RESUMEN
In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias Humanas , Células Ganglionares de la Retina , Humanos , Animales , Ratones , Células Madre Embrionarias Humanas/citología , Células Ganglionares de la Retina/citología , Trasplante de Células Madre/métodos , Organoides/citología , Organoides/trasplante , Técnicas de Cultivo de Célula/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Retina/citología , Células Madre Embrionarias/citologíaRESUMEN
During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.
Asunto(s)
Transdiferenciación Celular , Factores de Transcripción , Trofoblastos , Humanos , Trofoblastos/citología , Trofoblastos/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factor de Transcripción GATA3/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA2/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción AP-2/genética , Blastocisto/metabolismo , Blastocisto/citología , Embarazo , Diferenciación CelularRESUMEN
BACKGROUND: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. METHODS: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. RESULTS: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. CONCLUSIONS: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Proteínas de Homeodominio , Células Madre Embrionarias Humanas , Próstata , Factores de Transcripción , Humanos , Próstata/citología , Próstata/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Masculino , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Organoides/metabolismo , Organoides/citología , Ratones , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Animales , Línea CelularRESUMEN
CD98, also known as SLC3A2, is a multifunctional cell surface molecule consisting of amino acid transporters. CD98 is ubiquitously expressed in many types of tissues, but expressed at higher levels in cancerous tissues than in normal tissues. CD98 is also upregulated in most hepatocellular carcinoma (HCC) patients; however, the function of CD98 in HCC cells has been little studied. In this study, we generated a panel of monoclonal antibodies (MAbs) against surface proteins on human embryonic stem cells (hESCs). NPB15, one of the MAbs, bound to hESCs and various cancer cells, including HCC cells and non-small cell lung carcinoma (NSCLC) cells, but not to peripheral blood mononuclear cells (PBMCs) and primary hepatocytes. Immunoprecipitation and mass spectrometry identified the target antigen of NPB15 as CD98. CD98 depletion decreased cell proliferation, clonogenic survival, and migration and induced apoptosis in HCC cells. In addition, CD98 depletion decreased the expression of cancer stem cell (CSC) markers in HCC cells. In tumorsphere cultures, the expression of CD98 interacting with NPB15 was significantly increased, as were known CSC markers. After cell sorting by NPB15, cells with high expression of CD98 (CD98-high) showed higher clonogenic survival than cells with low expression of CD98 (CD98-low) in HCC cells, suggesting CD98 as a potential CSC marker on HCC cells. The chimeric version of NPB15 was able to induce antibody-dependent cellular cytotoxicity (ADCC) against HCC cells in vitro. NPB15 injection showed antitumor activity in an HCC xenograft mouse model. The results suggest that NPB15 may be developed as a therapeutic antibody for HCC patients.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma Hepatocelular , Proteína-1 Reguladora de Fusión , Neoplasias Hepáticas , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Animales , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/inmunología , Proteína-1 Reguladora de Fusión/metabolismo , Proteína-1 Reguladora de Fusión/inmunología , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/inmunología , Proliferación Celular , Línea Celular Tumoral , Apoptosis , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/inmunología , Cadena Pesada de la Proteína-1 Reguladora de FusiónRESUMEN
Epidermal transplantation is a common and widely used surgical technique in clinical medicine. Derivatives of embryonic stem cells have the potential to serve as a source of transplantable cells. However, allograft rejection is one of the main challenges. To investigate the immunogenicity of keratinocytes derived from human embryonic stem cells (ESKCs), we conducted a series of in vivo and in vitro experiments. The results showed that ESKCs have low HLA molecule expression, limited antigen presentation capabilities, and a weak ability to stimulate the proliferation and secretion of inflammatory factors in allogeneic PBMCs in vitro. In humanized immune mouse models, ESKCs elicited weak transplant rejection responses in the host. Overall, we found that ESKCs have low immunogenicity and may have potential applications in the field of regenerative medicine.
Asunto(s)
Células Madre Embrionarias Humanas , Queratinocitos , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/citología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/inmunología , Células Madre Embrionarias Humanas/metabolismo , Animales , Ratones , Proliferación Celular , Rechazo de Injerto/inmunología , Antígenos HLA/inmunología , Antígenos HLA/metabolismoRESUMEN
Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.
