RESUMO
Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.
Assuntos
Redes Reguladoras de Genes , Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Fosforilação , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Inhalation anesthesia stands as a pivotal modality within clinical anesthesia practices. Beyond its primary anesthetic effects, inhaled anesthetics have non-anesthetic effects, exerting bidirectional influences on the physiological state of the body and disease progression. These effects encompass impaired cognitive function, inhibition of embryonic development, influence on tumor progression, and so forth. For many years, inhaled anesthetics were viewed as inhibitors of stem cell fate regulation. However, there is now a growing appreciation that inhaled anesthetics promote stem cell biological functions and thus are now regarded as a double-edged sword affecting stem cell fate. In this review, the effects of inhaled anesthetics on self-renewal and differentiation of neural stem cells (NSCs), embryonic stem cells (ESCs), and cancer stem cells (CSCs) were summarized. The mechanisms of inhaled anesthetics involving cell cycle, metabolism, stemness, and niche of stem cells were also discussed. A comprehensive understanding of these effects will enhance our comprehension of how inhaled anesthetics impact the human body, thus promising breakthroughs in the development of novel strategies for innovative stem cell therapy approaches.
Assuntos
Anestésicos Inalatórios , Diferenciação Celular , Células-Tronco Neurais , Humanos , Diferenciação Celular/efeitos dos fármacos , Anestésicos Inalatórios/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Animais , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Autorrenovação Celular/efeitos dos fármacosRESUMO
Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.
Assuntos
Proteínas de Peixes , Peixes , Proteína Homeobox Nanog , Animais , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Evolução Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development.
Assuntos
Imagem com Lapso de Tempo , Inativação do Cromossomo X , Inativação do Cromossomo X/genética , Animais , Feminino , Camundongos , Imagem com Lapso de Tempo/métodos , Diferenciação Celular/genética , Análise de Célula Única/métodos , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Cromossomo X/genética , Genes ReporterRESUMO
Repetitive sequences play an indispensable role in gene expression, transcriptional regulation, and chromosome arrangements through trans and cis regulation. In this review, focusing on recent advances, we summarize the epigenetic regulatory mechanisms of repetitive sequences in embryonic stem cells. We aim to bridge the knowledge gap by discussing DNA damage repair pathway choices on repetitive sequences and summarizing the significance of chromatin organization on repetitive sequences in response to DNA damage. By consolidating these insights, we underscore the critical relationship between the stability of repetitive sequences and early embryonic development, seeking to provide a deeper understanding of repetitive sequence stability and setting the stage for further research and potential therapeutic strategies in developmental biology and regenerative medicine.
Assuntos
Células-Tronco Embrionárias , Sequências Repetitivas de Ácido Nucleico , Humanos , Animais , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Sequências Repetitivas de Ácido Nucleico/genética , Epigênese Genética , Cromatina/metabolismo , Cromatina/genética , Reparo do DNA , Dano ao DNA , Instabilidade GenômicaRESUMO
BACKGROUND: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium. METHODS: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions. RESULTS: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions. CONCLUSIONS: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.
Assuntos
Células-Tronco Embrionárias , Animais , Meios de Cultura Livres de Soro/farmacologia , Suínos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Diferenciação Celular , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células , Blastocisto/citologia , Blastocisto/metabolismo , Células CultivadasRESUMO
The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-Δ alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. p75NTR function is therefore largely dispensable for normal development, growth and basal homeostasis in the rat. However, the availability of constitutive and conditional p75NTREx2-Δ rats provides new opportunities to investigate specific roles of p75NTR upon injury and during tissue repair.
Assuntos
Ratos Transgênicos , Animais , Ratos , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Fertilidade/genética , Feminino , Encéfalo/metabolismo , Encéfalo/crescimento & desenvolvimento , Masculino , Éxons/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Embrionárias/metabolismo , Receptores de Fatores de CrescimentoRESUMO
DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the "PRC2-AgeIndex," defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.
Assuntos
Envelhecimento , Biomarcadores , Metilação de DNA , Epigênese Genética , Complexo Repressor Polycomb 2 , Rejuvenescimento , Animais , Envelhecimento/metabolismo , Envelhecimento/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Rejuvenescimento/fisiologia , Biomarcadores/metabolismo , Humanos , Camundongos , Senescência Celular/genética , Ilhas de CpG , Células-Tronco Embrionárias/metabolismo , Masculino , FemininoRESUMO
Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication.
