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The effect of 9-cis-retinoic acid (9-cis-RA) on the growth of eight gastric cancer cell lines was related to their transcription levels of mRNAs for retinoid receptors. Northern blot analysis showed that seven (TMK-1,MKN-1,-28,-45,-74,HSC-39,KATO-III) out of eight gastric cancer cell lines synthesized mRNAs for retinoic acid receptors (RARs) and retinoid X receptor-alpha (RXR-alpha). MKN-7 cells did not transcribe either RARs or RXR-alpha at the mRNA level although they appeared to have no alterations at the gene level. The growth of all of the cell lines except for MKN-7 cells was inhibited by 1 x 10(-6) M 9-cis-RA. Cell cycle distribution analysis revealed that G0-G1 arrest was not induced by exposure to 9-cis-RA in the sensitive TMK-1 and KATO-III cells or the resistant MKN-7 cells. Interestingly,9-cis-RA temporarily increased the amount of the cyclin dependent kinase (cdk) inhibitor,Waf1/Cip1/Sdi1/p21 protein,and also reduced the amount of cdk-7,epidermal growth factor receptor (EGFR) and cyclin D1 proteins,followed by reduction in phosphorylation of the product of the retinoblastoma tumor suppressor gene (Rb) in the sensitive TMK-1 cells,but not in the resistant MKN-7 cells. These results suggest that 9-cis-RA has a cytostatic effect on gastric cancer cells that synthesize the receptor molecules through cell cycle regulatory machinery. View Publication

Summary Rett syndrome (RTT) is caused by mutations of MECP2,a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show,using an isogenic human embryonic stem cell model of RTT,that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells. View Publication

Background Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling,cardiotoxicity screening,drug discovery,and cell-based therapies. Despite the fundamental importance of Ca2+-induced Ca2+ release in excitation-contraction coupling,the mechanistic basis of Ca2+ handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. Objectives To study Ca2+ sparks as unitary events of Ca2+ handling for mechanistic insights. Methods To avoid ambiguities owing to the heterogeneous nature,we experimented with hESC-VCMs,purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction. Results Ca2+ sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine,tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model,we further showed that RyRs could be sensitized by Ca2+. Increasing external Ca2+ dramatically escalated the basal Ca2+ and spark frequency. Furthermore,RyR-mediated Ca2+ release sensitized nearby RyRs,leading to compound Ca2+ sparks. Depolarization or L-type Ca2+ channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca2+-dependent cytosolic Ca2+ increase and reduced the sarcoplasmic reticulum content. By contrast,removal of external Na+ or the addition of the Na+-Ca2+ exchanger inhibitor (KB-R7943 and SN-6) had no effect,suggesting that the Na+-Ca2+ exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca2+ uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca2+ waves. Conclusion Taken collectively,our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca2+-induced Ca2+ release. However,the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development. View Publication

A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover,the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end,a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly,this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide,which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage,over 85% viability,and maintained a normal karyotype and the expression of pluripotency markers such as Nanog,Oct4,and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers,the cells adopted respective fates displaying relevant markers. Lastly,engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence,we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable,xeno-free propagation and differentiation of human stem cells intended for therapies. View Publication

AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy,particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity,critical elements for their survival and differentiation,have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and,more generally,to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes,a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand,A-769662,a well-characterized AMPK activator,was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice,providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation,we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved,whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation,p27 being involved in this regulation. View Publication

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals,thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide,the use of human cell-teratogenicity assays has just started to be explored. Herein,a human pluripotent stem cell test (hPST) for identifying teratogens is described,benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically,71 drug-like compounds with known in vivo effects,including thalidomide,were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore,15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally,to assess the suitability of the hPST for high-throughput screens,a small library of 300 kinase inhibitors was tested,demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus,the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models. View Publication

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo�? and radiotherapy. Here,we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen�?resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely,ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2�?expressing cells,and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure,and also in the primary tumours of these patients,compared to those of responders. Together,these results suggest that development of tamoxifen resistance is driven by Sox2�?dependent activation of Wnt signalling in cancer stem/progenitor cells. View Publication

Cellular plasticity contributes to the regenerative capacity of plants,invertebrates,teleost fishes and amphibians. In vertebrates,differentiated cells are known to revert into replicating progenitors,but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells,we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast,direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming,the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states,notably cancer. View Publication

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition,recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL,classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore,the murine WEHI-3B(D+) cells and human leukemic cells classified as M2,M3,and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600. View Publication

It has been recently reported that the regulatory circuitry formed by OCT4,miR-302,and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C,a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs,directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression,decreased BMP signaling,and enhanced TGF$\$ JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown,but not the control,cells within 3 days,accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together,our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF. View Publication

Many solid cancers display cellular hierarchies with self-renewing,tumorigenic stemlike cells,or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies,they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs,we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays,but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines,including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion,and targeting IL-17A signaling impaired CIC growth. Notably,IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. View Publication