技术资料

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6,enhances the level of cyclin D-Cdk6 complexes,and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here,we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which,unexpectedly,it was unable to do despite high levels of Cdk6. Instead,reduction of Fbxo7 expression increased proliferation,decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27,and increased levels of S phase cyclins and Cdk2 activity. Also,Fbxo7 protein levels correlated inversely with those of CD43,suggesting direct regulation of its expression and,therefore,of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle,indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however,decreased expression of Cdk6 also enhanced levels of CD43,indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations,consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells. View Publication

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine,numerous umbilical cord blood banks have been established. In this study,we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs,MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs),slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring,we successfully isolated MSCs which expressed CD13,CD44 and CD90 while CD34,CD45 and CD133 negative,had typical fibroblast-like shape,and was able to differentiate into adipocytes; EPCs which were CD34,and CD90 positive,CD13,CD44,CD45 and CD133 negative,adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. View Publication

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLL(N) and MLL(C)) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study,we used a genetically engineered mouse line that lacked MLL(C) to show that the MLL(N)-MLL(C) holocomplex is responsible for MLL functions in various developmental processes. The stability of MLL(N) is dependent on its intramolecular interaction with MLL(C),which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLL(N). Free MLL(N) is destroyed by a mechanism that targets the FYRN domain,whereas free MLL(C) is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia,and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore,this should be a loss-of-function mutant allele,suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited. View Publication

BACKGROUND Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however,the optimal cell type and long-term efficacy are unknown. In this study,we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34/M-cad BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34/M-cad BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS Colony-forming cell assays and flow cytometry analysis showed that CD34/M-cad BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE/ mice,CD34/M-cad BMCs alleviated ischemia and significantly improved blood flow compared with CD34/M-cad BMCs,CD34/M-cad BMCs,or unselected BMCs. Significantly more arterioles were seen in CD34/M-cad cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore,histologic assessment and morphometric analyses of hindlimbs treated with GFP CD34/M-cad cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP CD34/M-cad cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34/M-cad progenitor cells. A cytokine antibody array revealed that CD34/M-cad cell-conditioned medium contained higher levels of cytokines in a unique pattern,including bFGF,CRG-2,EGF,Flt-3 ligand,IGF-1,SDF-1,and VEGFR-3,than did CD34/M-cad cell-conditioned medium. The proangiogenic cytokines secreted by CD34/M-cad cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34/M-cad cells during hypoxia. CONCLUSION CD34/M-cad BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE/ mice by consistently improving blood flow and promoting arteriogenesis. Additionally,CD34/M-cad BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors. View Publication

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication

Methylation of cytosine is a DNA modification associated with gene repression. Recently,a novel cytosine modification,5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems,and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage,where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC,which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts,and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs,5-hmC is strongly enriched in bone marrow and brain,wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation,as has been reported previously,but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages,high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge,5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific. View Publication

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic,nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors,allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this,gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. View Publication

Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (mushroom alcohol")� View Publication

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off,depending upon the particular requirements of the study. View Publication