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Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Diferenciación Celular/genética , OrganoidesRESUMEN
Actin-interacting protein 1 (AIP1), also known as WD repeat-containing protein 1 (WDR1), is ubiquitous in eukaryotic organisms, and it plays critical roles in the dynamic reorganization of the actin cytoskeleton. However, the biological function and mechanism of AIP1 in mammalian oocyte maturation is still largely unclear. In this study, we demonstrated that AIP1 boosts ADF/Cofilin activity in mouse oocytes. AIP1 is primarily distributed around the spindle region during oocyte maturation, and its depletion impairs meiotic spindle migration and asymmetric division. The knockdown of AIP1 resulted in the gathering of a large number of actin-positive patches around the spindle region. This effect was reduced by human AIP1 (hAIP1) or Cofilin (S3A) expression. AIP1 knockdown also reduced the phosphorylation of Cofilin near the spindle, indicating that AIP1 interacts with ADF/Cofilin-decorated actin filaments and enhances filament disassembly. Moreover, the deletion of AIP1 disrupts Cofilin localization in metaphase I (MI) and induces cytokinesis defects in metaphase II (MII). Taken together, our results provide evidence that AIP1 promotes actin dynamics and cytokinesis via Cofilin in the gametes of female mice.
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Factores Despolimerizantes de la Actina/metabolismo , Citocinesis/fisiología , Metafase/fisiología , Oocitos/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos ICR , Fosforilación/fisiología , Huso Acromático/metabolismoRESUMEN
Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cellular events, and various actin-regulatory proteins modulate actin polymerization and depolymerization. Adenylyl cyclase-associated proteins (CAPs), highly conserved actin monomer-binding proteins, have been known to promote actin disassembly by enhancing the actin-severing activity of the ADF/cofilin protein family. In this study, we found that CAP1 regulated actin remodeling during mouse oocyte maturation. Efficient actin disassembly during oocyte maturation is essential for asymmetric division and cytokinesis. CAP1 knockdown impaired meiotic spindle migration and asymmetric division, and resulted in an accumulation of excessive actin filaments near the spindles. In contrast, CAP1 overexpression reduced actin mesh levels. CAP1 knockdown also rescued a decrease in cofilin family protein overexpression-mediated actin levels, and simultaneous expression of human CAP1 (hCAP1) and cofilin synergistically decreased cytoplasmic actin levels. Overexpression of hCAP1 decreased the amount of phosphorylated cofilin, indicating that CAP1 facilitated actin depolymerization via interaction with ADF/cofilin during mouse oocyte maturation. Taken together, our results provide evidence for the importance of dynamic actin recycling by CAP1 and cofilin in the asymmetric division of mouse female gametes.This article has an associated First Person interview with the first author of the paper.
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Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Destrina/metabolismo , Oocitos/metabolismo , Serina Endopeptidasas/metabolismo , Animales , División Celular/fisiología , Femenino , Ratones , Oocitos/citologíaRESUMEN
Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.
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Blastocisto/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Recombinasa Rad51/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Blastocisto/metabolismo , Reparación del ADN/efectos de los fármacos , Femenino , Embarazo , Recombinasa Rad51/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Meiotic oocytes lack classic centrosomes; therefore, bipolar spindle assembly depends on the clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. The bipolar spindle is an essential cellular component that ensures accurate chromosome segregation during anaphase. If the spindle does not form properly, it can result in aneuploidy or cell death. However, the molecular mechanism by which the bipolar spindle is established is not yet fully understood. Tumor suppressor p53-binding protein 1 (TP53BP1) is known to mediate the DNA damage response. Several recent studies have indicated that TP53BP1 has noncanonical roles in processes, such as spindle formation; however, the role of TP53BP1 in oocyte meiosis is currently unclear. Our results show that TP53BP1 knockdown affects spindle bipolarity and chromatin alignment by altering MTOC stability during oocyte maturation. TP53BP1 was localized in the cytoplasm and displayed an irregular cloud pattern around the spindle/chromosome region. TP53BP1 was also required for the correct localization of MTOCs into the two spindle poles during pro-meiosis I. TP53BP1 deletion altered the MTOC-localized Aurora Kinase A. TP53BP1 knockdown caused the microtubules to detach from the kinetochores and increased the rate of aneuploidy. Taken together, our data show that TP53BP1 plays crucial roles in chromosome stability and spindle bipolarity during meiotic maturation.
