RESUMO
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.
Assuntos
Autofagia , Diferenciação Celular , Células-Tronco Embrionárias Humanas , Proteostase , Humanos , Autofagia/genética , Células-Tronco Embrionárias Humanas/metabolismo , Diferenciação Celular/genética , Endopeptidases/metabolismo , Endopeptidases/genética , Técnicas de Silenciamento de GenesRESUMO
Mutations in STAMBP have been well-established to cause congenital human microcephaly-capillary malformation (MIC-CAP) syndrome, a rare genetic disorder characterized by global developmental delay, severe microcephaly, capillary malformations, etc. Previous biochemical investigations and loss-of-function studies in mice have provided insights into the mechanism of STAMBP, however, it remains controversial how STAMBP deficiency leads to malformation of those affected tissues in patients. In this study, we investigated the function and underlying mechanism of STAMBP during neural differentiation of human embryonic stem cells (hESCs). We found that STAMBP is dispensable for the pluripotency maintenance or neural differentiation of hESCs. However, neural progenitor cells (NPCs) derived from STAMBP-deficient hESCs fail to be long-term maintained/expanded in vitro. We identified the anti-apoptotic protein CFLAR is down-regulated in those affected NPCs and ectopic expression of CFLAR rescues NPC defects induced by STAMBP-deficiency. Our study not only provides novel insight into the mechanism of neural defects in STAMBP mutant patients, it also indicates that the death receptor mediated apoptosis is an obstacle for long-term maintenance/expansion of NPCs in vitro thus counteracting this cell death pathway could be beneficial to the generation of NPCs in vitro.
RESUMO
Embryonic stem cells (ESCs) are defined as stem cells with self-renewing and differentiation capabilities. These unique properties are tightly regulated and controlled by complex genetic and molecular mechanisms, whose understanding is essential for both basic and translational research. A large number of studies have mostly focused on understanding the molecular mechanisms governing pluripotency and differentiation of ESCs, while the regulation of proliferation has received comparably less attention. Here, we investigate the role of ZZZ3 (zinc finger ZZ-type containing 3) in human ESCs homeostasis. We found that knockdown of ZZZ3 negatively impacts ribosome biogenesis, translation, and mTOR signaling, leading to a significant reduction in cell proliferation. This process occurs without affecting pluripotency, suggesting that ZZZ3-depleted ESCs enter a "dormant-like" state and that proliferation and pluripotency can be uncoupled also in human ESCs.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA , Células-Tronco Embrionárias Humanas , Serina-Treonina Quinases TOR , Fatores de Transcrição , Humanos , Diferenciação Celular/genética , Homeostase , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Biossíntese de Proteínas , Ribossomos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME). Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.
RESUMO
Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.
Assuntos
Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Embrionárias , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de SinaisRESUMO
Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-ß, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.
Assuntos
Fluorocarbonos , Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Fluorocarbonos/toxicidade , Diferenciação Celular/efeitos dos fármacosRESUMO
In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.
Assuntos
Caspase 3 , Caspase 9 , Diferenciação Celular , Células-Tronco Embrionárias Humanas , Miócitos Cardíacos , Humanos , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Caspase 9/genética , Linhagem Celular , Ativação Enzimática , Células-Tronco Embrionárias Humanas/enzimologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/citologiaRESUMO
BACKGROUND: Retinal degenerative disorders (RDDs) cause vision loss by damaging retinal neurons and photoreceptors, affecting individuals of all ages. Cell-based therapy has emerged as an effective approach for the treatment of RDDs with promising results. This meta-analysis aims to comprehensively evaluate the efficacy of cell therapy in treating age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Stargardt macular degeneration (SMD) as the most prevalent RDDs. METHODS: PubMed, Scopus, Web of Science, and Embase were searched using keywords related to various retinal diseases and cell therapy treatments until November 25th, 2023. The studies' quality was evaluated using the Joanna Briggs Institute's (JBI) checklist for quasi-experimental studies. Visual acuity measured as LogMAR score was used as our main outcome. A three-level random-effect meta-analysis was used to explore the visual acuity in patients who received cell-based therapy. Heterogeneity among the included studies was evaluated using subgroup and sensitivity analyses. Moreover, meta-regression for the type of cells, year of publication, and mean age of participants were performed. RESULTS: Overall, 8345 studies were retrieved by the search, and 39 met the eligibility criteria, out of which 18 studies with a total of 224 eyes were included in the meta-analysis. There were 12 studies conducted on AMD, 7 on SMD, and 2 on RP. Cell therapy for AMD showed significant improvement in LogMAR (p < 0.05). Also, cell therapy decreased the LogMAR score in SMD and RP (p < 0.01 and p < 0.0001, respectively). Across all conditions, no substantial publication bias was detected (p < 0.05). CONCLUSION: The findings of the study highlight that the application of cell therapy can enhance the visual acuity in AMD, SMD, and RP.
