Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53.

Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.

A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.

Enterococcus faecium L-15 Extract Enhances the Self-Renewal and Proliferation of Mouse Skin-Derived Precursor Cells.

Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.

CXCR2 Ligands and mTOR Activation Enhance Reprogramming of Human Somatic Cells to Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology has great promise in regenerative medicine and disease modeling. In this study, we show that human placenta-derived cell conditioned medium stimulates chemokine (C-X-C motif) receptor 2 (CXCR2) in human somatic cells ectopically expressing the pluripotency-associated transcription factors Oct4, Sox2, Klf4, and cMyc (OSKM), leading to mechanistic target of rapamycin (mTOR) activation. This causes an increase in endogenous cMYC levels and a decrease in autophagy, thereby enhancing the reprogramming efficiency of human somatic cells into iPSCs. These findings were reproduced when human somatic cells after OSKM transduction were cultured in a widely used reprogramming medium (mTeSR) supplemented with CXCR2 ligands interleukin-8 and growth-related oncogene α or an mTOR activator (MHY1485). To our knowledge, this is the first report demonstrating that mTOR activation in human somatic cells with ectopic OSKM expression significantly enhances the production of iPSCs. Our results support the development of convenient protocols for iPSC generation and further our understanding of somatic cell reprogramming.

Variability and Heritability of Thiamine Pharmacokinetics With Focus on OCT1 Effects on Membrane Transport and Pharmacokinetics in Humans.

Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1-dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high-dose and low-dose (nutritional) intake, and heritability of thiamine and thiamine-phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR-2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.

Effects of Zuogui Wan on testis structure and expression of c-Kit and Oct4 in rats with impaired spermatogenesis.

CONTEXT: Zuogui Wan is a classic traditional Chinese prescription. Preliminary studies have confirmed that it could improve sperm quality significantly. OBJECTIVE: To investigate the effect of Zuogui Wan on testis structure and c-kitproto-oncogeneprotein (c-Kit) and octamer-binding transcription factor-4 (Oct4) expression in a rat model of impaired spermatogenesis. MATERIAL AND METHODS: Thirty-six Sprague-Dawley (SD) rats were divided into Blank control, Tripterygium glycosides (GTW) and Zuogui Wan groups (n = 12). GTW was used to generate models of impaired spermatogenesis. Then Zuogui Wan group was administered 6 g/kg/d of Zuogui Wan granules for 4 weeks. Changes in the pathological structure and ultrastructure were observed with optical microscope and transmission electron microscope. Expression of c-Kit and Oct4 were quantified by RT qPCR and Western blots. RESULTS: Both the pathological damage and the damages in the ultrastructure of spermatogenic epithelium had improved in Zuogui Wan group. Compared with the GTW model group (0.47 ± 0.19; 0.38 ± 0.14), c-Kit and Oct4 protein expression increased in the Zuogui Wan group (0.75 ± 0.27; 0.65 ± 0.23). C-Kit and Oct4 mRNA expression increased in Zuogui Wan group (1.06 ± 0.16; 1.85 ± 1.04) compared to the GTW model group (0.66 ± 0.23; 0.46 ± 0.29). CONCLUSIONS: Zuogui Wan is capable of restoring the damage to the testis structure and ultrastructure and regulates the expression of c-Kit and Oct4 at protein and mRNA levels, inhibiting apoptosis and promoting proliferation of spermatogenic cells.

Long noncoding RNAs sustain high expression levels of exogenous octamer-binding protein 4 by sponging regulatory microRNAs during cellular reprogramming.

Long noncoding RNAs (lncRNAs) modulate gene expression as competing endogenous RNAs (ceRNAs) that sponge regulatory microRNAs (miRNAs). During cellular reprogramming, genes associated with pluripotency establishment need to be up-regulated, and developmental genes need to be silenced. However, how ceRNAs control cellular reprogramming still awaits full elucidation. Here, we used doxycycline-inducible expression of the four transcription factors octamer-binding protein 4 (OCT4), SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc (c-Myc) to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Using RNA-Seq and bioinformatics approaches, we found that the expression levels of miRNAs from MEFs remain high from day 0 to 6 after the doxycycline induction. Many genes targeted by these miRNAs were up-regulated, and long intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs), which have complementary binding sites to these miRNAs, were highly expressed, indicating lincRNAs and circRNAs may function as ceRNAs. Intriguingly, knockdown of the linc/circRNAs that sponge the miRNAs, which target OCT4 down-regulated exogenous OCT4, decreased reprogramming efficiency, and resulted in low-grade iPSCs. Our results suggest that the ceRNA network plays an important role in cellular reprogramming.

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Effect of alanine supplementation during in vitro maturation on oocyte maturation and embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs.

