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1.
Methods Mol Biol ; 2774: 279-301, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38441772

RESUMO

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.


Assuntos
Megacariócitos , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Fibroblastos , Plaquetas , Diferenciação Celular/genética
2.
FASEB J ; 38(4): e23463, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38334393

RESUMO

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.


Assuntos
Células-Tronco Embrionárias Murinas , beta Catenina , Animais , Camundongos , beta Catenina/metabolismo , Diferenciação Celular/genética , Endoderma , Via de Sinalização Wnt/fisiologia
3.
Nat Commun ; 15(1): 1721, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409226

RESUMO

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.


Assuntos
Cromatina , Células-Tronco Embrionárias , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , S-Adenosilmetionina/metabolismo
4.
Nat Struct Mol Biol ; 31(3): 513-522, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38196033

RESUMO

Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.


Assuntos
Cromatina , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Mitose/genética , Sequências Reguladoras de Ácido Nucleico , Células-Tronco Embrionárias Murinas/metabolismo
5.
Int J Biochem Cell Biol ; 169: 106535, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38281697

RESUMO

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Impressão Genômica , Animais , Humanos , Camundongos , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Células-Tronco Embrionárias Murinas/metabolismo , Mutação
6.
Cells ; 13(2)2024 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-38247824

RESUMO

The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe the "WNT Switch" method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious.


Assuntos
Oftalmopatias Hereditárias , Células-Tronco Embrionárias Murinas , Miócitos Cardíacos , Degeneração Retiniana , Transtornos da Visão , Animais , Camundongos , Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos , Mamíferos
7.
Nat Metab ; 6(1): 127-140, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38172382

RESUMO

Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation.


Assuntos
Blastocisto , Células-Tronco Embrionárias , Camundongos , Animais , Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Aminoácidos/metabolismo , Mamíferos/metabolismo
8.
Nat Struct Mol Biol ; 31(1): 102-114, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38177678

RESUMO

As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.


Assuntos
Metilação de DNA , Células-Tronco Embrionárias , Animais , Camundongos , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias Murinas , Células Germinativas/metabolismo , Camadas Germinativas/metabolismo , Mamíferos/metabolismo
9.
Methods Mol Biol ; 2753: 217-230, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38285341

RESUMO

Animal-based test systems have traditionally been used to screen for the potential teratogenic activity of drugs. Still, their deficits in predicting precise human-specific outcomes and ethical concerns have led to a need for alternative approaches. In vitro, teratogenicity testing using cell cultures or other in vitro systems is a potential alternative. Of the different in vitro platforms, the mouse embryonic stem cell test (mEST) is currently the most widely used and validated in vitro test for assessing the potential effects of teratogens on early embryonic development. The mEST involves exposing mouse embryonic stem cells to the test compound and monitoring their differentiation for several days.Nevertheless, its predictive ability was comparatively lower when distinguishing weak developmental toxicants from non-toxic substances. Since then, several modifications and adaptations of the mEST protocol have been developed. This chapter describes an alternative method based on molecular approaches to predict embryotoxicity. This method, originated from the mEST, analyzes the expression of differentiation genes involved in the development of mesoderm, endoderm, and stoderm and allows screening embryo-toxicants with different mechanisms of action. The hanging drops embryoid bodies used in the original mEST protocol have been replaced with monolayer culture, and thus the process has been shortened. In general, the method shows higher predictability compared with the traditional ones.


Assuntos
Células-Tronco Embrionárias , Teratogênese , Feminino , Gravidez , Humanos , Animais , Camundongos , Células-Tronco Embrionárias Murinas , Teratógenos/toxicidade , Corpos Embrioides , Substâncias Perigosas
10.
Stem Cells Dev ; 33(3-4): 67-78, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38032751

