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1.
Biochem Biophys Res Commun ; 532(4): 541-547, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-32896380

RESUMO

The proper development of the cerebral cortex is essential for brain formation and functioning. O-GlcNAcylation, an important posttranslational modification, regulates the pathways critical for neuronal health and the survival of the cerebral cortex in neurodegenerative diseases. However, the role of O-GlcNAcylation in regulating cerebral cortical development at the embryonic and early postnatal (0-21 days) stages is still largely unknown. Here we report that the selective deletion of O-GlcNAc transferase (OGT) in neural stem cells (NSCs) in mice led to a series of severe brain developmental deficits, including dramatic shrinkage of cortical and hippocampal histoarchitecture, widespread neuronal apoptosis, decrease in cell proliferation, induction of endoplasmic reticulum (ER) stress, and inhibition of neuronal dendritic and axonal differentiation. The pathology of corticogenesis deficits caused by OGT deletion may largely rely on complicated biological processes, such as proliferation, apoptosis and differentiation. Our results suggest that dysfunctional O-GlcNAcylation in NSCs may be an important contributor to neurodevelopmental diseases.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , N-Acetilglucosaminiltransferases/fisiologia , Células-Tronco Neurais/enzimologia , Animais , Apoptose , Axônios/ultraestrutura , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/embriologia , Córtex Cerebral/enzimologia , Dendritos/ultraestrutura , Proteína 4 Homóloga a Disks-Large/metabolismo , Estresse do Retículo Endoplasmático , Masculino , Camundongos Knockout , Morfogênese , Células-Tronco Multipotentes/enzimologia , N-Acetilglucosaminiltransferases/genética , Neurônios/citologia , Neurônios/metabolismo
2.
Methods Mol Biol ; 2045: 93-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31020633

RESUMO

Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-ßgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Senescência Celular/genética , Citocinas/metabolismo , Dano ao DNA , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Inflamação/metabolismo , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/efeitos da radiação , Células-Tronco Multipotentes/enzimologia , Células-Tronco Multipotentes/fisiologia , Células-Tronco Multipotentes/efeitos da radiação , Fenótipo , Transdução de Sinais/genética , Telômero/genética , Telômero/metabolismo , Fluxo de Trabalho , beta-Galactosidase/metabolismo
3.
J Cell Physiol ; 233(2): 1752-1762, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28681925

RESUMO

Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.


Assuntos
Diferenciação Celular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células-Tronco Multipotentes/enzimologia , Osteoblastos/enzimologia , Osteogênese , Ligamento Periodontal/enzimologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Células-Tronco Multipotentes/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Ratos Sprague-Dawley , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transfecção , Via de Sinalização Wnt/efeitos dos fármacos
4.
Cell Death Differ ; 24(12): 2101-2116, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28800128

RESUMO

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1-2% of total c-kitpos myocardial cells, when stimulated with TGF-ß/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.


Assuntos
Células-Tronco Adultas/enzimologia , Células-Tronco Multipotentes/enzimologia , Miocárdio/enzimologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Células-Tronco Adultas/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Masculino , Camundongos , Células-Tronco Multipotentes/citologia , Miocárdio/citologia , Ratos , Ratos Wistar
5.
Sci Signal ; 8(401): pe3, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26535006

RESUMO

The phosphoinositide 3-kinase (PI3K) signaling pathway is among the most frequently altered in cancer. Now, two studies show that a mutated oncogenic PI3Kα, commonly found in breast cancer, leads to dedifferentiation or destabilization of luminal and basal epithelial lineages, which in turn leads to increased cancer cell heterogeneity.


