RESUMO
Insulin is essential for diverse biological processes in human pluripotent stem cells (hPSCs). However, the underlying mechanism of insulin's multitasking ability remains largely unknown. Here, we show that insulin controls hPSC survival and proliferation by modulating RNA translation via distinct pathways. It activates AKT signaling to inhibit RNA translation of pro-apoptotic proteins such as NOXA/PMAIP1, thereby promoting hPSC survival. At the same time, insulin acts via the mTOR pathway to enhance another set of RNA translation for cell proliferation. Consistently, mTOR inhibition by rapamycin results in eIF4E phosphorylation and translational repression. It leads to a dormant state with sustained pluripotency but reduced cell growth. Together, our study uncovered multifaceted regulation by insulin in hPSC survival and proliferation, and highlighted RNA translation as a key step to mediate mitogenic regulation in hPSCs.
Assuntos
Insulina , Células-Tronco Pluripotentes , Diferenciação Celular/genética , Proliferação de Células/genética , Humanos , Insulina/metabolismo , Células-Tronco Pluripotentes/metabolismo , RNA/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Thyroid hormone triiodothyronine (T3) is essential for embryogenesis and is commonly used during in vitro fertilization to ensure successful implantation. However, the regulatory mechanisms of T3 during early embryogenesis are largely unknown. METHOD: To study the impact of T3 on hPSCs, cell survival and growth were evaluated by measurement of cell growth curve, cloning efficiency, survival after passaging, cell apoptosis, and cell cycle status. Pluripotency was evaluated by RT-qPCR, immunostaining and FACS analysis of pluripotency markers. Metabolic status was analyzed using LC-MS/MS and Seahorse XF Cell Mito Stress Test. Global gene expression was analyzed using RNA-seq. To study the impact of T3 on lineage-specific differentiation, cells were subjected to T3 treatment during differentiation, and the outcome was evaluated using RT-qPCR, immunostaining and FACS analysis of lineage-specific markers. RESULTS: In this report, we use human pluripotent stem cells (hPSCs) to show that T3 is beneficial for stem cell maintenance and promotes trophoblast differentiation. T3 enhances culture consistency by improving cell survival and passaging efficiency. It also modulates cellular metabolism and promotes energy production through oxidative phosphorylation. T3 helps maintain pluripotency by promoting ERK and SMAD2 signaling and reduces FGF2 dependence in chemically defined culture. Under BMP4 induction, T3 significantly enhances trophoblast differentiation. CONCLUSION: In summary, our study reveals the impact of T3 on stem cell culture through signal transduction and metabolism and highlights its potential role in improving stem cell applications.
Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular/fisiologia , Cromatografia Líquida , Humanos , Espectrometria de Massas em Tandem , Hormônios TireóideosRESUMO
Pluripotent stem cells (PSCs) can be maintained in a continuum of cellular states with distinct features. Exogenous lipid supplements can relieve the dependence on de novo lipogenesis and shift global metabolism. However, it is largely unexplored how specific lipid components regulate metabolism and subsequently the pluripotency state. In this study, we report that the metabolic landscape of human PSCs (hPSCs) is shifted by signaling lipid lysophosphatidic acid (LPA), which naturally exists. LPA leads to a distinctive transcriptome profile that is not associated with de novo lipogenesis. Although exogenous lipids such as cholesterol, common free fatty acids, and LPA can affect cellular metabolism, they are not necessary for maintaining primed pluripotency. Instead, LPA induces distinct and reversible phenotypes in cell cycle, morphology, and mitochondria. This study reveals a distinct primed state that could be used to alter cell physiology in hPSCs for basic research and stem cell applications.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipogênese , Lisofosfolipídeos/farmacologia , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transcriptoma , Diferenciação Celular , Colesterol/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Células HEK293 , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Metabolic homeostasis is critical for cell pluripotency and differentiation in human embryonic stem cells (hESCs). It has been reported that metabolic changes specifically regulate cellular signaling during hESC differentiation. This protocol describes procedures for both cell culture and detection of intracellular and extracellular metabolites in hESCs by liquid chromatography-mass spectrometry. Metabolites in glycolysis, citric acid cycle, pentose phosphate pathway, and other metabolic processes can be detected using this approach. For complete details on the use and execution of this protocol, please refer to Song et al., (2019), Yang et al., (2019), Meng et al., (2018), and Chen et al., (2011b).