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Cromosomas de los Mamíferos/metabolismo , Cinetocoros/metabolismo , Meiosis , Oocitos/metabolismo , Huso Acromático/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Aurora Quinasa A/metabolismo , Citoplasma/metabolismo , Femenino , Ratones , Ratones Endogámicos ICR , Oocitos/citologíaRESUMEN
DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in homologous recombination (HR) repair of DNA breaks. HR depends on the formation of a RAD51 recombinase filament that facilitates strand invasion. However, the role of RAD51 during porcine oocyte maturation is unknown. The objective of this study was to investigate the expression and function of RAD51 during porcine oocyte maturation in vitro. RAD51 was mainly localized to the nucleus at the germinal vesicle (GV) stage, and was widely distributed in the cytoplasm between the GV breakdown (GVBD) and metaphase II stage. DNA damage induced by etoposide was accompanied by the formation of RAD51 foci that were colocalized with γH2AX. Inhibition of RAD51 increased DNA damage and induced metaphase I arrest along with spindle defects, chromosomal misalignment, and abnormal spindle assembly checkpoint (SAC) activity. Inhibition of RAD51 also increased ROS levels and led to an abnormal mitochondrial distribution. Our results indicate that RAD51 plays a critical role in maintaining chromosome integrity and mitochondrial activity during porcine oocyte maturation.
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Inestabilidad Genómica/genética , Técnicas de Maduración In Vitro de los Oocitos , Mitocondrias/metabolismo , Oocitos/fisiología , Oogénesis/genética , Recombinasa Rad51/fisiología , Animales , Células Cultivadas , Meiosis/genética , Mitocondrias/genética , Oocitos/citología , Recombinasa Rad51/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Porcinos , Distribución TisularRESUMEN
Widely used as a flame retardant, 2,2'4,4'-tetrabromodiphenyl ether (BDE-47) is a persistent environmental pollutant with toxicological effects, including hepatotoxicity, neurotoxicity, reproductive toxicity, and endocrine disruption. To investigate the toxicological effects of BDE-47 on early porcine embryogenesis in vitro, cultured porcine embryos were exposed to BDE-47 during early development. Exposure to 100 µM BDE-47 decreased the blastocyst rate and mRNA level of pluripotency genes but increased the level of LC3 and the expression of autophagy-related genes. After BDE-47 exposure, porcine embryos' antioxidant capability decreased; ROS levels increased, while glutathione (GSH) levels and the expression of antioxidant-related genes decreased. In addition, BDE-47 exposure reduced mitochondrial abundance and mitochondrial membrane potential levels, downregulated mitochondrial biogenesis-associated genes, decreased endoplasmic reticulum (ER) abundance, increased the levels of GRP78, a marker of ER stress (ERS), and upregulated the expression of ERS-related genes. However, ER damage and low embryo quality induced by BDE-47 exposure were reversed with the ERS inhibitor, the 4-phenylbutyric acid. In conclusion, BDE-47 inhibits the development of early porcine embryos in vitro by inducing mitochondrial dysfunction and ERS. This study sheds light on the mechanisms of BDE-47-induced embryonic toxicity.
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Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have presented a major and common health concern worldwide due to their increasing prevalence and progressive development of severe pathological conditions such as cirrhosis and liver cancer. Although a large number of drug candidates for the treatment of NASH have entered clinical trial testing, all have not been released to market due to their limited efficacy, and there remains no approved treatment for NASH available to this day. Recently, organoid technology that produces 3D multicellular aggregates with a liver tissue-like cytoarchitecture and improved functionality has been suggested as a novel platform for modeling the human-specific complex pathophysiology of NAFLD and NASH. In this review, we describe the cellular crosstalk between each cellular compartment in the liver during the pathogenesis of NAFLD and NASH. We also summarize the current state of liver organoid technology, describing the cellular diversity that could be recapitulated in liver organoids and proposing a future direction for liver organoid technology as an in vitro platform for disease modeling and drug discovery for NAFLD and NASH.
