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1.
Rinsho Ketsueki ; 58(8): 942-949, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-28883279

RESUMO

Repeated cell divisions induce DNA damage accumulation, which impairs stem cell function during aging. However, the general molecular mechanisms by which this occurs remain unclear. Herein, we show that the expression of protection of telomeres 1a (Pot1a), a component of shelterin, is crucial for prevention of telomeric DNA damage response (DDR) and maintenance of hematopoietic stem cell (HSC) activity during aging. We observed that HSCs express high levels of Pot1a during development, and this expression declines with aging. Knockdown of Pot1a induced an age-related phenotype, characterized by increased telomeric DDR and reduced long-term reconstitution activity. In contrast, treatment with exogenous Pot1a protein prevented telomeric DDR, which decreased stem cell activity and partially rejuvenated HSC activity. These results highlight a general, reversible mechanism by which aging compromises mammalian stem cell activity, with widespread implications for regenerative medicine.


Assuntos
Senescência Celular , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Telômero/genética , Envelhecimento , Animais , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Telômero/metabolismo
2.
Stem Cells ; 33(2): 479-90, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25329760

RESUMO

The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c-Myb in HSCs although remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-Myb(low) HSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myb(low) and c-Myb(high) HSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior.


Assuntos
Regulação da Expressão Gênica/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-myb/biossíntese , Animais , Genes Reporter , Camundongos , Proteínas Proto-Oncogênicas c-myb/genética
3.
Blood ; 121(11): 1995-2007, 2013 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-23315170

RESUMO

Prostaglandin E(2) (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3ß and ß-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs.


Assuntos
Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Células Cultivadas , Dinoprostona/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/farmacologia , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP2/fisiologia , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 34(4): 790-800, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24526691

RESUMO

OBJECTIVE: Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. APPROACH AND RESULTS: Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E-deficient mice (ApoE(-/-)/Angptl2(-/-)) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE(-/-) mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE(-/-)/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell-derived nitric oxide. Conversely, Angptl2(-/-) mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis. CONCLUSIONS: Endothelial cell-derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.


Assuntos
Angiopoietinas/metabolismo , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Mediadores da Inflamação/metabolismo , Vasculite/metabolismo , Idoso de 80 Anos ou mais , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/deficiência , Angiopoietinas/genética , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Transplante de Medula Óssea , Células Cultivadas , Quimiotaxia de Leucócito , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Dislipidemias/metabolismo , Dislipidemias/fisiopatologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Integrina alfa5beta1/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Óxido Nítrico/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Placa Aterosclerótica , Transdução de Sinais , Fatores de Tempo , Vasculite/genética , Vasculite/imunologia , Vasculite/patologia , Vasculite/prevenção & controle , Vasodilatação
5.
Biochem Biophys Res Commun ; 441(1): 196-201, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24140061

RESUMO

Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16(Ink4a) or p19(Arf) deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.


Assuntos
Proteínas de Transporte/metabolismo , Instabilidade Genômica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose , Células da Medula Óssea/metabolismo , Ciclo Celular , Ensaio de Unidades Formadoras de Colônias , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Proteínas de Ligação ao GTP , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Proteína Supressora de Tumor p53/metabolismo
6.
Biochem Biophys Res Commun ; 430(1): 20-5, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23149415

RESUMO

Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4 week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage(-)Sca-1(+)c-Kit(+) (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs.


Assuntos
Angiopoietina-1/metabolismo , Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Angiopoietina-1/genética , Animais , Vasos Sanguíneos/anormalidades , Células da Medula Óssea/citologia , Separação Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Perda do Embrião/genética , Perda do Embrião/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteínas Matrilinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Receptor TIE-2 , Transdução de Sinais
7.
Blood ; 117(16): 4169-80, 2011 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-21297001

RESUMO

Telomerase reverse transcriptase (TERT) contributes to the prevention of aging by a largely unknown mechanism that is unrelated to telomere lengthening. The current study used ataxia-telangiectasia mutated (ATM) and TERT doubly deficient mice to evaluate the contributions of 2 aging-regulating molecules, TERT and ATM, to the aging process. ATM and TERT doubly deficient mice demonstrated increased progression of aging and had shorter lifespans than ATM-null mice, while TERT alone was insufficient to affect lifespan. ATM-TERT doubly null mice show in vivo senescence, especially in hematopoietic tissues, that was dependent on p16(INK4a) and p19(ARF), but not on p21. As their HSCs show decreased stem cell activities, accelerated aging seen in these mice has been attributed to impaired stem cell function. TERT-deficient HSCs are characterized by reactive oxygen species (ROS) fragility, which has been suggested to cause stem cell impairment during aging, and apoptotic HSCs are markedly increased in these mice. p38MAPK activation was indicated to be partially involved in ROS-induced apoptosis in TERT-null HSCs, and BCL-2 is suggested to provide a part of the protective mechanisms of HSCs by TERT. The current study demonstrates that TERT mitigates aging by protecting HSCs under stressful conditions through telomere length-independent mechanisms.


