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1.
Blood Adv ; 8(1): 99-111, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-37939263

RESUMO

ABSTRACT: Aging leads to a decline in function of hematopoietic stem cells (HSCs) and increases susceptibility to hematological disease. We found CD61 to be highly expressed in aged murine HSCs. Here, we investigate the role of CD61 in identifying distinct subpopulations of aged HSCs and assess how expression of CD61 affects stem cell function. We show that HSCs with high expression of CD61 are functionality superior and retain self-renewal capacity in serial transplantations. In primary transplantations, aged CD61High HSCs function similarly to young HSCs. CD61High HSCs are more quiescent than their CD61Low counterparts. We also show that in aged bone marrow, CD61High and CD61Low HSCs are transcriptomically distinct populations. Collectively, our research identifies CD61 as a key player in maintaining stem cell quiescence, ensuring the preservation of their functional integrity and potential during aging. Moreover, CD61 emerges as a marker to prospectively isolate a superior, highly dormant population of young and aged HSCs, making it a valuable tool both in fundamental and clinical research.


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Envelhecimento , Células-Tronco Hematopoéticas/metabolismo , Integrina beta3/metabolismo
2.
Nat Aging ; 2: 851-866, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36438588

RESUMO

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.


Assuntos
Senescência Celular , Histonas , Humanos , Camundongos , Animais , Histonas/genética , Senescência Celular/genética , Tubercidina/farmacologia , Epigênese Genética
3.
Exp Hematol ; 94: 47-59.e5, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33333212

RESUMO

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression posttranscriptionally by binding to the 3' untranslated regions of their target mRNAs. The evolutionarily conserved microRNA-125a (miR-125a) is highly expressed in both murine and human hematopoietic stem cells (HSCs), and previous studies have found that miR-125 strongly enhances self-renewal of HSCs and progenitors. In this study we explored whether temporary overexpression of miR-125a would be sufficient to permanently increase HSC self-renewal or, rather, whether persistent overexpression of miR-125a is required. We used three complementary in vivo approaches to reversibly enforce expression of miR-125a in murine HSCs. Additionally, we interrogated the underlying molecular mechanisms responsible for the functional changes that occur in HSCs on overexpression of miR-125a. Our data indicate that continuous expression of miR-125a is required to enhance HSC activity. Our molecular analysis confirms changes in pathways that explain the characteristics of miR-125a overexpressing HSCs. Moreover, it provides several novel putative miR-125a targets, but also highlights the complex molecular changes that collectively lead to enhanced HSC function.


Assuntos
Células-Tronco Hematopoéticas/citologia , MicroRNAs/genética , Animais , Autorrenovação Celular , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Regulação para Cima
4.
Exp Hematol ; 91: 46-54, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32946982

RESUMO

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-resistant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rγ-/- (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patient-derived xenografts had significantly fewer clones compared with placebo-treated mice (100 ± 10 vs. 160 ± 15 clones, p = 0.0005), while clonal complexity in vincristine- and dexamethasone-treated xenografts was unaffected (115 ± 33 and 150 ± 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapy-mediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse.


Assuntos
Células Clonais/efeitos dos fármacos , Código de Barras de DNA Taxonômico , Resistencia a Medicamentos Antineoplásicos , Leucemia de Células B/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Adolescente , Animais , DNA de Neoplasias/genética , Dexametasona/farmacologia , Xenoenxertos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Seleção Genética , Análise de Célula Única , Vincristina/farmacologia
5.
Leukemia ; 34(7): 1974, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32005923

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

6.
Biol Blood Marrow Transplant ; 26(1): 16-25, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494231

RESUMO

Umbilical cord blood (UCB) provides an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Administration of sufficient donor HSCs is critical to restore recipient hematopoiesis and to maintain long-term polyclonal blood formation. However, due to lack of unique markers, the frequency of HSCs among UCB CD34+ cells is the subject of ongoing debate, urging for reproducible strategies for their counting. Here, we used cellular barcoding to determine the frequency and clonal dynamics of human UCB HSCs and to determine how data analysis methods affect these parameters. We transplanted lentivirally barcoded CD34+ cells from 20 UCB donors into Nod/Scid/IL2Ry-/- (NSG) mice (n = 30). Twelve recipients (of 8 UCB donors) engrafted with >1% GFP+ cells, allowing for clonal analysis by multiplexed barcode deep sequencing. Using multiple definitions of clonal diversity and strategies for data filtering, we demonstrate that differences in data analysis can change clonal counts by several orders of magnitude and propose methods to improve their consistency. Using these methods, we show that the frequency of NSG-repopulating cells was low (median ∼1 HSC/104 CD34+ UCB cells) and could vary up to 10-fold between donors. Clonal patterns in blood became increasingly consistent over time, likely reflecting initial output of transient progenitors, followed by long-term HSCs with stable hierarchies. The majority of long-term clones displayed multilineage output, yet clones with lymphoid- or myeloid-biased output were also observed. Altogether, this study uncovers substantial interdonor and analysis-induced variability in the frequency of UCB CD34+ clones that contribute to post-transplant hematopoiesis. As clone tracing is increasingly relevant, we urge for universal and transparent methods to count HSC clones during normal aging and upon transplantation.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Animais , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID
8.
Sci Rep ; 9(1): 4785, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886165