Assuntos
Técnicas de Cultura de Células/métodos , Espaço Extracelular , Células-Tronco Embrionárias Humanas , Espaço Intracelular , Metabolômica/métodos , Diferenciação Celular , Células Cultivadas , Cromatografia Líquida , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Glicólise/fisiologia , Células-Tronco Embrionárias Humanas/química , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espectrometria de Massas , Metaboloma/fisiologiaRESUMO
The human induced pluripotent stem cell line NCCDFWi001-A was derived from peripheral blood mononuclear cells (PBMC) of a 26-year-old female Marfan syndrome patient carrying two compound heterozygous variants FBN1c.2613A > C, (p.Leu871Phe) and c.684_736 + 4del. The established patient-derived iPSC showed expression of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.
Assuntos
Fibrilina-1/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome de Marfan/genética , Adulto , Animais , Linhagem Celular , Feminino , Humanos , MutaçãoRESUMO
During embryogenesis, various cell types emerge simultaneously from their common progenitors under the influence of intrinsic signals. Human embryonic stem cells can differentiate to diverse cell types of three embryonic lineages, making them an excellent system for understanding the regulatory mechanism that maintains the balance of different cell types in embryogenesis. In this report, we demonstrate that insulin-like growth factor (IGF) proteins are endogenously expressed during differentiation, and their temporal expression contributes to the cell fate diversity in mesoderm differentiation. Small molecule LY294002 inhibits the IGF pathway to promote cardiomyocyte differentiation while suppressing epicardial and noncardiac cell fates. LY294002-induced cardiomyocytes demonstrate characteristic cardiomyocyte features and provide insights into the molecular mechanisms underlying cardiac differentiation. We further show that LY294002 induces cardiomyocytes through CK2 pathway inhibition. This study elucidates the crucial roles of endogenous IGF in mesoderm differentiation and shows that the inhibition of the IGF pathway is an effective approach for generating cardiomyocytes.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Transdução de Sinais , Somatomedinas/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Células Cultivadas , Cromonas/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Morfolinas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Pyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Mesoderma/citologia , Ácido Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Linhagem da Célula , Ciclo do Ácido Cítrico/efeitos dos fármacos , Endoderma/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Nicotinamide, the amide form of vitamin B3, is widely used in disease treatments and stem cell applications. However, nicotinamide's impact often cannot be attributed to its nutritional functions. In a vitamin screen, we find that nicotinamide promotes cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibits the phosphorylation of myosin light chain, suppresses actomyosin contraction, and leads to improved cell survival after individualization. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and casein kinase 1. We demonstrate that nicotinamide affects human embryonic stem cell pluripotency and differentiation as a selective kinase inhibitor. The findings in this report may help researchers design better strategies to develop nicotinamide-related stem cell applications and disease treatments.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Niacinamida/farmacologia , Células-Tronco Pluripotentes/citologia , Inibidores de Proteínas Quinases/farmacologia , Actomiosina/metabolismo , Caseína Quinase I/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , NAD/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/enzimologia , Células-Tronco Pluripotentes/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The present study investigated the chemical constituents of the roots and rhizomes of Panax notoginseng. Compounds were isolated by various column chromatographic methods, and their structures were elucidated by the extensive analysis of spectroscopic data and chemical evidences. A novel 12, 23-epoxy dammarane-type saponin, named epoxynotoginsenoside A (1), together with four known compounds (2-5), was isolated and characterized.
Assuntos
Panax notoginseng/química , Saponinas/isolamento & purificação , Triterpenos/isolamento & purificação , Raízes de Plantas/química , Rizoma/química , Saponinas/química , Triterpenos/química , DamaranosRESUMO
A novel strategy for the qualitative and quantitative determination of 20(S)-protopanaxatriol saponins (PTS) and 20(S)-protopanaxadiol saponins (PDS) in Panax notoginseng, Panax ginseng and Panax quinquefolium, based on the overlapping peaks of main components of PTS (calibrated by ginsenoside Rg1) and PDS (calibrated by ginsenoside Rb1), was proposed. The analysis was performed by using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). Under specific chromatographic conditions, all samples showed two overlapping peaks containing several main ginsenosides belonging to PTS and PDS, respectively. The overlapping peaks were also identified by using HPLC-MS. Based on the sum and ratio of PTS and PDS, 60 tested Panax samples were divided into three main clusters according to their species. The findings suggested that this strategy provides a simple and rapid approach to quantify PTS and PDS in Panax herbs.