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1.
EMBO J ; 41(14): e110655, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35703167

RESUMO

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular , Células Endoteliais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Desenvolvimento Muscular , Fosfatidilinositol 3-Quinases/metabolismo , Nicho de Células-Tronco
2.
Arterioscler Thromb Vasc Biol ; 39(9): e195-e207, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31315437

RESUMO

OBJECTIVE: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aß (amyloid-ß) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. CONCLUSIONS: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.


Assuntos
Apolipoproteína E4/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Doença de Alzheimer/etiologia , Apolipoproteína E4/genética , Barreira Hematoencefálica , Genótipo , Humanos , Corpos de Weibel-Palade/fisiologia , Fator de von Willebrand/genética , Fator de von Willebrand/fisiologia
3.
Nat Med ; 22(8): 897-905, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27376579

RESUMO

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.


Assuntos
Envelhecimento/metabolismo , Fibronectinas/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Músculo Esquelético/metabolismo , Regeneração/genética , Nicho de Células-Tronco , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Integrinas/metabolismo , Camundongos , Músculo Esquelético/citologia , Reação em Cadeia da Polimerase
4.
Toxicol Appl Pharmacol ; 262(2): 167-76, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22564538

RESUMO

The acute toxicity of the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) varies widely among species and strains. Previous studies in rats have established that females are approximately 2-fold more sensitive to TCDD lethality than males. However, there is a surprising gap in the literature regarding possible gender-related sensitivity differences in mice. In the present study, by using three substrains of TCDD-sensitive C57BL/6 mice and transgenic mice on this background, we demonstrated that: 1) in contrast to the situation in rats, female mice are the more resistant gender; 2) the magnitude of the divergence between male and female mice depends on the substrain, but can amount to over 10-fold; 3) AH receptor protein expression levels or mutations in the primary structure of this receptor are not involved in the resistance of female mice of a C57BL/6 substrain, despite their acute LD50 for TCDD being over 5000 µg/kg; 4) transgenic mice that globally express the rat wildtype AH receptor follow the mouse type of gender difference; 5) in gonadectomized mice, ovarian estrogens appear to enhance TCDD resistance, whereas testicular androgens seem to augment TCDD susceptibility; and 6) the gender difference correlates best with the severity of liver damage, which is also reflected in hepatic histopathology and the expression of pro-inflammatory cytokines, especially IL-6. Hence, the two closely related rodent species most often employed in toxicological risk characterization studies, rat and mouse, represent opposite examples of the influence of gender on dioxin sensitivity, further complicating the risk assessment of halogenated aromatic hydrocarbons.


Assuntos
Fígado/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Androgênios/metabolismo , Animais , Estrogênios/metabolismo , Feminino , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Hidrocarboneto Arílico/genética , Fatores Sexuais , Estatísticas não Paramétricas
5.
Environ Toxicol Chem ; 31(3): 663-71, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22213473

RESUMO

The gene expression and induction of cytochrome P450 (CYP)-enzymes following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) peroral administration was studied in the livers of two wild vole species--the bank vole (Myodes glareolus) and the field vole (Microtus agrestis). The dioxin-sensitive C57BL/6 mouse was used as a reference. Doses of 0.05, 0.5, 5.0, and 50 µg/kg were applied to ascertain a dose-response relationship, and the dose of 50 µg/kg was applied to the study time course for up to 96 h. The cytochrome P450 1A1 (CYP1A1) mRNA expression showed an expected dose-dependent increase equally in both vole species. Bank voles expressed notably higher CYP2A mRNA levels as compared with field voles. Both species exhibited dose-dependent increases in putative CYP1A-, CYP2B-, and CYP2A-associated activities as measured by fluorometric assays for ethoxyresorufin-O-deethylase (EROD), penthoxyresorufin-O-depenthylase (PROD), and 7-ethoxycoumarin-O-deethylase (ECOD), respectively. Putative CYP2A-associated coumarin-7-hydroxylase (COH) activity showed a slight increase at the two highest doses of TCDD in field voles but not in bank voles, and their basal COH activity was only one-fourth or less of that in field voles. Overall, however, bank voles tended to exhibit higher CYP-associated enzyme activities measured at the two largest doses of TCDD than field voles. A western blot analysis of aryl hydrocarbon receptor (AhR) revealed that the two vole species had differential band patterns, suggesting dissimilar structures for their AhRs.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Fígado/enzimologia , Dibenzodioxinas Policloradas/toxicidade , Teratogênicos/toxicidade , O-Dealquilase 7-Alcoxicumarina/genética , O-Dealquilase 7-Alcoxicumarina/metabolismo , Animais , Arvicolinae , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6 , Relação Dose-Resposta a Droga , Feminino , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , RNA Mensageiro/metabolismo , Especificidade da Espécie
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