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Autism spectrum disorder (ASD) is a multifaceted neurodevelopmental disorder predominant in childhood. Despite existing treatments, the benefits are still limited. This study explored the effectiveness of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) loaded with miR-137 in enhancing autism-like behaviors and mitigating neuroinflammation. Utilizing BTBR mice as an autism model, the study demonstrated that intranasal administration of MSC-miR137-EVs ameliorates autism-like behaviors and inhibits pro-inflammatory factors via the TLR4/NF-κB pathway. In vitro evaluation of LPS-activated BV2 cells revealed that MSC-miR137-EVs target the TLR4/NF-κB pathway through miR-137 inhibits proinflammatory M1 microglia. Moreover, bioinformatics analysis identified that MSC-EVs are rich in miR-146a-5p, which targets the TRAF6/NF-κB signaling pathway. In summary, the findings suggest that the integration of MSC-EVs with miR-137 may be a promising therapeutic strategy for ASD, which is worthy of clinical adoption.
Assuntos
Comportamento Animal , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , NF-kappa B , Transdução de Sinais , Animais , Masculino , Camundongos , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/terapia , Vesículas Extracelulares/metabolismo , Inflamação/patologia , Lipopolissacarídeos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Autism spectrum disorder (ASD) is a group of complex neurodevelopmental disorders with abnormal behavior. However, the pathogenesis of ASD remains to be clarified. It has been demonstrated that miRNAs are essential regulators of ASD. However, it is still unclear how miR-92a-2-5p acts on the developing brain and the cell types directly. In this study, we used neural progenitor cells (NPCs) derived from ASD-hiPSCs as well as from neurotypical controls to examine the effects of miR-92a-2-5p on ASD-NPCs proliferation and neuronal differentiation, and whether miR-92a-2-5p could interact with genetic risk factor, DLG3 for ASD. We observed that miR-92a-2-5p upregulated in ASD-NPCs results in decreased proliferation and neuronal differentiation. Inhibition of miR-92a-2-5p could promote proliferation and neuronal differentiation of ASD-NPCs. DLG3 was negatively regulated by miR-92a-2-5p in NPCs. Our results suggest that miR-92a-2-5p is a strong risk factor for ASD and potentially contributes to neuropsychiatric disorders.
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Aberrant epigenetic reprogramming is one of the major barriers for somatic cell reprogramming. Although our previous study has indicated that H3K27me3 demethylase KDM6A can improve the nuclear reprogramming efficiency, the mechanism remains unclear. In this study, we demonstrate that the overexpression of Kdm6a may improve induced pluripotent stem cell (iPSC) reprogramming efficiency in a demethylase enzymatic activity-dependent manner. KDM6A erased H3K27me3 on pluripotency- and metabolism-related genes, and consequently facilitated changing the gene expression profile and metabolic pattern to an intermediate state. Furthermore, KDM6A may promote IL-6 expression, and the secreted IL-6 may further improve iPSC reprogramming efficiency. In addition, KDM6A may promote PTEN expression to decrease p-AKT and p-mTOR levels, which in turn facilitates reprogramming. Overall, our results reveal that KDM6A may promote iPSC reprogramming efficiency by accelerating changes in the gene expression profile and the metabolic pattern in a demethylation-activity-dependent manner. These results may provide an insight into the relationship between epigenomics, transcriptomics, metabolomics, and reprogramming.
Assuntos
Histona Desmetilases/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Interleucina-6/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Animais , Reprogramação Celular/fisiologia , Epigênese Genética , Feminino , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Transdução de SinaisRESUMO
Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.
Assuntos
Androgênios/metabolismo , Diferenciação Celular/genética , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Injeções de Esperma Intracitoplásmicas/métodos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Linhagem Celular , Corpos Embrioides/citologia , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Oócitos/citologia , Oócitos/metabolismo , Reprodutibilidade dos TestesRESUMO
Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.