Assuntos
Degeneração Macular , Retina , Humanos , Degeneração Macular/terapia , Acuidade Visual , Terapia Baseada em Transplante de Células e TecidosRESUMO
Human embryonic stem cells (hESCs) can proliferate infinitely (self-renewal) and give rise to almost all types of somatic cells (pluripotency). Hence, understanding the molecular mechanism of pluripotency regulation is important for applications of hESCs in regenerative medicine. Here we report that PATZ1 is a key factor that regulates pluripotency and metabolism in hESCs. We found that depletion of PATZ1 is associated with rapid downregulation of master pluripotency genes and prominent deceleration of cell growth. We also revealed that PATZ1 regulates hESC pluripotency though binding the regulatory regions of OCT4 and NANOG. In addition, we demonstrated PATZ1 is a key node in the OCT4/NANOG transcriptional network. We further revealed that PATZ1 is essential for cell growth in hESCs. Importantly, we discovered that depletion of PATZ1 drives hESCs to exploit glycolysis which energetically compensates for the mitochondrial dysfunction. Overall, our study establishes the fundamental role of PATZ1 in regulating pluripotency in hESCs. Moreover, PATZ1 is essential for maintaining a steady metabolic homeostasis to refine the stemness of hESCs.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes , Humanos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Zinco , Motivos AT-Hook , Diferenciação Celular/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco , Proteínas Repressoras/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismoRESUMO
Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.
Assuntos
Neoplasias Encefálicas , Células-Tronco Pluripotentes Induzidas , Criança , Humanos , Animais , Camundongos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasias Encefálicas/patologia , Encéfalo/patologia , MutaçãoRESUMO
BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.
Assuntos
Progesterona , Proibitinas , Gravidez , Feminino , Humanos , Progesterona/farmacologia , Progesterona/metabolismo , Decídua/metabolismo , Receptores de Progesterona/genética , Progestinas/metabolismo , Endométrio/metabolismo , Células Estromais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
In vitro modeling is a powerful approach to investigate the pathomechanisms driving human congenital conditions. Here we use human embryonic stem cells (hESCs) to model Nager and Rodriguez syndromes, two craniofacial conditions characterized by hypoplastic neural crest-derived craniofacial bones, caused by pathogenic variants of SF3B4, a core component of the spliceosome. We observed that siRNA-mediated knockdown of SF3B4 interferes with the production of hESC-derived neural crest cells, as seen by a marked reduction in neural crest gene expression. This phenotype is associated with an increase in neural crest cell apoptosis and premature neuronal differentiation. Altogether these results point at a role of SF3B4 in neural crest cell survival, maintenance, and differentiation. We propose that the dysregulation of these processes may contribute to Nager/Rodriguez syndrome associated craniofacial defects.
RESUMO
BACKGROUND: Clinically, recurrent spontaneous abortion (RSA) is a pregnancy illness that is difficult to treat. Impaired decidualization is a documented cause of RSA, but the etiology and mechanism are still unknown. cAMP-responsive element binding protein 5 (CREB5) is a member of the ATF/CREB family. CREB5 has been reported to be related to pathological pregnancy, but there are few related studies on this topic in patients with RSA, and the underlying mechanism is unclear. METHODS: We collected decidual tissues from RSA patients and healthy pregnant women to measure the expression level of CREB5, PRL, IGFBP1, ATG5, LC3B, and SQSTM/p62. Then, the changes in CREB5 expression and autophagy levels were measured in human endometrial stromal cells (hESCs) during decidualization. The expression levels of PRL and IGFBP1 were tested in sh-CREB5/ov-CREB5 hESCs after decidualization induction, and the autophagy level in sh-CREB5/ov-CREB5 hESCs was measured without decidualization induction. The decidualization ability of sh-CREB5 and ov-CREB5 hESCs treated with an autophagy inducer or inhibitor was measured. To investigate the effect of CREB5 in hESCs on the invasion and migration of HTR8/SVneo cells, we performed a coculture experiment. Finally, we examined the expression of CREB5 and autophagy key proteins in mouse decidual tissues by constructing an abortion mouse model. RESULTS: In our study, we found that the expression of CREB5 was unusually elevated in the uterine decidua of RSA patients, but the expression of PRL, IGFBP1, and autophagy were decreased. During the decidualization of hESCs, the expression of CREB5 gradually decreases in a time-dependent manner with increasing autophagy. Moreover, by knocking down or overexpressing CREB5 in hESCs, it was found that CREB5 can impair decidualization and reduce autophagy in hESCs. Furthermore, the damage caused by CREB5 in terms of decidualization can be reversed by the addition of an autophagy inducer (rapamycin). In addition, CREB5 can increase the secretion of proteins (IL-1ß and TGF-ß1) in hESCs to inhibit trophoblast invasion and migration. CONCLUSIONS: Our data support the supposition that CREB5 disturbs the decidualization of endometrial stromal cells and interactions at the maternal-fetal interface by inhibiting autophagy and that its abnormal upregulation and dysfunction may lead to RSA. It may function as a diagnostic and therapeutic target for RSA. Similarly, we found that in the spontaneous abortion mouse model, the expression of CREB5 in the decidual tissue of the abortion group was significantly increased, and autophagy was decreased.
