技术资料

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however,a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally,a decrease in colony-forming capacity was observed under increasing doses of imatinib. However,no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion,whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling. View Publication

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP),ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% +/- 14% of ITP megakaryocytes,which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% +/- 4% versus 0%). No difference,however,was observed in the number of bone marrow megakaryocyte colony-forming units (ITP,118 +/- 93/105 bone marrow cells; versus controls,128 +/- 101/105 bone marrow cells; P =.7). To demonstrate that circulating antibodies might affect megakaryocytes,suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally,the plasma glycocalicin index,a parameter of platelet and megakaryocyte destruction,was increased in ITP (57 +/- 70 versus 0.7 +/- 0.2; P =.009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P =.02; r = 0.7). In conclusion,most ITP megakaryocytes show ultrastructural features of (para-) apoptosis,probably due to action of factors present in ITP plasma. View Publication

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34(-) cell subsets that might reside at an earlier stage of differentiation than CD34(+) HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34(-)CD133(-)CD7(-)CD45(dim)lineage (lin)(-) HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133,2) generation of CFU-granulocyte-macrophage,burst-forming unit erythroid,and megakaryocytic aggregates,3) significant extended long-term culture-initiating cell activity,and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34(+)lin(-) cells,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs maintained with IL-15,but not with IL-2 or IL-7,proliferated vigorously and differentiated into a homogeneous population of CD7(+)CD45(bright)CD25(+)CD44(+) lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRgamma genes,IL-15-primed CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs failed to achieve full maturation,as manifested in their CD3(-)TCRalphabeta(-)gammadelta(-) phenotype. Conversely,culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34(-)lin(-) HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined. View Publication

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors,we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells,suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies,under the influence of Stat5 signaling,a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing,mainly myeloid,differentiation. When these cells are injected into lethally irradiated mice,they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal,and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells. View Publication

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study,it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and,unexpectedly,to skeletal muscle cells in mice. In fact,the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions,CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells,thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org. View Publication

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca),gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation,microphthalmia and craniofacial malformations that are not found in other Fanca mouse models,and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism,and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells,implicating a role for Fanca in meiotic recombination. However,the localization of Rad51,Brca1,Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together,our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination. View Publication

OVERVIEW: We show the existence of adult human mesenchymal progenitor cells (hMPCs) that can proliferate,in a cytokine-dependent manner,as individual cells in stirred suspension cultures (SSC) while maintaining their ability to form functional differentiated mesenchymal cell types. MATERIALS AND METHODS: Ficolled human bone marrow (BM)-derived cells were grown in SSC (and adherent controls) in the presence and absence of exogenously added cytokines. Phenotypic,gene expression,and functional assays for hematopoietic and nonhematopoietic cell populations were used to kinetically track cell production. Limiting-dilution analysis was used to relate culture-produced cells to input cell populations. RESULTS: Cytokine cocktail influenced total and progenitor cell expansion,as well as the types of cells generated upon plating. Flow cytometric analysis of CD117,CD123,and CD45 expression showed that cytokine supplementation influenced SSC output. The concomitant growth of CD45(+) and CD45(-) cells in the cultures that exhibited the greatest hMPC expansions suggests that the growth of these cells may benefit from interactions with hematopoietic cells. Functional assays demonstrated that the SSC-derived cells (input CFU-O number: 1990+/-377) grown in the presence of SCF+IL-3 resulted,after 21 days,in the generation of a significantly greater number (ptextless0.05) of bone progenitors (33,700+/-8763 CFU-O) than similarly initiated adherent cultures (214+/-75 CFU-O). RT-PCR analysis confirmed that the SSC-derived cells grown in osteogenic conditions express bone-specific genes (Cbfa1/Runx2,bone sialoprotein,and osteocalcin). CONCLUSIONS: Our approach not only provides an alternative strategy to expand adult BM-derived nonhematopoietic progenitor cell numbers in a scalable and controllable bioprocess,but also questions established biological paradigms concerning the properties of connective-tissue stem and progenitor cells. View Publication

Clinical observations suggest that in chronic myelogenous leukemia (CML),the Philadelphia chromosome (Ph+) clone has a growth advantage over normal hematopoiesis. Patients with CML have high levels of neutrophil elastase,which has recently been shown to antagonize the action of granulocyte-colony-stimulating factor (G-CSF) and other growth factors. We therefore compared the effect of elastase on the growth of normal and CML progenitor cells. In 10-day suspension cultures of normal or CML CD34+ cells supplemented with G-CSF,stem cell factor (SCF),and granulocyte macrophage-colony-stimulating factor (GM-CSF),CML cells had diminished sensitivity to the growth inhibitory effect of elastase. When equal numbers of CML and normal CD34+ cells were cocultured for 10 days,there was no change in the relative proportions of normal and leukemic cells (measured by fluorescence in situ hybridization [FISH] or flow cytometry). However,when elastase was added,CML cells predominated at the end of the culture period (78% vs 22% with 1 microg/mL and 80% vs 20% with 5 microg/mL elastase). CML neutrophils substituted effectively for elastase in suppressing the proliferation of normal CD34+ cells,but this effect was abrogated by serine protease inhibitors. These results suggest that elastase overproduction by the leukemic clone can change the growth environment by digesting growth factors,thereby giving advantage to Ph+ hematopoiesis. View Publication

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation,although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha,GATA1,GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells,indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes. View Publication