The objective of this study was to examine the effect of alanine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development in pigs. To this end, we investigated the nuclear maturation, intraoocyte glutathione (GSH) content of IVM oocytes, and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In addition, we analyzed the expression of genes associated with apoptosis and embryonic development in IVM oocytes, 4-cell stage embryos, and blastocysts produced via PA and SCNT. To determine the optimal concentration of alanine to promote the maturation and development of PA and SCNT embryos, various concentrations (0, 0.363, 1, 5, and 10 mM) of alanine were added to IVM medium during oocyte maturation. The proportion of metaphase II (MII) oocytes after IVM did not differ according to the concentration of alanine. However, significantly higher intraoocyte GSH content was observed in oocytes treated with 0.363 mM alanine compared with that in untreated oocytes. However, treatment of recipient oocytes with 5 or 10 mM alanine during IVM decreased the GSH content in mature oocytes compared to that in control oocytes. Oocytes matured in the presence of 0.363 mM alanine showed significantly increased rates of cleavage and blastocyst formation after PA and SCNT compared to untreated oocytes. PA and SCNT embryos from the 0.363 mM alanine-treated group of MII oocytes showed significantly higher transcript levels of POU5F1 and FGFR2, which are associated with oocyte quality and embryonic development, than the untreated group. Our results suggest that treatment of pig oocytes with 0.363 mM alanine during IVM improves embryonic developmental competence after PA and SCNT by increasing intraoocyte GSH content and increasing the mRNA expression of POU5F1 and FGFR2.

Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus.

BACKGROUND/AIMS: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. METHODS: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. RESULTS: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. CONCLUSION: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications.

Melatonin supplementation during prolonged in vitro maturation improves the quality and development of poor-quality porcine oocytes via anti-oxidative and anti-apoptotic effects.

Poor-quality oocytes (those with 1-2 layers of cumulus cells) typically possess low meiotic competence and development. Prolonging the duration of in vitro maturation (IVM; 52 hr) can enhance the maturation rate of poor-quality oocytes, but it does not improve subsequent embryonic development. This likely reflects the increased reactive oxygen species (ROS) production and apoptosis seen in these oocytes compared with the non-prolonged IVM (44 hr) group. Melatonin is a free radical scavenger, anti-oxidant and anti-apoptotic agent that reported to enhance the quality of embryos by inhibiting ROS generation and apoptosis. Therefore, we herein investigated whether melatonin combined with prolonged IVM (52 hr) could improve the quality and development of poor-quality oocytes. We supplemented IVM and/or in vitro culture (IVC) media with various concentrations (0, 10-7 , 10-6 , 10-5 M) of melatonin, and estimated parameters related to oocyte quality and development. The addition of melatonin (10-6 M) to a prolonged IVM system improved the oocyte quality and development compared with those of the melatonin-free poor-quality oocytes group, and that this was due to decreases in ROS generation, apoptosis, and DNA damage. When melatonin was added during both IVM (10-6 M) and IVC (10-6 M), we observed a cumulative positive influence on the embryonic development and quality; this treatment enhanced the expression level of Oct4 and decreased the levels of ROS, DNA damage, and apoptosis. Together, these findings suggest that the combination of melatonin plus prolonged IVM can improve the quality and development of poor-quality porcine oocytes via anti-oxidative and anti-apoptotic effects.

YY1 Positively Regulates Transcription by Targeting Promoters and Super-Enhancers through the BAF Complex in Embryonic Stem Cells.

Yin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.

Effects of prostaglandin E2 on clonogenicity, proliferation and expression of pluripotent markers in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Based on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE2 that could regulate mineralization of PDL cells, it was hypothesized that PGE2 had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE2. MATERIAL AND METHODS: Human PDL cells were cultured and treated with various doses of PGE2, and the aforementioned characteristics of PDL stemness were analyzed. RESULTS: The clonogenicity and proliferation were significantly enhanced by PGE2 at low concentrations (0.01, 0.1 and 1ng/ml; P<0.05), but only the proliferation was significantly diminished by PGE2 at a high concentration (100ng/ml; P<0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE2 treatment at 0.1 and 1ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE2 treatment at 1ng/ml (P<0.05). CONCLUSION: Our findings suggest that although a high dose of PGE2 (100ng/ml) inhibits proliferation of PDL cells, PGE2 at low doses appears to play a role in the maintenance of PDL stemness.

YM155 as an inhibitor of cancer stemness simultaneously inhibits autophosphorylation of epidermal growth factor receptor and G9a-mediated stemness in lung cancer cells.