Assuntos
Neoplasias da Mama , Mama/enzimologia , Células Epiteliais/enzimologia , Células-Tronco Multipotentes/enzimologia , Proteínas de Neoplasias , Células-Tronco Neoplásicas , Fosfatidilinositol 3-Quinases , Mama/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Células Epiteliais/patologia , Feminino , Humanos , Células-Tronco Multipotentes/patologia , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo
6.
Exp Hematol ; 42(10): 841-51, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25201757

RESUMO

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases in which cells of the myelo-erythroid lineage are overproduced and patients are predisposed to leukemic transformation. Hematopoietic stem cells are the suspected disease-initiating cells, and these cells must acquire a clonal advantage relative to nonmutant hematopoietic stem cells to perpetuate disease. In 2005, several groups identified a single gain-of-function point mutation in JAK2 that associated with the majority of MPNs, and subsequent studies have led to a comprehensive understanding of the mutational landscape in MPNs. However, confusion still exists as to how a single genetic aberration can be associated with multiple distinct disease entities. Many explanations have been proposed, including JAK2V617F homozygosity, individual patient heterogeneity, and the differential regulation of downstream JAK2 signaling pathways. Several groups have made knock-in mouse models expressing JAK2V617F and have observed divergent phenotypes, each recapitulating some aspects of disease. Intriguingly, most of these models do not observe a strong hematopoietic stem cell self-renewal advantage compared with wild-type littermate controls, raising the question of how a clonal advantage is established in patients with MPNs. This review summarizes the current molecular understanding of MPNs and the diversity of disease phenotypes and proposes that the increased proliferation induced by JAK2V617F applies a selection pressure on the mutant clone that results in highly diverse clonal evolution in individuals.


Assuntos
Transformação Celular Neoplásica/genética , Células Clonais/patologia , Células-Tronco Hematopoéticas/patologia , Modelos Genéticos , Transtornos Mieloproliferativos/patologia , Animais , Divisão Celular/genética , Células Clonais/enzimologia , Citocinas/fisiologia , Epigênese Genética/genética , Evolução Molecular , Dosagem de Genes , Técnicas de Introdução de Genes , Heterogeneidade Genética , Células-Tronco Hematopoéticas/enzimologia , Humanos , Janus Quinase 2/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Células-Tronco Multipotentes/enzimologia , Células-Tronco Multipotentes/patologia , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/classificação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Fenótipo , Mutação Puntual , Splicing de RNA/genética , Seleção Genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética
7.
Taiwan J Obstet Gynecol ; 53(2): 187-92, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25017264

RESUMO

OBJECTIVE: Stem cells offer great potential for clinical therapeutic use because of their ability to rejuvenate and to differentiate into numerous types of cells. We isolated multipotent cells from the human term placenta that were capable of differentiation into cells of all three germ layers. MATERIALS AND METHODS: We examined the ability of these placenta-derived multipotent cells (PDMCs) to differentiate into osteoblasts (OBs) or OB-like cells. The PDMCs were treated with osteogenic medium (OM) consisting of dexamethasone, ß-glycerol phosphate, and ascorbic acid. At sequential time intervals (0 day, 3 days, 6 days, 9 days, and 12 days) we measured several parameters. These included alkaline phosphatase (ALP) activity, alizarin red staining (ARS) to measure calcium deposition, messenger RNA (mRNA) expressions of osteogenesis-related transcription factor (Cbfa1), and calcium coordination protein (osteocalcin). These variables were used as indicators of PDMC osteodifferentiation. RESULTS: We showed that ALP activity in the early stage of differentiation and calcium deposition were both significantly increased in PDMCs after OM induction. Moreover, the Cbfa1 and osteocalcin gene expressions were upregulated. The results suggested that OM induced an osteodifferentiation potential in PDMCs. CONCLUSION: PDMC-derived osteocytes provide a useful model to evaluate the mechanisms of key biomolecules and bioengineering processes.


Assuntos
Diferenciação Celular , Células-Tronco Multipotentes/fisiologia , Osteogênese , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Expressão Gênica , Humanos , Células-Tronco Multipotentes/enzimologia , Osteocalcina/genética , Osteogênese/genética , Placenta , Gravidez , RNA Mensageiro/metabolismo , Regulação para Cima
8.
J Clin Invest ; 124(8): 3551-65, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24960165

