技术资料

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication

The c-Myb gene encodes a transcription factor required for proliferation and survival of normal myeloid progenitors and leukemic blast cells. Targeting of c-Myb by antisense oligodeoxynucleotides has suggested that myeloid leukemia blasts (including chronic myelogenous leukemia [CML]-blast crisis cells) rely on c-Myb expression more than normal progenitors,but a genetic approach to assess the requirement of c-Myb by p210(BCR/ABL)-transformed hematopoietic progenitors has not been taken. We show here that loss of a c-Myb allele had modest effects (20%-28% decrease) on colony formation of nontransduced progenitors,while the effect on p210(BCR/ABL)-expressing Lin(-) Sca-1(+) and Lin(-) Sca-1(+)Kit(+) cells was more pronounced (50%-80% decrease). Using a model of CML-blast crisis,mice (n = 14) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/w) marrow cells developed leukemia rapidly and had a median survival of 26 days,while only 67% of mice (n = 12) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/d) marrow cells died of leukemia with a median survival of 96 days. p210(BCR/ABL)-transduced c-Myb(w/w) and c-Myb(w/d) marrow progenitors expressed similar levels of the c-Myb-regulated genes c-Myc and cyclin B1,while those of Bcl-2 were reduced. However,ectopic Bcl-2 expression did not enhance colony formation of p210(BCR/ABL)-transduced c-Myb(w/d) Lin(-)Sca-1(+)Kit(+) cells. Together,these studies support the requirement of c-Myb for p210(BCR/ABL)-dependent leukemogenesis. View Publication

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently,HIV-1 mediates massive depletion of gut CD4+ T cells,which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120,a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells,engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1,the central integrin involved in the establishment of virological synapses,which facilitate efficient cell-to-cell spreading of HIV-1. View Publication

BACKGROUND Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However,because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation,progress in hESC technology development has been hindered. METHODOLOGY/PRINCIPAL FINDINGS Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation,we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2). CONCLUSIONS/SIGNIFICANCE The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes,enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues,and accelerate the pre-clinical evaluation of hESC-derived cells. View Publication

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL,CD8(+) CTL and NK cells,excluding transcripts expressed in B cells,monocytes and kidney. This generated three transcript sets: CD4(+)-associated,CD8(+)-associated and NK-associated. Surprisingly,many CATs were expressed in effector memory cells e.g. granzyme B/GZMB,interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example,cytotoxic molecule transcripts (perforin,granzymes,granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1,KLRC3,KLRD1,KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL,CD8(+) CTL and effector memory T cells. View Publication

In response to inflammatory stimuli,monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha),interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors,such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex,which plays a vital role in several cellular functions,including the regulation of transcription factors like NF-kappaB. In this study,we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132,a proteasome inhibitor,on the release of TNF-alpha,IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1,IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation,resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors. View Publication

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs),including polycythemia vera,essential thrombocythemia,and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice,which expressed a lower level of JAK2(V617F),showed moderate elevations of blood cell counts,whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid,megakaryocytic,and granulocytic hyperplasia in the bone marrow and spleen,displayed splenomegaly,and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood,spleen,and bone marrow,and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs. 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

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

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

Neuropilin-1 (Nrp1) is a multifunctional protein,identified principally as a receptor for the class 3 semaphorins and members of the vascular endothelial growth factor (VEGF) family,but it is capable of other interactions. It is a marker of regulatory T cells (Tr),which often carry Nrp1 and latency-associated peptide (LAP)-TGF-beta1 (the latent form). The signaling TGF-beta1 receptors bind only active TGF-beta1,and we hypothesized that Nrp1 binds the latent form. Indeed,we found that Nrp1 is a high-affinity receptor for latent and active TGF-beta1. Free LAP,LAP-TGF-beta1,and active TGF-beta1 all competed with VEGF165 for binding to Nrp1. LAP has a basic,arginine-rich C-terminal motif similar to VEGF and peptides that bind to the b1 domain of Nrp1. A C-terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP-TGF-beta1. We also analyzed the effects of Nrp1/LAP-TGF-beta1 coexpression on T cell function. Compared with Nrp1(-) cells,sorted Nrp1+ T cells had a much greater capacity to capture LAP-TGF-beta1. Sorted Nrp1(-) T cells captured soluble Nrp1-Fc,and this increased their ability to capture LAP-TGF-beta1. Conventional CD4+CD25(-)Nrp1(-) T cells coated with Nrp1-Fc/LAP-TGF-beta1 acquired strong Tr activity. Moreover,LAP-TGF-beta was activated by Nrp1-Fc and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin-1 peptide). Breast cancer cells,which express Nrp1,also captured and activated LAP-TGF-beta1 in a Nrp1-dependent manner. Thus,Nrp1 is a receptor for TGF-beta1,activates its latent form,and is relevant to Tr activity and tumor biology. View Publication

T-bet and STAT4 play critical roles in helper T cell differentiation,especially for Th1 cells. However,it is still unknown about the relative importance and redundancy of T-bet and STAT4 for Th1 differentiation. It is also unknown about their independent role of T-bet and STAT4 in the regulation of allergic airway inflammation. In this study,we addressed these issues by comparing T-bet-deficient (T-bet(-/-)) mice,STAT4(-/-) mice,and T-bet- and STAT4-double-deficient (T-bet(-/-)STAT4(-/-)) mice on the same genetic background. Th1 differentiation was severely decreased in T-bet(-/-) mice and STAT4(-/-) mice as compared with that in wild-type mice,but Th1 differentiation was still observed in T-bet(-/-) mice and STAT4(-/-) mice. However,Th1 cells were hardly detected in T-bet(-/-)STAT4(-/-) mice. In contrast,the maintenance of Th17 cells was enhanced in T-bet(-/-) mice but was reduced in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. In vivo,Ag-induced eosinophil and neutrophil recruitment into the airways was enhanced in T-bet(-/-) mice but was attenuated in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. Ag-induced IL-17 production in the airways was also diminished in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. These results indicate that STAT4 not only plays an indispensable role in T-bet-independent Th1 differentiation but also is involved in the maintenance of Th17 cells and the enhancement of allergic airway inflammation. View Publication