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1.
BMB Rep ; 53(10): 545-550, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32958120

RESUMO

Combination therapy using chloroquine (CQ) and azithromycin (AZM) has drawn great attention due to its potential anti-viral activity against SARS-CoV-2. However, clinical trials have revealed that the co-administration of CQ and AZM resulted in severe side effects, including cardiac arrhythmia, in patients with COVID-19. To elucidate the cardiotoxicity induced by CQ and AZM, we examined the effects of these drugs based on the electrophysiological properties of human embryonic stem cellderived cardiomyocytes (hESC-CMs) using multi-electrode arrays. CQ treatment significantly increased the field potential duration, which corresponds to prolongation of the QT interval, and decreased the spike amplitude, spike slope, and conduction velocity of hESC-CMs. AZM had no significant effect on the field potentials of hESC-CMs. However, CQ in combination with AZM greatly increased the field potential duration and decreased the beat period and spike slope of hESC-CMs when compared with CQ monotherapy. In support of the clinical data suggesting the cardiovascular side effects of the combination therapy of CQ and AZM, our results suggest that AZM reinforces the cardiotoxicity induced by CQ in hESC-CMs. [BMB Reports 2020; 53(10): 545-550].


Assuntos
Azitromicina/efeitos adversos , Cardiotoxicidade/etiologia , Cloroquina/efeitos adversos , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação , Animais , Arritmias Cardíacas/induzido quimicamente , Azitromicina/administração & dosagem , Diferenciação Celular , Cloroquina/administração & dosagem , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Humanos , Camundongos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico
2.
Nat Commun ; 11(1): 4265, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848154

RESUMO

Retinoids regulate a wide spectrum of cellular functions from the embryo throughout adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits make retinoids very attractive molecules for medical purposes. In light of some of the physicochemical limitations of retinoids, the development of drug delivery systems offers several advantages for clinical translation of retinoid-based therapies, including improved solubilization, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In this Review, we discuss advances in preclinical and clinical tests regarding retinoid formulations, specifically the ones based in natural retinoids, evaluated in the context of regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations of retinoid formulations, as well as prospects to push the field forward, will be presented.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Medicina Regenerativa/métodos , Retinoides/administração & dosagem , Animais , Encefalopatias/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Composição de Medicamentos , Sistemas de Liberação de Medicamentos/tendências , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Neoplasias/tratamento farmacológico , Medicina Regenerativa/tendências , Retinoides/química , Retinoides/uso terapêutico , Transdução de Sinais , Dermatopatias/tratamento farmacológico
3.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
4.
Life Sci ; 255: 117817, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32446845

RESUMO

Glucocorticoids can promote cardiomyocyte maturation. However, the mechanism underlying glucocorticoid-mediated cardiomyocyte maturation is still unclear. Mitophagy plays a key role in cardiomyocyte maturation. Based on current knowledge, our study evaluated the effects of the glucocorticoid dexamethasone (100 nM) on the maturation of mouse embryonic stem cell-derived cardiomyocytes and the role of mitophagy in this maturation. The results showed that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation, inhibit cardiomyocyte proliferation, and promote myocardial fiber arrangement. However, dexamethasone did not affect mitochondrial morphology in cardiomyocytes. Glucocorticoid receptor inhibitors (RU486, 1 nM) can inhibit dexamethasone-mediated cardiomyocyte maturation. Additionally, dexamethasone can promote mitophagy in embryonic stem cell-derived cardiomyocytes and induce LC3 and lysosomal aggregation in mitochondria. The inhibition of mitophagy can inhibit the cardiomyocyte maturation effect of dexamethasone. Furthermore, our research found that dexamethasone may mediate the occurrence of mitophagy in cardiomyocytes through Parkin. The siRNA-mediated inhibition of Parkin expression can inhibit mitochondrial autophagy caused by dexamethasone, thus inhibiting cardiomyocyte maturation. Overall, our study found that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation through Parkin-mediated mitophagy.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mitofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/administração & dosagem , Receptores de Glucocorticoides/metabolismo
5.
Curr Pharm Biotechnol ; 21(12): 1154-1164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297579