Assuntos
Aborto Habitual , Autofagia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Decídua , Feminino , Autofagia/fisiologia , Humanos , Gravidez , Decídua/metabolismo , Decídua/patologia , Aborto Habitual/metabolismo , Aborto Habitual/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Animais , Adulto , Camundongos , Células Estromais/metabolismo , Relações Materno-Fetais/fisiologia , Troca Materno-Fetal/fisiologia , Endométrio/metabolismo , Endométrio/patologia , Proteína A de Ligação a Elemento de Resposta do AMP CíclicoRESUMO
Exploring the mechanism of self-renewal and pluripotency maintenance of human embryonic stem cells (hESCs) is of great significance in basic research and clinical applications, but it has not been fully elucidated. Long non-coding RNAs (lncRNAs) have been shown to play a key role in the self-renewal and pluripotency maintenance of hESCs. We previously reported that the lncRNA ESRG, which is highly expressed in undifferentiated hESCs, can maintain the self-renewal and pluripotency of hPSCs. RNA pull-down mass spectrometry showed that ESRG could bind to other proteins, among which heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) attracted our attention. In this study, we showed that HNRNPA1 can maintain self-renewal and pluripotency of hESCs. ESRG bound to and stabilized HNRNPA1 protein through the ubiquitin-proteasome pathway. In addition, knockdown of ESRG or HNRNPA1 resulted in alternative splicing of TCF3, which originally and primarily encoded E12, to mainly encode E47 and inhibit CDH1 expression. HNRNPA1 could rescue the biological function changes of hESCs caused by ESRG knockdown or overexpression. Our results suggest that ESRG regulates the alternative splicing of TCF3 to affect CDH1 expression and maintain hESCs self-renewal and pluripotency by binding and stabilizing HNRNPA1 protein. This study lays a good foundation for exploring the new molecular regulatory mechanism by which ESRG maintains hESCs self-renewal and pluripotency.
Assuntos
Processamento Alternativo , Ribonucleoproteína Nuclear Heterogênea A1 , Células-Tronco Embrionárias Humanas , RNA Longo não Codificante , Humanos , Processamento Alternativo/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Autorrenovação Celular/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Diferenciação Celular/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genéticaRESUMO
Per- and polyfluoroalkyl substances (PFAS) are a class of highly stable chemicals, widely used in everyday products, and widespread in the environment, even in pregnant women. While epidemiological studies have linked prenatal exposure to PFAS with atopic dermatitis in children, little is known about their toxic effects on skin development, especially during the embryonic stage. In this study, we utilized human embryonic stem cells to generate non-neural ectoderm (NNE) cells and exposed them to six PFAS (perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid (PFBA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS) and perfluorobutyric acid (PFBS)) during the differentiation process to assess their toxicity to early skin development. Our results showed that PFOS altered the spindle-like morphology of NNE cells to a pebble-like morphology, and disrupted several NNE markers, including KRT16, SMYD1, and WISP1. The six PFAS had a high potential to cause hypohidrotic ectodermal dysplasia (HED) by disrupting the expression levels of HED-relevant genes. Transcriptomic analysis revealed that PFOS treatment produced the highest number (1156) of differentially expressed genes (DEGs) among the six PFAS, including the keratinocyte-related genes KRT6A, KRT17, KRT18, KRT24, KRT40, and KRT81. Additionally, we found that PFOS treatment disturbed several signaling pathways that are involved in regulating skin cell fate decisions and differentiation, including TGF-ß, NOTCH, Hedgehog, and Hippo signaling pathways. Interestingly, we discovered that PFOS inhibited, by partially interfering with the expression of cytoskeleton-related genes, the ciliogenesis of NNE cells, which is crucial for the intercellular transduction of the above-mentioned signaling pathways. Overall, our study suggests that PFAS can inhibit ciliogenesis and hamper the transduction of important signaling pathways, leading potential congenital skin diseases. It sheds light on the underlying mechanisms of early embryonic skin developmental toxicity and provides an explanation for the epidemiological data on PFAS. ENVIRONMENTAL IMPLICATION: We employed a model based on human embryonic stem cells to demonstrate that PFOS has the potential to elevate the risk of hypohidrotic ectodermal dysplasia. This is achieved by targeting cilia, inhibiting ciliogenesis, and subsequently disrupting crucial signaling pathways like TGF-ß, NOTCH, Hedgehog, and Hippo, during the early phases of embryonic skin development. Our study highlights the dangers and potential impacts of six PFAS pollutants on human skin development. Additionally, we emphasize the importance of closely considering PFHxA, PFBA, PFHxS, and PFBS, as they have shown the capacity to modify gene expression levels, albeit to a lesser degree.