RESUMO

Mutations within the gene encoding the DNA helicase RECQL4 underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though it is unclear how these mutations lead to disease. Here, we demonstrated that somatic deletion of Recql4 causes a rapid bone marrow failure in mice that involves cells from across the myeloid, lymphoid, and, most profoundly, erythroid lineages. Apoptosis was markedly elevated in multipotent progenitors lacking RECQL4 compared with WT cells. While the stem cell compartment was relatively spared in RECQL4-deficent mice, HSCs from these animals were not transplantable and even selected against. The requirement for RECQL4 was intrinsic in hematopoietic cells, and loss of RECQL4 in these cells was associated with increased replicative DNA damage and failed cell-cycle progression. Concurrent deletion of p53, which rescues loss of function in animals lacking the related helicase BLM, did not rescue BM phenotypes in RECQL4-deficient animals. In contrast, hematopoietic defects in cells from Recql4Δ/Δ mice were fully rescued by a RECQL4 variant without RecQ helicase activity, demonstrating that RECQL4 maintains hematopoiesis independently of helicase activity. Together, our data indicate that RECQL4 participates in DNA replication rather than genome stability and identify RECQL4 as a regulator of hematopoiesis with a nonredundant role compared with other RecQ helicases.


Assuntos
Hematopoese/fisiologia , RecQ Helicases/genética , RecQ Helicases/metabolismo , Síndrome de Rothmund-Thomson/enzimologia , Síndrome de Rothmund-Thomson/genética , Animais , Apoptose , Transplante de Medula Óssea , Dano ao DNA , Replicação do DNA , Modelos Animais de Doenças , Instabilidade Genômica , Hematopoese/genética , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Multipotentes/enzimologia , Células-Tronco Multipotentes/patologia , Mutação , Fenótipo , RecQ Helicases/deficiência
9.
Nat Commun ; 4: 1836, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23673621

RESUMO

Peptidylarginine deiminase 4 (PAD4) functions as a transcriptional coregulator by catalyzing the conversion of histone H3 arginine residues to citrulline residues. Although the high level of PAD4 expression in bone marrow cells suggests its involvement in haematopoiesis, its precise contribution remains unclear. Here we show that PAD4, which is highly expressed in lineage(-) Sca-1(+) c-Kit(+) (LSK) cells of mouse bone marrow compared with other progenitor cells, controls c-myc expression by catalyzing the citrullination of histone H3 on its promoter. Furthermore, PAD4 is associated with lymphoid enhancer-binding factor 1 and histone deacetylase 1 at the upstream region of the c-myc gene. Supporting these findings, LSK cells, especially multipotent progenitors, in PAD4-deficient mice show increased proliferation in a cell-autonomous fashion compared with those in wild-type mice. Together, our results strongly suggest that PAD4 regulates the proliferation of multipotent progenitors in the bone marrow by controlling c-myc expression.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Hidrolases/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/enzimologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Contagem de Células , Núcleo Celular/enzimologia , Proliferação de Células , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Hidrolases/deficiência , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Transporte Proteico , Proteína-Arginina Desiminase do Tipo 4 , Proteínas Proto-Oncogênicas c-myc/genética
10.
Biomaterials ; 34(13): 3223-30, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23410680

RESUMO

Current advances in stem cell biology have brought much hope for therapy of neuro-degenerative diseases. However, neural stem cells (NSCs) are rare adult stem cells, and the use of non-NSCs requires efficient and high-yielding lineage-specific differentiation prior to transplantation for efficacy. We report on the efficient differentiation of placental-derived multipotent cells (PDMCs) into a neural phenotype with use of Y-27632, a clinically compliant small molecular inhibitor of Rho kinase (ROCK) which is a major mediator of cytoskeleton dynamics. Y-27632 does not induce differentiation of PDMC toward the mesodermal lineages of adipogenesis and osteogenesis, but rather a neural-like morphology, with rapid development of cell extensions and processes within 24 h. Compared with conventional neurogenic differentiation agents, Y-27632 induces a higher percentage of neural-like cells in PDMCs without arresting proliferation or cell cycle dynamics. Y-27632-treated PDMCs express several neural lineage genes at the RNA and protein level, including nestin, MAP2, and GFAP. The effect of the ROCK inhibitor is cell-specific to PDMCs, and is mainly mediated through the ROCK2 isoform and its downstream target, myosin II. Our data suggest that ROCK inhibition and cytoskeletal rearrangement may allow for induction of a neural phenotype in PDMCs without compromising cell survival.