RESUMO

BACKGROUND: Stem cells are of two types: embryonic and adult stem cells and they act as a repair system by replenishing body tissue. Stem cells differentiate into different types of cells, such as neural, hematopoietic, adipose, etc. and are used for the treatment of various conditions like myocardial infarction, spinal cord injury, Parkinson's disease and diabetes. METHODS: This article focuses on recent research development that addresses the viability issues of stem cells. The efficiency of transplanted stem cells reduces due to conditions like hypoxia, inflammation, nutrient deprivation, immunogenicity, extracellular matrix loss on delivery and mechanical stress. RESULTS: To increase the viability of stem cells, techniques like scaffolds of stem cells with hydrogel or alginate, pre-conditioning, different routes of administration and encapsulation, are implemented. CONCLUSION: For the protection of stem cells against apoptosis, different pathways, namely Phosphoinositide 3-Kinase (PI3K/AKT), Hypoxia-Inducible Factor (HIF1), Mitogen-Activated Protein Kinases (MAPK) and Hippo, are discussed. DISCUSSION: Activation of the PI3K/AKT pathway decreases the concentration of apoptotic factors, while the HIF pathway protects stem cells against the micro-environment of tissue (hypoxia).


Assuntos
Células-Tronco Adultas/fisiologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criopreservação , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Transplante de Células-Tronco
6.
Biochem Pharmacol ; 177: 113984, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311348

RESUMO

Pluripotent stem cells are have therapeutic applications in regenerative medicine and drug discovery. However, the differentiation of stem cells in vitro hinders their large-scale production and clinical applications. The maintenance of cell pluripotency relies on a complex network of transcription factors; of these, octamer-binding transcription factor-4 (Oct4) plays a key role. This study aimed to construct an Oct4 gene promoter-driven firefly luciferase reporter and screen small-molecule compounds could maintain cell self-renewal and pluripotency. The results showed that ethyl-p-methoxycinnamate (EPMC) enhance the promoter activity of the Oct4 gene, increased the expression of Oct4 at both mRNA and protein levels, and significantly promoted the colony formation of P19 cells. These findings suggesting that EPMC could reinforce the self-renewal capacity of P19 cells. The pluripotency markers Oct4, SRY-related high-mobility-group-box protein-2, and Nanog were expressed at higher levels in EPMC-induced colonies. EPMC could promote teratoma formation and differentiation potential of P19 cells in vivo. It also enhanced self-renewal and pluripotency of human umbilical cord mesenchymal stem cells and mouse embryonic stem cells. Moreover, it significantly activated the nuclear factor kappa B (NF-κB) signaling pathway via the myeloid differentiation factor 88-dependent pathway. The expression level of Oct4 decreased after blocking the NF-κB signaling pathway, suggesting that EPMC promoted the expression of Oct4 partially through the NF-κB signaling pathway. This study indicated that EPMC could maintain self-renewal and pluripotency of stem cells.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Cinamatos/farmacologia , NF-kappa B/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/agonistas , NF-kappa B/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genética
7.
Cell Death Dis ; 11(1): 75, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001672

RESUMO

The bivalent domain (BD) at promoter region is an unique epigenetic feature poised for activation or repression during cell differentiation in embryonic stem cell. However, the function of BDs in already differentiated cells remains exclusive. By profiling the epigenetic landscape of endothelial cells during VEGFA (vascular endothelial growth factor A) stimulation, we discovered that BDs are widespread in endothelial cells and preferentially marked genes responsive to VEGFA. The BDs responsive to VEGFA have more permissive chromatin environment comparing to other BDs. The initial activation of bivalent genes depends on RNAPII pausing release induced by EZH1 rather than removal of H3K27me3. The later suppression of bivalent gene expression depended on KDM5A recruitment by its interaction with PRC2. Importantly, EZH1 promoted both in vitro and in vivo angiogenesis by upregulating EGR3, whereas KDM5A dampened angiogenesis. Collectively, this study demonstrates a novel dual function of BDs in endothelial cells to control VEGF responsiveness and angiogenesis.