Assuntos
Envelhecimento , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Telomerase/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Senescência Celular , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Telomerase/genética , Telômero/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Nat Med ; 12(4): 446-51, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16565722

RESUMO

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.


Assuntos
Senescência Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fosforilação , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
9.
Commun Biol ; 6(1): 996, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37773433

RESUMO

Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis.


Assuntos
Linfopoese , Telômero , Animais , Camundongos , Envelhecimento , Diferenciação Celular , Linfopoese/genética , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
10.
Biochem Biophys Res Commun ; 428(3): 354-9, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-23092738

RESUMO

Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad(+)c-Kit(+) and N-cad(+) endothelial protein C receptor (EPCR)(+) HSCs co-localized with Lyve-1(+) sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad(+) HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR(+) long-term (LT)-HSCs were enriched in the N-cad(+)Lin(-)Sca-1(+)c-Kit(+) (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad(+) LSK cells versus N-cad(-) LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad(+) LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.


Assuntos
Medula Óssea/fisiologia , Caderinas/metabolismo , Feto/fisiologia , Hematopoese Extramedular , Células-Tronco Hematopoéticas/fisiologia , Fígado/embriologia , Animais , Caderinas/análise , Movimento Celular , Regulação para Baixo , Células Endoteliais/fisiologia , Feminino , Glicoproteínas/análise , Glicoproteínas/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL
11.
Blood ; 116(4): 554-63, 2010 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-20427705

RESUMO

During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of N-cadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad-transduced lineage(-)Sca-1(+)c-Kit(+) cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad-mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation.


Assuntos
Caderinas/genética , Sobrevivência de Enxerto/genética , Transplante de Células-Tronco Hematopoéticas , Animais , Caderinas/fisiologia , Adesão Celular/genética , Adesão Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Sobrevivência Celular/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Nicho de Células-Tronco/fisiologia , Fatores de Tempo
12.
Blood ; 116(9): 1422-32, 2010 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-20472830

RESUMO

The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM(-)Sca-1(+) cells, osteoblast-enriched ALCAM(+)Sca-1(-), and ALCAM(-)Sca-1(-) cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM(+)Sca-1(-) cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM(-)Sca-1(+) fraction highly expressed cytokine-related genes, whereas the ALCAM(+)Sca-1(-) fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic marker(low/-) subpopulation in ALCAM(+)Sca-1(-) fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow.


Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Molécula de Adesão de Leucócito Ativado/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Biomarcadores/metabolismo , Western Blotting , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Nature ; 431(7011): 997-1002, 2004 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-15496926

RESUMO

The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16(INK4a)-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA , Deleção de Genes , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor
14.
Cell Syst ; 11(6): 640-652.e5, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33296684

RESUMO

Changes in stem cell activity may underpin aging. However, these changes are not completely understood. Here, we combined single-cell profiling with machine learning and in vivo functional studies to explore how hematopoietic stem cell (HSC) divisions patterns evolve with age. We first trained an artificial neural network (ANN) to accurately identify cell types in the hematopoietic hierarchy and predict their age from single-cell gene-expression patterns. We then used this ANN to compare identities of daughter cells immediately after HSC divisions and found that the self-renewal ability of individual HSCs declines with age. Furthermore, while HSC cell divisions are deterministic and intrinsically regulated in young and old age, they are variable and niche sensitive in mid-life. These results indicate that the balance between intrinsic and extrinsic regulation of stem cell activity alters substantially with age and help explain why stem cell numbers increase through life, yet regenerative potency declines.


Assuntos
Envelhecimento/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Aprendizado de Máquina/normas , Células-Tronco Hematopoéticas/metabolismo , Humanos
15.
iScience ; 23(11): 101654, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33103089

RESUMO

p32/C1qbp regulates mitochondrial protein synthesis and is essential for oxidative phosphorylation in mitochondria. Although dysfunction of p32/C1qbp impairs fetal development and immune responses, its role in hematopoietic differentiation remains unclear. Here, we found that mitochondrial dysfunction affected terminal differentiation of newly identified erythroid/B-lymphoid progenitors among CD45- Ter119- CD31- triple-negative cells (TNCs) in bone marrow. Hematopoietic cell-specific genetic deletion of p32/C1qbp (p32cKO) in mice caused anemia and B-lymphopenia without reduction of hematopoietic stem/progenitor cells. In addition, p32cKO mice were susceptible to hematopoietic stress with delayed recovery from anemia. p32/C1qbp-deficient CD51- TNCs exhibited impaired mitochondrial oxidation that consequently led to inactivation of mTORC1 signaling, which is essential for erythropoiesis. These findings uncover the importance of mitochondria, especially at the stage of TNCs during erythropoiesis, suggesting that dysregulation of mitochondrial protein synthesis is a cause of anemia and B-lymphopenia with an unknown pathology.