Assuntos
Amidas/farmacologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/enzimologia , Neurônios/citologia , Placenta/citologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Multipotentes/efeitos dos fármacos , Miosina Tipo II/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , Gravidez , Quinases Associadas a rho/metabolismo
11.
Acta Biomater ; 8(11): 3974-81, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22796654

RESUMO

Tissue engineering strategies based on multipotent stem cells (MSCs) hold significant promise for the repair or replacement of damaged smooth muscle tissue. To design scaffolds which specifically induce MSC smooth muscle lineage progression requires a deeper understanding of the relative influence of various microenvironmental signals on myogenesis. For instance, MSC myogenic differentiation has been shown to be promoted by increases in active RhoA and FAK, both of which can be induced via increased cell-substrate stress. Separate studies have demonstrated MSC myogenesis to be enhanced by uniaxial cell alignment. The goal of the present study was to compare the impact of increased peak cell-substrate stresses vs. increased uniaxial cell alignment on MSC myogenic differentiation. To this end, MSC fate decisions were compared within two distinct multicellular "forms". A "stripe" multicellular pattern was designed to induce uniaxial cell alignment. In contrast, a second multicellular pattern was designed with "loops" or curves, which altered cell directionality while simultaneously generating regional peak stresses significantly above that intrinsic to the "stripe" form. As anticipated, the higher peak stress levels of the "loop" pattern were associated with increased fractions of active RhoA and active FAK. In contrast, two markers of early smooth muscle lineage progression, myocardin and SM-α-actin, were significantly elevated in the "stripe" pattern relative to the "loop" pattern. These results indicate that scaffolds which promote uniaxial MSC alignment may be more inductive of myogenic differentiation than those associated with increased peak, cell-substrate stress but in which cell directionality varies.


Assuntos
Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Desenvolvimento Muscular , Estresse Mecânico , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Caderinas/metabolismo , Linhagem da Célula , Módulo de Elasticidade , Ensaio de Imunoadsorção Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Camundongos , Células-Tronco Multipotentes/enzimologia , Células NIH 3T3 , Fatores de Transcrição/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Biochem Biophys Res Commun ; 422(1): 121-7, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22564728

RESUMO

Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18α-glycyrrhetinic acid (18α-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.


Assuntos
Células-Tronco Adultas/citologia , Células da Medula Óssea/citologia , Senescência Celular , Células-Tronco Multipotentes/citologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Células Estromais/citologia , Células-Tronco Adultas/enzimologia , Células da Medula Óssea/enzimologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Leupeptinas/farmacologia , Células-Tronco Multipotentes/enzimologia , Inibidores de Proteassoma , Células Estromais/enzimologia
13.
J Cell Sci ; 125(Pt 2): 295-309, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22331353

RESUMO

Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.


Assuntos
Histona Acetiltransferases/genética , Células-Tronco Multipotentes/citologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Biomarcadores/metabolismo , Proliferação de Células , Proteína Glial Fibrilar Ácida , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Histona Acetiltransferases/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/enzimologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Multipotentes/enzimologia , Células-Tronco Multipotentes/metabolismo , Proteínas do Tecido Nervoso/análise , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética
14.
Stem Cells Dev ; 21(8): 1287-98, 2012 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21882976

RESUMO

Conventional human induced pluripotent stem cells (hiPSCs), reprogrammed from somatic cells by induced expression of Oct4, Sox2, Klf4, and c-Myc, are phenotypically different from mouse embryonic stem cells (ESCs). In mice, culture in N2B27 serum-free 2i media (mitogen-activated protein kinase/extracellular signal-regulated kinase and glycogen synthase kinase 3 inhibitors; PD0325901 and CHIR99021) plus leukemia inhibitory factor (LIF) (2i+LIF medium) enriches for germline competent ESCs. Here, we demonstrate that flat-shaped hiPSC colonies can be reprogrammed into bowl-shaped multi-potent stem cells (2i-hiPSCs) by using 2i+LIF medium. Mechanical dissociation of 2i-hiPSC colonies enables stable maintenance for >20 passages. Importantly, gene expression profiling demonstrated that 2i-hiPSCs more closely resemble primitive neural stem cells (PNSCs). Notably, this 2i-induced phenotype was generated from conventional hiPSCs, but not human ESCs (hESCs), thus correlating with the observation of neuroectodermal SOX1-positive colonies in conventional hiPSCs, but not hESCs in 2i+LIF medium. Thus, 2i-hiPSCs, which are nonteratoma forming PNSCs, may represent a safe source of cells for neural research and regenerative medicine.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Meios de Cultura/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fator Inibidor de Leucemia/farmacologia , Camundongos , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/enzimologia
15.
Bone ; 50(2): 499-509, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21839191