Assuntos
Células Endoteliais/metabolismo , Histonas/metabolismo , Neovascularização Fisiológica/genética , Regiões Promotoras Genéticas/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/efeitos dos fármacos , Epigênese Genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Domínios Proteicos/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno , RNA-Seq , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Regulação para Cima
8.
Toxicol Appl Pharmacol ; 392: 114929, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105654

RESUMO

We investigated the responses of microRNAs (miRNAs) using mouse embryonic stem cells (mESCs) exposed to nine chemicals (bis(2-ethylhexyl)phthalate, p-cresol, p-dichlorobenzene, phenol, pyrocatecol, chloroform, tri-n-butyl phosphate, trichloroethylene, and benzene), which are listed as "Class I Designated Chemical Substances" from the Japan Pollutant Release and Transfer Register. Using deep sequencing analysis (RNA-seq), several miRNAs were identified that show a substantial response to general chemical toxicity (i.e., to these nine chemicals considered as a group) and several miRNA biomarkers that show a substantial and specific response to benzene. The functions of the identified miRNAs were investigated in accordance with Gene Ontology terms of their predicted target genes, indicating regulation of cellular processes. We compared the results with those for the long non-coding RNAs (ncRNAs) and mRNAs reported in our previous studies in addition to previously identified miRNAs that are either up- or down-regulated in response to the benzene as stimuli. We also observed that the changes in expression of miRNAs were smaller than those for long ncRNAs and mRNAs. Taken together the current and previous results revealed that toxic chemical stimuli regulate the expression of miRNAs. We believe that the use of miRNAs, including the thus identified miRNAs, as biomarkers contribute to predicting the potential toxicity of particular chemicals or identifying human individuals that have been exposed to chemical hazards.


Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Substâncias Perigosas/toxicidade , MicroRNAs/metabolismo , Análise de Sequência de RNA/métodos , Animais , Biomarcadores , Substâncias Perigosas/química , Camundongos , Estrutura Molecular , Testes de Toxicidade
9.
Sci Rep ; 10(1): 232, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937797

RESUMO

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Tubercidina/análogos & derivados , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Tubercidina/farmacologia
10.
Toxicology ; 432: 152380, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31981723

RESUMO

Bisphenol AF (BPAF) is a derivative of bisphenol A (BPA) that is widely used in fluorinated polymers, fluorinated rubber, electronic equipment, plastic optical fibers, etc. Studies have shown that BPAF exposure is associated with a number of diseases; however, little is known about the effects of BPAF on cardiomyocytes. We investigated the impact of chronic exposure to BPAF on cardiomyocytes derived from embryonic stem cells (ESCs). The present study showed that chronic exposure to various concentrations of BPAF (0, 8, 200 and 1000 ng/ml) induces cardiomyocyte hypertrophy. The ratios of microfilaments to mitochondrial length and the ratio of microfilaments to cell nuclei and MYH7b levels indicate that BPAF exposure alters the morphology of the cells and mitochondria. Furthermore, BPAF exposure at concentrations from 8 to 1000 ng/ml results in an increase in G protein-coupled estrogen receptor (GPER) expression. Additionally, our results suggest that these effects of BPAF mediate cardiomyocyte hypertrophy apparently due to an increase in the production of reactive nitrogen species (RNS) via an increase in endothelial NO synthase (eNOS). These results imply that ESC-based myocardial differentiation can be an excellent cellular model to study BPAF-induced cardiotoxicity at the cellular and molecular levels.


