Derivation of the MUSIe002-A human embryonic stem cell line.

The MUSIe002-A cell line was established from in vitro fertilization of human sperm and oocytes donated for research with informed consent. This cell line exhibited normal human embryonic stem cell (hESC) characteristics, including typical cell morphology, expression of all pluripotent stem cell markers, and potential to differentiate into three germ layers. A karyotyping analysis revealed 46 XY chromosome and cells that did not have mycoplasma contamination. MUSIe002-A represents a valuable unlimited cell source and is of potential interest for human in vitro stem cell based-models, genetic modifications, and stem cell-based therapy of human disease.

Generation of a serine/threonine-protein kinase LATS1 gene-edited iPSC MUSIi012-A-3.

In mammals, there are a number of kinases, including serine/threonine-protein kinase LATS1, that act as a core kinase of the Hippo pathway and that negatively regulate the Hippo effector protein YAP and its paralog TAZ. Using CRISPR/Cas9 technology, we established a stable LATS1 knockdown (LATS1-KD) iPSC from the MUSIi012-A cell line. The LATS1-KD iPSC MUSIi012-A-3 that was developed maintained both the normal karyotype and the pluripotent phenotype, and retained the ability to differentiate into all three embryonic germ layers.

YAP-depleted iPSC MUSIi012-A-2 maintained all normal stem cell characteristics.

Yes-associated protein (YAP) is an important transcriptional coactivator in the Hippo signaling pathway. Using CRISPR/Cas9 technology, we established a stable YAP-knockdown (YAP-KD) induced pluripotent stem cell (iPSC) from the MUSIi012-A cell line. The YAP-KD iPSC MUSIi012-A-2 maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and it maintained the normal karyotype.

Derivation of human embryonic stem cell line MUSIe001-A from an embryo with homozygous α0-thalassemia (SEA deletion).

MUSIe001-A cell line was derived from a Southeast Asian (SEA) type deletion α0-thalassemia embryo. The SEA deletion embryo was donated for research with informed consent. This cell line shows normal hESC morphology, expresses all pluripotent markers, and has the potential to differentiate into all three germ layers in vitro and in vivo. The MUSIe001-A line has normal karyotype and is free from mycoplasma contamination. PCR analysis confirmed the MUSIe001-A cell line to be a SEA type deletion. MUSIe001-A is a valuable proof of principle model for gene therapy that will facilitate the development of new treatments for affected foetuses.

Generation of human induced pluripotent stem cell line carrying SCN5AC2204>T Brugada mutation (MUSli009-A-1) introduced by CRISPR/Cas9-mediated genome editing.

Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.

Derivation of a MUSIi012-A iPSCs from mobilized peripheral blood stem cells.

CD34+ cells were isolated from mobilized peripheral blood of a healthy donor and reprogrammed by nucleofection with episomal plasmids carrying l-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53. The obtained MUSIi012-A cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers, and a normal karyotype.

High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation.

BACKGROUND: Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used. METHODS: We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells. RESULTS: This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen-thawed embryos with better derivation efficiency of more than 50% (range 50-100%; median 70%). CONCLUSIONS: We successfully developed a better hESC derivation methodology using the "MTP" culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.

Partial Substitution of Glucose with Xylitol Suppressed the Glycolysis and Selectively Inhibited the Proliferation of Oral Cancer Cells.

Suitable diet for cancer survivors remains an unresolved challenge. Increased glucose utilization is a hallmark of various cancers. Therefore, alternative carbohydrate supplying normal tissue but retarding cancer growth is needed. This study investigated the effect of sugar alcohols on the proliferation of oral cancer cells compared to nontransformed cells and explored the mechanism. Six oral squamous cell carcinoma (CAL-27, FaDu, SCC4, SCC9, SCC15, and SCC25) and one nontransformed oral keratinocyte (OKF6/TERT2) lines were cultured in media containing 1 mg/ml glucose and 5.8 mg/ml xylitol or sorbitol, yielding equal energy input to control group (4.5 mg/ml glucose). Partial substitution of glucose with sugar alcohols especially xylitol significantly suppressed proliferation of oral cancer but not nontransformed cells. Despite the addition of isocaloric quantities of the sugars, cancer cells exposed to low glucose plus xylitol had retarded ATP generation and decreased activity of phosphofructokinase (PFK), the rate-limiting enzyme in glycolysis. Furthermore, D-xylulose, its key metabolic intermediate, enhanced the anticancer effect of xylitol. These findings suggested a selective anticancer activity of xylitol and the potential mechanism involving inhibition of glucose utilization. Partial substitution of glucose with xylitol may be a proper nutrient for oral cancer survivors, deserving further investigation in animal and clinical settings.

Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death.

BACKGROUND: A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy. RESULTS: In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy. CONCLUSIONS: These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.

Eryngium foetidum suppresses inflammatory mediators produced by macrophages.

