技术资料

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that,unlike steady-state erythropoiesis,erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM),severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments,donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context,stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system,EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion,and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x),in contrast,was not significantly induced via WT-EpoR,EpoR-HM,or EpoR-H alleles. In Kit+ CD71+ erythroblasts,EpoR-PY343 signals furthermore enhanced SCF growth effects,and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts,oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis,therefore,requires stage-specific EpoR-PY343-Stat5 signals,some of which selectively bolster SCF and oncostatin-M action. View Publication

RATIONALE: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations,increased production of proinflammatory cytokines and markers of bone resorption have been reported. OBJECTIVE: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before,during,and after intravenous antibiotic treatment of infective exacerbations in patients with CF. METHODS: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14,CD33,CD34,and CD45 expression. RESULTS: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated,p = 0.025),proliferation (28.5%,p textless 0.001),and the numbers of circulating potential osteoclast precursors (6.5%,p textless 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline. CONCLUSIONS: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF. View Publication

We have previously demonstrated that mouse embryonic stem (ES) cells differentiated on three-dimensional (3D),highly porous,tantalum-based scaffolds (Cytomatrixtrade mark) have significantly higher hematopoietic differentiation efficiency than those cultured under conventional two-dimensional (2D) tissue culture conditions. In addition,ES cell-seeded scaffolds cultured inside spinner bioreactors showed further enhancement in hematopoiesis compared to static conditions. In the present study,we evaluated how these various biomaterial-based culture conditions,e.g. 2D vs. 3D scaffolds and static vs. dynamic,influence the global gene expression profile of differentiated ES cells. We report that compared to 2D tissue culture plates,cells differentiated on porous,Cytomatrixtrade mark scaffolds possess significantly higher expression levels of extracellular matrix (ECM)-related genes,as well as genes that regulate cell growth,proliferation and differentiation. In addition,these differences in gene expression were more pronounced in 3D dynamic culture compared to 3D static culture. We report specific genes that are either uniquely expressed under each condition or are quantitatively regulated,i.e. over expressed or inhibited by a specific culture environment. We conclude that that biomaterial-based 3D cultures,especially under dynamic conditions,might favor efficient hematopoietic differentiation of ES cells by stimulating increased expression of specific ECM proteins,growth factors and cell adhesion related genes while significantly down-regulating genes that act to inhibit expression of these molecules. View Publication

Differentiation of embryonic stem (ES) cells typically requires cell-cell aggregation in the form of embryoid bodies (EBs). This process is not very well controlled and final cell numbers can be limited by EB agglomeration and the inability to drive differentiation towards a desired cell type. This study compares three-dimensional (3D) fibrin culture to conventional two-dimensional (2D) suspension culture and to culture in a semisolid methylcellulose medium solution. Two types of fibrin culture were evaluated,including a PEGylated fibrin gel. PEGylation with a difunctional PEG derivative retarded fibrinogen migration during through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a result of crosslinking,similarly,degradation was slowed in the PEGylated gel. ES cell proliferation was higher in both the fibrin and PEGylated fibrin gels versus 2D and methylcellulose controls. FACS analysis and real-time-PCR revealed differences in patterns of differentiation for the various culture systems. Culture in PEGylated fibrin or methylcellulose culture demonstrated features characteristic of less extensive differentiation relative to fibrin and 2D culture as evidenced by the transcription factor Oct-4. Fibrin gels showed gene and protein expression similar to that in 2D culture. Both fibrin and 2D cultures demonstrated statistically greater cell numbers positive for the vascular mesoderm marker,VE-cadherin. View Publication

The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Ralpha and beta(c) subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT(388)IL3). Among 19 patient samples,the relative level of the IL-3Ralpha was higher than the IL-3Rbeta(c) and highest in CD34(+)CD38(-)CD71(-) cells,enriched for candidate leukemia stem cells,compared with cell fractions depleted of such progenitors. Overall,the amount of IL-3Rbeta(c) subunit did not vary among sorted subpopulations. However,expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rbeta(c) expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34(+)CD38(-)CD71(-) cells from different samples; this value was correlated (r = .76,P = .05) with the ability of DT(388)IL3 to kill AML progenitors that engraft in beta(2)-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus,quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors. View Publication