RESUMO

Expansion of hematopoietic stem cells (HSCs) is a 'holy grail' of regenerative medicine, as successful stem cell transplantations depend on the number and quality of infused HSCs. Although many attempts have been pursued to either chemically or genetically increase HSC numbers, neither clonal analysis of these expanded cells nor their ability to support mature blood lineages has been demonstrated. Here we show that miR-125a, at the single cell level, can expand murine long-term repopulating HSCs. In addition, miR-125a increases clone longevity, clone size and clonal contribution to hematopoiesis. Unexpectedly, we found that miR-125a expanded HSCs clones were highly homogenously distributed across multiple anatomical sites. Interestingly, these miR-125a overexpressing cells had enhanced mobility and were more frequently detected in the spleen. Our study reveals a novel, cell-intrinsically controlled mechanism by which HSC migration is regulated.


Assuntos
Movimento Celular , Autorrenovação Celular , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Baço/citologia
9.
Cell Rep ; 26(7): 1906-1918.e8, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759399

RESUMO

In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7-binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Células-Tronco/metabolismo , Animais , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células HEK293 , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 1/genética , Células-Tronco/citologia , Transcrição Gênica
10.
Cell Stem Cell ; 19(3): 383-96, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27424784

RESUMO

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.


Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Autorrenovação Celular/genética , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Marcação por Isótopo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/transplante , Reprodutibilidade dos Testes , Fatores de Tempo
11.
Stem Cell Reports ; 4(1): 74-89, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25434821

RESUMO

Accurate monitoring of tumor dynamics and leukemic stem cell (LSC) heterogeneity is important for the development of personalized cancer therapies. In this study, we experimentally induced distinct types of leukemia in mice by enforced expression of Cbx7. Simultaneous cellular barcoding allowed for thorough analysis of leukemias at the clonal level and revealed high and unpredictable tumor complexity. Multiple LSC clones with distinct leukemic properties coexisted. Some of these clones remained dormant but bore leukemic potential, as they progressed to full-blown leukemia after challenge. LSC clones could retain multilineage differentiation capacities, where one clone induced phenotypically distinct leukemias. Beyond a detailed insight into CBX7-driven leukemic biology, our model is of general relevance for the understanding of tumor dynamics and clonal evolution.


Assuntos
Evolução Clonal/genética , Leucemia/genética , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/genética , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Perfilação da Expressão Gênica , Imunofenotipagem , Leucemia/patologia , Camundongos , Células-Tronco Neoplásicas/patologia , Fenótipo
12.
Exp Hematol ; 42(10): 909-18.e1, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25092555

RESUMO

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype.


Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/fisiologia , Animais , Transplante de Medula Óssea , Divisão Celular , Linhagem da Célula , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , MicroRNAs/genética , Mutagênese Sítio-Dirigida , Mielopoese/genética , Oligonucleotídeos/farmacologia , Mutação Puntual , Quimera por Radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade
13.
J Exp Med ; 211(3): 487-97, 2014 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-24567446

RESUMO

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make-up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long-term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols.


Assuntos
Medula Óssea/metabolismo , Movimento Celular/fisiologia , Citocinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Código de Barras de DNA Taxonômico , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Estatísticas não Paramétricas
14.
Nat Cell Biol ; 15(4): 353-62, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23502315

RESUMO

The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs.


Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Leucemia Experimental/patologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Apoptose , Western Blotting , Imunoprecipitação da Cromatina , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Experimental/etiologia , Leucemia Experimental/mortalidade , Ligases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Transporte da Membrana Mitocondrial , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Blood ; 119(2): 377-87, 2012 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-22123844

