参考文献

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

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts,and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer,retaining a pre-inactivation X chromosome state,and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF,naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells,they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression,pronounced tendency for X chromosome inactivation in most female human ES cells,increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells,from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells,and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively,our findings establish new avenues for regenerative medicine,patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. View Publication

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions,where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh),whose posttranslational lipid modifications and assembly into multimers enhance its biological potency,potentially through receptor clustering. Investigations of Shh typically utilize recombinant,monomeric protein,and thus the impact of multivalency on ligand potency is unexplored. Among its many activities,Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies,compared to the monomeric Shh,increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%,respectively. Thus,multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients. View Publication

We describe here the effects of three drugs that are either approved or have the potential for treating multiple sclerosis (MS) patients through the in vitro activities of human natural killer (NK) cells and dendritic cells (DCs). Our results indicate that 1,25(OH)2D3,the biologically active metabolite of vitamin D3,calcipotriol and FTY720 augment IL-2-activated NK cell lysis of K562 and RAJI tumor cell lines as well as immature (i) and mature (m) DCs,with variable efficacies. These results are corroborated with the ability of the drugs to up-regulate the expression of NK cytotoxicity receptors NKp30 and NKp44,as well as NKG2D on the surfaces of NK cells. Also,they down-regulate the expression of the killer inhibitory receptor CD158. The three drugs down-regulate the expression of CCR6 on the surface of iDCs,whereas vitamin D3 and calcipotriol tend to up-regulate the expression of CCR7 on mDCs,suggesting that they may influence the migration of DCs into the lymph nodes. Finally,vitamin D3,calcipotriol and FTY720 enhance NK17/NK1 cell lysis of K562 cells,suggesting that a possible mechanism of action for these drugs is via activating these newly described cells. In conclusion,our results show novel mechanisms of action for vitamin D3,calcipotriol and FTY720 on cells of the innate immune system. View Publication

$\$-Thalassemia ($\$-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in $\$-globin gene (HBB). It is estimated that 4.5% of the population in the world carry $\$-Thal mutants (1),posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However,homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here,we describe a robust process combining efficient generation of integration-free $\$-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly,the gene-corrected $\$-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal $\$-globin. Our studies provide an efficient and universal strategy to correct different types of $\$-globin mutations in $\$-Thal iPSCs for disease modeling and applications. View Publication

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS),as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear,with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed,and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased,leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α,suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6,and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. 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

Exposure to ionizing radiation was shown to result in an increased risk of breast cancer. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs). In order to assess whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs,we measured by flow cytometry the proportion of CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR)-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells,progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the downregulation of miR-22 and miR-29c expression),which resulted in increased proportions of radiation-resistant tumor-initiating CSCs. View Publication

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials,we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg,cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence,their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose,the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4-40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages,the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4,SSEA3,and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM,however,hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications,providing their degradation rate is moderate. Additionally,the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro,and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. View Publication

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented,but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally,the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor,such as microbial agents,pollutants,or allergens. Briefly,nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates,and then transferred onto cell culture inserts. Upon reaching confluency,cells continue to be cultured at the air-liquid interface (ALI),for several weeks,which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium,with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents,transduction with lentiviral vectors,exposure to gases,or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways,functional changes,morphology,etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo. View Publication