OBJECTIVE: This study assessed anti-inflammatory and antioxidant activities of E. foetidum leaf extract on LPS-activated murine macrophages. METHODS: RAW264.7 cells were pretreated with or without E. foetidum extract for 1 h prior to incubation with LPS for 24 h. Anti-inflammatory activity was evaluated with reference to iNOS, COX-2, TNF-α and IL-6 gene expression. In addition, NO and intracellular ROS generation were determined by Griess method and fluorescence intensity and activation of MAPKs and IκB by Western blotting. RESULTS: Prior treatment with E. foetidum leaf extract inhibited elevation of IL-6, TNF-α, iNOS and COX-2, together with their cognate mRNAs in a dose-dependent manner. NO and intracellular ROS contents were similarly reduced. These effects were due to inhibition of LPS-induced phosphorylation of JNK and p38 as well as IκB. E. foetidum ethanol extract was shown to contain lutein, ß-carotenes, chlorogenic acid, kaempferol and caffeic acid, compounds known to exert these bioactive properties. CONCLUSIONS: E. foetidum leaf extract possesses suppressive effects against pro-inflammatory mediators. Thus, E. foetidum has a high potential to be used as a food supplement to reduce risk of cancer associated with inflammation.

Moringa oleifera pod inhibits inflammatory mediator production by lipopolysaccharide-stimulated RAW 264.7 murine macrophage cell lines.

Pro-inflammatory mediators produced during inflammatory response have been demonstrated to initiate and aggravate pathological development of several chronic diseases. Plant bioactive constituents have been reported to exert anti-inflammatory activities. Various parts of Moringa oleifera have long been used as habitual diets and traditional remedy along the tropical region. Anti-inflammatory activity of boiled M. oleifera pod extract was assessed by measuring pro-inflammatory mediator expression in the lipopolysaccharide-induced murine RAW264.7 macrophage cells. Prior treatment with 31-250 µg/mL M. oleifera extract for 1 h inhibited elevation of mRNA and protein level of interleukine-6, tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclooxygenease-2, induced by lipopolysaccharide for 24 h in a dose-dependent manner. The suppressive effect was mediated partly by inhibiting phosphorylation of inhibitor kappa B protein and mitogen-activated protein kinases. These results indicate that the anti-inflammatory activity from bioactive compounds present in the M. oleifera pod constituents may contribute to ameliorate the pathogenesis of inflammatory-associated chronic diseases.

Anti-inflammatory activities of red curry paste extract on lipopolysaccharide-activated murine macrophage cell line.

OBJECTIVE: This study investigated anti-inflammatory and antioxidant activities of an ethanol extract from Thai red curry paste. METHODS: The RAW264.7 murine macrophage cell line was incubated with the extract (65-260 µg/mL) with or without lipopolysaccharide. The anti-inflammatory activities of the extract were examined by measuring inducible nitric oxide synthase, cyclo-oxygenase-2, tumor necrosis factor-α, and interleukin-6 mRNA and protein level by reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay, respectively. Nitric oxide production and intracellular reactive oxygen species generation were determined by the Griess method and fluorescence intensity. The activation of mitogen-activated protein kinases and inhibitor κB were determined by western blot. RESULTS: Exposure of cells with the extract significantly suppressed lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase, cyclo-oxygenase-2, tumor necrosis factor-α, and interleukin-6 expressions (P < 0.05) by dose-dependently without cytotoxic effect. Intracellular reactive oxygen species significantly decreased (P < 0.05) in lipopolysaccharide-induced RAW264.7 cells. The inhibitory effect was mediated partly by inhibiting activation of inhibitor κB-α and mitogen-activated protein kinases. CONCLUSION: These results suggest that the anti-inflammatory and antioxidant properties of Thai red curry paste stem from bioactive compounds present in the spice and herb constituents. The health benefits of Thai red curry paste warrant further investigations in vivo.

Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells.

The HepG2 cell line is a useful tool for studying dengue virus-cell interactions but as it grows in clumps rather than monolayers, it does not readily adapt itself to the standard plaque assay technique. We therefore sought to develop an indirect plaque assay methodology. Initially HepG2 cells were infected with dengue virus serotype 2 and post-infection incubated for between 0 and 16 h before being treated with trypsin to separate the cells, followed by dilution and plating onto pre-grown monolayers of Vero cells in six well plates. After 7 days incubation and crystal violet staining, plaques were observed at all time points, although there was a relationship between number of plaques and post-infection incubation time, with the longest post-infection incubation time giving the highest number of plaques. To validate the assay with respect to virus input, the experiment was repeated at both the 0 and 16 h post-infection incubation times with different virus: cell levels. At both post-infection incubation times the response of input virus to plaque number was linear. This is a useful adaptation of the plaque assay methodology and one that may be applicable to other virus/cell line combinations.