Assuntos
Ácidos Alcanossulfônicos , Displasia Ectodérmica Anidrótica Tipo 1 , Poluentes Ambientais , Fluorocarbonos , Criança , Humanos , Feminino , Gravidez , Animais , Ouriços , Ácidos Alcanossulfônicos/toxicidade , Alcanossulfonatos , Poluentes Ambientais/toxicidade , Fluorocarbonos/toxicidade , Fator de Crescimento Transformador beta , MicrotúbulosRESUMO
Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future.
Assuntos
Células-Tronco Embrionárias Humanas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Camundongos SCID , Camundongos Endogâmicos NOD , Diferenciação Celular , Fígado , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.
Assuntos
Células-Tronco Embrionárias Humanas , Fator 4 Semelhante a Kruppel , Humanos , Diferenciação Celular , Genes Homeobox , Células Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Fator 4 Semelhante a Kruppel/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.
Assuntos
Proteínas de Ciclo Celular , Células-Tronco Embrionárias Humanas , Proteínas de Ligação a RNA , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Células-Tronco Embrionárias Humanas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Histonas/genética , Histonas/metabolismoRESUMO
Organoids can meet the needs between the use of cell culture and in vivo work, bringing together aspects of multicellular tissues, providing a more similar in vitro system for the study of various components, including host-interactions with pathogens and drug response. Organoids are structures that resemble organs in vivo, originating from pluripotent stem cells (PSCs) or adult stem cells (ASCs). There is great interest in deepening the understanding of the use of this technology to produce information about fungal infections and their treatments. This work aims the use 2D human lung organoid derived from human embryonic stem cells (hESCs), to investigate Cryptococcus neoformans-host interactions. C. neoformans is an opportunistic fungus acquired by inhalation that causes systemic mycosis mainly in immunocompromised individuals. Our work highlights the suitability of human minilungs for the study of C. neoformans infection (adhesion, invasion and replication), the interaction with the surfactant and induction of the host's alveolar pro-inflammatory response.
Assuntos
Criptococose , Cryptococcus neoformans , Células-Tronco Embrionárias Humanas , Humanos , Cryptococcus neoformans/fisiologia , Criptococose/microbiologia , Pulmão/microbiologia , Técnicas de Cultura de CélulasRESUMO
BACKGROUND: Research on human pluripotent stem cells (hPSCs) has shown tremendous progress in cell-based regenerative medicine. Corneal endothelial dysfunction is associated with the loss and degeneration of corneal endothelial cells (CECs), rendering cell replacement a promising therapeutic strategy. However, comprehensive preclinical assessments of hPSC-derived CECs for this cell therapy remain a challenge. RESULTS: Here we defined an adapted differentiation protocol to generate induced corneal endothelial cells (iCECs) consistently and efficiently from clinical-grade human embryonic stem cells (hESCs) with xeno-free medium and manufactured cryopreserved iCECs. Cells express high levels of typical CECs markers and exhibit transendothelial potential properties in vitro typical of iCECs. After rigorous quality control measures, cells meeting all release criteria were available for in vivo studies. We found that there was no overgrowth or tumorigenicity of grafts in immunodeficient mice. After grafting into rabbit models, the surviving iCECs ameliorated edema and recovered corneal opacity. CONCLUSIONS: Our work provides an efficient approach for generating iCECs and demonstrates the safety and efficacy of iCECs in disease modeling. Therefore, clinical-grade iCECs are a reliable source for future clinical treatment of corneal endothelial dysfunction.