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1.
Biomolecules ; 11(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34439829

RESUMO

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.


Assuntos
Antirreumáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fator Inibidor de Leucemia/farmacologia , Mutação , Retina/efeitos dos fármacos , Retinite Pigmentosa/tratamento farmacológico , Rodopsina/genética , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/imunologia , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Retina/imunologia , Retina/patologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/imunologia , Retinite Pigmentosa/patologia , Rodopsina/deficiência , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Transgenes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
2.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445681

RESUMO

Parthenogenetic embryos have been widely studied as an effective tool related to paternal and maternal imprinting genes and reproductive problems for a long time. In this study, we established a parthenogenetic epiblast-like stem cell line through culturing parthenogenetic diploid blastocysts in a chemically defined medium containing activin A and bFGF named paAFSCs. The paAFSCs expressed pluripotent marker genes and germ-layer-related genes, as well as being alkaline-phosphatase-positive, which is similar to epiblast stem cells (EpiSCs). We previously showed that advanced embryonic stem cells (ASCs) represent hypermethylated naive pluripotent embryonic stem cells (ESCs). Here, we converted paAFSCs to ASCs by replacing bFGF with bone morphogenetic protein 4 (BMP4), CHIR99021, and leukemia inhibitory factor (LIF) in a culture medium, and we obtained parthenogenetic advanced stem cells (paASCs). The paASCs showed similar morphology with ESCs and also displayed a stronger developmental potential than paAFSCs in vivo by producing chimaeras. Our study demonstrates that maternal genes could support parthenogenetic EpiSCs derived from blastocysts and also have the potential to convert primed state paAFSCs to naive state paASCs.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Partenogênese/fisiologia , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Técnicas de Cultura Embrionária/métodos , Feminino , Fatores de Crescimento de Fibroblastos/farmacologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/citologia , Partenogênese/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia
3.
Cell Prolif ; 54(8): e13090, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34197016

RESUMO

OBJECTIVES: Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC-specific growth factors and signaling inhibitors. METHODS: The culture medium of Tet-On-OSKM/Oct4-GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3ß inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA-Seq was conducted to analyze gene expression. RESULTS: Via continuous expression of OSKM, mouse ESCs (OSKM-ESCs) and the resulting iPSCs (OSKM-iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM-iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM-iPSCs contributed to germline transmission. CONCLUSIONS: Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission, with potential applications in other species.


Assuntos
Autorrenovação Celular , Reprogramação Celular , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Transcriptoma/efeitos dos fármacos
4.
PLoS One ; 16(2): e0243727, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33534866

RESUMO

In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.


Assuntos
Bovinos/embriologia , Criopreservação/veterinária , Embrião de Mamíferos/embriologia , Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Insulin-Like I , Fator Inibidor de Leucemia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Criopreservação/métodos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/ultraestrutura , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/farmacologia , Fator Inibidor de Leucemia/administração & dosagem , Fator Inibidor de Leucemia/farmacologia , Gravidez
5.
Cell Mol Life Sci ; 78(6): 2781-2795, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33034697

RESUMO

Autosomal-dominant leukodystrophy (ADLD) is a rare fatal neurodegenerative disorder with overexpression of the nuclear lamina component, Lamin B1 due to LMNB1 gene duplication or deletions upstream of the gene. The molecular mechanisms responsible for driving the onset and development of this pathology are not clear yet. Vacuolar demyelination seems to be one of the most significant histopathological observations of ADLD. Considering the role of oligodendrocytes, astrocytes, and leukemia inhibitory factor (LIF)-activated signaling pathways in the myelination processes, this work aims to analyze the specific alterations in different cell populations from patients with LMNB1 duplications and engineered cellular models overexpressing Lamin B1 protein. Our results point out, for the first time, that astrocytes may be pivotal in the evolution of the disease. Indeed, cells from ADLD patients and astrocytes overexpressing LMNB1 show severe ultrastructural nuclear alterations, not present in oligodendrocytes overexpressing LMNB1. Moreover, the accumulation of Lamin B1 in astrocytes induces a reduction in LIF and in LIF-Receptor (LIF-R) levels with a consequential decrease in LIF secretion. Therefore, in both our cellular models, Jak/Stat3 and PI3K/Akt axes, downstream of LIF/LIF-R, are downregulated. Significantly, the administration of exogenous LIF can partially reverse the toxic effects induced by Lamin B1 accumulation with differences between astrocytes and oligodendrocytes, highlighting that LMNB1 overexpression drastically affects astrocytic function reducing their fundamental support to oligodendrocytes in the myelination process. In addition, inflammation has also been investigated, showing an increased activation in ADLD patients' cells.