16.
Biochem Biophys Res Commun ; 378(3): 467-72, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19032938

RESUMO

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rgamma(null) (NOG) mice. Hypoxic culture (1% O(2)) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34(+)CD38(-) cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.


Assuntos
Diferenciação Celular , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Anaerobiose , Animais , Antígenos CD34/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Sangue Fetal/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Mutantes , Transplante Heterólogo , Regulação para Cima
17.
Int J Hematol ; 107(6): 646-655, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29550946

RESUMO

In order to maintain the homeostasis of the hematopoietic system, hematopoietic stem cells (HSCs) need to be maintained while slowly dividing over their lifetime. However, repeated cell divisions lead to the gradual accumulation of DNA damage and ultimately impair HSC function. Since telomeres are particularly fragile when subjected to replication stress, cells have several defense machinery to protect telomeres. Moreover, HSCs must protect their genome against possible DNA damage, while maintaining telomere length. A group of proteins called the shelterin complex are deeply involved in this two-way role, and it is highly resistant to the replication stress to which HSCs are subjected. Most shelterin-deficient experimental models suffer acute cytotoxicity and severe phenotypes, as each shelterin component is essential for telomere protection. The Tin2 point mutant mice show a dyskeratosis congenita (DC) like phenotype, and the Tpp1 deletion impairs the hematopoietic system. POT1/Pot1a is highly expressed in HSCs and contributes to the maintenance of the HSC pool during in vitro culture. Here, we discuss the role of shelterin molecules in HSC regulation and review current understanding of how these are regulated in the maintenance of the HSC pool and the development of hematological disorders.


Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas , Proteínas de Ligação a Telômeros/fisiologia , Telômero , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Aminopeptidases/genética , Animais , Divisão Celular/genética , Dano ao DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Disceratose Congênita , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Mutação , Serina Proteases/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/deficiência
18.
Nat Commun ; 8(1): 804, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28986560

RESUMO

Repeated cell divisions and aging impair stem cell function. However, the mechanisms by which this occurs are not fully understood. Here we show that protection of telomeres 1A (Pot1a), a component of the Shelterin complex that protects telomeres, improves haematopoietic stem cell (HSC) activity during aging. Pot1a is highly expressed in young HSCs, but declines with age. In mouse HSCs, Pot1a knockdown increases DNA damage response (DDR) and inhibits self-renewal. Conversely, Pot1a overexpression or treatment with POT1a protein prevents DDR, maintained self-renewal activity and rejuvenated aged HSCs upon ex vivo culture. Moreover, treatment of HSCs with exogenous Pot1a inhibits the production of reactive oxygen species, suggesting a non-telomeric role for Pot1a in HSC maintenance. Consistent with these results, treatment with exogenous human POT1 protein maintains human HSC activity in culture. Collectively, these results show that Pot1a/POT1 sustains HSC activity and can be used to expand HSC numbers ex vivo.Repeated cell divisions induce DNA damage in haematopoietic stem cells (HSC) and telomeres are sensitive to this damage. Here, the authors show in murine HSCs that the telomere binding protein POT1a inhibited the production of reactive oxygen species, and rejuvenated aged HSCs.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Células Cultivadas , Senescência Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Complexo Shelterina , Telômero/metabolismo , Telômero/fisiologia , Proteínas de Ligação a Telômeros
19.
Ann N Y Acad Sci ; 1266: 72-7, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22901259

RESUMO

Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches are critical for the maintenance of stem cell properties. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is expressed in hematopoietic stem/progenitor cells (HSPCs) and plays a critical role in the regulation of HSPC engraftment. Furthermore, overexpression of N-cadherin in HSCs promoted quiescence and preserved HSC activity during serial bone marrow (BM) transplantation (BMT). Inhibition of N-cadherin by the transduction of N-cadherin short hairpin (sh) RNA (shN-cad) reduced the lodgment of donor HSCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. shN-cad-transduced cells were maintained in the spleen for six months after BMT, indicating that N-cadherin expression in HSCs is specifically required in the BM. These findings suggest that N-cadherin-mediated cell adhesion is functionally essential for the regulation of HSPC activities in the BM niche.


Assuntos
Caderinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco/fisiologia , Animais , Transplante de Medula Óssea , Caderinas/antagonistas & inibidores , Caderinas/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Células NIH 3T3 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Nicho de Células-Tronco/genética , Estresse Fisiológico
20.
Cell Stem Cell ; 9(3): 247-61, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21885020

RESUMO

Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Combined deficiency of p57 and p27 in HSCs resulted in nuclear import of an Hsc70/cyclin D1 complex, concomitant with Rb phosphorylation, and elicited severe defects in maintaining HSC quiescence. Taken together, these data suggest that regulation of cytoplasmic localization of Hsc70/cyclin D1 complex by p57 and p27 is a key intracellular mechanism in controlling HSC dormancy.


Assuntos
Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Senescência Celular/genética , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Transporte Proteico
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