Assuntos
Blastômeros/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica/genética , Células-Tronco Embrionárias Murinas/metabolismo , Partenogênese/genética , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Agregação Celular/genética , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Feminino , Imunofluorescência , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Pig pluripotent cells may represent an advantageous experimental tool for developing therapeutic application in the human biomedical field. However, it has previously been proven to be difficult to establish from the early embryo and its pluripotency has not been distinctly documented. In recent years, induced pluripotent stem (iPS) cell technology provides a new method of reprogramming somatic cells to pluripotent state. The generation of iPS cells together with or without certain small molecules has become a routine technique. However, the generation of iPS cells from pig embryonic tissues using viral infections together with small molecules has not been reported. Here, we reported the generation of induced pig pluripotent cells (iPPCs) using the iPS technology in combination with valproic acid (VPA). VPA treatment significantly increased the expression of pluripotent genes and played an important role in early reprogramming. We showed that iPPCs resembled pig epiblast cells in their morphology and pluripotent markers, such as OCT4, NANOG, and SSEA1. It had a normal karyotype and could form embryoid bodies, which express three germ layer markers in vitro. In addition, the iPPCs might directly differentiate into neural progenitors after being induced with the retinoic acid and extracellular matrix. Our study established a reasonable method to generate pig pluripotent cells, which might be a new donor cell source for human neural disease therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Suínos , Porco MiniaturaRESUMO
Background: Sepsis is a life-threatening disease characterized by multiple organ failure due to excessive activation of the inflammatory response and cytokine storm. Despite recent advances in the clinical use of anti-cytokine biologics, sepsis treatment efficacy and improvements in mortality remain unsatisfactory, largely due to the mechanistic complexity of immune regulation and cytokine interactions. Methods: In this study, a broad-spectrum anti-inflammatory and endotoxin neutralization strategy was developed based on autologous "cryo-shocked" neutrophils (CS-Neus) for the management of sepsis. Neutrophils were frozen to death using a novel liquid nitrogen "cryo-shock" strategy. The CS-Neus retained the source cell membrane structure and functions related to inflammatory site targeting, broad-spectrum inflammatory cytokines, and endotoxin (LPS) neutralizing properties. This strategy aimed to disable harmful pro-inflammatory functions of neutrophils, such as cytokine secretion. Autologous cell-based therapy strategies were employed to avoid immune rejection and enhance treatment safety. Results: In both LPS-induced sepsis mouse models and clinical patient-derived blood samples, CS-Neus treatment significantly ameliorated cytokine storms by removing inflammatory cytokines and endotoxin. The therapy showed notable anti-inflammatory therapeutic effects and improved the survival rate of mice. Discussion: The results of this study demonstrate the potential of autologous "cryo-shocked" neutrophils as a promising therapeutic approach for managing sepsis. By targeting inflammatory organs and exhibiting anti-inflammatory activity, CS-Neus offer a novel strategy to combat the complexities of sepsis treatment. Further research and clinical trials are needed to validate the efficacy and safety of this approach in broader populations and settings.
RESUMO
H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.
Assuntos
Reprogramação Celular , Epigênese Genética , Transição Epitelial-Mesenquimal , Células-Tronco Pluripotentes Induzidas , RNA Longo não Codificante , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transição Epitelial-Mesenquimal/genética , Animais , Reprogramação Celular/genética , Camundongos , Técnicas de Silenciamento de Genes , Masculino , Metilação de DNA/genéticaRESUMO
The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.
Assuntos
Blastocisto/metabolismo , Nucléolo Celular/metabolismo , Metilação de DNA , DNA Ribossômico/metabolismo , Genes de RNAr , Técnicas de Transferência Nuclear , Processamento Pós-Transcricional do RNA , Animais , Feminino , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Nucleofosmina , Fatores de TempoRESUMO
AIMS: Maternal immune activation (MIA) via infection during pregnancy is known to be an environmental risk factor for neurodevelopmental disorders and the development of autism spectrum disorders (ASD) in the offspring, but it still remains elusive that the molecular relevance between infection-induced abnormal neurodevelopmental events and an increased risk for ASD development. MAIN METHODS: Fully considering the extremely high genetic heterogeneity of ASD and the universality of risk-gene with minimal effect-sizes, the gene and pathway-based association analysis was performed with the transcriptomic and DNA methylation landscapes of temporal human embryonic brain development and ASD, and the time-course transcriptional profiling of MIA. We conducted the transcriptional profiling of mouse abnormal neurodevelopment two days following induced MIA via LPS injection at E10.5. KEY FINDINGS: A novel evidence was proved that illustrated altering four immune and metabolism-related risk pathways, including starch and sucrose metabolism, ribosome, protein processing in endoplasmic reticulum, and retrograde endocannabinoid signaling pathway, which were prominent involvement in the process of MIA regulating abnormal fetal brain development to induce an increased risk of ASD. Here, we have observed that almost all key genes within these risk pathways are significantly differentially expressed at embryonic days (E) 10.5-12.5, which is considered to be the optimal coincidence window of mouse embryonic brain development to study the intimate association between MIA and ASD using mouse animal models. SIGNIFICANCE: There search establishes that MIA causes dysregulation of immune and metabolic pathways, which leads to abnormal embryonic neurodevelopment, thus promoting development of ASD symptoms in offspring.