Assuntos
Astrócitos/metabolismo , Doenças Desmielinizantes/patologia , Lamina Tipo B/metabolismo , Transdução de Sinais , Astrócitos/citologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Mediadores da Inflamação/metabolismo , Lamina Tipo B/genética , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptores de OSM-LIF/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
Life Sci ; 264: 118701, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33130086

RESUMO

AIMS: Deriving canine-induced pluripotent stem cells (ciPSCs) have paved the way for developing novel cell-based disease models and transplantation therapies in the dog. Though ciPSCs have been derived in the presence of Leukemia inhibitory factor (LIF) as well in the presence of basic fibroblast growth factor (bFGF), the positioning of ciPSCs in the naïve or the primed state of pluripotency remains elusive. This study aims to understand whether canine iPSCs belong to naïve or prime state in comparison to mouse (m) iPSCs and human (h) iPSCs. MAIN METHODS: In the present study, we derived ciPSCs in presence of LIF and compared their state of pluripotency with that of miPSCs and hiPSCs by culturing them in the presence of LIF, bFGF, and LIF + bFGF. Gene expression level at transcript level was performed by RT-PCR and qRT-PCR and at the protein level was analysed by immunofluorescence. We also attempted to understand the pluripotency state using lipid body analysis by bodipy staining and blue fluorescence emission. KEY FINDINGS: In contrast to miPSCs, the naïve pluripotent stem cells, ciPSCs showed the expression of FGF5 similar to that of primed pluripotent stem cell, hiPSCs. Compared to miPSCs, ciPSCs cultured in presence of LIF showed enhanced expression of primed pluripotent marker FGF5, similar to hiPSCs cultured in presence of bFGF. Upon culturing in hiPSC culture condition, ciPSCs showed enhanced expression of core pluripotency genes compared to miPSCs cultured in similar condition. However, ciPSCs expressed naïve pluripotent marker SSEA1 similar to miPSCs and lacked the expression of primed state marker SSEA4 unlike hiPSCs. Interestingly, for the first time, we demonstrate the ciPSC pluripotency using lipid body analysis wherein ciPSCs showed enhanced bodipy staining and blue fluorescence emission, reflecting the primed state of pluripotency. ciPSCs expressed higher levels of fatty acid synthase (FASN), the enzyme involved in the synthesis of palmitate, similar to that of hiPSCs and higher than that of miPSCs. As ciPSCs exhibit characteristic properties of both naïve and primed pluripotent state, it probably represents a unique intermediary state of pluripotency that is distinct from that of mice and human pluripotent stem cells. SIGNIFICANCE: Elucidating the pluripotent state of ciPSCs assists in better understanding of the reprogramming events and development in different species. The study would provide a footprint of species-specific differences involved in reprogramming and the potential implication of iPSCs as a tool to analyse evolution.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Cães , Fluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Lipídeos/química , Camundongos
7.
Theriogenology ; 159: 13-19, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33113439

RESUMO

Poor development of oocytes from prepubertal animals is a major factor that hinders the application of the technology, juvenile in vitro embryo transfer (JIVET). The aim of this study was to explore the possibility of improving the developmental competence of prepubertal oocytes by supplementing the oocyte in vitro maturation (IVM) medium with antioxidants and cytokines. Effects of two antioxidants, melatonin and sericin, were first examined. The results showed that melatonin had no significant beneficial roles on the lamb oocyte development, while 0.5% sericin supplemented during IVM significantly increased the blastocyst rate of lamb oocytes (46.5% vs 19.2% in control, P < 0.05). Next, effects of two kinds of combined supplements, insulin-transferrin-selenium (ITS) and fibroblast growth factor 2(FGF2)-leukemia inhibitory factor (LIF)-insulin-like growth factor1 (IGF1)(FLI) were tested. The results indicated that addition of FLI, but not ITS, in the IVM medium, significantly improved the blastocyst development of lamb oocytes (43.9% in FLI group vs 21.6% in control, P < 0.05). Further comparison showed that the developmental competence of oocytes was not significantly different among supplementation with sericin or FLI alone or both, all of which generated similar outcomes of blastocyst yield to the supplementation with adult follicular fluid. Finally, 27 blastocysts produced from lamb oocytes matured in the presence of sericin and FLI were transferred into 18 recipients, of which 9 were pregnant. This study suggests that the developmental competence of prepubertal oocytes can be improved by supplementing IVM medium with relevant agents like sericin and cytokines.


