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1.
J Cell Sci ; 129(4): 788-803, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26759175

RESUMO

Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.


Assuntos
Histona Desmetilases/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Núcleo Celular/enzimologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Dano ao DNA , Humanos , Camundongos , Transporte Proteico
2.
Transgenic Res ; 26(5): 709-713, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28730514

RESUMO

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.


Assuntos
Biotecnologia/tendências , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Biotecnologia/métodos , Europa (Continente) , Humanos
3.
Transfus Med Hemother ; 43(4): 247-254, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27721700

RESUMO

Hematopoietic stem cells (HSCs) are the best characterized adult stem cells and the only stem cell type in routine clinical use. The concept of stem cell transplantation laid the foundations for the development of novel cell therapies within, and even outside, the hematopoietic system. Here, we report on the history of hematopoietic cell transplantation (HCT) and of HSC isolation, we briefly summarize the capabilities of HSCs to reconstitute the entire hemato/lymphoid cell system, and we assess current indications for HCT. We aim to draw the lines between areas where HCT has been firmly established, areas where HCT can in the future be expected to be of clinical benefit using their regenerative functions, and areas where doubts persist. We further review clinical trials for diverse approaches that are based on HCT. Finally, we highlight the advent of genome editing in HSCs and critically view the use of HSCs in non-hematopoietic tissue regeneration.

4.
Mol Med ; 21: 185-96, 2015 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-25811991

RESUMO

Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.


Assuntos
Diferenciação Celular , Citotoxicidade Imunológica , Células-Tronco Embrionárias/citologia , Expressão Gênica , Antígenos HLA-G/genética , Células Matadoras Naturais/imunologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/metabolismo , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Regulação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos HLA-G/imunologia , Antígenos HLA-G/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Células-Tronco Neurais/citologia
5.
Mol Med ; 19: 399-408, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24408113

RESUMO

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1-3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of ß-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.


Assuntos
Eritropoetina/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese , Animais , Proliferação de Células , Sobrevivência Celular , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/fisiologia , Oligodendroglia/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Potenciais Sinápticos
6.
Cytotherapy ; 14(5): 570-83, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22300364

RESUMO

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. METHODS: Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. RESULTS: Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. CONCLUSIONS: We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.


Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células , Meios de Cultura , Células-Tronco Mesenquimais/citologia , Soro , Tecido Adiposo/metabolismo , Adulto , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Feminino , Fibroblastos/citologia , Humanos , Pessoa de Meia-Idade
7.
Cytotherapy ; 12(7): 899-908, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20210674

RESUMO

BACKGROUND: DNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH3(+) groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci. METHODS: We analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin(+)) and progenitor lineage marker-negative (Lin⁻) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence. RESULTS: We observed that Lin⁻ and Lin(+) cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4(-/-) mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4. CONCLUSIONS: Overall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.


Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Acetilação/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Histona Desacetilases/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/genética
8.
Cells Tissues Organs ; 191(3): 167-74, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19776550

RESUMO

We report flow cytometric protocols that quantitatively display the levels of different histone modifications on the single cell level. This method allows the rapid and simultaneous analysis of the global levels of chromatin marks in combination with other flow cytometric features. We show that chromatin flow cytometry identifies changes in epigenetic marks, such as histone acetylation and methylation, in drug-treated and differentiated mouse embryonic stem cell populations.


Assuntos
Cromatina/genética , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Citometria de Fluxo/métodos , Acetilação/efeitos dos fármacos , Animais , Azepinas/farmacologia , Diferenciação Celular , Células Cultivadas , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Metilação/efeitos dos fármacos , Camundongos , Quinazolinas/farmacologia
9.
Stem Cells ; 26(6): 1474-83, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18369101

RESUMO

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article.


Assuntos
Androgênios/fisiologia , Blastocisto/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Animais , Blastocisto/citologia , Encéfalo/citologia , Encéfalo/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Divisão Celular , Feminino , Genes Reporter , Genoma , Impressão Genômica , Masculino , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genética , Zigoto
10.
Stem Cells ; 26(4): 920-6, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18203677

RESUMO

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin-modifying agents trichostatin A (TSA) and 5-Aza-2'-deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long-term, multilineage, and transplantable. Here, we have analyzed the TSA/AzaC-induced changes in gene expression by global gene expression profiling. TSA/AzaC caused both up- and downregulation of genes, without increasing the total number of expressed genes. Chromosome analysis showed no hot spot of TSA/AzaC impact on a particular chromosome or chromosomal region. Hierarchical cluster analysis revealed common gene expression patterns among neurosphere cells treated with TSA/AzaC, embryonic stem (ES) cells, and hematopoietic stem cells. Furthermore, our analysis identified several stem cell genes and pluripotency-associated genes that are induced by TSA/AzaC in neurosphere cells, including Cd34, Cd133, Oct4, Nanog, Klf4, Bex1, and the Dppa family members Dppa2, 3, 4, and 5. Sox2 and c-Myc are constitutively expressed in neurosphere cells. We propose a model in which TSA/AzaC, by removal of epigenetic inhibition, induces the reactivation of several stem cell and pluripotency-associated genes, and their coordinate expression enlarges the differentiation potential of somatic precursor cells.


