技术资料

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity,and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs,but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA,human pDCs upregulate the mannose receptor (MR,CD206). To examine the functional outcome of this upregulation,inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha,viability,and upregulation of several pDC-activation markers: CD40,CD86,HLA-DR,CCR7,and PD-L1. The level of activation negatively correlated with degree of mannosylation,however,subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore,two of the infectious HIV-1 strains induced profound necrosis in pDCs,also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs. View Publication

Cancer stem cells (CSCs) have been identified in a variety of cancers and emerged as a new target for cancer therapy. CSCs are resistant to many current cancer treatments,including chemotherapy and radiation therapy. Therefore,eradication of this cell population is a primary objective in cancer therapy. Here,we report gold nanorods (AuNRs) mediated photothermal treatment can selectively eliminate CSCs in MCF-7 breast cancer cells. It significantly reduced the aldehyde dehydrogenase positive (ALDH(+)) cells subpopulation and the mammosphere formation ability of treated cells. Also,the gene expression of stem cell markers was decreased. Cellular uptake assay revealed that polyelectrolyte conjugated AuNRs could be internalized by CSCs much more and faster than non cancer stem cells (NCSCs),which might be the main reason for the selective elimination of CSCs. We further loaded salinomycin (SA),a CSCs inhibitor with polyelectrolyte conjugated AuNRs to get a synergistic CSCs inhibition. Enhanced inhibition of CSCs was obtained by NIR light triggered drug release and hyperthermia. This CSCs-targeted thermo-chemotherapy platform provides a new combinatorial strategy for efficient inhibition of CSCs,which is promising to improve cancer treatment and may overcome the chemoresistance and recurrence of cancer. 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

Cardiomyocytes (CM) derived from human embryonic stem cells (hESC) are used for cardio-toxicity evaluation and tested in many preclinical trials for their potential use in regenerative therapeutics. As more efficient CM differentiation protocols are developed,reliable automated platforms for characterization and detection are needed. An automated time-resolved video analysis and management system (TVAMS) has been developed for the evaluation of hESC differentiation to CM. The system was used for monitoring the kinetics of embryoid bodies (EB) generation (numbers and size) and differentiation into beating EBs (percentage beating area and beating EB count) in two differentiation protocols. We show that the percentage beating areas of EBs (from total area of the EBs) is a more sensitive and better predictor of CM differentiation efficiency than percentage of beating EBs (from total EBs) as the percentage beating areas of EBs correlates with cardiac troponin-T and myosin heavy chain expression levels. TVAMS can also be used to evaluate the effect of drugs and inhibitors (e.g. isoproterenol and ZD7288) on CM beating frequency. TVAMS can reliably replace the commonly practiced,time consuming,manual counting of total and beating EBs during CM differentiation. TVAMS is a high-throughput non-invasive video imaging platform that can be applied for the development of new CM differentiation protocols,as well as a tool to conduct CM toxicology assays. View Publication

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells,suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists,which bind to extracellular BMP ligands and prevent their association with BMP receptors,dramatically reduced lung tumor growth. However,clinical application of protein-based BMP antagonists is limited by short half-lives,poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study,we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study,we demonstrated that DMH1,one of such inhibitors,potently reduced lung cell proliferation,promoted cell death,and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling,as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1,Id2 and Id3. Additionally,DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion,our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment. View Publication

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8,which is ˜99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD,both have been reported to migrate,on Western blotting (WB),at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore,detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies,immunoprecipitation,mass spectrometry,and studies using recombinant proteins,here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ˜22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate,on denaturing SDS-PAGE,at ˜28 kD to 180 kD. Interestingly,different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins,which can spontaneously form high M.W protein species. Finally,we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether,the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells. View Publication

BACKGROUND: Transforming growth factor-β (TGF-β) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers,such as smooth muscle alpha actin,SM22α,myocardin,and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression,calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt,BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely,addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2,calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover,TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway. View Publication

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development,several issues regarding their pluripotency,differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study,we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that,upon spontaneous differentiation,iPS-MBMC present high amounts of terminal $\$-galactopyranoside residues,pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation,prompting an ectoderm commitment. Exposed $\$-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface,which could modulate intercellular or intracellular interactions. Together,our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and,therefore,the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation. View Publication

The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs,how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny,and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d,the in vitro assessment requires another day,and the in vivo assessment requires up to 12 additional weeks. View Publication

BACKGROUND Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers,identify the same population of cells,or equate to therapeutic response is controversial. METHODS We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo,comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin,docetaxol and radiotherapy. RESULTS CD24,CD44,ALDH and SOX2 expression,the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo,cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers,although ER-negative cells accumulate. CONCLUSIONS Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications,rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer. View Publication

Renal cell therapy has shown clinical efficacy in the treatment of acute renal failure (ARF) and promise for treatment of end-stage renal disease (ESRD) by supplementing conventional small solute clearance (hemodialysis or hemofiltration) with endocrine and metabolic function provided by cells maintained in an extracorporeal circuit. A major obstacle in the widespread adoption of this therapeutic approach is the lack of a cryopreservable system to enable distribution,storage,and therapeutic use at point of care facilities. This report details the design,fabrication,and assessment of a Bioartificial Renal Epithelial Cell System (BRECS),the first all-in-one culture vessel,cryostorage device,and cell therapy delivery system. The BRECS was loaded with up to 20 cell-seeded porous disks,which were maintained by perfusion culture. Once cells reached over 5 A- 10(6) cells/disk for a total therapeutic dose of approximately 10(8) cells,the BRECS was cryopreserved for storage at -80°C or -140°C. The BRECS was rapidly thawed,and perfusion culture was resumed. Near precryopreservation values of cell viability,metabolic activity,and differentiated phenotype of functional renal cells were confirmed post-reconstitution. This technology could be extended to administer other cell-based therapies where metabolic,regulatory,or secretion functions can be leveraged in an immunoisolated extracorporeal circuit. View Publication