RESUMO
Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1-5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down's syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.
Assuntos
Engenharia Genética , Técnicas In Vitro , Oócitos , Cromossomo X , Animais , Feminino , Masculino , Camundongos , Oócitos/metabolismo , Oócitos/fisiologia , Cromossomo X/genética , Cromossomo Y/genética , Células-Tronco Pluripotentes/metabolismo , Síndrome de Down/genética , Síndrome de Down/terapia , Fertilização , Infertilidade/terapia , Homossexualidade Masculina , Transtornos dos Cromossomos Sexuais/complicações , Transtornos dos Cromossomos Sexuais/genética , Transtornos dos Cromossomos Sexuais/terapia , Engenharia Genética/métodosRESUMO
Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.
Assuntos
Caderinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nicho de Células-Tronco , Via de Sinalização Wnt , Animais , Caderinas/genética , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFATC/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Ciclo Celular/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/metabolismoRESUMO
A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.
Assuntos
Células Endoteliais , Fator de Células-Tronco , Camundongos , Animais , Fator de Células-Tronco/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea/metabolismo , Osso e Ossos , Nicho de Células-Tronco , Células da Medula Óssea/metabolismoRESUMO
There is much interest in understanding the signals in the bone marrow niche that keep hematopoietic stem cells (HSCs) in a quiescent state. In the current issue of Cell Stem Cell, Fleming et al. (2008) report that blocking Wnt signaling in the niche increases the number of proliferating HSCs and reduces their ability to reconstitute the hematopoietic system of irradiated recipient mice. These findings show that Wnt/beta-catenin activity is crucial for the maintenance of HSC quiescence in the bone marrow niche.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Wnt/metabolismo , Animais , Medula Óssea/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Proteínas Wnt/antagonistas & inibidoresRESUMO
Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.
Assuntos
Citometria de Fluxo/métodos , Hematopoese , Neutrófilos/citologia , Cultura Primária de Células/métodos , Animais , Células Cultivadas , Lectinas/metabolismo , Neutrófilos/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Modern single cell experiments have revealed unexpected heterogeneity in apparently functionally 'pure' cell populations. However, we are still lacking a conceptual framework to understand this heterogeneity. Here, we propose that cellular memories-changes in the molecular status of a cell in response to a stimulus, that modify the ability of the cell to respond to future stimuli-are an essential ingredient in any such theory. We illustrate this idea by considering a simple age-structured model of stem cell proliferation that takes account of mitotic memories. Using this model we argue that asynchronous mitosis generates heterogeneity that is central to stem cell population function. This model naturally explains why stem cell numbers increase through life, yet regenerative potency simultaneously declines.
Assuntos
Mitose , Células-Tronco/fisiologia , Modelos BiológicosRESUMO
Expansion of stem cell numbers without reduction in their regenerative potential is crucial for therapeutic applications. However, the repeated cell divisions and aging impair stem cell function. We found that Pot1a, a component of the shelterin that protects telomeres, involves the maintenance of hematopoietic stem cell (HSC) activity during aging. Pot1a maintained the self-renewal activity of HSCs through the prevention of DNA damage responses in HSCs and suppression of the production of reactive oxygen species. Furthermore, the exogenous Pot1a expanded the HSC number and rejuvenated aged HSCs function upon ex vivo culture. Consistent with these results, treatment with exogenous human POT1 protein maintains human HSC activity in culture. Collectively, these results show that Pot1a or POT1 sustains HSC activity and can be used to expand HSC numbers ex vivo.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Envelhecimento , Divisão Celular , Humanos , Manutenção , Complexo Shelterina , Telômero , Proteínas de Ligação a TelômerosRESUMO
Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2 The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs.
Assuntos
Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Medula Óssea/patologia , Retrovirus Endógenos/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Deleção de Genes , Inativação Gênica , Gluconeogênese/genética , Homeostase/genética , Leucemia/genética , Leucemia/patologia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
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/metabolismoRESUMO
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/genéticaRESUMO
Hematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate into all blood lineage cells. The fate decisions of HSCs (self-renewal versus differentiation) are made through the process of cell division and are often compared to "birth" and "death". Stem cells give rise to undifferentiated stem cells (birth) or differentiate into progenitor cells (death). This process is regulated by asymmetric/symmetric divisions of HSCs. It has been proposed that fate determination occurs as a stochastic process and that individual stem cell dynamics are randomly regulated. The behavior of HSCs is known to be regulated by the cell intrinsic factor and extrinsic (microenvironmental) stimuli. Therefore, it is possible that the signals from a specific microenvironment (niche) have the potential to control or modulate stem cell dynamics. This review focuses on the functions of the HSC niche and the application of single cell analysis for understanding the mechanisms underlying the HSC decision-making process.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Animais , Divisão Celular , Linhagem da Célula , HumanosRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
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 SinaisRESUMO
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/metabolismoRESUMO
Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.
Assuntos
Dosagem de Genes/fisiologia , Duplicação Gênica/fisiologia , Perda de Heterozigosidade/fisiologia , Proteínas de Membrana/genética , Transtornos Mieloproliferativos/genética , Tirosina Quinase 3 Semelhante a fms/fisiologia , Alelos , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células da Medula Óssea/fisiologia , Proliferação de Células , Células Cultivadas , Técnicas de Introdução de Genes , Perda de Heterozigosidade/genética , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Sequências de Repetição em Tandem/genética , Sequências de Repetição em Tandem/fisiologia , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
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 & inibidoresRESUMO
The transversus abdominis plane (TAP) block is a newly described technique introducing a local anesthetic agent between the internal oblique and the transversus abdominis muscles of the abdominal wall, which is safer and more reliable analgesia in recent years by ultrasound technique. We report the perioperative management of transversus abdominis plane block with catheterization for a patient with severe cardiac dysfunction and chronic kidney failure, who underwent bilateral inguinal hernioplasty. A bilateral TAP block was first performed with 0.5% ropivacaine 20 ml under ultrasonographic visualization on right side, and after sixty-minutes the other side injection was performed through the indwelling catheter. During the operation, the patient received a target-controlled infusion of 0.4-0.6 microg x ml(-1) propofol. The perioperative courses were uneventful and there was no adverse effect including central nervous system (CNS) symptoms.
Assuntos
Cardiopatias/complicações , Hérnia Inguinal/cirurgia , Falência Renal Crônica/complicações , Bloqueio Nervoso/métodos , Músculos Abdominais , Idoso de 80 Anos ou mais , Cateterismo , Humanos , MasculinoRESUMO
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.