Assuntos
Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Células Cultivadas , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Prosencéfalo/citologia , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/fisiologia
11.
Leuk Res ; 32(5): 781-9, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17980910

RESUMO

While critical steps in the regulation of leukemia cell development have been intensively studied in recent years, less is known about the interactions of leukemic cells with their stroma. Previously, we have shown that human acute myeloid leukemia (AML) cells differentiate upon injection into murine blastocysts. We here describe that human AML Kasumi-1 cells, cocultured with murine aorta-gonad-mesonephros (AGM) region-derived DAS104-4 stromal cells, decrease proliferation and colony formation efficiency; and up-regulate myelo-monocytic cell surface markers. Gene expression analysis showed decreased transcription of the AML1-ETO fusion gene and increased transcription of p16 (INK4A), p21 (WAF1) and C/EBPalpha genes. Coculture can induce myeloid differentiation also in patient-derived AML cells. Our findings strengthen the notion that the embryonic milieu can regulate the proliferation and differentiation of leukemic cells.


Assuntos
Aorta/embriologia , Gônadas/embriologia , Leucemia Mieloide Aguda/patologia , Mesonefro/embriologia , Células Estromais/fisiologia , Aorta/citologia , Antígeno CD11b/análise , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Gônadas/citologia , Humanos , Receptores de Lipopolissacarídeos/análise , Mesonefro/citologia , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1
12.
Eur J Heart Fail ; 10(2): 119-24, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18279767

RESUMO

OBJECTIVE: Bone marrow (BM)-derived haematopoietic stem cells have been proposed as a potential cell source to functionally engraft the myocardium and to improve cardiac function after myocardial infarction (MI). However, experimental and clinical data are inconsistent. Since the specific characteristics of different BM cell subsets could influence their therapeutic potential we determined the effect of different BM cell populations on left ventricular remodelling after MI. METHODS AND RESULTS: MI was induced in female mice by coronary artery ligation. Surviving mice were randomised to receive either: total BM, mature Lin(+) or primitive Lin(-) cells from male mice, or saline, via intracardiac injection. Injected cells were detected in the infarct and border zone by PCR for Y-chromosomal sequences. Serial transthoracic echocardiography was performed 1, 21, and 42 days after MI. Over a period of 6 weeks, mortality was not different between the groups. After MI, animals exhibited left ventricular dilatation, as expected. Left ventricular remodelling was not influenced by Lin(+) or Lin(-) BM cells but was partially improved by unfractionated BM cell injection. Paracrine secretion of cytokines (e.g. IL-6, GM-CSF) was differentially regulated in supernatants of cultured BM cells. SUMMARY: Treatment with unfractionated BM cells, but not Lin(+), or Lin(-) cells partially improved cardiac remodelling and function after MI. This may be mediated by paracrine effects.


Assuntos
Transplante de Medula Óssea , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Remodelação Ventricular , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/fisiopatologia , Comunicação Parácrina/fisiologia , Análise de Sobrevida
13.
Cells Tissues Organs ; 188(1-2): 103-15, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18600024

RESUMO

Hematopoietic stem cells maintain the development of all mature blood cells throughout life due to their sustained self-renewal capacity and multilineage differentiation potential. During development into specific cell lineages, the options of stem cells and multipotent progenitor cells become increasingly restricted concomitant with a successive decline in self-renewal potential. Here we describe an Flt3+CD11b+ multipotent progenitor that can be amplified in vitro with a specific combination of cytokines to yield homogeneous populations in high cell numbers. By employing gene expression profiling with DNA microarrays, we studied the transcription factor repertoire of Flt3+CD11b+ progenitors and related it to the transcription factor repertoire of hematopoietic stem cells and embryonic stem cells. We report here on overlapping and nonoverlapping expression patterns of transcription factors in these cells and thus provide novel insights into the dynamic networks of transcriptional regulators in embryonic and adult stem cells. Additionally, the results obtained open the perspective for elucidating lineage and 'stemness' determinants in hematopoiesis.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular , Análise por Conglomerados , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fatores de Transcrição/metabolismo
14.
Oncogene ; 24(4): 561-72, 2005 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-15580301

