RESUMO
The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another-so far unknown-pathway regulates HB differentiation.
Assuntos
Embrião de Mamíferos/metabolismo , Camadas Germinativas/metabolismo , Proteína Homeobox Nanog/genética , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/metabolismo , Sistemas CRISPR-Cas , Bovinos , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Edição de Genes , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Homeobox Nanog/deficiência , Proteína Homeobox Nanog/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOXF/metabolismo , Transdução de SinaisRESUMO
When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.
Assuntos
Padronização Corporal , Gástrula/crescimento & desenvolvimento , Tecido Nervoso/crescimento & desenvolvimento , Organogênese , Proteínas Wnt/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Gástrula/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Hidrogéis/farmacologia , Camundongos , Tecido Nervoso/efeitos dos fármacos , Organogênese/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Wnt3a-coated beads can induce asymmetric divisions of mouse embryonic stem cells (mESCs), resulting in one self-renewed mESC and one differentiating epiblast stem cell. This provides an opportunity for studying histone inheritance pattern at a single-cell resolution in cell culture. Here, we report that mESCs with Wnt3a-bead induction display nonoverlapping preexisting (old) versus newly synthesized (new) histone H3 patterns, but mESCs without Wnt3a beads have largely overlapping patterns. Furthermore, H4K20me2/3, an old histone-enriched modification, displays a higher instance of asymmetric distribution on chromatin fibers from Wnt3a-induced mESCs than those from non-induced mESCs. These locally distinct distributions between old and new histones have both cellular specificity in Wnt3a-induced mESCs and molecular specificity for histones H3 and H4. Given that post-translational modifications at H3 and H4 carry the major histone modifications, our findings provide a mammalian cell culture system to study histone inheritance for maintaining stem cell fate and for resetting it during differentiation.
Assuntos
Histonas/metabolismo , Células-Tronco Embrionárias Murinas , Proteína Wnt3A/farmacologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Desenvolvimento Embrionário , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Wnt3A/metabolismoRESUMO
We recently reported that epiblast stem cells (EpiSCs)-like cells could be derived from preimplantation embryos (named as AFSCs). Here, we established AFSCs from pre-implantation embryos of multiple mouse strains and showed that unlike EpiSCs, the derivation efficiency of AFSCs was affected by the genetic background. We then used AFSCs lines to dissect the roles of Activin A (Act A) and basic fibroblast growth factor and reported that Act A alone was capable of maintaining self-renewal but not developmental potential in vivo. Finally, we established a novel experimental system, in which AFSCs were efficiently converted to multipotent progenitor stem cells using Act A and bone morphogenetic protein 4 (named as ABSCs). Importantly, these ABSCs contributed to neural mesodermal progenitors and lateral plate mesoderm in postimplantation chimeras. Taken together, our study established a robust experimental system for the generation of specific multipotent progenitor stem cells that was self-renewable and capable of contributing to embryonic and extra-embryonic tissues.
Assuntos
Ativinas/farmacologia , Camadas Germinativas/efeitos dos fármacos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Ativinas/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/crescimento & desenvolvimento , Camundongos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Dual inhibition (2i) of Ras-MEK-ERK and GSK3ß pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFß/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFß/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo , Amidas/farmacologia , Animais , Benzamidas/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Inibidores Enzimáticos/farmacologia , Feminino , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Coelhos , Tiossemicarbazonas/farmacologia , Proteínas ras/antagonistas & inibidoresRESUMO
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/farmacologiaRESUMO
Mouse primordial germ cells (PGCs), originate from the early post-implantation epiblast in response to BMP4 secreted by the extraembryonic ectoderm. However, how BMP4 acts here has remained unclear. Recent work has identified the transcription factor (TF), OTX2 as a key determinant of the segregation of the germline from the soma. OTX2 is expressed ubiquitously in the early post-implantation epiblast, decreasing rapidly in cells that initiate the PGC programme. Otx2 mRNA is also rapidly repressed by BMP4 in vitro, in germline competent cells. Supporting a model in which BMP4 represses Otx2, enforcing sustained OTX2 expression in competent cells blocks germline entry. In contrast, Otx2-null epiblast cells enter the germline with increased efficiency in vitro and in vivo and can do so independently of BMP4. Also, Otx2-null cells can initiate germline entry even without the crucial PGC TF, BLIMP1. In this review, we survey recent advances and propose hypotheses concerning germline entry.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Células Germinativas/metabolismo , Camadas Germinativas/metabolismo , Fatores de Transcrição Otx/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/genética , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição Otx/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismoRESUMO
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 , SuínosRESUMO
Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Interferon Tipo I/metabolismo , Antivirais/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/fisiologia , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , Ectoderma/fisiologia , Ectoderma/virologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Endoderma/fisiologia , Endoderma/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/virologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camadas Germinativas/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/virologia , Fator 4 Semelhante a Kruppel , RNA Viral/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO's capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Aminopiridinas/farmacologia , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem Celular , Fatores de Transcrição E2F/antagonistas & inibidores , Fatores de Transcrição E2F/metabolismo , Técnicas de Silenciamento de Genes , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/fisiologia , Humanos , Hidroxiquinolinas/farmacologia , Células-Tronco Pluripotentes/fisiologia , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismo , Transdução de Sinais/genética , Fatores de TempoRESUMO
The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.