Assuntos
Fator 2 de Crescimento de Fibroblastos , Sericinas , Animais , Blastocisto , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator de Crescimento Insulin-Like I , Fator Inibidor de Leucemia/farmacologia , Oócitos , Gravidez , Sericinas/farmacologia , Ovinos , Carneiro Doméstico
8.
Int J Mol Sci ; 21(19)2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32992968

RESUMO

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.


Assuntos
Criopreservação , Meios de Cultura/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Oócitos/efeitos dos fármacos , Vitrificação/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Feminino , Fertilização In Vitro , Humanos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia , Gravidez , Proteínas Recombinantes/farmacologia
9.
Cell Reprogram ; 22(5): 244-253, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32936029

RESUMO

Mouse embryonic stem cells (mESCs) go through self-renewal in the existence of the cytokine leukemia inhibitory factor (LIF). LIF is added to the mouse stem cells culture medium, and its removal results in fast differentiation. Dimethyl sulfoxide (DMSO) is one of the most used solvents in drug test. We exposed 4-day mESC cultures to different concentrations of DMSO (0.1%, 0.5%, 1.0%, and 2.0%) to identify the safest dose exhibiting efficacy as a solvent. mESCs grown under general pluripotency conditions in the absence of LIF were treated with DMSO. In addition, as a control for differentiation, mESCs were grown in the absence of LIF. DMSO upregulated the mRNA expression level of pluripotency markers. Moreover, DMSO reduced the mRNA expression levels of ectodermal marker (ß-tubulin3), mesodermal marker (Hand1), and endodermal markers (Foxa2 and Sox17) in mESCs. These results indicate that DMSO treatment enhances the pluripotency and disrupts the differentiation of mESCs. We also show that members of the Tet oncogene family are critical to inhibiting the differentiation and methylation of mESCs. DMSO is appropriate to sustain the pluripotency of mESCs in the absence of LIF, and that mESCs can be sustained in an undifferentiated state using DMSO. Therefore, DMSO may, in part, function as a substitute for LIF.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Fator Inibidor de Leucemia/farmacologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia
10.
Stem Cells ; 38(9): 1091-1106, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32478947

RESUMO

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage-induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53-/- ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content; annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage-dependent G2-checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53-/- ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53-dependent formation of haploid cells, increased genomic stability, and suppression of the G2-checkpoint. Minimal induction of DNA damage now resulted in p53-facilitated apoptosis, but regulation of pluripotent gene expression remained p53-independent. Primary embryonic fibroblasts underwent p53-dependent total cell cycle arrest (a prelude to cell senescence), and p53-independent apoptosis occurred in the presence of 10-fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.


Assuntos
Apoptose , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Contagem de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Haploidia , Fator Inibidor de Leucemia/farmacologia , Camundongos , Proteínas Supressoras de Tumor/metabolismo
11.
Biomed Res Int ; 2020: 1308526, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32509845

RESUMO

The mammalian central nervous system (CNS) has a limited ability to renew the damaged cells after a brain or spinal cord injury whether it is nonhuman primates like monkeys or humans. Transplantation of neural stem cells (NSCs) is a potential therapy for CNS injuries due to their pluripotency and differentiation abilities. Cytokines play an important role in CNS development and repair of CNS injuries. However, the detailed cytokine signaling response in monkey neural stem cells is rarely studied. In our previous research, we isolated NSCs from the adult monkey brain and found the effects of cytokines on monkey NSCs. Now, we further analyzed the regulation mechanisms of cytokines to the proliferation of monkey NSCs such as bone morphogenic protein 4 (BMP4), BMP4/leukaemia inhibitory factor (LIF), or retinoic acid (RA)/Forskolin. The data showed that BMP4 inhibited cell proliferation to arrest, but it did not affect the stemness of NSCs. BMP4/LIF promoted the astrocyte-like differentiation of monkey NSCs, and RA/forskolin induced the neuronal differentiation of monkey NSCs. BMP4/LIF and RA/forskolin induced monkey NSC differentiation by regulating Notch signaling. These results provide some theoretical evidence for NSC therapy to brain or spinal cord injury in regenerative medicine.


