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Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However,tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs,especially those in cancer patients. To circumvent these issues,we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable,but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition,invasion,stemness,and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ,downstream protumor factors,and hyaluronan and its cofactor TSG6,which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf" therapies and bioengineering applications." View Publication

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore,we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs,RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation,while down-regulated miRNAs such as miR-367,miR-18b,and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development,cell cycle progression,cell death,and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis,eye differentiation and development. View Publication

Gastric diseases,including peptic ulcer disease and gastric cancer,affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis,and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF,WNT,BMP,retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains,proliferative zones containing LGR5-expressing cells,surface and antral mucous cells,and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease,we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor,activation of signalling and induction of epithelial proliferation. Together,these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication

Clinical trials are currently used to test therapeutic efficacies for lung cancer,infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA,protein,morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates,volumes and cryoprotectant formulation were optimized to maintain tissue architecture,decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis,showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology,RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types,including alveolar epithelial cells,fibroblasts and stem cells,from the tissue for at least three months after cryopreservation. This new method should provide a uniform,cost-effective approach to the banking of biospecimens,with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. View Publication

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

OBJECTIVE The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However,the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. METHODS The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR,immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. RESULTS The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (Ptextless0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (Ptextless0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486,P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza),the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased,the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. CONCLUSION KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis. View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication

Findings from studies on stem cells have been applied to cancer stem cell (CSC) research,but little is known about the relationship between ES cell-related cell surface markers and CSCs. In this study,we focused on stage-specific embryonic antigen 3 (SSEA-3),a marker of mesenchymal stem cells and Muse cells in colorectal cancer (CRC). Expression of SSEA-3 in human CRC cell lines and clinical specimens,specifically the relationship of SSEA-3 expression and the representative CSC markers (CD44,CD166,ALDH,CD24 and CD26) as well as with mesenchymal stem cell/Muse cell marker (CD105) were assessed. To characterize SSEA-3-expressing cells,tumorigenicity,sphere formation ability,expression of iPS genes (Oct4,NANOG,SOX2 and c-Myc),cell proliferation and cell cycle status were assessed. SSEA-3 expression was identified in Caco-2,DLD-1,HT-29,SW480 and HCT116,but not in CaR-1 cells. No significant relationship between SSEA-3 and other stem cell markers was detected. SSEA-3+ cells showed increased tumorigenicity in vivo,but lower sphere formation ability in vitro than SSEA-3-. iPS gene expression was not correlated with SSEA-3 expression status. SSEA-3+ cells showed higher proliferative ability than SSEA-3- through enhanced cell cycles by decreased expression of p21Cip1/Waf1 and p27Kip1. Immunofluorescence analysis in clinical specimens indicated that expression of SSEA-3 is limited to stromal cells in normal mucosa but broad in poorly differentiated adenocarcinoma. These observations indicated that SSEA-3+ cells in CRC have immature phenotype but decreased self-renewal ability and may function as tumor transient amplifying cells or delayed contributing tumor-initiating cells. View Publication