Assessment of Genetic Stability in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Using Droplet Digital PCR.

Unintended genetic modifications that occur during the differentiation and proliferation of human induced pluripotent stem cells (hiPSCs) can lead to tumorigenicity. This is a crucial concern in the development of stem cell-based therapies to ensure the safety and efficacy of the final product. Moreover, conventional genetic stability testing methods are limited by low sensitivity, which is an issue that remains unsolved. In this study, we assessed the genetic stability of hiPSCs and hiPSC-derived cardiomyocytes using various testing methods, including karyotyping, CytoScanHD chip analysis, whole-exome sequencing, and targeted sequencing. Two specific genetic mutations in KMT2C and BCOR were selected from the 17 gene variants identified by whole-exome and targeted sequencing methods, which were validated using droplet digital PCR. The applicability of this approach to stem cell-based therapeutic products was further demonstrated with associated validation according to the International Council for Harmonisation (ICH) guidelines, including specificity, precision, robustness, and limit of detection. Our droplet digital PCR results showed high sensitivity and accuracy for quantitatively detecting gene mutations, whereas conventional qPCR could not avoid false positives. In conclusion, droplet digital PCR is a highly sensitive and precise method for assessing the expression of mutations with tumorigenic potential for the development of stem cell-based therapeutics.

Effects of Gallotannin-Enriched Extract of Galla Rhois on the Activation of Apoptosis, Cell Cycle Arrest, and Inhibition of Migration Ability in LLC1 Cells and LLC1 Tumors.

Gallotannin (GT) and GT-enriched extracts derived from various sources are reported to have anti-tumor activity in esophageal, colon and prostate tumors, although their anti-tumor effects have not been determined in lung carcinomas. To investigate the anti-tumor activity of GT-enriched extract of galla rhois (GEGR) against lung carcinomas, alterations in the cytotoxicity, apoptosis activation, cell cycle progression, migration ability, tumor growth, histopathological structure, and the regulation of signaling pathways were analyzed in Lewis lung carcinoma (LLC1) cells and LLC1 tumor bearing C57BL/6NKorl mice, after exposure to GEGR. A high concentration of GT (69%) and DPPH scavenging activity (IC50=7.922 µg/ml) was obtained in GEGR. GEGR treatment exerted strong cytotoxicity, cell cycle arrest at the G2/M phase and subsequent activation of apoptosis, as well as inhibitory effects on the MAPK pathway and PI3K/AKT mediated cell migration in LLC1 cells. In the in vivo syngeneic model, exposure to GEGR resulted in suppressed growth of the LLC1 tumors, as well as inhibition of NF-κB signaling and their inflammatory cytokines. Taken together, our results provide novel evidence that exposure to GEGR induces activation of apoptosis, cell cycle arrest, and inhibition of cell migration via suppression of the MAPK, NF-κB and PI3K/AKT signaling pathways in LLC1 cells and the LLC1 syngeneic model.

Correction to: Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment.

[This corrects the article DOI: 10.1186/s42826-020-00063-z.].

Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice.

Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.

Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment.

Differences in responsiveness of BALB/c substrains have been investigated in various fields, including diabetes induction, corpus callosum deficiency, virus-induced demyelinating disease, aggressive behavior and osteonecrosis. However, induction efficacy of skin tumor remains untried. We therefore investigated the influence of BALB/c substrain backgrounds on the skin tumor induction efficacy in response to DMBA (7,12-Dimethylbenz[a]anthracene) and TPA (12-O-tetradecanoylphorbol-13-acetate) cotreatment. Alterations in the levels of tumor growth related factors, histopathological structure, and the expression to tumor related proteins were measured in three BALB/c substrains (BALB/cKorl, BALB/cA and BALB/cB) after exposure to DMBA (25 µg/kg) and three different doses of TPA (2, 4 and 8 µg/kg). The average number and induction efficacy of tumors in response to DMBA+TPA treatment were significantly greater in the BALB/cKorl substrain than in BALB/cA and BALB/cB. However, cotreatment with DMBA+TPA induced similar responses for body and organ weights of all three substrains. Few differences were detected in the serum analyzing factors, while similar responsiveness was observed for blood analyzing factors after DMBA+TPA treatment. Furthermore, the three BALB/c substrains exhibited similar patterns in their histopathological structure in DMBA+TPA-induced tumors. The expression levels of apoptotic proteins and tumor related proteins were constantly maintained in all three BALB/c substrains treated with DMBA+TPA. In addition, the responsiveness to cisplatin treatment was overall very similar in the three BALB/c substrains with DMBA+TPA-induced tumors. Taken together, these results indicate that genetic background of the three BALB/c substrains does not have a major effect on the DMBA+TPA-induced skin carcinogenesis and therapeutic responsiveness of cisplatin, except induction efficacy.