RESUMO

HOXB4 overexpression mediates increased self-renewal of haematopoietic stem cells (HSCs) ex vivo. Since HOXB4-expanded HSCs retain normal differentiation potential and there is no leukaemia development from transduced HSCs, HOXB4 represents a promising tool for human HSC therapy. However, the increased proliferation capacity of HOXB4 overexpressing fibroblasts resulting from upregulation of JunB, Fra-1 and cyclin D1 protein levels may indicate a potential risk associated with the HOXB4 overexpression approach. This prompted us to investigate the proliferation rate, differentiation and expression of cell cycle regulators directly in bone marrow cultures overexpressing HOXB4. Here we show that in comparison to neo-transduced control bone marrow cultures, HOXB4-overexpressing cultures had a more homogenous morphology and increased numbers of haematopoietic progenitor cells capable to generate primitive colonies in vitro. In contrast, neo-transduced bone marrow cells in long-term cultures showed hallmarks of myeloid differentiation and a reduced secondary colony forming activity. We further show that multilineage repopulating activity in vivo, which was present only in HOXB4 long-term cultures, declined over time. HOXB4 overexpression in vitro did not result in an increase but in a stabilization of the proliferation rate (1.4-1.8 cell divisions per day), while the proliferation rate of control neo-transduced bone marrow cultures gradually declined. Correspondingly, increased HOXB4 expression was paralleled by decreased expression levels of cyclins, CDKs and AP-1 family members. These results suggest that the growth rate of HOXB4- compared to neo-transduced bone marrow cells remains constant in long-term cultures along with a suppression of myeloid differentiation. In contrast to HOXB4 overexpression in fibroblasts, bone marrow cells engineered to overexpress HOXB4 do not upregulate AP-1 complex members or cyclins indicating that HOXB4 acts in a cell type-specific way.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Homeodomínio/metabolismo , Animais , Transplante de Medula Óssea , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Proteínas de Homeodomínio/genética , Camundongos , Fator de Transcrição AP-1/genética , Fatores de Transcrição , Transcrição Gênica , Transdução Genética
15.
Mech Ageing Dev ; 127(7): 600-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16620920

RESUMO

BCL-2 functions as a death repressor molecule in an evolutionary conserved cell death pathway. Inactivation of bcl-2 in mice results in pleiotropic effects including postnatal growth retardation, massive apoptosis in lymphoid tissues, polycystic kidney disease (PKD) and shortened lifespan. To evaluate the influence of the affected bcl-2 deficient kidneys on the postnatal development and lifespan of bcl-2 knockout mice we used "the rescue of (n-1) affected tissues" strategy. According to this strategy bcl-2 heterozygous animals were crossed with H2K-hbcl-2 transgenic mice expressing human BCL-2 in most tissues and organs excluding the kidney. Overexpression of hBCL-2 in bcl-2-/- mice rescues growth retardation, normalizes and protects the hematolymphoid system from gamma-radiation. However, the hbcl-2 transgene is not expressed in kidneys and the rescued mice have PKD and a shortened lifespan. Thus, our results indicated that PKD is the main reason of early mortality in bcl-2 deficient mice. Moreover, we have created mouse model, similar to the kidney specific knockout of bcl-2. Such models can be useful to study the influence of bcl-2 or other gene deficiency in individual organs (or tissues) on development and ageing of whole organism.


Assuntos
Apoptose/genética , Longevidade/genética , Doenças Renais Policísticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Insuficiência Renal/genética , Animais , Cruzamentos Genéticos , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Heterozigoto , Humanos , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/patologia , Insuficiência Renal/patologia
16.
Stem Cells Dev ; 25(12): 922-33, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27100571

RESUMO

Polycomb proteins such as the B lymphoma Mo-MLV insertion region 1 homolog (BMI1) are essential chromatin factors for the self-renewal and differentiation of embryonic and adult stem cells. BMI1 also plays a critical role in osteogenesis as Bmi1-deficient mice display a skeletal phenotype caused by the exhaustion of the mesenchymal stem cell pool. In this study, we have studied the role of BMI1 in the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs). BMI1 protein, but not RNA levels, increases during in vitro osteogenic differentiation of hASCs. Overexpression of BMI1 leads to an osteogenic priming of hASCs under nondifferentiating conditions and enhanced osteogenesis upon differentiation, along with increased BMP2 and WNT11 expressions. Conversely, knockdown of BMI1 expression reduces osteogenic differentiation. Furthermore, our studies indicate that during osteogenic differentiation of hASCs, BMI1 is a downstream target of GSK3 signaling. BMI1, therefore, acts as a pro-osteogenic differentiation factor in hASCs and hence it is a promising target for active modulation of hASC-derived osteogenesis.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Complexo Repressor Polycomb 1/metabolismo , Fosfatase Alcalina/metabolismo , Matriz Óssea/metabolismo , Calcificação Fisiológica , Células Cultivadas , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Transdução de Sinais
17.
Oncogene ; 22(57): 9185-91, 2003 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-14668800

