 (Ac-P143) protein are important for induction of apoptosis and rRNA degradation, respectively. However, it remains unexplored whether other residues of Ac-P143 protein also participate in antiviral immune responses. Here, we conducted transient expression analysis using a number of Ac-P143 protein deletion and truncation mutants and found that some of the N-terminal 413 residues (amino acids 1-413), besides previously identified residues between amino acids 514 and 599, are indispensable, whereas C-terminal 622 residues (amino acids 600-1221) are dispensable, for Ac-P143 protein to induce apoptosis or rRNA degradation. In addition, we found that the N-terminal 413 sequence (amino acids 1-413) of Ac-P143 protein can be substituted with corresponding BmNPV P143 (Bm-P143) protein sequence. Further analysis demonstrated that mutant Ac-P143 protein consisting of 275 residues (amino acids 325-599), but not 274 residues (amino acids 326-599) lacking glutamine residue at position 325 (Q325), is sufficient for triggering apoptosis and rRNA degradation of B. mori cells. These 275 residues are located outside the region of DNA helicase motifs of Ac-P143 protein, indicating that induction of apoptosis or rRNA degradation occurs independently of viral DNA replication-related function of the Ac-P143 protein. Moreover, Ac-P143(325-599/Q325A) and Ac-P143(1-599/Q325A) proteins harboring Q325A substitution retain the ability to induce apoptosis and rRNA degradation in B. mori cells. These findings suggest that the Ac-P143 protein needs minimal sequence length starting from the Q325 residue that contains a specific effector domain to induce apoptosis and rRNA degradation.


Asunto(s)
Apoptosis , Bombyx/virología , Nucleopoliedrovirus/patogenicidad , Estabilidad del ARN , ARN Ribosómico/metabolismo , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Bombyx/citología , Bombyx/inmunología , Caspasas/metabolismo , Línea Celular , ADN Viral/genética , Mutación , Nucleopoliedrovirus/inmunología , Replicación Viral
18.
Bio Protoc ; 10(9): e3601, 2020 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-33659567

RESUMEN

The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as pre-HSCs. Both HECs and pre-HSCs show endothelial surface phenotype and require stromal support to detect their hematopoietic activity. The method utilizing stromal cell culture followed by modified semisolid clonal culture enables us to detect the number of colony forming units for B-1/B-2 progenitors originally derived from HECs/pre-HSCs, which will reflect the potential of B-1 biased or multi-lineage repopulating HSCs.

19.
Nat Commun ; 10(1): 5649, 2019 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-31827082

RESUMEN

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.


Asunto(s)
Epigénesis Genética , Enfermedades Hematológicas/metabolismo , Hematopoyesis , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/fisiopatología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilación , Ratones Endogámicos C57BL , Mutación , Unión Proteica
20.
Stem Cell Reports ; 13(1): 21-30, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31231025

RESUMEN

Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45-VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate.


Asunto(s)
Subgrupos de Linfocitos B/metabolismo , Desdiferenciación Celular , Diferenciación Celular , Células Endoteliales/citología , Células Madre Hematopoyéticas/citología , Animales , Subgrupos de Linfocitos B/citología , Biomarcadores , Linaje de la Célula , Técnicas de Cocultivo , Embrión de Mamíferos , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Ratones , Modelos Biológicos
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