Assuntos
Transtorno do Espectro Autista , Efeitos Tardios da Exposição Pré-Natal , Gravidez , Feminino , Camundongos , Animais , Humanos , Transtorno do Espectro Autista/genética , Comportamento Animal , Modelos Animais de Doenças , Transcriptoma , Efeitos Tardios da Exposição Pré-Natal/genéticaRESUMO
Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Partenogênese , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Blastocisto/metabolismo , Contagem de Células , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Cariótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Injeções de Esperma Intracitoplásmicas , Teratoma/metabolismo , Teratoma/patologia , Ativação TranscricionalRESUMO
Rare and undiagnosed diseases tend to be diverse, misdiagnosed, and difficult to diagnose. In some cases, the disease is progressive and life-threatening. Yet, to date, an estimated 95% of rare diseases have no approved therapy. Therefore, rare and undiagnosed diseases are considered the ultimate challenges for understanding human diseases. Here, we review the research progress, research frontiers, and important scientific issues related to rare and undiagnosed diseases. We mainly focus on five topics: (1) the identification and functional analysis of disease-causing genes; (2) the construction of cells, organoids, and animal models for mechanism validation; (3) subtyping and diagnosis; (4) treatment and drug screening based on causative genes and mutations; and (5) new technologies and methods for studying rare and undiagnosed diseases. In this review, we briefly update and discuss the pathogenic mechanisms and precision medicine for rare and undiagnosed diseases.
RESUMO
Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker-positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein-protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites.
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Reprogramação Celular , MicroRNAs , Animais , Diferenciação Celular/genética , Reprogramação Celular/genética , Fibroblastos/metabolismo , Mesoderma , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismoRESUMO
In this study, we generated embryonic stem cells from parthenogenetic embryos (PESCs), and induced them to differentiate to motor neurons, which could be an alternative source of histocompatible cells for replacement of therapy and theoretical foundation for studying the relationship of genome imprint and neural differentiation. The parthenogenetic activation rate of B6D2F1 mouse oocytes was 93.26%. We established eight parthenogenetic embryonic stem cell lines and the establishment rate from parthenogenetic embryos was 23.53%. The pluripotency marker Oct4, the cell surface marker SSEA-1, and alkaline phosphatase exhibited in PESCs. Karyotype analysis showed normal 40 chromosomes when examined at passages 10 and 30, which was in accordance with their oocyte origins. Three germinal layers were differentiated in vivo and in vitro. Mouse PESCs, which were treated by tretinoin and sonic hedgehog with extracellular matrix, could generate motor neurons that expressed the specific markers such as HB9 and Olig2.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Neurônios Motores/citologia , Partenogênese , Animais , Técnicas de Cultura de Células , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Antígenos CD15/genética , Antígenos CD15/metabolismo , Camundongos , Neurônios Motores/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
The study of human brain physiology, including cellular interactions in normal and disease conditions, has been a challenge due to its complexity and unavailability. Induced pluripotent stem cell (iPSC) study is indispensable in the study of the pathophysiology of neurological disorders. Nevertheless, monolayer systems lack the cytoarchitecture necessary for cellular interactions and neurological disease modeling. Brain organoids generated from human pluripotent stem cells supply an ideal environment to model both cellular interactions and pathophysiology of the human brain. This review article discusses the composition and interactions among neural lineage and non-central nervous system cell types in brain organoids, current studies, and future perspectives in brain organoid research. Ultimately, the promise of brain organoids is to unveil previously inaccessible features of neurobiology that emerge from complex cellular interactions and to improve our mechanistic understanding of neural development and diseases.
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Células-Tronco Pluripotentes Induzidas , Doenças do Sistema Nervoso , Encéfalo , Humanos , Organoides , TecnologiaRESUMO
Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells.
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Diferenciação Celular/fisiologia , Metilação de DNA , Epigênese Genética/fisiologia , Regulação da Expressão Gênica/fisiologia , Neurônios/efeitos dos fármacos , Tretinoína/metabolismo , Animais , Azacitidina/análogos & derivados , Decitabina , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/metabolismo , Tretinoína/farmacologiaRESUMO
The parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos have the totipotency and proliferation capacity similar to those of the fertilized embryonic stem cells (fESCs). Therefore, the establishment of pESCs line avoids destroy of embryo and kence may make pESCs less concerns with political and ethical issues. These cells are characterized by their histocompatibility with the oocyte donor and therefore is more suitable for cell and tissue replacement therapy. In addition, because of the typical imprinting status, pESCs also provide a valuable in vitro model system for studying the molecular mechanisms in genomic imprinting.