Assuntos
Citocinas/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/farmacologia , Encéfalo/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Fator Inibidor de Leucemia/farmacologia , Macaca fascicularis , Masculino , Tretinoína/farmacologia
12.
Differentiation ; 112: 67-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045848

RESUMO

To induce and maintain naïve pluripotency in mouse embryonic and induced pluripotent stem cells (ESCs/iPSCs), chemically defined N2B27 medium with PD0325901, CHIR99021, and leukemia inhibitory factor (2i/LIF) is a classic and simple condition. However, this method cannot be simply extrapolated to human ESCs/iPSCs that are principally stabilized in primed pluripotency and become primitive neuroepithelium-like cells in N2B27+2i/LIF culture. Here, we assessed iPSC reprogramming of fibroblasts from chimpanzee, our closest living relative, in N2B27+2i/LIF culture. Under this condition, chimpanzee cells formed alkaline phosphatase-positive dome-shaped colonies. The colony-forming cells could be stably expanded by serial passaging without a ROCK inhibitor. However, their gene expression was distinct from iPSCs and neuroepithelium. They expressed the OCT3/4 transgene and a subset of transcripts associated with pluripotency, mesenchymal-epithelial transition, and neural crest formation. These cells exhibited a differentiation potential into the three germ layers in vivo and in vitro. The current study demonstrated that iPSC reprogramming in N2B27+2i/LIF culture converted chimpanzee fibroblasts into a multipotent cancerous state with unique gene expression, but not fully pluripotent stem cells.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Multipotentes/citologia , Animais , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Transição Epitelial-Mesenquimal/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/crescimento & desenvolvimento , Humanos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , Crista Neural/citologia , Pan troglodytes , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia
13.
Folia Biol (Praha) ; 66(5-6): 155-160, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-34087971

RESUMO

Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-ß pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-ß inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.


Assuntos
Quinase 3 da Glicogênio Sintase , Células-Tronco Neurais , Animais , Proteínas Morfogenéticas Ósseas , Diferenciação Celular , Células-Tronco Embrionárias , Fator Inibidor de Leucemia/farmacologia , Camundongos
14.
Cells ; 8(10)2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635340

RESUMO

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Ovário/citologia , Ovário/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzenossulfonatos/farmacologia , Western Blotting , Bovinos , Cromatina/metabolismo , Cisplatino/farmacologia , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Hidrazonas/farmacologia , Marcação In Situ das Extremidades Cortadas , Fator Inibidor de Leucemia/farmacologia , Masculino , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Citocinas/metabolismo
15.
Front Immunol ; 10: 1164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191537

RESUMO

Background and Purpose: The gp130 family of cytokines signals through receptors dimerizing with the gp130 subunit. Downstream signaling typically activates STAT3 but also SHP2/Ras/MAPK pathways. Oncostatin M (OSM) is a unique cytokine in this family since the receptor (OSMR) activates a non-redundant signaling pathway by recruitment of the adapter Shc1. We have studied the functional relevance of Shc1 for OSM-induced bone resorption. Experimental Approach: Osteoblasts were stimulated with OSM and STAT3 and Shc1 activations were studied using real-time PCR and Western blots. The role of STAT3 and Shc1 for OSM-induced RANKL expression and osteoclast formation was studied by silencing their mRNA expressions. Effects of OSM were compared to those of the closely related cytokine leukemia inhibitory factor (LIF). Key Results: OSM, but not LIF, induced the mRNA and protein expression of Shc1 and activated phosphorylation of Shc1 in the osteoblasts. Silencing of Shc1 decreased OSM-induced activation of STAT3 and RANKL expression. Silencing of STAT3 had no effect on activation of Shc1, but prevented the OSM-mediated increase of RANKL expression. Silencing of either Shc1 or STAT3 in osteoblasts decreased formation of osteoclasts in OSM-stimulated co-cultures of osteoblasts and macrophages. In agreement with these observations, OSM was a more potent and robust stimulator than LIF of RANKL formation and bone resorption in mouse calvariae and osteoclast formation in bone marrow cultures. Conclusions and Implications: Activation of the Shc1-dependent STAT3 signaling is crucial for OSM-induced osteoclast formation. Inhibition of Shc1 is a potential mechanism to specifically inhibit OSM-induced bone resorption.