RESUMO

The histamine H3 receptor, prominently expressed in neurons with a minor presence in glial cells, acts as both an autoreceptor and an alloreceptor, controlling the release of histamine and other neurotransmitters. The receptor impacts various essential physiological processes. Our team's initial investigations had demonstrated that the histamine H3 receptor antagonists could facilitate nerve regeneration by promoting the histamine H1 receptors on primary neural stem cells (NSCs) in the traumatic brain injury mouse, which suggested the potential of histamine H3 receptor as a promising target for treating neurological disorders and promoting nerve regeneration. Pitolisant (PITO) is the only histamine H3 receptor antagonist approved by the Food and Drug Administration (FDA) for treating narcolepsy. However, there is no report on Pitolisant in neural development or regeneration, and it is urgent to be further studied in strong biological activity models in vitro. The embryonic stem (ES) cells were differentiated into neural cells in vitro, which replicated the neurodevelopmental processes that occur in vivo. It also provided an alternative model for studying neurodevelopmental processes and testing drugs for neurological conditions. Therefore, we aimed to elucidate the regulatory role of Pitolisant in the early differentiation of ES cells into neural cells. Our results demonstrated that Pitolisant could promote the differentiation of ES cells toward NSCs and stimulated the formation of growth cones. Furthermore, Pitolisant was capable of inducing the polarization of NSCs through the cAMP-LKB1-SAD/MARK2 pathway, but had no significant effect on later neuronal maturation. Pitolisant altered mitochondrial morphology and upregulated the levels of mitochondrion-related proteins TOM20, Drp1, and p-Drp1, and reversed the inhibitory effect of Mdivi-1 on mitochondrial fission during the early neural differentiation of ES cells. In addition, Pitolisant induced the increase in cytosolic Ca2+. Our study provided an experimental foundation for the potential application of histamine H3 receptor-targeted modulators in the field of neuroregeneration.


Assuntos
Histamina , Piperidinas , Receptores Histamínicos H3 , Camundongos , Animais , Histamina/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Agonistas dos Receptores Histamínicos/uso terapêutico , Receptores Histamínicos H3/metabolismo
11.
Cell Prolif ; 57(2): e13534, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37592709

RESUMO

A minority of mouse embryonic stem cells (ESCs) display totipotent features resembling 2-cell stage embryos and are known as 2-cell-like (2C-like) cells. However, how ESCs transit into this 2C-like state remains largely unknown. Here, we report that the overexpression of negative elongation factor A (Nelfa), a maternally provided factor, enhances the conversion of ESCs into 2C-like cells in chemically defined conditions, while the deletion of endogenous Nelfa does not block this transition. We also demonstrate that Nelfa overexpression significantly enhances somatic cell reprogramming efficiency. Interestingly, we found that the co-overexpression of Nelfa and Bcl2 robustly activates the 2C-like state in ESCs and endows the cells with dual cell fate potential. We further demonstrate that Bcl2 overexpression upregulates endogenous Nelfa expression and can induce the 2C-like state in ESCs even in the absence of Nelfa. Our findings highlight the importance of BCL2 in the regulation of the 2C-like state and provide insights into the mechanism underlying the roles of Nelfa and Bcl2 in the establishment and regulation of the totipotent state in mouse ESCs.


Assuntos
Células-Tronco Embrionárias , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
12.
J Mol Cell Biol ; 15(8)2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-37604781

RESUMO

Endogenous retroviruses (ERVs) are important components of transposable elements that constitute ∼40% of the mouse genome. ERVs exhibit dynamic expression patterns during early embryonic development and are engaged in numerous biological processes. Therefore, ERV expression must be closely monitored in cells. Most studies have focused on the regulation of ERV expression in mouse embryonic stem cells (ESCs) and during early embryonic development. This review touches on the classification, expression, and functions of ERVs in mouse ESCs and early embryos and mainly discusses ERV modulation strategies from the perspectives of transcription, epigenetic modification, nucleosome/chromatin assembly, and post-transcriptional control.


Assuntos
Retrovirus Endógenos , Animais , Camundongos , Retrovirus Endógenos/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas
13.
Arch Toxicol ; 98(2): 551-565, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38085275

RESUMO

The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.


Assuntos
Petróleo , Hidrocarbonetos Policíclicos Aromáticos , Camundongos , Animais , Petróleo/toxicidade , Petróleo/análise , Peixe-Zebra , Células-Tronco Embrionárias Murinas
14.
Cell Rep ; 43(1): 113615, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38159280

RESUMO

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.


