技术资料

Enforced expression of the HOXB4 transcription factor and downregulation of p21(Cip1/Waf) (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild-type (wt) and p21-/- mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4),or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture,the number of primary CFU-GM (colony-forming unit granulocyte-macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21-/- BM (p21B4) cells compared with MIGB4-transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo,we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4,in combination with suppression of p21 expression,could be a useful strategy for effective and robust expansion of HSCs. View Publication

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here,we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition,SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore,through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether,our results indicate that even an extremely homogeneous HSC population,based on phenotype and dye efflux,cannot reconstitute mice at absolute efficiencies. View Publication

Erythropoietin (Epo) stimulation of its receptor's downstream signaling pathways and optimum function of GATA-1 transcription factor are both essential for normal erythroid cell development. Epo-receptor (EpoR) signaling and GATA-1 regulate proliferation,survival,differentiation,and maturation of erythroid cells. Whether any signal that is generated by EpoR targets GATA-1 or affects GATA-1 transcriptional activity is not known. Here,we demonstrate that stimulation of EpoR results in phosphorylation of GATA-1 at serine 310 (S310) in primary fetal liver erythroid progenitors and in cultured erythroid cells. We show that phosphorylation of GATA-1 is important for Epo-induced maturation of fetal liver erythroid progenitor cells. The PI3-kinase/AKT signaling pathway is identified as a mediator of Epo-induced phosphorylation of GATA-1. AKT serine threonine kinase phosphorylates GATA-1S310 in vitro and in erythroid cells and enhances GATA-1 transcriptional activity. These data demonstrate that EpoR signaling phosphorylates GATA-1 and modulates its activity via the PI3-kinase/AKT signaling pathway. View Publication

Mutations of MYH9,the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA),cause a complex clinical phenotype characterized by macrothrombocytopenia and granulocyte inclusion bodies,often associated with deafness,cataracts and/or glomerulonephritis. The pathogenetic mechanisms of these defects are either completely unknown or controversial. In particular,it is a matter of debate whether haploinsufficiency or a dominant-negative effect of mutant allele is responsible for hematological abnormalities. We investigated 11 patients from six pedigrees with different MYH9 mutations. We evaluated NMMHC-IIA levels in platelets and granulocytes isolated from peripheral blood and in megakaryocytes (Mks) cultured from circulating progenitors. NMMHC-IIA distribution in Mks and granulocytes was also assessed. We demonstrated that all the investigated patients had a 50% reduction of NMMHC-IIA expression in platelets and that a similar defect was present also in Mks. In subjects with R1933X and E1945X mutations,the whole NMMHC-IIA of platelets and Mks was wild-type. No NMMHC-IIA inclusions were observed at any time of Mk maturation. In granulocytes,the extent of NMMHC-IIA reduction in patients with respect to control cells was significantly greater than that measured in platelets and Mks,and we found that wild-type protein was sequestered within most of the NMMHC-IIA inclusions. Altogether these results indicate that haploinsufficiency of NMMHC-IIA in megakaryocytic lineage is the mechanism of macrothrombocytopenia consequent to MYH9 mutations,whereas in granulocytes a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies. The finding that the same mutations act through different mechanisms in different cells is surprising and requires further investigation. View Publication

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study,we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway,which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells,human CD34+,and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells,but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42),nuclear polyploidization levels,cell size,or cell survival. The downstream effectors of mTOR,p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1),are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion,these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors,without affecting differentiation or cell survival. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context,little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional,biochemical,and microarray analyses. When deprived of natural ligands,GM-CSF,interleukin-3,or erythropoietin,AP20187 prevented apoptosis of transduced TF1 cells,induced dose-dependent proliferation,and supported long-term growth. In addition,AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably,mRNAs affiliated with heat stress,including Hsp70 and Hsp105,were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence,in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor,or KDR,signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes. View Publication