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Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , Hígado/patología , Cirrosis Hepática/etiología , Descubrimiento de Drogas , Organoides/patologíaRESUMEN
Brain organoids have been considered as an advanced platform for in vitro disease modeling and drug screening, but numerous roadblocks exist, such as lack of large-scale production technology and lengthy protocols with multiple manipulation steps, impeding the industrial translation of brain organoid technology. Here, we describe the high-speed and large-scale production of midbrain organoids using a high-throughput screening-compatible platform within 30 days. Micro midbrain organoids (µMOs) exhibit a highly uniform morphology and gene expression pattern with minimal variability. Notably, µMOs show dramatically accelerated maturation, resulting in the generation of functional µMOs within only 30 days of differentiation. Furthermore, individual µMOs display highly consistent responsiveness to neurotoxin, suggesting their usefulness as an in vitro high-throughput drug toxicity screening platform. Collectively, our data indicate that µMO technology could represent an advanced and robust platform for in vitro disease modeling and drug screening for human neuronal diseases.
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Here we described two human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) of idiopathic autism spectrum disorder (ASD) patients through forced expression of OCT4, SOX2, KLF4, and c-MYC. The hiPSC lines displayed morphology, gene expression patterns, and pluripotential differentiation potentials similar to those of human embryonic stem cells (hESCs). The hiPSC lines from idiopathic ASD patients might be useful to unveil the underlying mechanism of idiopathic ASD and finding its therapeutics.
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Trastorno del Espectro Autista , Células Madre Pluripotentes Inducidas , Diferenciación Celular , Humanos , Factor 4 Similar a Kruppel , Leucocitos MononuclearesRESUMEN
WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.
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Actinas/metabolismo , Oocitos/metabolismo , Polimerizacion , Huso Acromático/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Ratones , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Oogénesis/fisiologíaRESUMEN
Imperatorin (IMP) exhibits a variety of pharmacological properties, including antioxidant, anti-inflammatory, antibacterial, anti-cancer, and anti-hypertension activities. However, its effects on animal reproduction systems, especially oocyte development, maturation, and aging are not yet clear. In this study, the effects of IMP on oocyte development and aging as well as the underlying molecular mechanisms were explored. Oocytes were cultured for an additional 24 h for aging. Results revealed that the blastocyst formation and hatching rates of embryos, which were parthenogenetically activated aged oocytes, were significantly increased with IMP treatment (40 µM). Simultaneously, well-distributed cortical granules but no significant difference in zona pellucida hardness were observed after IMP treatment. During this stage, intracellular reactive oxygen species, apoptosis, and autophagy levels were decreased, while mitochondrial membrane potential, glutathione level, and activity of superoxide dismutase and catalase were increased. IMP-treated aged oocytes also showed significantly higher expression of MOS, CCNB1, BMP15, and GDF9 than non-IMP-treated aged oocytes although their levels were still lower than those in the fresh oocytes. These results suggest that IMP can effectively ameliorate the quality of aged porcine oocytes by reducing oxidative stress and protecting mitochondrial function.
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Leonurine (LEO) is pseudoalkaloid that has been isolated from motherwort. It has been found to have various biological activities, including an antioxidant capacity. This study aimed to confirm whether LEO could be used in porcine in vitro culture (IVC) medium for its antioxidant effect and related molecular mechanisms. The results showed that embryos in IVC medium supplemented with 40 µM LEO had an increased blastocyst formation rate, total cell number, and proliferation capacity and a low apoptosis rate. LEO supplementation decreased reactive oxygen species levels and increased glutathione levels. Moreover, LEO-treated embryos exhibited improved intracellular mitochondrial membrane potential and reduced autophagy. In addition, pluripotency related gene was up-regulated while apoptosis and autophagy related genes were down-regulated with LEO supplementation. These results suggest that LEO has a beneficial effect on pre-implantation embryo development by reducing oxidative stress and enhancing mitochondrial function.