Assuntos
Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo
15.
FEBS Open Bio ; 14(2): 309-321, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38098212

RESUMO

The linker histone H1 C-terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1-4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1-4 with a photoactivatable GFP (PA-GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1-4 protein.


Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Cromatina , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo
16.
Dev Cell ; 59(2): 175-186.e8, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38159568

RESUMO

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.


Assuntos
Proteínas Hedgehog , Células-Tronco Embrionárias Murinas , Camundongos , Animais , Proteínas Hedgehog/metabolismo , Glândulas Mamárias Animais , Células Epiteliais , Diferenciação Celular , Organoides
17.
Exp Cell Res ; 435(1): 113902, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38145818

RESUMO

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Neurônios/metabolismo , Linhagem Celular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-38083144

RESUMO

Accurate single cell segmentation provides means to monitor the behavior of single cell within a population of cells. Time-lapse fluorescence images are used to reveal heterogeneous nature of single mouse embryonic stem cell (ESC) colony and monitor fluctuations of the cell states. Automatic quantification of speed and status shifts of the ESCs depends on accurate single cell segmentation that is used to calculate the 3D center of every cell and track this cell for the quantification. This study proposes a new 3D U-net to accurately detect center of each single cell in 3D confocal images. The dimension of input 3D images to the U-net is flexible so that multiple center detections from different image directions can be implemented simultaneously to improve the center detection accuracy. This study showed that our method can improve accuracy for cell center detection and thus the quantification for ESC speeds and status shifts.


Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador , Animais , Camundongos , Processamento de Imagem Assistida por Computador/métodos , Células-Tronco Embrionárias Murinas , Imageamento Tridimensional/métodos , Microscopia de Fluorescência
19.
J Vis Exp ; (202)2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38145374

RESUMO

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.


Assuntos
Células-Tronco Embrionárias Murinas , Ácidos Nucleicos , Animais , Camundongos , Ácidos Nucleicos/metabolismo , Transfecção , Células-Tronco Embrionárias/metabolismo , Plasmídeos/genética , Mamíferos/genética
20.
Stem Cell Res Ther ; 14(1): 371, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38110996

RESUMO

BACKGROUND: Morbidity and mortality associated with cardiovascular diseases, such as myocardial infarction, stem from the inability of terminally differentiated cardiomyocytes to regenerate, and thus repair the damaged myocardial tissue structure. The molecular biological mechanisms behind the lack of regenerative capacity for those cardiomyocytes remains to be fully elucidated. Recent studies have shown that c-Jun serves as a cell cycle regulator for somatic cell fates, playing a key role in multiple molecular pathways, including the inhibition of cellular reprogramming, promoting angiogenesis, and aggravation of cardiac hypertrophy, but its role in cardiac development is largely unknown. This study aims to delineate the role of c-Jun in promoting early-stage cardiac differentiation. METHODS: The c-Jun gene in mouse embryonic stem cells (mESCs) was knocked out with CRISPR-Cas9, and the hanging drop method used to prepare the resulting embryoid bodies. Cardiac differentiation was evaluated up to 9 days after c-Jun knockout (ko) via immunofluorescence, flow cytometric, and qPCR analyses. RESULTS: Compared to the wild-type control group, obvious beating was observed among the c-Jun-ko mESCs after 6 days, which was also associated with significant increases in myocardial marker expression. Additionally, markers associated with mesoderm and endoderm cell layer development, essential for further differentiation of ESCs into cardiomyocytes, were also up-regulated in the c-Jun-ko cell group. CONCLUSIONS: Knocking out c-Jun directs ESCs toward a meso-endodermal cell lineage fate, in turn leading to generation of beating myocardial cells. Thus, c-Jun plays an important role in regulating early cardiac cell development.


Assuntos
Corpos Embrioides , Miócitos Cardíacos , Proteínas Proto-Oncogênicas c-jun , Animais , Camundongos , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias Murinas , Miocárdio , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo
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