Assuntos
Ciclo Celular , Replicação do DNA , Células-Tronco Embrionárias , Origem de Replicação , Animais , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Camundongos , Ciclo Celular/genética , Fase S/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Instabilidade Genômica , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , HumanosRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) is a highly conserved nuclear protein in multicellular organisms that by modulating chromatin opening facilitates gene expression during development. All reported Parp1 null knockout mouse strains are viable with no developmental anomalies. It was believed that functional redundancy with other PARP family members, mainly PARP2, explains such a controversy. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains, making the absence of developmental problems in Parp1 mice knockouts unlikely. Contrary to prior assumptions, in our analysis of the best-investigated Parp1 knockout mouse strain, we identified persistent mRNA expression, albeit at reduced levels. Transcript analysis revealed an alternatively spliced Parp1 variant lacking exon 2. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. The decreased level of poly(ADP-ribose) (pADPr) was detected in Parp1 knockout embryonic stem (ES) cells with western blotting analysis, but immunofluorescence staining did not detect any difference in distribution or level of pADPr in nuclei of knockout ES cells. pADPr level in double Parp1 Parg mutant ES cells greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Poli(ADP-Ribose) Polimerase-1 , Animais , Camundongos , Processamento Alternativo , Células-Tronco Embrionárias/metabolismo , Loci Gênicos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Vascular tissue engineering is a promising approach for regenerating damaged blood vessels and developing new therapeutic approaches for heart disease treatment. To date, different sources of cells have been recognized that offer assistance within the recovery of heart supply routes and veins with distinctive capacities and are compelling for heart regeneration. However, some challenges still remain that need to be overcome to establish the full potential application of these cells. In this paper, we review the different cell sources used for vascular tissue engineering, focusing on extraembryonic tissue-derived cells (ESCs), and elucidate their roles in cardiovascular disease. In addition, we highlight the intricate interplay between mechanical and biochemical factors in regulating mesenchymal stem cell (MSC) differentiation, offering insights into optimizing their application in vascular tissues.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Regeneração , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Regeneração/fisiologia , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/fisiologia , Vasos Sanguíneos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Doenças Cardiovasculares/terapia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologiaRESUMO
Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI.
Assuntos
Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Pluripotentes Induzidas/metabolismo , Ratos , Ratos Sprague-Dawley , Células Piramidais/metabolismo , Células Piramidais/patologia , Camundongos , Células-Tronco Neurais/transplante , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Regeneração Nervosa , Axônios/metabolismoRESUMO
Transposable elements (TEs) have emerged as important factors in establishing the cell type-specific gene regulatory networks and evolutionary novelty of embryonic and placental development. Recently, studies on the role of TEs and their dysregulation in cancers have shed light on the transcriptional, transpositional, and regulatory activity of TEs, revealing that the activation of developmental transcriptional programs by TEs may have a role in the dedifferentiation of cancer cells to the progenitor-like cell states. This essay reviews the recent evidence of the cis-regulatory TEs (henceforth crTE) in normal development and malignancy as well as the key transcription factors and regulatory pathways that are implicated in both cell states, and presents existing gaps remaining to be studied, limitations of current technologies, and therapeutic possibilities.
Assuntos
Elementos de DNA Transponíveis , Células-Tronco Embrionárias , Neoplasias , Placenta , Humanos , Elementos de DNA Transponíveis/genética , Placenta/metabolismo , Placenta/citologia , Animais , Feminino , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Neoplasias/genética , Neoplasias/patologia , Gravidez , Elementos Facilitadores Genéticos/genética , Desdiferenciação Celular/genética , Redes Reguladoras de Genes , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
Embryonic and adult stem cells enable development and regeneration. Embryonic cells, like adult stem cells, can enter dormancy as part of their lifecycle. Recent evidence suggests that this cellular transition to dormancy requires active rewiring of metabolism. The dormancy-induced metabolic switches in embryonic and adult stem cells are explored here.
Assuntos
Células-Tronco Embrionárias , Animais , Humanos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologiaRESUMO
Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1-10 µl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.
Assuntos
Eletroporação , Transfecção , Transfecção/métodos , Eletroporação/métodos , Animais , Camundongos , Linhagem Celular , Humanos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismoRESUMO
Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead , Mesoderma , Síndrome da Persistência do Padrão de Circulação Fetal , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Animais , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Camundongos , Elementos Facilitadores Genéticos/genética , Mesoderma/metabolismo , Mesoderma/embriologia , Pulmão/patologia , Células Endoteliais/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Células-Tronco Embrionárias/metabolismo , Alvéolos Pulmonares/anormalidadesRESUMO
Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.
Assuntos
Duplicação Gênica , Edição de Genes , Genoma Humano , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , DNA/genética , Animais , Células-Tronco Embrionárias/metabolismo , Cromossomos Humanos/genéticaRESUMO
The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.
Assuntos
Hematopoese , Camundongos Knockout , Animais , Hematopoese/genética , Camundongos , Humanos , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Mutação , Anemia/genética , Masculino , Células-Tronco Embrionárias/metabolismoRESUMO
The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.
Assuntos
Diferenciação Celular , Reprogramação Celular , Fator 3 de Transcrição de Octâmero , Oxirredução , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Animais , Camundongos , Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , HumanosRESUMO
Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.