Assuntos
Células-Tronco Pluripotentes/citologia , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/farmacologia , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Inhibition of Wnt/ß-catenin signaling facilitates the derivation of mouse epiblast stem cells (EpiSCs), as well as dramatically promotes EpiSC self-renewal. The specific mechanism, however, is still unclear. Here, we showed that IWR1, a Wnt/ß-catenin signaling inhibitor, allowed long-term self-renewal of EpiSCs in serum medium in combination with ROCK inhibitor Y27632. Through transcriptome data analysis, we arrived at a set of candidate transcription factors induced by IWR1. Among these, Forkhead box D3 (Foxd3) was most abundant. Forced expression of Foxd3 could recapitulate the self-renewal-promoting effect of IWR1 in EpiSCs. Conversely, knockdown of Foxd3 profoundly compromised responsiveness to IWR1, causing extinction of pluripotency markers and emergence of differentiation phenotype. Foxd3 thus is necessary and sufficient to mediate self-renewal downstream of Wnt/ß-catenin signaling inhibitor. These findings highlight an important role for Foxd3 in regulating EpiSCs and will expand current understanding of the primed pluripotency.
Assuntos
Autorrenovação Celular/efeitos dos fármacos , Fatores de Transcrição Forkhead/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , Amidas/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Piridinas/farmacologia , Proteínas Repressoras/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. METHODS: Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. RESULTS: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. CONCLUSIONS: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.
Assuntos
Autofagia/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Sirolimo/farmacologia , Autofagia/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/fisiologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/fisiologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/fisiologia , Humanos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Proteína Homeobox Nanog/genética , Fatores de Transcrição Otx/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Compartimento Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimera/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Fator Inibidor de Leucemia/farmacologia , Mesoderma/citologia , Camundongos , Mutação/genética , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição Otx/genética , Ligação Proteica/efeitos dos fármacosRESUMO
The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/É as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs.
Assuntos
Caseína Quinase I/antagonistas & inibidores , Células-Tronco Embrionárias/efeitos dos fármacos , Camadas Germinativas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pteridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Caseína Quinase I/metabolismo , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/metabolismo , Células HCT116 , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Pteridinas/química , Bibliotecas de Moléculas Pequenas/química , Células-Tronco/metabolismo , Relação Estrutura-AtividadeRESUMO
The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Oligopeptídeos/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Camadas Germinativas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteína de Leucina Linfoide-Mieloide/deficiência , Proteína de Leucina Linfoide-Mieloide/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.
Assuntos
Elementos Facilitadores Genéticos/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Camadas Germinativas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição/genética , Ativinas/farmacologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA , Epigênese Genética , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica , Proteínas de Ligação a RNA , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. METHODS: Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. RESULTS: The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). CONCLUSIONS: Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Teratogênicos/farmacologia , Transcriptoma/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Humanos , Regulação para Cima/efeitos dos fármacosRESUMO
Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
Assuntos
Clobetasol/farmacologia , Miconazol/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/patologia , Humanos , Lisofosfatidilcolinas , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores de Glucocorticoides/metabolismo , Regeneração/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Autophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of the chick embryo using both in vivo and in vitro approaches, especially in the EMT process, and we found that autophagy gene Atg7 was expressed on the apical side of the ectoderm and endoderm. Over-expression of Atg7 could enhance the expression of Atg8 and the E-cadherin, the latter of which is a crucial marker of the EMT process. We also found that the disturbance of autophagy could retard the development of chick embryos in HH4 with shorter primitive steak than that in the control group, which is a newly formed structure during EMT process. So we assumed that autophagy could affect EMT process by adhesion molecule expression. Moreover, more molecules, such as slug, chordin, shh et., which were all involved in EMT process, were detected to address the mechanism of this phenomena. We established that the inhibition of autophagy could cause developmental delay by affecting EMT process in gastrulation of chick embryos.