Assuntos
Compostos Benzidrílicos/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fenóis/toxicidade , Transdução de Sinais/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Calmodulina/biossíntese , Calmodulina/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/efeitos dos fármacos
11.
Antioxid Redox Signal ; 32(1): 35-59, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31656084

RESUMO

Aims: The naive or primitive states of stem cells (SCs) residing in specific niches are unstable and difficult to preserve in vitro. Vitamin C (VitC), in addition to suppressing oxygen radicals, exerts pleiotropic effects to preserve the core functions of SCs. However, this compound is labile and readily oxidized, resulting in cellular toxicity and preventing its reliable application in this context. We found that a VitC derivative, ascorbic acid 2-glucoside (AA2G), stably maintains the naive pluripotency of murine embryonic SCs (mESCs) and the primitiveness of human mesenchymal SCs (hMSCs) without cellular toxicity. Results: The beneficial effects of AA2G and related molecular mechanisms were evaluated in mESCs, induced pluripotent-SCs (iPSCs), and hMSCs. AA2G was stable in aqueous solution and barely induced cellular toxicity in cultured SCs, unlike VitC. AA2G supplementation recapitulated the well-known effects of VitC, including induction of ten-eleven translocation-dependent DNA demethylation in mESCs and suppression of p53 during generation of murine iPSCs. Furthermore, supplementation of hMSCs with AA2G improved therapeutic outcomes in an asthma mouse model by promoting their self-renewal, engraftment, and anti-inflammatory properties. Particularly, activation of the cAMP-responsive element-binding protein-1 (CREB1) pathway contributed to the ability of AA2G to maintain naive pluripotency of mESCs and functionality of hMSCs. Innovation and Conclusion: Given its long-lasting effects and low cellular toxicity, AA2G supplementation is useful to support the naive pluripotency of mESCs and the primitiveness of hMSCs, affecting their developmental potency and therapeutic efficacy. Furthermore, we demonstrate the significance of the CREB1 pathway in the mechanism of action of AA2G.


Assuntos
Ácido Ascórbico/análogos & derivados , Asma/terapia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Mesenquimais/citologia , Animais , Ácido Ascórbico/farmacologia , Asma/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Nicho de Células-Tronco
12.
Endocrinology ; 161(1)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31758175

RESUMO

Gonadotropin-releasing hormone (Gnrh) plays important roles in reproduction by stimulating luteinizing hormone release, and subsequently ovulation and sperm release, ultimately controlling reproduction in many species. Here we report on a new role for this decapeptide. Surprisingly, Gnrh3-null zebrafish generated by CRISPR/Cas9 exhibited a male-biased sex ratio. After the dome stage, the number of primordial germ cells (PGCs) in gnrh3-/- fish was lower than that in wild-type, an effect that was partially rescued by gnrh3 overexpression. A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis revealed no detectable apoptosis of PGCs in gnrh3-/- embryos. Proliferating PGCs could be detected in wild-type embryos, while there was no detectable signal in gnrh3-/- embryos. Compared with wild type, the phosphorylation of AKT was not significantly different in gnrh3-/- embryos, but the phosphorylation of ERK1/2 decreased significantly. Treatment with a Gnrh analog (Alarelin) induced ERK1/2 phosphorylation and increased PGC numbers in both wild-type and gnrh3-/- embryos, and this was blocked by the MEK inhibitor PD0325901. The relative expression of sox9a, amh, and cyp11b were significantly upregulated, while cyp19a1a was significantly downregulated at 18 days post-fertilization in gnrh3-/- zebrafish. Taken together, these results indicate that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path.


Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Diferenciação Sexual/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Benzamidas/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Embrião não Mamífero/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Razão de Masculinidade , Proteínas de Peixe-Zebra/genética
13.
Food Chem Toxicol ; 135: 111015, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31812737

