3D hepatic organoid production from human pluripotent stem cells.

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.

Diosmetin Promotes Early Embryonic Development in Pigs by Alleviating Oxidative Stress.

Diosmetin (DIOS), a natural flavonoid monomer derived from lemons and present in various plants such as spearmint and spider moss, exhibits antioxidant, anti-inflammatory, and antiaging properties. Nonetheless, its impact on early embryonic development in pigs remains unexplored. This study aimed to determine the influence of DIOS supplementation in an in vitro culture (IVC) medium on porcine embryo development and to elucidate the underlying mechanisms. Findings revealed that embryos cultured in IVC medium with 0.1 µM DIOS demonstrated an increased blastocyst formation rate, higher total cell number, reduced LC3B and CASPASE3 levels, elevated Nrf2 levels, decreased ROS, and enhanced GSH and mitochondrial membrane potential at the 4-cell embryonic stage. Additionally, the expression of proapoptotic genes (CAS3, CAS8, and BAX) and autophagy-related genes (BECLIN1, ATG5, LC3B, and P62) was downregulated, whereas the expression of embryonic development-related genes (CDK1 and CDK2), antioxidant-related genes (SOD1 and SOD2), and mitochondrial biogenesis-related genes (NRF2) was upregulated. These findings suggest that DIOS promotes early embryonic development in pigs by mitigating oxidative stress and enhancing mitochondrial function, thereby reducing autophagy and apoptosis levels.

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing in vitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing in vitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.

Xanthoangelol promotes early embryonic development of porcine embryos by relieving endoplasmic reticulum stress and enhancing mitochondrial function.

RESEARCH QUESTION: Does the addition of an antioxidant agent, xanthoangelol (XAG), to the culture medium improve in-vitro development of porcine embryos? DESIGN: Early porcine embryos were incubated in the presence of 0.5 µmol/l XAG in in-vitro culture (IVC) media and analysed using various techniques, including immunofluorescence staining, reactive oxygen species (ROS) detection, TdT-mediated dUTP nick-end labelling (TUNEL), and reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). RESULTS: The addition of 0.5 µmol/l XAG to IVC media increased the rate of blastocyst formation, total cell number, glutathione concentrations and proliferative capacity, while reducing reactive oxygen species concentrations, apoptosis and autophagy. In addition, upon XAG treatment, the abundance of mitochondria and mitochondrial membrane potential significantly increased (both P < 0.001), and the genes related to mitochondrial biogenesis (TFAM, NRF1 and NRF2) were significantly up-regulated (all P < 0.001). XAG treatment also significantly increased the endoplasmic reticulum abundance (P < 0.001) and reduced the concentrations of endoplasmic reticulum stress (ERS) marker GRP78 (P = 0.003) and expression of the ERS-related genes EIF2α, GRP78, CHOP, ATF6, ATF4, uXBP1 and sXBP 1 (all P < 0.001). CONCLUSION: XAG promotes early embryonic development in porcine embryos in vitro by reducing oxidative stress, enhancing mitochondrial function and relieving ERS.

Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress.

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 µM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.

Protective effect of onion peel extract on ageing mouse oocytes.

Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 µg/ml). The addition of 100 µg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1-induced enhancement of mitochondrial activity and alleviation of proapoptotic events.

Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development-related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 µM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan-activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency-related factor levels were elevated (NANOG and octamer-binding transcription factor 4 [OCT4]), antioxidant-related genes were upregulated (nuclear factor-erythroid 2-related factor 2 [NRF2]), and apoptosis- (caspase 3 [CAS3]) and autophagy-related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

Oroxin A reduces oxidative stress, apoptosis, and autophagy and improves the developmental competence of porcine embryos in vitro.

Oroxin A (OA) is a flavonoid isolated from Oroxylum indicum (L.) Kurz that has various biological activities, including antioxidant activities. This study aimed to examine the viability of using OA in an in vitro culture (IVC) medium for its antioxidant effects and related molecular mechanisms on porcine blastocyst development. In this study, we investigated the effects of OA on early porcine embryo development via terminal deoxynucleotidyl transferase dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, quantitative reverse transcription PCR, and immunocytochemistry. Embryos cultured in the IVC medium supplemented with 2.5 µM of OA had an increased blastocyst formation rate, total cell number, and proliferation capacity, along with a low apoptosis rate. OA supplementation decreased reactive oxygen species levels while increasing glutathione levels. OA-treated embryos exhibited an improved intracellular mitochondrial membrane potential and reduced autophagy. Moreover, levels of pluripotency- and antioxidant-related genes were upregulated, whereas those of apoptosis- and autophagy-related genes were downregulated by OA addition. In conclusion, OA improves preimplantation embryonic development by reducing oxidative stress and enhancing mitochondrial function.

The PI3Kα inhibitor DFX24 suppresses tumor growth and metastasis in non-small cell lung cancer via ERK inhibition and EPHB6 reactivation.

EPHB6 is a metastasis inhibitory gene that is frequently decreased or deficiency in non-small cell lung cancer (NSCLC), which contributed to the subsequent development of distant metastasis. These suggested the possibility that reactivation of EPHB6 might prevent the metastasis of NSCLC. Nevertheless, EPHB6 expression might also promote cancer cell growth and inhibit cell apoptosis by activating Akt and ERK pathway, apart from inhibition of migration and invasion. In the present study, we developed a novel quinazolin-4(3H)-one analog (DFX24) as a potential PI3Kα inhibitor, which inhibited both cell proliferation and metastasis of NSCLC cell lines. Investigation to the molecular mechanisms revealed DFX24 inhibited the cell growth and metastasis via inhibition of PI3Kα and ERK activity, as well as the increase in EPHB6 expression. In addition, DFX24 also induced cell cycle arrest and tumor cell apoptosis by inhibiting PI3K/Akt pathway and activating mitochondria-dependent pathway, respectively. These findings suggested that DFX24 might be considered as a novel drug candidate and may provide a potential therapy for NSCLC.