RESUMO

Multipotent mesenchymal cells (MMCs) differentiate into osteoblasts or adipocytes through RUNX2 and PPARγ2, respectively. Strontium ranelate has been shown to promote osteoblastogenesis and prevent adipogenesis in long-term experiments using MMCs. The present study involved in-vitro and in-vivo investigations of whether Sr might first be an inhibitor of adipogenesis, thus explaining late osteoblastogenesis. It was established in vivo that Sr reduces adipogenesis in mice treated only for 3 weeks with a 6 mmol/kg/day dose of Sr while the trabecular bone volume is increased. In order to decipher molecular mechanisms during inhibition of adipogenesis, we used murine MMCs C3H10T1/2 cultured under adipogenic conditions (AD) and treated Sr of a concentration up to 3 mM. It was shown that early on (day 1), Sr dose-dependently reduced PPARγ2 and CEBPα mRNA without affecting the RUNX2 gene expression whereas it repressed ALP mRNA. Later (day 5), PPARγ2 and CEBPα mRNA remained inhibited by Sr, preventing adipocyte lipid accumulation, while Runx2 and ALP mRNA were increased. Moreover, under the mentioned conditions, Sr was able to quickly induce the Cyclin D1 gene expression, proliferation and fibronectin fibrillogenesis, both involved in the inhibition of adipogenesis. The inhibition of the ERK pathway by U0126 blunted the Sr-induced PPARγ2 repression while restoring the lipid accumulation. These results demonstrated that Sr was capable of rapidly reducing adipogenesis by a selective PPARγ2 repression that can be explained by its ability to promote MMC proliferation.


Assuntos
Adipogenia/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Medula Óssea/fisiologia , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Estrôncio/farmacologia , Adipogenia/genética , Adiposidade/genética , Animais , Medula Óssea/anatomia & histologia , Medula Óssea/diagnóstico por imagem , Medula Óssea/efeitos dos fármacos , Osso e Ossos/anatomia & histologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Butadienos/farmacologia , Linhagem da Célula/genética , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/enzimologia , Nitrilas/farmacologia , Tamanho do Órgão/efeitos dos fármacos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radiografia , Rosiglitazona , Tiazolidinedionas/farmacologia
16.
Cell Transplant ; 20(8): 1221-30, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21176405

RESUMO

Multipotent mesenchymal stromal cells (MSCs) are bone marrow-derived cells of nonhematopoietic origin with immunoregulatory properties. Although some functions of MSCs have been identified, there are still features that are not explained thus far. The aim of the present study was to identify novel factors involved in MSC-mediated inhibition of T-cell proliferation. We here demonstrate that the surface molecule CD39 is coexpressed in concert with CD73 on murine MSCs catalyzing the generation of adenosine, which can directly act on activated T cells via the adenosine A2A receptor. Blocking of the adenosine pathway either by the A2A receptor antagonist SCH58261 or the specific CD39 inhibitor polyoxotungstate 1 (POM-1) blocked MSC-mediated suppression of T-cell proliferation almost completely. We conclude that CD39/CD73 coexpression is a novel important component of the immunoregulatory functions of murine MSCs. Our findings may both be important to improve our understanding of MSC function and for the development of immunomodulatory cellular therapies.


