Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.

AIM: To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells. METHODS: Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging. RESULTS: We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging. CONCLUSION: The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

Is the Linear No-Threshold Dose-Response Paradigm Still Necessary for the Assessment of Health Effects of Low Dose Radiation?

Inevitable human exposure to ionizing radiation from man-made sources has been increased with the proceeding of human civilization and consequently public concerns focus on the possible risk to human health. Moreover, Fukushima nuclear power plant accidents after the 2011 East-Japan earthquake and tsunami has brought the great fear and anxiety for the exposure of radiation at low levels, even much lower levels similar to natural background. Health effects of low dose radiation less than 100 mSv have been debated whether they are beneficial or detrimental because sample sizes were not large enough to allow epidemiological detection of excess effects and there was lack of consistency among the available experimental data. We have reviewed an extensive literature on the low dose radiation effects in both radiation biology and epidemiology, and highlighted some of the controversies therein. This article could provide a reasonable view of utilizing radiation for human life and responding to the public questions about radiation risk. In addition, it suggests the necessity of integrated studies of radiobiology and epidemiology at the national level in order to collect more systematic and profound information about health effects of low dose radiation.

Wild ginseng cambial meristematic cells ameliorate hepatic steatosis and mitochondrial dysfunction in high-fat diet-fed mice.

OBJECTIVES: The aim of this study was to determine the protective mechanisms of wild ginseng cambial meristematic cells (CMCs) on non-alcoholic fatty liver disease in high-fat diet (HFD)-fed mice. METHODS: Male C57BL/6 mice received either normal-fat diet or HFD for 10 weeks along with wild ginseng CMCs (75, 150 and 300 mg/kg) or vehicle (0.5% carboxyl methyl cellulose) by oral administration once a day. Triglyceride and total cholesterol contents were measured in liver and serum samples. Parameters for hepatic lipid metabolism and mitochondria biogenesis were assessed. KEY FINDINGS: Treatment with wild ginseng CMCs markedly attenuated body weight, serum and hepatic lipid contents, and serum aminotransferase activity. While wild ginseng CMCs attenuated the increases in sterol regulatory element-binding transcription factor 1 (SREBP-1) and carbohydrate-responsive element-binding protein (ChREBP) expression, it enhanced the increases in carnitine palmitoyltransferase 1A (CPT1A) and peroxisome proliferator-activated receptor alpha (PPAR-α) expression. HFD decreased glutamate dehydrogenase activity and glutathione content, and increased lipid peroxidation, which were all attenuated by wild ginseng CMCs. Furthermore, wild ginseng CMCs enhanced mitochondrial biogenesis-related factors, including peroxisome proliferator-activated receptor-γ co activator 1α (PGC1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). CONCLUSIONS: Wild ginseng CMCs protect against HFD-induced liver injury, which prevents lipid accumulation and mitochondrial oxidative stress, and enhances mitochondrial biogenesis.

Potentiation of natural killer (NK) cell activity by methanol extract of cultured cambial meristematic cells of wild ginseng and its mechanism.

AIMS: As an alternative strategy to obtain large amounts of ginseng extract with high yield of ginsenosides, we have utilized culture of cambial meristematic cells (CMCs) from wild ginseng. The anti-tumor effects of methanol extract of ginseng CMCs (MEGC) and their action mechanisms were investigated. MAIN METHODS: Mice were intraperitoneally administered with MEGC, and we explored NK cell activity, suppression of in vivo growth of tumor cells and relevant molecule expression. KEY FINDINGS: MEGC significantly potentiated NK cell activity and suppressed in vivo growth of B16 melanoma cells. However, we observed no increase in NK cell number and unaltered expression of NK cell-activating (NKG2D) and inhibitory (Ly49, CD94/NKG2A) receptors as well as NK cell activation markers (CD25, CD69, CD119, and CD212) in MEGC-treated group compared to the controls. Instead, MEGC significantly enhanced IL-2 responsiveness in the early effector phase and the constitutive expression of granzyme B. SIGNIFICANCE: Our data indicate that culture of CMCs is an attractive alternative method for sustainable production of ginseng extracts and clinical use. In addition, we have unraveled a novel mechanism underlying the potentiation of NK cell activity and antitumor effect of ginseng extract, in which it upregulates the constitutive expression of cytotoxic mediator(s) and IL-2 responsiveness.

