技术资料

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study,the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also,however,CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies,mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes,it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes,the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization. View Publication

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied,the developmental capacity of AG ESCs is much less clear. Here,we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed,with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs,we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Expansion of the CGG.CCG-repeat tract in the 5' UTR of the FMR1 gene to textgreater200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS),the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase,SIRT1,plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I,II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase,hMOF. DNA methylation,on the other hand,is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However,since DNA methylation inhibitors require DNA replication in order to be effective,SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious. View Publication

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4,Sox2,Klf4,and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs,we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first,followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene,marking fully reprogrammed cells,was only observed late in the process. Importantly,the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming. View Publication

Ectopic expression of the transcription factors Oct4,Sox2,c-Myc,and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors,we have shown that exogenous factors are required for about 10 days,after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points,reactivation of endogenous Oct4,Sox2,telomerase,and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells. View Publication

The generation of amyloid-beta peptide (Abeta) and its accumulation in amyloid plaques are generally recognized as key characteristics of Alzheimer's disease. A number of reports have indicated that Abeta can regulate the proliferation of neural precursor cells and adult neurogenesis,suggesting that this may underpin the cognitive decline and compromised olfaction also associated with the condition. Here we report that Abeta(1-42) treatment both in vitro and in vivo,as well as endogenous generation of Abeta in C100 and APP/PS1 transgenic models of Alzheimer's disease,stimulate neurogenesis of young adult subventricular zone precursors. The neurogenic effect of Abeta(1-42) was found to require expression of the p75 neurotrophin receptor (p75(NTR)) by the precursor cells,and activation of p75(NTR) by metalloprotease cleavage. However,precursors from 12-month-old APP/PS1 mice failed to respond to Abeta(1-42). Our results suggest that overstimulation of p75(NTR)-positive progenitors during early life might result in depletion of the stem cell pool and thus a more rapid decline in basal neurogenesis. This,in turn,could lead to impaired neurogenic function in later life. View Publication

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore,we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content,membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase,ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSC(lo)ALDH(br) cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall,9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison,primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover,we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We,therefore,conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly,the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity. View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5,using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells,including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells,expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs. View Publication

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4,Sox2,Klf4,and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However,reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes,which gave rise to adult chimeras with germline contribution,and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency. View Publication

This unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity,establishment of stable hybridoma lines,cloning of these B cell lines by limiting dilution to obtain monoclonal lines,and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semi-solid methylcellulose-based medium (ClonaCell-HY). View Publication

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335,1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078,2005). In the present study,the kinase selectivity of GW2580 was further characterized,and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes,with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%,indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model,GW2580 dosed twice a day (b.i.d.) from days 0 to 21,7 to 21,or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology,histology and bone mineral content measurements. In contrast,GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion,GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats. View Publication