Asunto(s)
Astrocitos , Proteína 2 de Unión a Metil-CpG , Mitocondrias , Mutación , Neuronas , Especies Reactivas de Oxígeno , Síndrome de Rett , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Mitocondrias/metabolismo , Astrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Humanos , Neuronas/metabolismo , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patología , Células Madre Embrionarias Humanas/metabolismo , Línea CelularRESUMEN
BACKGROUND: Cardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells. METHODS AND RESULTS: Initially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro. CONCLUSION: In conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias Humanas , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Animales , Ratas , Linaje de la Célula , Células CultivadasRESUMEN
Human pluripotent stem cells (hPSCs) are pivotal in regenerative medicine, yet their in vitro expansion often leads to genetic abnormalities, raising concerns about their safety in clinical applications. This study analyzed ten human embryonic stem cell lines across multiple passages to elucidate the dynamics of chromosomal abnormalities and single-nucleotide variants (SNVs) in 380 cancer-related genes. Prolonged in vitro culture resulted in 80% of the lines acquiring gains of chromosome 20q or 1q, both known for conferring an in vitro growth advantage. 70% of lines also acquired other copy number variants (CNVs) outside the recurrent set. Additionally, we detected 122 SNVs in 88 genes, with all lines acquiring at least one de novo SNV during culture. Our findings showed higher loads of both CNVs and SNVs at later passages, which were due to the cumulative acquisition of mutations over a longer time in culture, and not to an increased rate of mutagenesis over time. Importantly, we observed that SNVs and rare CNVs followed the acquisition of chromosomal gains in 1q and 20q, while most of the low-passage and genetically balanced samples were devoid of cancer-associated mutations. This suggests that recurrent chromosomal abnormalities are potential drivers for the acquisition of other mutations.
Asunto(s)
Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN , Mutación , Neoplasias , Células Madre Pluripotentes , Humanos , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Células Madre Pluripotentes/metabolismo , Variaciones en el Número de Copia de ADN/genética , Polimorfismo de Nucleótido Simple/genética , Línea Celular , Células Madre Embrionarias Humanas/metabolismo , Técnicas de Cultivo de Célula/métodosRESUMEN
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Asunto(s)
Diferenciación Celular , Endodermo , Epigénesis Genética , Células Madre Embrionarias Humanas , Humanos , Endodermo/citología , Endodermo/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Faringe/citología , Faringe/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , RatonesRESUMEN
Individuals with the Autism Susceptibility Candidate 2 (AUTS2) gene disruptions exhibit symptoms such as intellectual disability, microcephaly, growth retardation, and distinct skeletal and facial differences. The role of AUTS2 in neurodevelopment has been investigated using animal and embryonic stem cell models. However, the precise molecular mechanisms of how AUTS2 influences neurodevelopment, particularly in humans, are not thoroughly understood. Our study employed a 3D human cerebral organoid culture system, in combination with genetic, genomic, cellular, and molecular approaches, to investigate how AUTS2 impacts neurodevelopment through cellular signaling pathways. We used CRISPR/Cas9 technology to create AUTS2-deficient human embryonic stem cells and then generated cerebral organoids with these cells. Our transcriptomic analyses revealed that the absence of AUTS2 in cerebral organoids reduces the populations of cells committed to the neuronal lineage, resulting in an overabundance of cells with a transcription profile resembling that of choroid plexus (ChP) cells. Intriguingly, we found that AUTS2 negatively regulates the WNT/ß-catenin signaling pathway, evidenced by its overactivation in AUTS2-deficient cerebral organoids and in luciferase reporter cells lacking AUTS2. Importantly, treating the AUTS2-deficient cerebral organoids with a WNT inhibitor reversed the overexpression of ChP genes and increased the downregulated neuronal gene expression. This study offers new insights into the role of AUTS2 in neurodevelopment and suggests potential targeted therapies for neurodevelopmental disorders.
Asunto(s)
Diferenciación Celular , Proteínas del Citoesqueleto , Neuronas , Organoides , Factores de Transcripción , Vía de Señalización Wnt , Humanos , Organoides/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Sistemas CRISPR-Cas , beta Catenina/metabolismo , beta Catenina/genética , Células Madre Embrionarias Humanas/metabolismo , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
Nucleoli are fundamentally essential sites for ribosome biogenesis in cells and formed by liquid-liquid phase separation (LLPS) for a multilayer condensate structure. How the nucleoli integrity is maintained remains poorly understood. Here, we reveal that METTL3/METTL14, the typical methyltransferase complex catalyzing N6-methyladnosine (m6A) on mRNAs maintain nucleoli integrity in human embryonic stem cells (hESCs). METTL3/METTL14 deficiency impairs nucleoli and leads to the complete loss of self-renewal in hESCs. We further show that SUV39H1/H2 protein, the methyltransferases catalyzing H3K9me3 were dramatically elevated in METTL3/METTL14 deficient cells, which causes an accumulation and infiltration of H3K9me3 across the whole nucleolus and impairs the LLPS. Mechanistically, METTL3/METTL14 complex serves as an essential adapter for CRL4 E3 ubiquitin ligase targeting SUV39H1/H2 for polyubiquitination and proteasomal degradation and therefore prevents H3K9me3 accumulation in nucleoli. Together, these findings uncover a previously unknown role of METTL3/METTL14 to maintain nucleoli integrity by facilitating SUV39H1/H2 degradation in human cells.
Asunto(s)
Nucléolo Celular , Metiltransferasas , Proteínas Represoras , Humanos , Metiltransferasas/metabolismo , Metiltransferasas/genética , Nucléolo Celular/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Histonas/metabolismo , Ubiquitinación , Células Madre Embrionarias Humanas/metabolismo , Proteolisis , Células HEK293 , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , N-Metiltransferasa de Histona-LisinaRESUMEN
Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.
