技术资料

In metazoans,the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes,by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific,while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps,we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover,cLADs are depleted of synteny breakpoints,pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically,the overall sequence conservation is low for cLADs. Instead,cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this A/T rule" in embryonic stem cells� View Publication

The differentiation of eosinophils from hematopoietic precursors and their subsequent maturation,chemotaxis,and activation is primarily regulated by interleukin-5 (IL-5). To examine the effect of chronic IL-5 exposure on hematopoiesis,IL-5 transgenic (IL-5trg) mice and wild-type BALB/c (WT) mice were examined. In comparison to WT mice,a significant alteration in bone marrow hematopoiesis was observed in IL-5trg mice. Although the total number of myeloid progenitors in the bone marrow of IL-5trg mice was not significantly altered,the number of long-term culture-initiating cells (LTC-ICs) was 1.5-fold lower than that observed in WT mice. Furthermore,IL-5trg mice failed to demonstrate hematopoietic activity in long-term bone marrow cultures,which correlated with a significant decrease in the number of bone marrow mesenchymal/stromal progenitor (MSP) cells in these mice. In comparison to WT mice,a 10-fold decrease was observed in the number of fibroblast colony-forming units (CFU-Fs) in IL-5trg bone marrow. Hematopoietic activity of IL-5trg bone marrow cells was rescued by cultivation on preestablished layers of bone marrow-derived stromal cells. However,in contrast to bone marrow,increased hematopoietic activity was observed in the spleen and peripheral blood of IL-5trg mice. Likewise,the numbers of LTC-ICs and granulocyte-macrophage,macrophage,eosinophil,B-lymphocyte progenitors in the peripheral blood and spleen of IL-5trg mice were approximately 20-fold higher than in WT mice. A significant increase in CFU-F numbers was also observed in the spleens of IL-5trg mice compared with WT mice. Overall,our results suggest that constitutive overexpression of IL-5 can potentially induce colonization of spleen with MSP cells,which provides the necessary microenvironment for establishment of hematopoiesis in extramedullary sites. View Publication

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid,T,and B lineage lymphoid cells in culture,but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term,stromal-free propagation,and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture,it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition,we demonstrate how it may be experimentally manipulated to generate valuable cell lines. View Publication

This work describes a genetic approach to isolate small,artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric,44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small,dimeric TM proteins that were structurally unrelated to erythropoietin (EPO),but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore,this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus,we have changed the specificity of a protein so that it no longer recognizes its natural target but,instead,modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word traptamer" for these transmembrane aptamers." View Publication

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However,it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9,HS401 and HS360) on short (2 hours),intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore,the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs,were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover,transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further,MYC protein expression in hypoxia was affected by silencing HIF2α,but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. View Publication

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice,with a corresponding retention of progenitors in blood. Remarkably,the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells,studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin,studied in parallel. Furthermore,the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies,in contrast to antibodies against alpha4 integrin,did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells. View Publication

Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle,fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented,little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here,we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts,physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin,a marker of regenerating skeletal muscle,to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC,human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle. View Publication

Heterogeneity is an often unappreciated characteristic of stem cell populations yet its importance in fate determination is becoming increasingly evident. Although gene expression noise has received greater attention as a source of non-genetic heterogeneity,the effects of stochastic partitioning of cellular material during mitosis on population variability have not been researched to date. We examined self-renewing human embryonic stem cells (hESCs),which typically exhibit a dispersed distribution of the pluripotency marker NANOG. In conjunction with our experiments,a multiscale cell population balance equation (PBE) model was constructed accounting for transcriptional noise and stochastic partitioning at division as sources of population heterogeneity. Cultured hESCs maintained time-invariant profiles of size and NANOG expression and the data were utilized for parameter estimation. Contributions from both sources considered in this study were significant on the NANOG profile,although elimination of the gene expression noise resulted in greater changes in the dispersion of the NANOG distribution. Moreover,blocking of division by treating hESCs with nocodazole or colcemid led to a 39% increase in the average NANOG content and over 68% of the cells had higher NANOG level than the mean NANOG expression of untreated cells. Model predictions,which were in excellent agreement with these findings,revealed that stochastic partitioning accounted for 17% of the total noise in the NANOG profile of self-renewing hESCs. The computational framework developed in this study will aid in gaining a deeper understanding of how pluripotent stem/progenitor cells orchestrate processes such as gene expression and proliferation for maintaining their pluripotency or differentiating along particular lineages. Such models will be essential in designing and optimizing efficient differentiation strategies and bioprocesses for the production of therapeutically suitable stem cell progeny. View Publication

The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process,few avoid denaturation of proteins or allow control over protein orientation,both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel,which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption. View Publication

To harness the potential of human pluripotent stem cells (hPSCs),an abundant supply of their progenies is required. Here,hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors,applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration,whereas the aggregate size was no prevailing factor across culture platforms. However,in bioreactors,the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists,suggesting their decisive role in cell-fate determination. Furthermore,metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors,the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation. View Publication