Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells Abstract Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines. The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation. StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

Correction to: A1CF-Axin2 signal axis regulates apoptosis and migration in Wilms tumor-derived cells through Wnt/β-catenin pathway In their paper “A1CF-Axin2 signal axis regulates apoptosis and migration in Wilms tumor-derived cells through Wnt/β-catenin pathway” (In Vitro Cell. Devel. Biol. Anim. 55: 252-259, 2019) Ni et al., regarded the G-401 cell line as being derived from a Wilms’ tumor.

Cytotoxic and cytostatic effects of four Annonaceae plants on human cancer cell lines Abstract Several species of the Annonaceae plants have been used as complementary medicine for cancer-associated illnesses in some ethnic groups of northern Thailand. This study investigated the cytotoxic and cytostatic activity of methanolic extracts derived from the stems of these plants, including Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders, and Dasymaschalon sp. Cell death induction of seven human cancer cell lines and cell cycle analyses were assessed by Annexin V and/or propidium iodide (PI) staining and analyzed by flow cytometry. Treatment of cancer cell lines with the extract of four Annonaceae plants resulted in various cytotoxic activities depending on cell type. The extract of U. longipes exhibited the highest cytotoxic activity capable of inducing cell death of several cancer cell lines, particularly against hepatocellular carcinoma cell lines (HepG2 and Hep3B). This extract was capable of inducing cell cycle arrest at the SubG1 phase. Phytochemical screening of all the extracts revealed the presence of alkaloids, sterols, tannins, anthraquinone glycoside, coumarin, and flavonoids. Determination of active compounds by high-performance liquid chromatography standards revealed bullatacin and asiminecin in all the extracts. The extract of Annonaceae stem or its compounds may provide an opportunity for the development of new therapies against cancer.

LncRNA KCNQ1OT1 acting as a ceRNA for miR-4458 enhances osteosarcoma progression by regulating CCND2 expression Abstract Osteosarcoma is prevalent worldwide and characterized as a challenging health burden. It has been increasingly indicated that long non-coding RNAs (lncRNAs) are significant in pathological processes of numerous cancers, exerting oncogenic or tumor-suppressive function. However, the participation of KCNQ1OT1 in osteosarcoma has not been elaborated. In this study, we focus on interrogating the function of KCNQ1OT1 and its underlying mechanism in osteosarcoma. Our work demonstrated the upregulation of KCNQ1OT1 in osteosarcoma through qRT-PCR. Besides, loss of function assay (CCK-8, transwell migration) indicated KCNQ1OT1 promoted cell proliferation, migration in osteosarcoma. Mechanically, KCNQ1OT1 acting as sponge for miR-4458 antagonized its tumor-suppressive impact on CCND2 expression. The anti-apoptotic nature of KCNQ1OT1 was also unveiled via caspase-3 activity assay. Overexpressed KCNQ1OT1 acted as competing endogenous RNA (ceRNA) for miR-4458 and subsequently reinforced target gene CCND2. Collectively, the results of rescue experiments suggested that the oncogenic role of KCNQ1OT1 was performed through sponging miR-4458 and upregulating CCND2 during osteosarcoma development, providing a novel perspective of intervention in osteosarcoma management.

PTEN-mediated mitophagy and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury Abstract Autophagy plays a critical role in cardiac hypoxia/reoxygenation (H/R). Studies indicated that the phosphatase and tensin homolog (PTEN) influences level of autophagy. This study aims to explore the role of PTEN mediating a specific autophagy, mitophagy, in cardiac H/R injury. H9c2 cells were cultured and suffered hypoxia and reoxygenation treatment. To inhibit function of PTEN protein, bpv (phen) was added into medium throughout the process of H/R injury. In addition, we overexpressed the apurinic/apyrimidinic endonuclease 1 (APE1) in H/R-injured H9c2 cells. Then the cell viability, apoptosis, and release of Cytochrome C were determined through CCK-8 assay, flow cytometry, and western blotting, respectively. The results indicated that H/R significantly induced autophagy, as identified by an increased level of microtubule-associated protein 1 light chain 3 beta (LC3B) and a decreased level of sequestosome 1 (P62). After stimulation of bpv (phen), PTEN-induced putative kinase protein 1 (PINK1)/Parkin-mediated mitophagy was inhibited, while apoptosis and releases of Cytochrome C were both significantly increased, indicating an exacerbated H/R injury. Furthermore, the overexpression of APE1 attenuated the apoptosis and releases of Cytochrome C induced by H/R injury, and promoted PINK1/Parkin-mediated mitophagy. Our findings provide an insight into the PTEN and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury, which may be through inducing the PINK1/Parkin-mediated mitophagy.

Testosterone promotes GPX5 expression of goat epididymal epithelial cells cultured in vitro Abstract Androgens are involved in maintaining epididymal structure and function. In the present study, primary culture of goat EECs and effect of testosterone on expression of glutathione peroxidase-5 (GPX5) in goat epididymal epithelial cells (EECs) were investigated. The EECs isolated from 12-mo-old goat caput epididymis were cultured with testosterone in vitro, and expression of glutathione peroxidase-5 (GPX5) and androgen receptors (ARs) was analyzed. Our results showed that testosterone effectively increased EEC proliferation activity, and EECs cultured with testosterone could maintain molecular markers for up to 12 passages. Compared with the control group, 100 nM testosterone significantly increased the mRNA and protein expression of GPX5 (P < 0.05) and ARs (P < 0.01 and P < 0.05, respectively) in EECs, and this effect was blocked by the AR blocker enzalutamide. In conclusion, testosterone can promote the expression of GPX5 in EECs by up-regulating AR expression. We established an effective culture system for goat EECs which can be for further investigation on the regulation of epithelial function.

