技术资料

PURPOSE FK506-binding protein like (FKBPL) and its peptide derivative,AD-01,have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here,we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC). EXPERIMENTAL DESIGN Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75),primary patient samples,and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays,quantitative PCR (qPCR),and immunofluorescence. RESULTS AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers,Nanog,Oct4,and Sox2,were also significantly reduced. Furthermore,we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor,DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally,FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs,highlighting a role for endogenous FKBPL in stem cell signaling. CONCLUSIONS AD-01 has dual antiangiogenic and anti-BCSC activity,which will be advantageous as this agent enters clinical trial. View Publication

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics,and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is,however,traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer,and as a result,possible xenogeneic contamination,contribution of unlabeled amino acids by the feeders,interlaboratory variability of MEF preparation,and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used,chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison,WI) licensed to STEMCELL Technologies (Vancouver,Canada)]. This medium,together with adjustments to the culturing protocol,facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View Publication

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition,including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients,we demonstrate that cell viability can be preserved throughout differentiation. In addition,modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique,as well as gene expression patterns,indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm. View Publication

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells,the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here,we attempted to reprogram E-PZ cells by forced expression of Oct4,Sox2,c-Myc,and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81,SSEA-3,Nanog,Sox2,and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5,CK14,and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal,mesodermal,and endodermal cells in vitro,although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly,E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44,p63,MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR),and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA),a hallmark of secretory cells,suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally,when injected into mice,E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme,E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore,provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. View Publication

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

Ovarian cancer is one of the most lethal female malignancies and epigenetic abnormalities are thought to play a vital role in the pathogenesis,development and progression of ovarian cancer. Our goal was to investigate whether the combination of trichostatin A (TSA) and 5-aza-2'-deoxycytidine (decitabine) was superior to single agent on tumorigenicity of ovarian cancer cells. We found that tumorigenicity and metastasis of SKOV3 cells were significantly suppressed by the combination of TSA and decitabine in xenograft mouse models. Migration capacity was markedly suppressed through the induction of E-cadherin and suppression of N-cadherin when treated with TSA and decitabine. Invasion was also suppressed at least partially through inhibition of MMP-2 and MMP-9 with the combined treatment. The combination drugs markedly inhibited spheroid formation and significantly impaired migration and invasion capacity of spheroid derived cells through inhibition of Twist,N-cadherin,MMP-2,MMP-9 and induction of E-cadherin. Epigenetically,the activity of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) were markedly inhibited when TSA was used in combination with decitabine,especially the expression of DNMT3A/3B and HDAC1/2. Acetylation of histone H3 and H4 were more markedly stimulated with the combination than with either agent alone. The expression level of lysine-specific demethylase-1 (LSD1) was also suppressed. The transcription activity marker dimethylated-H3K4 was induced,but the dimethylated-H3K9 was suppressed by exposure to the combined drugs. These results suggest that the combination of TSA and decitabine significantly suppresses tumorigenicity by inhibiting migration and invasion of ovarian cancer cells via regulating the expression of the cadherins and MMPs,which may be epigenetically regulated by DNA methylation and histone modification. View Publication

Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here,we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells,human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h,the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell–cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels,inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity,highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary,we have developed microengineered three-dimensional vascular structures that can be used as a model to test the effects of drugs and study the interaction between different human vascular cell types. In the future,the model may be integrated into larger tissue constructs to advance the development of organs-on-chips. View Publication

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of,for example,ion selectivity,gating mechanism,composition,or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source,keratinocytes from plucked human hair. This comparison revealed that 26&x25; of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6&x25; were downregulated. Additionally,iPSCs express a much higher number of ion channels compared to keratinocytes. Further,to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny,namely,neurons and cardiomyocytes derived from iPS cells. To conclude,hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms. View Publication

Retinoic acid (RA),a vitamin A metabolite,modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition,RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody,demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved,we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells. View Publication

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly,several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and,as a result,undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably,we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings,which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization,suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. View Publication

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC),which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation,and this hydroxymethylation change is critical for optimal epigenetic reprogramming,but does not compromise primed pluripotency. Compared with hESCs,we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions,most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly,these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences. View Publication

A satisfactory in vitro model of vocal fold mucosa does not exist,thus precluding a systematic,controlled study of vocal fold biology and biomechanics. We sought to create a valid,reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design,hESCs were differentiated into cells that coexpressed the simple epithelial cell marker,keratin 18 (K18),and the transcription factor,p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air–liquid interface. After that time,the engineered mucosa demonstrated a stratified,squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified,squamous epithelial markers,keratin 13 (K13) and 14 (K14),as well as tight junctions,adherens junctions,gap junctions,and desmosomes. Despite the presence of components critical for epithelial structural integrity,the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity,this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered,terminally differentiated epithelium. View Publication