RESUMO

It has been suggested that the embryonic microenvironment can control the survival and the transformed phenotype of tumour cells. Here, we addressed the hypothesis that the murine embryonic microenvironment can induce the differentiation of human tumour cells. To examine such interactions, we injected human leukaemic cells into preimplantation murine blastocysts at embryonic day 3.5 of gestation (E3.5). Microinjection of human KG-1 myeloid leukaemia cells and primary human acute myeloid leukaemia (AML) cells led to the generation of chimaeric embryos and adults. We observed that in E12.5 murine embryos, KG-1 cells were preferentially detected in yolk sac and peripheral blood, while primary AML cells mainly seeded the aorta gonad mesonephros region of chimaeric embryos. Analysis of the donor contribution in 15 different adult tissues showed that progeny of primary AML cells seeded to various haematopoietic and nonhaematopoietic tissues. Chimaeric embryos and adults showed no apparent tumour formation. Furthermore, analysis of chimaeric E12.5 embryos revealed that the progeny of human KG-1 cells activated erythroid-specific human globin and glycophorin A expression. In summary, our data indicate that human AML cells activate markers of erythroid differentiation after injection into early murine embryos.


Assuntos
Blastocisto/fisiologia , Leucemia Mieloide Aguda/patologia , Quimeras de Transplante/fisiologia , Transplante Heterólogo/fisiologia , Animais , Biomarcadores/análise , Diferenciação Celular , Idade Gestacional , Glicoforinas/análise , Hematopoese , Humanos , Rim/embriologia , Leucemia Mieloide Aguda/fisiopatologia , Camundongos , Microinjeções , Células Tumorais Cultivadas
18.
Leuk Res ; 29(10): 1191-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15941586

RESUMO

To better characterize acute myeloid leukemia (AML) development in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we transplanted samples from patients with AML or KG-1 and EOL-1 cell lines. We found 9/12 primary AML samples and both cell lines to engraft within 2-8 weeks, with 5-80% human cells in bone marrow. Compared with freshly isolated AML cells, percentages of human CD33+, CD38+, CD31+ CD13+ or CD15+ subpopulations increased after transplantation, whereas percentages of CD34+ cells decreased. Engrafted mice frequently showed expression of human endothelial cell markers. Thus, transplantation of human AML into NOD/SCID mice reveals expression of hematopoietic and endothelial differentiation markers.


Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Leucemia Mieloide/patologia , Transplante Heterólogo/patologia , Doença Aguda , Adulto , Idoso , Animais , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/patologia , Citometria de Fluxo , Humanos , Leucemia Mieloide/classificação , Leucemia Mieloide/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo/imunologia , Células Tumorais Cultivadas
19.
Exp Hematol ; 32(7): 673-82, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15246164

RESUMO

OBJECTIVE: In contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, somatic stem cells have been thought to be more limited in their differentiation potential in that they are committed to generate only cells of their tissue of origin. Unexpectedly, some recent data suggest that somatic stem cells isolated from one tissue can also generate cells of heterologous tissues and organs, implying that somatic stem cells have a greater potential for differentiation. METHODS: To explore further the developmental potential of murine neural stem cells (NSCs) we injected cultured NSCs as neurospheres into preimplantation blastocysts and determined the seeding by donor cells in tissues of developing chimeric fetal and adult animals. RESULTS: We frequently detected progeny of injected NSCs both in embryos and in adult animals. In embryos we observed transient seeding of donor cells to hematopoietic tissues and generation of NSC-derived cells that express globin genes and an erythroid-specific cell-surface marker. In adults progeny of NSCs were mostly detected in neural tissues. The observed low level of chimerism of wild-type NSCs was increased if we injected stem cells expressing a bcl-2 transgene, without changing the seeding pattern. CONCLUSION: These results suggest that cultured NSCs, following their injection into blastocysts, generate at mid-gestation erythroid-like cells but later, in adult chimeric mice, engraftment mainly persisted in neural tissues.


Assuntos
Blastocisto/citologia , Córtex Cerebral/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Animais , Blastocisto/fisiologia , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco , Quimeras de Transplante
20.
Stem Cell Rev Rep ; 11(1): 50-61, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25134795

RESUMO

Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Complexo Repressor Polycomb 2/genética , Interferência de RNA , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lisina/metabolismo , Metilação , Camundongos Knockout , Microscopia de Fluorescência , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
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