RESUMO

Bisphenol A (BPA) and its derivatives, including bisphenols S (BPS), F (BPF), E (BPE), B (BPB), Z (BPZ), and AF (BPAF), are widely used in consumer products. Moreover, they are typically detected in the environment, food, and humans. Previous studies have linked BPA to several health risks, but it is still unclear whether BPA replacements are safe. In this study, we developed an in vitro model based on human embryonic stem cells (hESCs) to explore the potential neural toxicity of these compounds. We observed that the bisphenols affected the viability of hESCs and hESC-derived neural stem cells (NSCs) at high concentrations, with BPS being the least cytotoxic and BPAF the strongest cytotoxic compound. At human-relevant concentrations, the bisphenols did not significantly interfere with gene expression and protein levels during hESC differentiation into the neural epithelium, as well as during specification of neuron-like cells from NSCs. Nevertheless, monitoring of cell morphology changes indicated that exposure to BPA and its derivatives impaired neurite length in neuron-like cells. Thus, our findings provide insights into the molecular mechanisms of bisphenol-dependent neurotoxicity at low nanomolar levels and support the view that BPA substitutes may not be sufficiently safe for widespread use as industrial chemicals.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Neuritos , Neurônios/efeitos dos fármacos , Fenóis/toxicidade , Compostos Benzidrílicos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Disruptores Endócrinos/química , Humanos , Fenóis/química
14.
Sci China Life Sci ; 63(7): 1026-1036, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31872377

RESUMO

The cellular consequences of aneuploidy are largely dependent on the cell types examined. Aneuploid yeasts and mouse embryonic fibroblasts exhibit cell proliferation defects and can be selectively inhibited by compounds that cause proteotoxic or energy stress. By contrast, most aneuploid pluripotent stem cells proliferate rapidly and reach higher saturation densities. The responses of aneuploid pluripotent stem cells to the stress-inducing compounds remain uncharacterized. Here, we tested the response of aneuploid embryonic stem cells to several compounds that caused proteotoxic, energy and genotoxic stress using previously established mouse embryonic stem cell lines trisomic for chromosome 6, 8, 11, or 15. Not all trisomic embryonic stem cells were selectively inhibited by compounds that cause proteotoxic or energy stress. However, most of these cells exhibited increased sensitivity to genotoxins. They displayed elevated DNA damage response as characterized by increased γH2A.X foci under genotoxic stress. Further investigations indicated that elevated autophagy levels might contribute to the increased cytotoxic effects of genotoxins on trisomic embryonic stem cells. Our study laid the foundation for eliminating aneuploidy that might be an effective approach for controlling cancer progression.


Assuntos
Aneuploidia , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Mutagênicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Cromossomos/genética , Cromossomos/metabolismo , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Mutação , Transdução de Sinais
15.
J Med Life ; 12(3): 225-229, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31666821

RESUMO

Almost 30 years have passed since the term 'tissue engineering' was created to represent a new concept that focuses on the regeneration of neotissues from cells with the support of biomaterials and growth factors. This interdisciplinary engineering has attracted much attention as a new therapeutic means that may overcome the drawbacks involved in the current artificial organs and organ transplantation that have also been aiming at replacing lost or severely damaged tissues or organs. However, the tissues regenerated by tissue engineering and widely applied to patients are still minimal, including skin, bone, cartilage, capillary, and periodontal tissues. What are the reasons for such slow advances in clinical applications of tissue engineering? This article gives a brief overview of the current state of tissue engineering, covering the fundamentals and applications. The fundamentals of tissue engineering involve cell sources, scaffolds for cell expansion and differentiation, as well as carriers for growth factors. Animal and human trials are a major part of the applications. Based on these results, some critical problems to be resolved for the advances of tissue engineering are addressed from the engineering point of view, emphasizing the close collaboration between medical doctors and biomaterials scientists.


Assuntos
Engenharia Tecidual/tendências , Animais , Materiais Biocompatíveis/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Humanos , Tecidos Suporte/química
16.
EBioMedicine ; 50: 343-354, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31707150