α-Linolenic Acid-Enriched Cold-Pressed Perilla Oil Suppress High-Fat Diet-Induced Hepatic Steatosis through Amelioration of the ER Stress-Mediated Autophagy.

Perilla oil has been considered to have excellent potential for treating various diseases due to its contents of beneficial fatty acids, such as α-linolenic acid, oleic acid and linoleic acid. The therapeutic effects and molecular mechanism of an α-linolenic acid-enriched cold-pressed perilla oil (LEP) on hepatic steatosis of an obesity model were investigated by analyzing alterations in fat accumulation and endoplasmic reticulum (ER) stress-mediated autophagy, in high-fat diet (HFD)-induced obesity C57BL/6N mice treated with LEP for 16 weeks. Although no significant alterations were detected in body weight and most organ weights, the liver weight and accumulation of lipid droplets in the liver section were significantly lower in HFD + LEP treated group as compared to the HFD + Vehicle treated group. Reduced mRNA expression levels of adipogenesis and lipogenesis regulating factors, including the peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer-binding protein (C/EBP)α, fatty acid synthase (FAS), and adipocyte fatty acid-binding protein 2 (aP2) were observed after LEP treatment for 16 weeks, while the levels of lipolysis were remarkably increased in the same group. Moreover, the LEP-treated groups showed suppression of ER stress-regulating factors, such as the C/EBP homologous protein (CHOP), eukaryotic translation initiation factor 2α (eIF2α), inositol-requiring protein 1 (IRE1)α, and Jun-N-terminal kinase (JNK) during anti-hepatic steatosis effects. The expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) protein and phosphatidylinositol-3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) pathway for the autophagy response showed a significant decrease in the HFD+LEP-treated group. Furthermore, ER stress-mediated autophagy was accompanied with enhanced phosphorylation of extracellular signal-regulated kinase (ERK), JNK, and p38 protein in the mitogen-activated protein (MAP) kinase signaling pathway. Taken together, the results of the present study indicate that treatment with LEP inhibits hepatic steatosis in the HFD-induced obese model through regulation of adipogenesis and lipolysis. We believe our results are the first to show that the anti-hepatic steatosis activity of α-linolenic acid from cold-pressed perilla oil might be tightly correlated with the amelioration of ER stress-mediated autophagy.

Therapeutic Effects of Cold-Pressed Perilla Oil Mainly Consisting of Linolenic acid, Oleic Acid and Linoleic Acid on UV-Induced Photoaging in NHDF Cells and SKH-1 Hairless Mice.

Positive physiological benefits of several plant oils on the UV-induced photoaging have been reported in some cell lines and model mice, but perilla oil collected from the seeds of Perilla frutescens L. has not been investigated in this context. To study the therapeutic effects of cold-pressed perilla oil (CPO) on UV-induced photoaging in vitro and in vivo, UV-induced cellular damage and cutaneous photoaging were assessed in normal human dermal fibroblasts (NHDFs) and HR-1 hairless mice. CPO contained five major fatty acids including linolenic acid (64.11%), oleic acid (16.34%), linoleic acid (11.87%), palmitic acid (5.06%), and stearic acid (2.48%). UV-induced reductions in NHDF cell viability, ROS production, SOD activity, and G2/M cell cycle arrest were remarkably improved in UV + CPO treated NHDF cells as compared with UV + Vehicle treated controls. Also, UV-induced increases in MMP-1 protein and galactosidase levels were remarkably suppressed by CPO. In UV-radiated hairless mice, topical application of CPO inhibited an increase in wrinkle formation, transepidermal water loss (TEWL), erythema value, hydration and melanin index on dorsal skin of UVB-irradiated hairless mice. CPO was observed to similarly suppress UV-induced increases in epidermal thickness, mast cell numbers, and galactosidase and MMP-3 mRNA levels. These results suggest CPO has therapeutic potential in terms of protecting against skin photoaging by regulating skin morphology, histopathology and oxidative status.

Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice.

The antibacterial, anti-inflammatory, anti-metastatic/anti-invasion activities and laxative activity of Galla Rhois (GR) are well-known, although the neuropreservation effects of their extracts are still to be elucidated. To investigate the novel therapeutic effects and molecular mechanism of GR on alleviation of cognitive impairment, two different dosages of gallotannin-enriched GR (GEGR) were administered to Korl:ICR mice for three weeks, and to induce memory impairment, scopolamine (SP) was administered during the last seven days of the GEGR treatment period. GEGR showed the high level of the free radical scavenging activity to DPPH and suppressive activity to reactive oxygen species (ROS) in B35 cells as well as enhanced SOD and CAT activity in brains of the SP-induced model. Latency time for memory impairment assessed by the passive avoidance test significantly protected in the SP+GEGR treated group as compared to the SP+Vehicle treated group. Moreover, similar protective effects were observed on the secretion of BDNF in SP+GEGR treated mice. The expression of TrkB receptor, and phosphorylation of PI3K on the TrkB receptor signaling pathway were dramatically protected in the SP-induced model after GEGR treatment, whereas the expression of p75NTR receptor, the phosphorylation of JNK, and expression of Bax/Bcl-2 on the p75NTR receptor signaling pathway was significantly protected in the same group. Furthermore, the GEGR treated SP-induced model showed decreased number of dead neural cells and suppressed acetylcholine esterase (AChE) activity and inhibited inflammatory responses. Taken together, these results indicate that the anti-oxidant activity of GEGR contributes to improving the neuronal cell function and survival during cognitive impairment in the SP-induced model through regulation of BDNF secretion and their receptor signaling pathway.

Laxative Effect of Spicatoside A by Cholinergic Regulation of Enteric Nerve in Loperamide-Induced Constipation: ICR Mice Model.

Researches on spicatoside A (SpiA)-containing natural products suggest the possibility of SpiA as a potential laxative to alleviate chronic constipation. However, no studies have been conducted with single compound administration of SpiA. To verify the laxative effects and mechanism of action of SpiA on chronic constipation, we investigated alterations in the excretion parameters, histological structure, and cholinergic regulation of the enteric nerve in the colons of Institute of Cancer Research (ICR) mice with loperamide (Lop)-induced constipation after exposure to 20 mg/kg of SpiA. Decrease in the number, weight and water contents of stools in the Lop+Vehicle treated group significantly recovered after SpiA treatment, and alterations in the histological structure and transmission electron microscopy (TEM) images were improved in the Lop+SpiA treated group. Similar recovery effects were observed in the ability for mucin secretion and expression of the membrane water channel gene (aquaporin 8, AQP8). Furthermore, significant improvements were observed in the acetylcholinesterase (AChE) activity and acetylcholine receptors' (AChRs) downstream signaling pathway after treatment of SpiA. The levels of gastrointestinal (GI) hormones including cholecystokinin (CCK) and gastrin were also remarkably enhanced in the Lop+SpiA treated group as compared to the Lop+Vehicle treated group. The expression of receptor tyrosine kinase (C-kit) and protein gene product 9.5 (PGP9.5) in Cajal and neural cells, as well as the phosphorylation of myosin light chain (MLC) in smooth muscle cells, were recovered after SpiA exposure. Taken together, the results of the present study provide the first strong evidence that SpiA improves chronic constipation through muscarinic cholinergic regulation of the enteric nerve in a Lop-induced constipation ICR mice model.

Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks.

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1ß, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.