Assuntos
Células-Tronco Embrionárias/citologia , Impressão Genômica , Partenogênese/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histocompatibilidade , Partenogênese/genética , Células-Tronco Pluripotentes/citologiaRESUMO
Induced pluripotent stem cell (iPSC) technology refers to the reprogramming of terminally differentiated somatic cells into pluripotent stem cells by introducing specific transcription factors that are known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. In this study, we reprogrammed the primary fibroblasts isolated from the Daxxflox/flox mice, which carry the Oct4-green fluorescent protein reporter, and employed wild-type littermates as a control to induce iPSCs, then knocked out Daxx by infecting with Cre virus at the cellular level. The pluripotency and self-renewal capacity of iPSCs were determined. In addition, Daxx deletion altered the pluripotency marker (Nanog, Oct4) expression and displayed neural differentiation defects. Particularly, by performing transcriptome analysis, we observed that numerous ribosome biogenesis-related genes were altered, and quantitative polymerase chain reaction revealed that the expression of rDNA-related genes, 47S and 18S, was elevated after Daxx deletion. Finally, we illustrated that the expression of the neurodevelopment-related gene was upregulated both in iPSCs and differentiated neurospheres. Taken together, we demonstrated that Daxx knockout promotes the expression of rDNA, pluripotency, and neurodevelopment genes, which may improve the differentiation abilities of mouse iPSCs (miPSCs).
Assuntos
Diferenciação Celular/genética , Proteínas Correpressoras/genética , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Chaperonas Moleculares/genética , Animais , Técnicas de Cultura de Células , Células Cultivadas , Reprogramação Celular/genética , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Knockout , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
PURPOSE: Death domain-associated protein (DAXX) is a multifunctional nuclear protein involved in apoptosis, transcription, deoxyribonucleic acid damage response, and tumorigenesis. However, the role of DAXX in breast cancer development and progression remains elusive. In this study, we examined the expression patterns and function of DAXX in human breast cancer samples and cell lines. METHODS: Immunohistochemistry was used to analyze the expression and localization patterns of DAXX. Additionally, we investigated whether DAXX played an intrinsic role in the cellular response to damage induced by ultraviolet (UV) irradiation in MDA-MB-231 breast cancer cells (isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma). RESULTS: Our results showed that nucleus size, chromatin organization, and DAXX localization were altered in breast cancer tissues compared with those in control tissues. Compared with cytoplasmic and nuclear expression in benign breast tissues, DAXX was colocalized with promyelocytic leukemia in nuclei with a granular distribution. Endogenous DAXX messenger ribonucleic acid levels were upregulated upon UV radiation in MDA-MB-231 cells. DAXX-deficient cells tended to be more sensitive to irradiation than control cells. Conversely, DAXX-overexpressing cells exhibited reduced phosphorylated histone H2AX (γ-H2AX) accumulation, increased cell survival, and resistance to UV-induced damage. The protective effects of DAXX may be related to the activation of the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (ATM-CHK2)-cell division cycle 25c (CDC25c) signaling pathways in Gap2/Mitosis (G2/M) checkpoint and ultimately cell cycle arrest at G2/M phase. CONCLUSIONS: Taken together, these results suggested that DAXX may be an essential component in breast cancer initiation, malignant progression, and radioresistance.
Assuntos
Neoplasias da Mama/genética , Dano ao DNA/genética , Domínio de Morte/genética , Imuno-Histoquímica/métodos , Adulto , Proliferação de Células , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Pessoa de Meia-IdadeRESUMO
Immune checkpoint inhibitors (ICIs) and immunotherapy have proven to be a transformative therapy for many forms of cancer treatment. While many antibodies targeting the PD-1, PD-L1, and CTLA-4 pathways have been approved for clinical use by the FDA, it is clear that a single ICI is not sufficient to eradicate disease. ICI combination strategies are being extensively investigated to advance cancer treatment to next curative stage. Among the immune checkpoint inhibitors being actively investigated, the potential of VISTA (V-domain Ig suppressor of T cell activation), a unique B7 family member that functions as both ligand and receptor, is being actively pursued. This article summarizes the expression and immunomodulatory effects of VISTA in autoimmune diseases and cancer, and assesses its potential as an additional component of immune checkpoint cancer therapy.