Assuntos
Fator Inibidor de Leucemia/farmacologia , Oncostatina M/farmacologia , Osteoclastos/efeitos dos fármacos , Ligante RANK/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Interferência de RNA , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo
16.
Immunopharmacol Immunotoxicol ; 41(3): 455-462, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31142168

RESUMO

Objective: Dendritic cells (DCs) are professional antigen presenting cells majorly modulated by various environmental factors. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine from interleukin-6 family. Previous studies demonstrate that LIF is associated with several tolerogenic events; yet the exact effect of this cytokine on the generation and function of DCs was not explicitly identified. Materials and methods: To clarify the role of LIF in DCs development, immature DCs were differentiated from mouse bone marrow (BM) in a GM-CSF and IL-4 containing medium with or without LIF. Afterwards, in maturation process, the differentiated DCs were exposed to TNF-α in the presence or absence of LIF. Results: Immature DCs differentiated in the presence of LIF, proved a significant enhancement in the expression of MHCII, CD40, or CD86 molecules and in the antigen uptake function. LIF treatment of normal DCs while stimulating for maturation, caused a significant decrement in the expression of phenotypic markers as well as an increment in the antigen uptake function in comparison with TNF-α-only stimulated cells; however, the reduced ability for induction of allogenic T-cell proliferation proved no statistical significance. Conclusions: Our results can reflect a role for LIF in the generation and particularly maturation of DCs. It can be assumed that LIF rather modulates the maturation level, leading to the development of semi-mature and tolerogenic DCs. According to the high levels of LIF in immune-privileged sites like brain and uterine, it seems that the cytokine may account for the formation of local DCs that help the establishment of immunosuppressive environments.


Assuntos
Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Fator Inibidor de Leucemia/imunologia , Animais , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/imunologia
17.
Wound Repair Regen ; 27(5): 477-487, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31107586

RESUMO

Severe uterine injuries may lead to infertility or pregnancy complications. There is a lack of effective methods to restore the structure and function of seriously injured uteri. Leukemia inhibitory factor (LIF), which plays a crucial role in blastocyst implantation, promotes the process of regeneration after injury in several different tissues. In this study, we explored the effect of LIF on the regeneration of rat uterine horns following full-thickness injury. One hundred and twenty four female Sprague-Dawley rats were assigned to three groups, including a sham-operated group (n = 34 uterine horns), a PBS/collagen group (n = 90 uterine horns), and a LIF/collagen group (n = 124 uterine horns). The regenerated uterine horns were collected at 1, 2, 4, 8, or 12 weeks after the surgery. The results showed that LIF/collagen scaffolds increased the number of endometrial cells and neovascularization 2 weeks after uterine full-thickness defect in excision sites (p < 0.001 vs PBS/collagen). Eight weeks after the surgery, the number of endometrial glands was dramatically higher in the LIF/collagen scaffolds group (35.2 ± 4.1/field) than in the PBS/collagen scaffolds (15.1 ± 1.4/field). The percentage of a-smooth muscle actin (a-SMA)-positive areas in the LIF/collagen scaffolds (88.8% ± 9.8%) was also significantly higher than that in the PBS/collagen group (52.9% ± 3.7%). Moreover, LIF improved the pregnancy rate and fetus number. We also found that LIF inhibited the infiltration of inflammatory cells and down-regulated the pro-inflammatory cytokine IL-12 expression while up-regulating the anti-inflammatory cytokine IL-10 expression in the injured part of the uterine horns. Our results indicate that LIF promotes regeneration of the uterus after injury, and this is at least partially due to its immunomodulatory properties. In addition, it is worth to explore further the possibility for LIF/collagen to be an alternative therapeutic approach for uterine damage in the clinic in near future.