Assuntos
Adenosina/biossíntese , Antígenos CD/metabolismo , Apirase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Linfócitos T/citologia , 5'-Nucleotidase/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Imunossupressores/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitógenos/farmacologia , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/enzimologia , Fenótipo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta/farmacologia
17.
Biochem Biophys Res Commun ; 400(1): 27-33, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20691159

RESUMO

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)(+) mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1(+) embryonic stem cells. Here, we transplanted these Flk1(+) mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4weeks after when sorted Flk1(+) mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1(+) mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1(+) mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to ß-galactosidase staining assay. In conclusion, Flk1(+) mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.


Assuntos
Células Germinativas/citologia , Células-Tronco Multipotentes/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Testículo/citologia , Animais , Diferenciação Celular , Senescência Celular , Modelos Animais de Doenças , Células Germinativas/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Células-Tronco Multipotentes/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Testículo/enzimologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise
18.
Bioessays ; 32(7): 626-37, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20586054

RESUMO

Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants act in this germ line maintenance and function. The most universal of these genes are Vasa and Vasa-like DEAD-box RNA helicase genes. However, recent evidence indicates that Vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of Vasa function and its regulation during development, addressing Vasa's emerging role in multipotent cells. We also explore the evolutionary diversification of the N-terminal domain of this gene and how this impacts the association of Vasa with nuage-like perinuclear structures.


Assuntos
RNA Helicases DEAD-box/genética , Células Germinativas/enzimologia , Células-Tronco Multipotentes/enzimologia , Animais , RNA Helicases DEAD-box/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Interferência de RNA
19.
J Cell Physiol ; 224(1): 178-86, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20232315

RESUMO

Adult human bone marrow-derived multipotent progenitor cells (MPCs) are able to differentiate into a variety of specialized cell types, including chondrocytes, and are considered a promising candidate cell source for use in cartilage tissue engineering. In this study, we examined the regulation of MPC chondrogenesis by mitogen-activated protein kinases in an attempt to better understand how to generate hyaline cartilage in the laboratory that more closely resembles native tissue. Specifically, we employed the high-density pellet culture model system to assess the roles of ERK5 and ERK1/2 pathway signaling in MPC chondrogenesis. Western blotting revealed that high levels of ERK5 phosphorylation correlate with low levels of MPC chondrogenesis and that as TGF-beta 3-enhanced MPC chondrogenesis proceeds, phospho-ERK5 levels steadily decline. Conversely, levels of phospho-ERK1/2 paralleled the progression of MPC chondrogenesis. siRNA-mediated knockdown of ERK5 pathway components MEK5 and ERK5 resulted in increased MPC pellet mRNA transcript levels of the cartilage-characteristic marker genes SOX9, COL2A1, AGC, L-SOX5, and SOX6, as well as enhanced accumulation of SOX9 protein, collagen type II protein, and Alcian blue-stainable proteoglycan. In contrast, knockdown of ERK1/2 pathway members MEK1 and ERK1 decreased expression of all chondrogenic markers tested. Finally, overexpression of MEK5 and ERK5 also depressed MPC chondrogenesis, as indicated by diminished activity of a co-transfected collagen II promoter-luciferase reporter construct. In conclusion, our results suggest a novel role for the ERK5 pathway as an important negative regulator of adult human MPC chondrogenesis and illustrate that the ERK5 and ERK1/2 kinase cascades play opposing roles regulating MPC cartilage formation.


Assuntos
Células-Tronco Adultas/enzimologia , Células da Medula Óssea/enzimologia , Condrogênese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células-Tronco Multipotentes/enzimologia , Células-Tronco/enzimologia , Idoso , Idoso de 80 Anos ou mais , Cartilagem/metabolismo , Diferenciação Celular , Células Cultivadas , Condrogênese/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Fosforilação , Interferência de RNA , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta3/metabolismo
20.
Proc Natl Acad Sci U S A ; 107(9): 4147-52, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20150512

RESUMO

Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.


Assuntos
Células-Tronco Multipotentes/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Multipotentes/enzimologia , Células-Tronco Multipotentes/metabolismo , Osteoprotegerina/metabolismo , Oximas/farmacologia , PPAR gama/antagonistas & inibidores , Células Estromais/enzimologia , Células Estromais/metabolismo , Engenharia Tecidual , beta Catenina/metabolismo
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