Hoechst-effluxing cells (side population cells) are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. To identify potential stem and progenitor cells during lung development,we generated gene expression profiles for CD45- and CD45+ side population cells in the embryonic day 17.5 lung. We found that side population cells comprise 1% of total embryonic day 17.5 lung cells (55% CD45+,45% CD45-). Gene profiling data demonstrated an overrepresentation of endothelial genes within the CD45- side population. We used expression of several distinct genes to identify two types of CD45- side population cells: 1) von Willebrand factor+/smooth muscle actin+ cells that reside in the muscular layer of select large vessels and 2) von Willebrand factor+/intercellular adhesion molecule+ cells that reside within the endothelial layer of select small vessels. Gene profiling of the CD45+ side population indicated an overrepresentation of genes associated with myeloid cell differentiation. Consistent with this,culturing CD45+ side population cells was associated with induction of mature dendritic markers (CD86). The microarray results suggested that expression of myeloperoxidase and proteinase-3 might be used to identify CD45+ side population cells. By immunohistochemistry,we found that myeloperoxidase+/proteinase-3+ cells represent a small subset of total CD45+ cells in the embryonic day 17.5 lung and that they reside in the mesenchyme and perivascular regions. This is the first detailed information regarding the phenotype and localization of side population cells in a developing organ. View Publication

The hematopoietic stem cell (HSC) gives rise to all mature,terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure,characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn,these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2. View Publication

Although significant advances have been made over the last decade with respect to our understanding of stem cell biology,progress has been limited in the development of successful techniques for clinically significant ex vivo expansion of hematopoietic stem and progenitor cells. We here describe the effect of Notch ligand density on induction of Notch signaling and subsequent cell fate of human CD34+CD38- cord blood progenitors. Lower densities of Delta1(ext-IgG) enhanced the generation of CD34+ cells as well as CD14+ and CD7+ cells,consistent with early myeloid and lymphoid differentiation,respectively. However,culture with increased amounts of Delta1(ext-IgG) induced apoptosis of CD34+ precursors resulting in decreased cell numbers,without affecting generation of CD7+ cells. RNA interference studies revealed that the promotion of lymphoid differentiation was primarily mediated by Delta1 activation of Notch1. Furthermore,enhanced generation of NOD/SCID repopulating cells was seen following culture with lower but not higher densities of ligand. These studies indicate critical,quantitative aspects of Notch signaling in affecting hematopoietic precursor cell-fate outcomes and suggest that density of Notch ligands in different organ systems may be an important determinant in regulating cell-fate outcomes. Moreover,these findings contribute to the development of methodology for manipulation of hematopoietic precursors for therapeutic purposes. View Publication

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function,anemia,and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression,studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils,even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils,and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b,CD35,and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry,whereas a second subset remained negative,consistent with a bimodal expression. Finally,human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst,compared with human control IgG in stem cell-derived neutrophils. Taken together,these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors. View Publication

Although there is evidence that endothelial cells are important targets for human pathogenic Bartonella species,the primary niche of infection is unknown. Here we elucidated whether human CD34+ hematopoietic progenitor cells (HPCs) internalize B. henselae and may serve as a potential niche of the pathogen. We showed that B. henselae does not adhere to or invade human erythrocytes. In contrast,B. henselae invades and persists in HPCs as shown by gentamicin protection assays,confocal laser scanning microscopy (CLSM),and electron microscopy (EM). Fluorescence-activated cell sorting (FACS) analysis of glycophorin A expression revealed that erythroid differentiation of HPCs was unaffected following infection with B. henselae. The number of intracellular B. henselae continuously increased over a 13-day period. When HPCs were infected with B. henselae immediately after isolation,intracellular bacteria were subsequently detectable in differentiated erythroid cells on day 9 and day 13 after infection,as shown by CLSM,EM,and FACS analysis. Our data provide,for the first time,evidence that a bacterial pathogen is able to infect and persist in differentiating HPCs,and suggest that HPCs might serve as a potential primary niche in Bartonella infections. View Publication