技术资料

Stem cell (SC) lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS) SC-containing neurosphere cultures for studying heritable neurodegenerative disease,we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD) mutation,rTg(tau(P301L))4510,with those expressing comparable levels of wild type human tau,rTg(tau(wt))21221. rTg(tau(P301L))4510 mice express the human tau(P301L) variant in their forebrains and display cellular,histological,biochemical and behavioral abnormalities similar to those in human FTD,including age-dependent differences in tau phosphorylation that distinguish them from rTg(tau(wt))21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tau(P301L))4510 mice and from rTg(tau(wt))21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice,validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tau(P301L))4510 cultures was hypophosphorylated in comparison with rTg(tau(wt))21221 as was seen in young adult mice. In addition,there were fewer human tau-expressing cells in rTg(tau(P301L))4510 than in rTg(tau(wt))21221 cultures. Following differentiation,neuronal filopodia-spine density was slightly greater in rTg(tau(P301L))4510 than rTg(tau(wt))21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns,the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms. View Publication

Histone deacetylase enzymes (HDACs) are emerging as a promising biological target for cancer and inflammation. Using a fluorescence assay,we tested the in vitro HDAC inhibitory activity of twenty-one natural chalcones,a widespread group of natural products with well-known anti-inflammatory and antitumor effects. Since HDACs regulate the expression of the transcription factor NF-κB,we also evaluated the inhibitory potential of the compounds on NF-κB activation. Only four chalcones,isoliquiritigenin (no. 10),butein (no. 12),homobutein (no. 15) and the glycoside marein (no. 21) showed HDAC inhibitory activity with IC50 values of 60-190 µM,whereas a number of compounds inhibited TNFα-induced NF-κB activation with IC50 values in the range of 8-41 µM. Interestingly,three chalcones (nos. 10,12 and 15) inhibited both TNFα-induced NF-κB activity and total HDAC activity of classes I,II and IV. Molecular modeling and docking studies were performed to shed light into dual activity and to draw structure-activity relationships among chalcones (nos. 1-21). To the best of our knowledge this is the first study that provides evidence for HDACs as potential drug targets for natural chalcones. The dual inhibitory potential of the selected chalcones on NF-κB and HDACs was investigated for the first time. This study demonstrates that chalcones can serve as lead compounds in the development of dual inhibitors against both targets in the treatment of inflammation and cancer. View Publication

A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4),which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However,there is little data on the precise mechanism,by which the PDE4 inhibitor regulates osteoblast gene expression. In this study,we investigated the combined ability of BMP-2 and cilomilast,a second-generation PDE4 inhibitor,to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (cilomilast or rolipram),BMP-2,and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover,expression of Runx2,osterix,and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover,according to the H89 inhibition experiments,Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2,and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor. View Publication

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the two C1q receptors found on the DC surface - gC1qR and cC1qR - lack a direct conduit into intracellular elements,we postulated that the receptors must form complexes with transmembrane partners. Here we show that DC-SIGN,a C-type lectin expressed on DCs,binds directly to C1q,as assessed by ELISA,flow cytometry and immuno-precipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific,and intact C1q,as well as the globular portion of C1q,bound to DC-SIGN. While IgG significantly reduced the binding; the Arg residues (162-163) of the C1q-A-chain,considered to contribute to C1q-IgG interaction,were not required for C1q binding to DC-SIGN. Binding was significantly reduced in the absence of Ca(2+) and by pre-incubation of DC-SIGN with mannan,suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket,which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. Thus the data suggest that C1q/gC1qR may regulate DC differentiation and function through DC-SIGN-mediated induction of cell signaling pathways. View Publication

PURPOSE: We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines,the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524,2011). In this report,the ability of PP2 and PP1,the more specific Src inhibitor SU6656,and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1,Colo 357) and primary NSCLC (Tu459) cell lines were investigated. METHODS: The effects of PP2,PP1,SU6656,and SB431542 on TGF-β1-dependent cell scattering/EMT,cell migration/invasion,and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR,respectively. RESULTS: In all three cell lines tested,PP1,PP2,and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT,migration,and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast,SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1,and to a greater extent PP2,also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. CONCLUSIONS: These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC. View Publication

Cellular reprogramming from adult somatic cells into an embryonic cell-like state,termed induced pluripotency,has been achieved in several cell types. However,the ability to reprogram human amniotic epithelial cells (hAECs),an abundant cell source derived from discarded placental tissue,has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs),but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore,AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation,including NEUROD1 and SOX17,markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs,we analyzed global DNA methylation,global histone acetylation,and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts,hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise,quantitative gene expression analyses show that hAECs endogenously express OCT4,SOX2,KLF4,and c-MYC,all four factors used in cellular reprogramming. Thus,hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. View Publication

Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs),yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast,highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations,one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1),have little intravenous engraftment potential,and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However,varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated,turning MSCs into variety of cell lineages of mesodermal,ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial,these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently,using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function,as well as with specific signalling pathways that promote neural fate commitment,we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However,the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs,comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs,accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright textcopyright 2012 John Wiley & Sons,Ltd. View Publication

Y-box binding protein-1 (YB-1) is the first reported oncogenic transcription factor to induce the tumor-initiating cell (TIC) surface marker CD44 in triple-negative breast cancer (TNBC) cells. In order for CD44 to be induced,YB-1 must be phosphorylated at S102 by p90 ribosomal S6 kinase (RSK). We therefore questioned whether RSK might be a tractable molecular target to eliminate TICs. In support of this idea,injection of MDA-MB-231 cells expressing Flag-YB-1 into mice increased tumor growth as well as enhanced CD44 expression. Despite enrichment for TICs,these cells were sensitive to RSK inhibition when treated ex vivo with BI-D1870. Targeting RSK2 with small interfering RNA (siRNA) or small molecule RSK kinase inhibitors (SL0101 and BI-D1870) blocked TNBC monolayer cell growth by ∼100%. In a diverse panel of breast tumor cell line models RSK2 siRNA predominantly targeted models of TNBC. RSK2 inhibition decreased CD44 promoter activity,CD44 mRNA,protein expression,and mammosphere formation. CD44(+) cells had higher P-RSK(S221/227),P-YB-1(S102),and mitotic activity relative to CD44(-) cells. Importantly,RSK2 inhibition specifically suppressed the growth of TICs and triggered cell death. Moreover,silencing RSK2 delayed tumor initiation in mice. In patients,RSK2 mRNA was associated with poor disease-free survival in a cohort of 244 women with breast cancer that had not received adjuvant treatment,and its expression was highest in the basal-like breast cancer subtype. Taking this further,we report that P-RSK(S221/227) is present in primary TNBCs and correlates with P-YB-1(S102) as well as CD44. In conclusion,RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs. View Publication

PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However,little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.backslashnbackslashnMETHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition,expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2),tumour protein p63 (p63),paired box 6 (Pax6),cytokeratin 3 (AE5),cytokeratin 10,and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes,osteocytes,and chondrocytes.backslashnbackslashnRESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2,p63,Pax6,AE-5,and keratocan sulfate. After passaged,a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60,Tra-1-81,and transcription factors like octamer-binding transcription factor 4 (Oct4),SRY(sex determining region Y)-box 2 (Sox2),and Nanog. Early passaged cells when induced were able to differentiate into adipocytes,osteocytes and chondrocytes.backslashnbackslashnCONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However,despite the expression of SSEA-4,these cells did not express any other markers of ESC. Therefore,we conclude that the cells did not show properties of ESC. View Publication