Asunto(s)
Baculoviridae , Sistemas CRISPR-Cas , Diferenciación Celular , Edición Génica , Vectores Genéticos , Células Madre Pluripotentes , Microglobulina beta-2 , Humanos , Microglobulina beta-2/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Baculoviridae/genética , Edición Génica/métodos , Vectores Genéticos/genética , Diferenciación Celular/genética , Técnicas de Inactivación de Genes/métodos , Animales , Fibroblastos/metabolismo , Fibroblastos/citología , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , RatonesRESUMEN
Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sistemas CRISPR-Cas , Marcación de Gen , Células Madre Embrionarias Humanas , Humanos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Técnicas de Sustitución del Gen , Genes Reporteros , Diferenciación Celular , Proteína Fluorescente RojaRESUMEN
The KCNQ1 gene encodes a voltage-gated potassium channel required for cardiac action potentials. Mutations in this gene have been associated with hereditary long QT syndrome 1, Jervell and Lange-Nielsen syndromes, and familial atrial fibrillation. The NM_000218.3(KCNQ1): c.604 + 2T > C mutation has been categorized as the causative variant leading to LQT1. In this study, we generated a KCNQ1 (c.644 + 2T > C) mutation human embryonic stem cell line WAe009-A-1L based on CRISPR base editing system. WAe009-A-1L cell has the potential to differentiate cardiomyocytes and would be used as an in vitro disease model for mechanism exploration and drug screening.
Asunto(s)
Edición Génica , Células Madre Embrionarias Humanas , Canal de Potasio KCNQ1 , Mutación , Humanos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Edición Génica/métodos , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Línea Celular , Sistemas CRISPR-Cas , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Diferenciación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway-Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.
Asunto(s)
Autofagia , Diferenciación Celular , Células Madre Embrionarias Humanas , Proteostasis , Humanos , Autofagia/genética , Células Madre Embrionarias Humanas/metabolismo , Diferenciación Celular/genética , Endopeptidasas/metabolismo , Endopeptidasas/genética , Técnicas de Silenciamiento del GenAsunto(s)
Investigaciones con Embriones , Embrión de Mamíferos , Guías como Asunto , Células Madre Embrionarias Humanas , Confianza , Humanos , Investigaciones con Embriones/ética , Investigaciones con Embriones/legislación & jurisprudencia , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Células Madre Embrionarias Humanas/citología , Laboratorios/ética , Laboratorios/normas , Modelos BiológicosRESUMEN
YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Diferenciación Celular , Células Madre Embrionarias Humanas , Espermatogénesis , Factores de Transcripción , Proteínas Señalizadoras YAP , Masculino , Humanos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genéticaRESUMEN
Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Retina , Factores de Transcripción , Animales , Humanos , Ratones , Elementos de Facilitación Genéticos/genética , Evolución Molecular , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Microftalmía/genética , Microftalmía/patología , Neurogénesis/genética , Organoides/metabolismo , Retina/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explore the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibit growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single-cell transcriptome levels. This mutation induces premature stem-like cell exhaustion, accelerates the maturation of GABAergic neurons, and disrupts neurodevelopmental and synaptic pathways. Additionally, it specifically hinders CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrors the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.
Asunto(s)
Factor de Unión a CCCTC , Trastornos del Neurodesarrollo , Organoides , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Humanos , Animales , Ratones , Trastornos del Neurodesarrollo/genética , Organoides/metabolismo , Mutación , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Masculino , Cromatina/metabolismo , Cromatina/genética , Femenino , Encéfalo/metabolismo , Encéfalo/patología , Mutación Puntual , Células Madre Embrionarias Humanas/metabolismoRESUMEN
Previous retrospective cohort studies have found that, compared with oxygen tension in the uterus and fallopian tubes (2â¯%-8â¯%), exposure of pre-implantation embryos to atmospheric oxygen tension (AtmO2, 20â¯%) during assisted reproductive technology(ART) can affect embryo quality, pregnancy outcomes and offspring health. However, current research on the effects and mechanisms of AtmO2 on the development of embryos and offspring is mainly limited to animal experiments. Human embryonic stem cells (hESCs) play a special and irreplaceable role in the study of early human embryonic development. In this study, we used hESCs as a model to elucidate the possible effects and mechanisms of AtmO2 exposure on human embryonic development. We found that exposure to AtmO2 can reduce cell viability, produce oxidative stress, increase DNA damage, initiate DNA repair, activate autophagy, and increase cell apoptosis. We also noticed that approximately 50â¯% of hESCs survived, adapted and proliferated through high expression of self-renewal and pluripotency regulatory factors, and affected embryoid body differentiation. These data indicate that hESCs experience oxidative stress, accumulation of DNA damage, and activate DNA damage response under the selective pressure of AtmO2.Some hESCs undergo cell death, whereas other hESCs adapt and proliferate through increased expression of self-renewal genes. The current findings provide in vitro evidence that exposure to AtmO2 during the early preimplantation stage negatively affects hESCs.