Mesenchymal stem cells (MSCs) are widespread in adult organisms and may be involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. Thus,it is important to discover the factors controlling MSC renewal and differentiation. Here we report that adult MSCs express functional Toll-like receptors (TLRs),confirmed by the responses of MSCs to TLR ligands. Pam3Cys,a prototypic TLR-2 ligand,augmented interleukin-6 secretion by MSC,induced nuclear factor kappa B (NF-kappaB) translocation,reduced MSC basal motility,and increased MSC proliferation. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We show herein that Pam3Cys inhibited MSC differentiation into osteogenic,adipogenic,and chondrogenic cells while sparing their immunosuppressive effect. Our study therefore shows that a TLR ligand can antagonize MSC differentiation triggered by exogenous mediators and consequently maintains the cells in an undifferentiated and proliferating state in vitro. Moreover,MSCs derived from myeloid factor 88 (MyD88)-deficient mice lacked the capacity to differentiate effectively into osteogenic and chondrogenic cells. It appears that TLRs and their ligands can serve as regulators of MSC proliferation and differentiation and might affect the maintenance of MSC multipotency. View Publication

BACKGROUND: The purpose of this study was to determine whether murine mesenchymal stem cells (MSC) are able to home to the viable myocardium when injected intravenously and attenuate cardiac dysfunction and ventricular remodeling associated with myocardial infarction. METHODS AND RESULTS: Murine bone marrow cells were negatively selected for lineage markers and adherent MSC differentiated into adipocytes and osteocytes following treatment in culture. Two weeks after coronary occlusion that resulted in a permanent transmural infarct we observed a significant drop in LV systolic pressure,dP/dt(max),dP/dt(min),ESPVR and E(max) and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSC 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size,or end-diastolic volume. Injected MSC pre-labeled with fluorescent paramagnetic microspheres were observed scattered in noninfarcted regions of the myocardium. Flow cytometry of whole heart digests after intravenous injection of MSC labeled with either fluorescent microspheres or fluorescent PKH26 dye demonstrated that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart (20x) than those from uninfarcted controls. CONCLUSION: We conclude that intravenously injected MSC were able to home to viable myocardium and preserve systolic function by 2 weeks following ligation. The preserved contractility is likely an MSC-mediated paracrine response since infarct morphology was unchanged and labeled cells observed at two weeks exhibited the same characteristics as the injected MSC. These data underscore the importance of using MSC as a potential therapeutic intervention in preserving cardiac function following infarction. View Publication

OBJECTIVE: In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs,involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring,which is time-consuming and error-prone. As a consequence,automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. METHODS: CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. RESULTS: The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations,compact colonies in some cases were missed. CONCLUSIONS: The industry's scoring methods all rely on structure alone and process 2D data. Instead,the classifier here fuses data from a whole stack and is capable,in principle,of high-throughput screening. View Publication

Hematopoietic stem cells are resistant to HIV-1 infection. Here,we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21),a known regulator of stem cell pool size,restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs,p27(Kip1) and p18(INK4C),had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase,SIVmac-251,and the other cell-intrinsic inhibitors of HIV-1,Trim5alpha,PML,Murr1,and IFN-alpha,were unaffected by p21. Therefore,p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected sanctuary" in HIV disease." View Publication

Conventionally,mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9,CD10,CD13,CD73,CD105,CD166,and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4),Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free,basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast,the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9),an increased Oct-4 and nestin mRNA expression,and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9,nanog-3,and Oct-4. In contrast to PL-MSC,only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions,these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm),glucagon and insulin expressing pancreatic-like cells (endoderm),as well as cells expressing the neuronal markers neuron-specific enolase,glutamic acid decarboxylase-67 (GAD),or class III beta-tubulin,and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion,using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9,Oct-4,nanog-3,and nestin and are able of multi-lineage differentiation. View Publication

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM,but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition,immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides,there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM,their role in disease resistance in SM remains unclear. View Publication