Assuntos
Fator Inibidor de Leucemia/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Útero/patologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Tecidos Suporte , Útero/imunologia , Útero/lesões
18.
FASEB J ; 33(8): 9350-9361, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31125263

RESUMO

The establishment of ungulate embryonic stem cells (ESCs) has been notoriously difficult via a conventional approach. We combined a traditional ESC culture method with reprogramming factors to assist the establishment of porcine naive-like ESCs (nESCs). Pig embryonic fibroblasts were transfected with a tetracycline-inducible vector carrying 4 classic mouse reprogramming factors, followed by somatic cell nuclear transfer and culturing to the blastocyst stage. Then, the inner cell mass was isolated and seeded in culture medium. The naive-like ESCs had characteristic verys similar to those of mouse ESCs and showed no signs of altered morphology or differentiation, even after 130 passages. They depended on leukemia inhibitory factor signals for maintenance of pluripotency, and the female cell lines had low expression of the X-inactive specific transcript gene and no histone H3 lysine 27 trimethylation spot. Notably, the ESCs differentiated into 3 germ layers in vitro and could be induced to undergo directional neural and kidney precursor differentiation under defined conditions, and the ESCs could keep proliferating after doxycycline was removed. nESCs can be established, and the well-characterized ESC lines will be useful for the research of transgenic pig models for human disease.-Zhang, M., Wang, C., Jiang, H., Liu, M., Yang, N., Zhao, L., Hou, D., Jin, Y., Chen, Q., Chen, Y., Wang, J., Dai, Y., Li, R. Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.


Assuntos
Reprogramação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Animais , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Imuno-Histoquímica , Fator Inibidor de Leucemia/farmacologia , Camundongos , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Suínos
19.
Int J Neuropsychopharmacol ; 22(6): 402-414, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31125414

RESUMO

BACKGROUND: Leukemia inhibitory factor, a novel myokine, is known to be associated with neural function, but the underlying molecular mechanism remains unclear. METHODS: HT-22 mouse hippocampal cells, primary hippocampal cells, and Drosophila Alzheimer's disease model were used to determine the effect of leukemia inhibitory factor on neurons. Immunoblot analysis and immunofluorescence method were used to analyze biological mechanism. RESULTS: Leukemia inhibitory factor increased Akt phosphorylation in a phosphoinositide-3-kinase-dependent manner in hippocampal cells. Leukemia inhibitory factor also increased the phosphorylation of the mammalian target of rapamycin and the downstream S6K. Leukemia inhibitory factor stimulated the phosphorylation of signal transducer and activator of transcription via extracellular signal-regulated kinases. Leukemia inhibitory factor increased c-fos expression through both Akt and extracellular signal-regulated kinases. Leukemia inhibitory factor blocked amyloid ß-induced neural viability suppression and inhibited amyloid ß-induced glucose uptake impairment through the block of amyloid ß-mediated insulin receptor downregulation. Leukemia inhibitory factor blocked amyloid ß-mediated induction of the autophagy marker, microtubule-associated protein 1A/1B-light chain 3. Additionally, in primary prepared hippocampal cells, leukemia inhibitory factor stimulated Akt and extracellular signal-regulated kinase, demonstrating that leukemia inhibitory factor has physiological relevance in vivo. Suppression of the autophagy marker, light chain 3II, by leukemia inhibitory factor was observed in a Drosophila model of Alzheimer's disease. CONCLUSIONS: These results demonstrate that leukemia inhibitory factor protects against amyloid ß-induced neurotoxicity via Akt/extracellular signal-regulated kinase-mediated c-fos induction, and thus suggest that leukemia inhibitory factor is a potential drug for Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Hipocampo/citologia , Fator Inibidor de Leucemia/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/toxicidade , Animais , Animais Geneticamente Modificados , Células Cultivadas , Drosophila , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 3/biossíntese , Hipocampo/metabolismo , Fator Inibidor de Leucemia/biossíntese , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-fos/biossíntese , Receptor de Insulina/biossíntese , Receptor de Insulina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
20.
Stem Cells ; 37(9): 1223-1237, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31132299

RESUMO

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.


Assuntos
Giro Denteado/citologia , Hipocampo/citologia , Mitógenos/farmacologia , Fator de Crescimento Neural/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator Inibidor de Leucemia/farmacologia , Camundongos Transgênicos , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo
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