Adventitious root cultures of Oplopanax elatus inhibit LPS-induced inflammation via suppressing MAPK and NF-κB signaling pathways Abstract Bioreactor-cultured adventitious roots (ARs) of the endangered medicinal plant Oplopanax elatus Nakai is a novel alternative plant material. To utilize ARs in the product production, the present study investigated the anti-inflammatory effect of O. elatus ARs. In the in vivo experiment, lipopolysaccharide (LPS)-induced acute lung injury disease model was established and several inflammatory indexes were determined. For the LPS-stimulated mice, after pretreatment of AR crude extract (200 mg/kg), cell infiltration in lungs was decreased, the production of proinflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, and 1β in the bronchoalveolar lavage fluid was evidently reduced, which indicated that O. elatus ARs had an anti-inflammatory effect. In the in vitro experiment, ethyl acetate (EtOAc) fractions (12.5, 25, and 50 μg/mL) were used to treat LPS-induced peritoneal macrophages (PMs) of mice. The production of NO, prostaglandin E2, TNF-α, IL-6, and IL-1β in LPS-stimulated PMs was obviously inhibited (p < 0.05) after pretreatment with EtOAc fractions, and the expression of the inducible nitric oxide synthase and cyclooxygenase were also suppressed. To clarify the anti-inflammatory mechanism, effects of EtOAc fraction on changes of proteins related to the pathways of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were investigated. The phosphorylation of extracellular regulated protein kinases, c-jun n-terminal kinase, and p38 MAPK in LPS-induced PMs was inhibited after pretreatment of EtOAc fractions. In addition, EtOAc fractions enhanced inhibitor of nuclear factor-kappa B-α expression and decreased nuclear translocation of p65 NF-κB. Thus, EtOAc from O. elatus ARs is involved in regulating MAKP and NF-κB signaling pathways to inhibit LPS-induced inflammation.

SQSTM1/p62 is involved in docosahexaenoic acid–induced cellular autophagy in glioblastoma cell lines Abstract Docosahexaenoic acid (DHA) is the most abundant n-3 polyunsaturated fatty acid in the human brain and works as an anticancer agent to induce cell cycle arrest and apoptosis in glioblastoma multiforme (GBM) cell lines. However, little is known about the connection between DHA and autophagy in GBM cells. We found that high-dose DHA caused cellular autophagy in cultured U251 and U118 GBM cell lines, but there was no effect with a low dose. Moreover, after treatment with a high dose of DHA at 12, 24, and 48 h, the protein expression of SQSTM1/p62 decreased in DHA-treated U251 cells at 12 and 24 h, but increased at 48 h, while in DHA-treated U118 cells, the protein expression increased at all time points. Interestingly, the level of SQSTM1/p62 mRNA was elevated in both DHA-treated U251 and U118 cells at all time points, indicating that DHA activated SQSTM1/p62 transcription in both cell lines. Furthermore, downregulation of SQSTM1/p62 by siRNA attenuated DHA-induced cellular autophagy in both cell lines. This report confirms that high-dose DHA induces cellular autophagy in GBM cells, and demonstrates that SQSTM1/p62 acts as a regulator and participates in DHA-induced autophagy.

Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) Abstract The fall armyworm, Spodoptera frugiperda (Sf), is a polyphagous lepidopteran herbivore that consumes more than 80 plant species, including many economically important crops, such as corn, soybeans, and sorghum. While already a serious pest in the Americas, it was recently introduced into Africa, India, and China. Because of its high economic costs in the New World and the continent-wide damage potentials in Africa, research to develop advanced pest management technologies is necessary. We are supporting this need by developing novel, next-generation insect cell lines from targeted tissues. Cell lines, such as these, will boost insecticide discovery programs and lead to innovative pest management solutions. Here, we report on the establishment of 16 new cell lines from larval S. frugiperda tissues: nine from the central nervous system, three from the aorta, and four from the testes. We confirmed the identities of the cell lines by DNA amplification fingerprinting polymerase chain reaction, determined their doubling times from growth curves, and described cell types via microscopy. We also developed 16 sublines from three neuronal cell lines.

Inhibitory effects of Semaphorin 3F as an alternative candidate to anti-VEGF monoclonal antibody on angiogenesis Abstract Vascular endothelial growth factor (VEGF) inhibition forms the basis for anti-angiogenic therapies. With the methods based on the monoclonal antibody-mediated typical VEGF blockade, pathological angiogenesis in the tumor microenvironment is inhibited and the limitation of tumor growth is provided; however, the existing tumor tissue cannot be intervened. In this study, the anti-angiogenic effects of Semaphorin (SEMA) 3F, which has frequently been reported to have tumor suppressive properties, on a chick chorioallantoic membrane model as well as in vitro cell-cell interactions were investigated and comparatively assessed using anti-VEGF antibody. Vascular endothelial cells and chick embryos were stimulated with 10–16 ng/mL VEGF165 prior to SEMA 3F administration in order to generate pathological vascularization conditions. Both in vitro and in ovo results revealed that SEMA 3F suppressed VEGF165-induced abnormal vascularization more effectively than anti-VEGF. Moreover, the required dose of SEMA 3F was significantly lower than that of anti-VEGF (103 times less under in ovo conditions). In light of these results, SEMA 3F is recommended as an important therapeutic agent for the prevention of pathological angiogenesis. SEMA 3F may offer an effective and efficient anti-angiogenic intervention that can be administered at a lower dose alternative to typical VEGF blocking agents.