D-chiro-inositol and pinitol extend the life span of Drosophila melanogaster.

D-chiro-inositol, a member of the inositol family, and pinitol, a 3-methoxy analogue of D-chiro-inositol, have been proposed to have antidiabetic, antiinflammatory, anticancer and stamina enhancing effects. We found that supplementing the diet of Drosophila with D-chiro-inositol and pinitol extended adult longevity in both male and female flies. Life span extension was accompanied by protection against oxidative and starvation stresses, improvement in health span, and no reduction in fecundity. Pinitol increased the fly life span, both in dietary restriction and in ad libitum conditions, suggesting that pinitol increased life span in a manner that was independent of the dietary restriction pathway. Nuclear localization of dFOXO increased in D-chiro-inositol and pinitol-fed flies when compared with controls. Pinitol treatment significantly activated JNK and S6K, but not AKT, indicating that the activation of dFOXO by pinitol is acquired by the activation of S6K and JNK signaling. Hence, our study indicated that D-chiro-inositol and pinitol could be novel food-derived antiaging compounds.

Centella asiatica protects against UVB-induced HaCaT keratinocyte damage through microRNA expression changes.

This study aimed to evaluate the protective effects of Centella asiatica (C. asiatica) against ultraviolet B (UVB) damage in human keratinocytes using microRNA (miRNA) expression profiling analysis. Titrated extract of C. asiatica (TECA) demonstrated low cytotoxicity in normal human HaCaT keratinocytes only at low doses (<5 µg/ml). UVB (50 mJ/cm2) irradiation significantly decreased cell viability, and TECA treatment decreased the UVB toxicity. By using miRNA microarrays, we determined that 72 miRNAs had an altered expression following TECA treatment in UVB-irradiated keratinocytes (46 upregulated and 26 downregulated). Using an miRNA target gene prediction tool and Gene Ontology (GO) analysis, we determined that miRNAs with altered expression were functionally related with the inhibition of apoptosis and cell proliferation. Overall, these results provide meaningful information to facilitate the understanding of TECA-mediated UVB protection in human keratinocytes.

The effects of low-dose ionizing radiation in the activated rat basophilic leukemia (RBL-2H3) mast cells.

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, ß-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.

Plant natural products: history, limitations and the potential of cambial meristematic cells.

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.

Suppression of lipopolysaccharide-induced expression of inflammatory indicators in RAW 264.7 macrophage cells by extract prepared from Ginkgo biloba cambial meristematic cells.

CONTEXT: Ginkgo biloba L. (Ginkgoaceae) leaves have been used as an herbal medicine that has a complex range of biological activities. However, when we consider that biological activity of plant extracts is highly variable according to the source, location, and harvest season, technology to obtain the natural products with homogeneity is extremely important. OBJECTIVE: We established the technology to obtain the cambial meristematic cells (CMCs) of Ginkgo biloba, which were expanded in vitro with homogeneity through a suspension culture and then determined the anti-inflammatory activity of fractionated samples prepared from the ethanol extract of CMCs. MATERIALS AND METHODS: We determined the anti-inflammatory activity of samples using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Especially, influence of sample treatment on the expression of various indicators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, mitogen-activated protein (MAP) kinases, transcription factor, and cytokines, involved in inflammatory activity was assessed. RESULTS: A fractionated sample demonstrated 53.4% inhibition of LPS-induced NO production from the cells. Additionally, when fractionated samples were treated, iNOS and COX-2 expressions were almost completely suppressed. Fractionated samples also inhibited the phosphorylation of LPS-induced extracellular signal-regulated (ERK) and p38 MAP kinases more than 60%. IκB phosphorylation and subsequent nuclear factor (NF)-κB activation were also suppressed by fractionated samples. The expression of pro-inflammatory cytokines, IL-6 and tumor necrosis factor (TNF)-α, was significantly inhibited by the sample treatment. DISCUSSION AND CONCLUSION: Fractionated samples from the ethanol extract of Ginkgo biloba CMCs could potentially be the source of a powerful anti-inflammatory substance.

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression.

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05Gy did not affect cell death, but high-dose radiation (> 0.2Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-kappaB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-kappaB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.