RESUMO

Hematopoietic stem/progenitor cell (HSPC) traits differ between genetically distinct mouse strains. For example, DBA/2 mice have a higher HSPC frequency compared with C57BL/6 mice. We performed a genetic screen for micro-RNAs that are differentially expressed between LSK, LS(-)K(+), erythroid and myeloid cells isolated from C57BL/6 and DBA/2 mice. This analysis identified 131 micro-RNAs that were differentially expressed between cell types and 15 that were differentially expressed between mouse strains. Of special interest was an evolutionary conserved miR cluster located on chromosome 17 consisting of miR-99b, let-7e, and miR-125a. All cluster members were most highly expressed in LSKs and down-regulated upon differentiation. In addition, these microRNAs were higher expressed in DBA/2 cells compared with C57BL/6 cells, and thus correlated with HSPC frequency. To functionally characterize these microRNAs, we overexpressed the entire miR-cluster 99b/let-7e/125a and miR-125a alone in BM cells from C57BL/6 mice. Overexpression of the miR-cluster or miR-125a dramatically increased day-35 CAFC activity and caused severe hematopoietic phenotypes upon transplantation. We showed that a single member of the miR-cluster, namely miR-125a, is responsible for the majority of the observed miR-cluster overexpression effects. Finally, we performed genome-wide gene expression arrays and identified candidate target genes through which miR-125a may modulate HSPC fate.


Assuntos
Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , MicroRNAs/genética , Células Mieloides/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Células Eritroides/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células Mieloides/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS Genet ; 5(10): e1000692, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19834560

RESUMO

Genetical genomics is a strategy for mapping gene expression variation to expression quantitative trait loci (eQTLs). We performed a genetical genomics experiment in four functionally distinct but developmentally closely related hematopoietic cell populations isolated from the BXD panel of recombinant inbred mouse strains. This analysis allowed us to analyze eQTL robustness/sensitivity across different cellular differentiation states. Although we identified a large number (365) of "static" eQTLs that were consistently active in all four cell types, we found a much larger number (1,283) of "dynamic" eQTLs showing cell-type-dependence. Of these, 140, 45, 531, and 295 were preferentially active in stem, progenitor, erythroid, and myeloid cells, respectively. A detailed investigation of those dynamic eQTLs showed that in many cases the eQTL specificity was associated with expression changes in the target gene. We found no evidence for target genes that were regulated by distinct eQTLs in different cell types, suggesting that large-scale changes within functional regulatory networks are uncommon. Our results demonstrate that heritable differences in gene expression are highly sensitive to the developmental stage of the cell population under study. Therefore, future genetical genomics studies should aim at studying multiple well-defined and highly purified cell types in order to construct as comprehensive a picture of the changing functional regulatory relationships as possible.


Assuntos
Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Locos de Características Quantitativas , Animais , Feminino , Marcadores Genéticos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos
17.
Stem Cells ; 24(6): 1564-72, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16527900

RESUMO

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.


Assuntos
Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Proliferação de Células , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Feminino , Substâncias de Crescimento/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Tolerância a Radiação , Proteínas Recombinantes/farmacologia
18.
Blood ; 107(5): 2170-9, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16293602

RESUMO

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.


Assuntos
Senescência Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas/metabolismo , Animais , Transplante de Medula Óssea , Divisão Celular/fisiologia , Sobrevivência Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco Hematopoéticas/citologia , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Camundongos , Complexo Repressor Polycomb 2 , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Retroviridae , Transdução Genética
19.
Nat Genet ; 37(3): 225-32, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15711547

RESUMO

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.


Assuntos
Genoma Humano , Células-Tronco Hematopoéticas/citologia , Proteínas de Transporte/genética , Humanos , Dados de Sequência Molecular , Locos de Características Quantitativas , RNA Mensageiro/genética
20.
Stem Cells ; 23(1): 82-92, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15625125

RESUMO

Adult somatic stem cells possess extensive self-renewal capacity, as their primary role is to replenish aged and functionally impaired tissues. We have previously shown that the stem cell pool in short-lived DBA/2 (D2) mice is reduced during aging, in contrast to long-lived C57BL/6 (B6) mice. This suggests the existence of a genetically determined mitotic clock operating in stem cells, which possibly limits organismal aging. In the study reported here, unfractionated bone marrow (BM) cells or highly purified Lin(-)Sca-1(+)c-kit(+) (LSK) cells were serially transplanted in lethally irradiated D2 and B6 mice. In both strains, serial transplantation resulted in a substantial loss of stem cell activity. However, as we estimate that in B6 mice, the maximum number of population doublings of primitive cells is approximately 30, in D2 mice this is only approximately 20, resulting in a 1,000-fold difference in expansion potential, irrespective of whether whole bone marrow or purified hematopoietic stem cells (HSCs) were transplanted. Interestingly, recipients reconstituted with serially transplanted BM cells were able to accept a freshly isolated graft without any further conditioning. Finally, we show that whereas transplantation of BM cells into healthy, nonconditioned, young B6 recipients does not lead to engraftment, young BM cells do engraft and provide multilineage reconstitution in nonirradiated aged mice. Our data clearly establish the relevance of an intrinsic, genetically controlled program associated with impaired stem cell functioning during aging.


Assuntos
Senescência Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Animais , Contagem de Células , Estudos de Avaliação como Assunto , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fatores de Tempo
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