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Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Animales , Blastocisto , Técnicas de Cultivo de Embriones/veterinaria , Ácido Gálico/análogos & derivados , Mitocondrias , Especies Reactivas de Oxígeno , PorcinosRESUMEN
INTRODUCTION: The preferable pharmacokinetics of rhenium-186 (186Re)-monoaminemonoamidedithiol-conjugated or 186Re-mercaptoacetyltriglycine-conjugated bisphosphonates (BPs) suggested that the molecular design would be applicable to other radionuclides such as 68Ga, 99mTc, 153Sm and 177Lu. In this study, a key factor affecting the pharmacokinetics of a chelate-conjugated BP was investigated to estimate the validity and the applicability of molecular design. METHODS: Chemically inert and well-characterized tricarbonyl[186Re][(cyclopentadienylcarbonyl amino)-acetic acid]rhenium ([186Re]CpTR-Gly) was conjugated with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate and purified by high-performance liquid chromatography (HPLC) to prepare [186Re](1-{3-[tricarbonyl(cyclopentadienylcarbonyl amino)-acetylamido]-1-hydroxy-1-phosphono-propyl}-phosphonic acid)rhenium ([186Re]CpTR-Gly-APD). Plasma stability, plasma protein binding, hydroxyapatite (HA) binding and the pharmacokinetics of [186Re]CpTR-Gly-APD were compared with those of 186Re 1-hydroxyethylidene-1,1-diphosphonate (HEDP). The effect of HEDP coadministration and preadministration on the pharmacokinetics of [186Re]CpTR-Gly-APD was also determined. RESULTS: The HPLC-purified [186Re]CpTR-Gly-APD showed higher plasma stability, higher HA binding, higher bone accumulation and lower plasma protein binding than did 186Re-HEDP. However, HA binding of [186Re]CpTR-Gly-APD decreased to levels slightly higher than that of 186Re-HEDP at similar HEDP concentrations. Bone accumulation of [186Re]CpTR-Gly-APD also decreased to levels similar to that of 186Re-HEDP when [186Re]CpTR-Gly-APD was coinjected with HEDP equivalent to that in 186Re-HEDP. In contrast, HEDP pretreatment did not impair bone accumulation of the two 186Re-labeled compounds. However, a delay in blood clearance and an increase in renal radioactivity levels were observed particularly with 186Re-HEDP. CONCLUSIONS: Although 186Re-HEDP possessed HA binding and bone accumulation similar to those of [186Re]CpTR-Gly-APD, the specific activity of 186Re-labeled BPs was found to play a crucial role in bone accumulation and blood clearance. Thus, the molecular design of chelate-conjugated BP would be useful for the development of bone-seeking radiopharmaceuticals with a variety of radionuclides by selecting chelating molecules that provide high specific activities.
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Difosfonatos/farmacocinética , Radioisótopos/farmacocinética , Renio/farmacocinética , Animales , Quelantes/química , Difosfonatos/uso terapéutico , Evaluación Preclínica de Medicamentos , Tasa de Depuración Metabólica , Ratones , Especificidad de Órganos , Radioisótopos/química , Radioisótopos/uso terapéutico , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Radiofármacos/uso terapéutico , Renio/química , Renio/uso terapéutico , Distribución TisularRESUMEN
Urushi wax is a natural gum base used as a food additive. In order to evaluate the quality of urushi wax as a food additive and to obtain information useful for setting official standards, we investigated the constituents and their concentrations in urushi wax, using the same sample as scheduled for toxicity testing. After methanolysis of urushi wax, the composition of fatty acids was analyzed by GC/MS. The results indicated that the main fatty acids were palmitic acid, oleic acid and stearic acid. LC/MS analysis of urushi wax provided molecular-related ions of the main constituents. The main constituents were identified as triglycerides, namely glyceryl tripalmitate (30.7%), glyceryl dipalmitate monooleate (21.2%), glyceryl dioleate monopalmitate (2.1%), glyceryl monooleate monopalmitate monostearate (2.6%), glyceryl dipalmitate monostearate (5.6%), glyceryl distearate monopalmitate (1.4%). Glyceryl dipalmitate monooleate isomers differing in the binding sites of each constituent fatty acid could be separately determined by LC/MS/MS.
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Ácidos Grasos/análisis , Aditivos Alimentarios/química , Ceras/química , Cromatografía Liquida , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Triglicéridos/análisisRESUMEN
Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.