Cancer stem cell survival is the leading factor for tumor recurrence after tumor-suppressive treatments. Therefore, specific and efficient inhibitors of cancer stemness must be discovered for reducing tumor recurrence. YM155 has been indicated to significantly reduce stemness-derived tumorsphere formation. However, the pharmaceutical mechanism of YM155 against cancer stemness is unclear. This study investigated the potential mechanism of YM155 against cancer stemness in lung cancer. Tumorspheres derived from epidermal growth factor receptor (EGFR)-mutant HCC827 and EGFR wild-type A549 cells expressing higher cancer stemness markers (CD133, Oct4, and Nanog) were used as cancer stemness models. We observed that EGFR autophosphorylation (Y1068) was higher in HCC827- and A549-derived tumorspheres than in parental cells; this autophosphorylation induced tumorsphere formation by activating G9a-mediated stemness. Notably, YM155 inhibited tumorsphere formation by blocking the autophosphorylation of EGFR and the EGFR-G9a-mediated stemness pathway. The chemical and genetic inhibition of EGFR and G9a revealed the significant role of the EGFR-G9a pathway in maintaining the cancer stemness property. In conclusion, this study not only revealed that EGFR could trigger tumorsphere formation by elevating G9a-mediated stemness but also demonstrated that YM155 could inhibit this formation by simultaneously blocking EGFR autophosphorylation and G9a activity, thus acting as a potent agent against lung cancer stemness.

In vitro induction of human embryonal carcinoma differentiation by a crude extract of Rhazya stricta.

BACKGROUND: Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells. METHODS: Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography - mass spectrometry (GC-MS). RESULTS: Chloroform stem extract induced differentiation of NT2 cells at 5 µg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times. CONCLUSION: Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases.

YY1 Is Required for Posttranscriptional Stability of SOX2 and OCT4 Proteins.

Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein-protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

BACKGROUND: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. METHODS AND RESULTS: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 µg kg-1 min-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. CONCLUSIONS: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Phenethyl isothiocyanate suppresses cancer stem cell properties in vitro and in a xenograft model.

BACKGROUND: Cancer stem cells (CSCs) are a subset of cells within the bulk of a tumor that have the ability to self-renew and differentiate, and are thus associated with cancer invasion, metastasis, and recurrence. Phenethyl isothiocyanate (PEITC) is a natural compound found in cruciferous vegetables such as broccoli and is used as a cancer chemopreventive agent; however, its effects on CSCs are little known. PURPOSE: To evaluate the effect of PEITC on CSCs in this study by examining CSC properties. METHODS: NCCIT human embryonic carcinoma cells were treated with PEITC, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by luciferase assay and western blot. Effect of PEITC on self-renewal capacity and clonogenicity were assessed with the sphere formation, soft agar assay, and clonogenic assay in an epithelial cell adhesion molecule (EpCAM)-expressing CSC model derived from HCT116 colon cancer cells using a cell sorting system. The effect of PEITC was also investigated in a mouse xenograft model obtained by injecting nude mice with EpCAM-expressing cells. RESULTS: We found that PEITC treatment suppressed expression of the all three pluripotency factors in the NCCIT cells, in which pluripotency factors are highly expressed. Moreover, PEITC suppressed the self-renewal capacity and clonogenicity in the EpCAM-expressing CSC model. EpCAM was used as a specific CSC marker in this study. Importantly, PEITC markedly suppressed both tumor growth and expression of three pluripotency factors in a mouse xenograft model. CONCLUSION: These results demonstrate that PEITC might be able to slow down or prevent cancer recurrence by suppressing CSC stemness.

Suppression of cancer stem-like phenotypes in NCI-H460 lung cancer cells by vanillin through an Akt-dependent pathway.

Cancer stem cells (CSCs) have been reported as a major cause of cancer metastasis and the failure of cancer treatment. Cumulative studies have indicated that protein kinase B (Akt) and its downstream signaling pathway, including CSC markers, play a critical role in the aggressive behavior of this cancer. In this study, we investigated whether vanillin, a major component in Vanilla planifolia seed, could suppress cancer stemness phenotypes and related proteins in the human non-small cell lung cancer NCI­H460 cell line. A non-toxic concentration of vanillin suppressed spheroid and colony formation, two hallmarks of the cancer stemness phenotype, in vitro in NCI­H460 cells. Western blot analysis revealed that the CSC markers CD133 and ALDH1A1 and the associated transcription factors, Oct4 and Nanog, were extensively downregulated by vanillin. Vanillin also attenuated the expression and activity of Akt, a transcription regulator upstream of CSCs, an action that was confirmed by treatment with the Akt inhibitor perifosine. Furthermore, the ubiquitination of Akt was elevated in response to vanillin treatment prior to proteasomal degradation. This finding indicates that vanillin can inhibit cancer stem cell-like behavior in NCI­H460 cells through the induction of Akt-proteasomal degradation and reduction of downstream CSC transcription factors. This inhibitory effect of vanillin may be an alternative approach in the treatment against lung cancer metastasis and its resistance to chemotherapy.