RESUMO
In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.
Assuntos
Diferenciação Celular , Redes Reguladoras de Genes , Células-Tronco Embrionárias Murinas , Proteína Homeobox Nanog , Animais , Camundongos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Camundongos KnockoutRESUMO
In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal, and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the antioxidant system and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.
Assuntos
Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , Animais , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heme Oxigenase-1/metabolismo , Luciferases/genética , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células NIH 3T3 , Proteína Homeobox Nanog/antagonistas & inibidores , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Regiões Promotoras Genéticas , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.
Assuntos
Glutationa Redutase/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Genes Reporter , Glutationa Redutase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/antagonistas & inibidores , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.
Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Linhagem da Célula/fisiologia , Implantação do Embrião/fisiologia , Microscopia Intravital , Camundongos , Microscopia Confocal , Imagem com Lapso de TempoRESUMO
Neurogenesis in the substantia nigra (SN) has been a controversial issue. Here we report that neurogenesis can be induced in the adult rodent SN by transplantation of embryoid body cells (EBCs) derived from mouse embryonic stem cells. The detection of Sox2+ dividing (BrdU+) putative host neural precursor cells (NPCs) between 1 and 6â¯days post-transplantation (dpt) supported the neurogenic capacity of the adult SN. In agreement with the awakening of NPCs by EBCs, only host cells from implant-bearing SN were able to generate neurosphere-like aggregates in the presence of Egf and Fgf2. Later, at 15 dpt, a significant number of SN Dcx+ neuroblasts were detected. However, a continuous BrdU administration after transplantation showed that only a fraction (about 20-30%) of those host Dcx+ progeny derived from dividing cells and few BrdU+ cells, some of them NeuN+, survived up to 30 dpt. Unexpectedly, 25-30% of Dcx+ or Psa-Ncam+ cells at 15 dpt displayed astrocytic markers such as Gfap and S100b. Using a genetic lineage tracing strategy, we demonstrated that a large proportion of host Dcx+ and/or Tubb3+ neuroblasts originated from Gfap+ cells. Remarkably, new blood vessels formed in association with the neurogenic process that, when precluded, caused a reduction in neuroblast production. Accordingly, two proteins secreted by EBCs, Fgf2 and Vegf, were able to promote the emergence of Dcx+/Psa-Ncam+, Tubb3+ and NeuN+/BrdU+ cells in vivo in the absence of EBCs. We propose that the adult SN is a mostly silent neurogenic niche with the ability to generate new neurons by typical and atypical mechanisms.
Assuntos
Células-Tronco Embrionárias Murinas/transplante , Neurogênese/fisiologia , Neurônios/fisiologia , Substância Negra/fisiologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Proteína Duplacortina , Fator 2 de Crescimento de Fibroblastos/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Embryonic stem cells (ESC) are widely used due to their unlimited capacity of differentiation into different cell lineages, which makes ESC a viable choice as a toxicology test model. Toxicological analysis using embryonic stem cells (ESC) has become an important tool in toxicology procedures. Regarding toxicological analysis methods, flow cytometry (FC) is one technique designed to detect and evaluate cells in suspension, for example, ESC suspension, thus making possible to study different biological, physical, and/or chemical characteristics of cells. Thus, FC can be very useful for cell toxicology and tumorigenic analyses.
Assuntos
Diferenciação Celular , Citometria de Fluxo/métodos , Células-Tronco Embrionárias Murinas/patologia , Teratogênicos/toxicidade , Testes de Toxicidade/métodos , Toxicologia/métodos , Animais , Linhagem da Célula , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacosRESUMO
Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo-matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.
Assuntos
Antígeno B7-H1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Antígeno B7-H1/deficiência , Antígeno B7-H1/genética , Diferenciação Celular , Feminino , Células-Tronco Fetais/citologia , Células-Tronco Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transplante de Células-Tronco Hematopoéticas , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Gravidez , Regulação para CimaRESUMO
Extracellular vesicles (EVs) released by mouse embryonic stem cells (mESCs) are considered a source of bioactive molecules that modulate their microenvironment by acting on intercellular communication. Either intracellular endosomal machinery or their derived EVs have been considered a relevant system of signal circuits processing. Herein, we show that these features are found in mESCs. Ultrastructural analysis revealed structures and organelles of the endosomal system such as coated pits and endocytosis-related vesicles, prominent rough endoplasmic reticulum and Golgi apparatus, and multivesicular bodies (MVBs) containing either few or many intraluminal vesicles (ILVs) that could be released as exosomes to extracellular milieu. Besides, budding vesicles shed from the plasma membrane to the extracellular space is suggestive of microvesicle biogenesis in mESCs. mESCs and mouse blastocyst express specific markers of the Endosomal Sorting Complex Required for Transport (ESCRT) system. Ultrastructural analysis and Nanoparticle Tracking Analysis (NTA) of isolated EVs revealed a heterogeneous population of exosomes and microvesicles released by mESCs. These vesicles contain Wnt10b and the Notch ligand Delta-like 4 (DLL4) and also the co-chaperone stress inducible protein 1 (STI1) and its partner Hsp90. Wnt10b and Dll4 colocalize with EVs biogenesis markers in mESCs. Overall, the present study supports the function of the mESCs endocytic network and their EVs as players in stem cell biology.
Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Células-Tronco Embrionárias Murinas/ultraestrutura , Corpos Multivesiculares/metabolismoRESUMO
O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-ß produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação ß-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação ß-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-ß através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1
Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic ß-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the ß-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of ß-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of ß-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients
Assuntos
Terapias Complementares , Células-Tronco Embrionárias Murinas , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Transplante das Ilhotas Pancreáticas , Laminina , Células Secretoras de InsulinaRESUMO
Purpose: To evaluate the effects of mesenchymal stem cells on liver regeneration in rats following a 70% hepatectomy. Methods: Forty rats were subjected to 70% hepatectomy and then ~106 mesenchymal stem cells (test group), or saline solution (control group), were infused into their livers via the portal vein. Each treatment group was divided into early and late subgroups (euthanized 3 d and 5 d following the operation, respectively). Group comparisons of Albumin, aminotransaminases (AST, ALT), and Alcaline Phosphatase (AP) levels, proliferative index (ki-67+ straining), and mitotic cell counts were conducted. Results: No significant differences in liver regeneration rate, number of mitoses, proliferative index, or serum levels of albumin, AST, or AP were observed. ALT levels were higher in the test group than in the control group (p .05). Conclusions: Mesenchymal stem-cell therapy did not improve liver regeneration rate 3 d or 5 d after 70% hepatectomy in rats. Likewise, the therapy appeared not to affect liver function, proliferative index, or number of mitoses significantly.(AU)