RESUMO

BACKGROUND: Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly due to impaired decidualization. The endocytic and transcytotic activity in the endometrium are crucial for decidualization. The most representative endocytic gene is the C-terminal Eps15 homology domain-containing 1 (EHD1), but whether EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains unclear. METHODS: A transcriptomic analysis was performed to evaluate the differentially expressed genes between the fertile control and RIF group. The expression and location of EHD1 in endometrial tissues were further examined by IHC, qRT-PCR and Western blotting. The transduction of an EHD1 recombinant adenovirus into human endometrial stromal cells was performed to investigate relevant decidualization marker genes. Additionally, a microarray analysis following the adenovirus-mediated overexpression of EHD1 was conducted to identify EHD1-related changes in HESCs, and the potential molecular mechanisms were further confirmed through immunofluorescence and coimmunoprecipitation analyses. FINDINGS: An RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. The analysis of the menstrual cycle showed that expression of EHD1 increased in the mid-proliferative phase and showed a gradual decrease in the mid-secretory and decidual phases. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin and insulin-like growth factor binding protein-1 and the formation of the cytoskeleton. The mechanistic analysis revealed the EHD1 regulated LRP5/6 protein function through the endocytic pathway, and subsequently suppressed the Wnt4/ß-catenin pathway during decidualization. In addition, a Wnt4 agonist improved an impaired decidualization process. INTERPRETATION: Regulation of the EHD1-Wnt4 pathway might serve as a promising therapeutic strategy for improving endometrial receptivity in RIF women.


Assuntos
Aborto Habitual/genética , Aborto Habitual/metabolismo , Decídua/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteína Wnt4/metabolismo , beta Catenina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aborto Habitual/diagnóstico , Adulto , Biomarcadores , Decídua/efeitos dos fármacos , Decídua/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adulto Jovem
17.
Sci Rep ; 9(1): 14106, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575920

RESUMO

Embryonic stem cell (ESC) derived tissue is a promising tool to be used in different clinical, preclinical and also scientific settings, for example as in vivo biological pacemaker, preclinical drug safety screening tool or ultimately as part of a cell replacement therapy. However, before ESC derived tissue can be used routinely for these purposes in humans, further studies are needed. In this context, the aims of the present study were to examine the effect of antiarrhythmic drugs on human ESC (hESC) und human induced pluripotent stem cell (hiPSC) derived cardiomyocytes by analyzing the beat rate variability (BRV), which can be considered as the in vitro equivalent of the heart rate variability (HRV) in vivo. Short-term recordings of extracellular field potentials of spontaneously beating cardiomyocytes derived from hESCs and hiPSCs were made using Microelectrode Arrays (MEA). The effect of Flecainide, Ivabradine and Metoprolol was tested. The offline analysis of the BRV was mainly focused on time domain methods. Additionally a non-linear analysis method was used. The evaluation of the Poincaré-Plots of the measurements without pharmacological intervention revealed that the vast majority of the scatter plots have a similar, ellipsoid shape. Flecainide and Ivabradine influenced BRV parameters significantly, whereas Metoprolol did not alter the BRV markedly. We detected remarkable similarities between the BRV of hESC and hiPSC derived cardiomyocytes in vitro and the HRV in vivo. The effect of antiarrhythmic drugs on spontaneously beating cardiomyocytes derived from hESC and hiPSC was generally consistent with clinical experiences and also with our previous study based on murine ESC derived cardiomyocytes. In conclusion, our study points out the great potential of hESC and hiPSC derived tissue to be used routinely for many different applications in medicine and science.


Assuntos
Antiarrítmicos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos
18.
Biomater Sci ; 7(12): 5467-5481, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31656967

RESUMO

Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.


Assuntos
Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Animais , Linhagem Celular , Humanos , Miócitos Cardíacos/efeitos dos fármacos
19.
Biochem Biophys Res Commun ; 520(2): 257-262, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31594640

RESUMO

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.


Assuntos
Fusão Celular/instrumentação , Citoplasma , Células-Tronco Embrionárias/citologia , Animais , Fusão Celular/métodos , Tamanho Celular , Células Cultivadas , Demecolcina/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Desenho de Equipamento , Regulação da Expressão Gênica/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Camundongos , Células-Tronco Pluripotentes/fisiologia
20.
Stem Cell Rev Rep ; 15(6): 892-899, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31520298

RESUMO

We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.


Assuntos
5'-Nucleotidase/antagonistas & inibidores , Adenosina/deficiência , Apirase/antagonistas & inibidores , Medula Óssea/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antígenos CD , Medula Óssea/metabolismo , Movimento Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Vasodilatadores/metabolismo
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