技术资料

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

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

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

OBJECTIVE: The aim of this study was the preclinical evaluation of imatinib mesylate (Gleevec,formerly STI571) in conjunction with arsenic trioxide (As2O3,Trisenox) for the treatment of chronic myelogenous leukemia (CML). MATERIALS AND METHODS: Tetrazolium-based cell line proliferation assays (MTT assays) were performed to determine the cytotoxicity of As2O3 alone and in combination with imatinib. Cell lines tested in this study were Bcr-Abl-expressing cells (K562,MO7p210,32Dp210) and parental cells (MO7e,32D). Isobologram analysis was performed manually and using the median effect method. In vitro cytotoxicity also was determined in colony-forming assays using CML patient cells. Western blot analysis was performed to detect Bcr-Abl protein levels in K562 cells exposed to As2O3 at graded concentrations. Bcr-Abl protein level kinetics were correlated with cell viability (trypan blue count) and activated caspase-3 detected by flow cytometry. RESULTS: We show additive to synergistic cytotoxicity in Bcr-Abl+ cell lines depending on inhibitory concentrations and cell type. Results obtained by colony-forming assays confirmed the findings in cell line proliferation assays. Flow cytometric detection of activated caspase-3 revealed synergistic activity in K562 cells. Treatment of K562 cells with As2O3 alone led to down-regulation of Bcr-Abl protein within 24 hours,even at low doses. The decline of Bcr-Abl preceded activation of caspase-3 and the loss of viable cells. CONCLUSIONS: Favorable cytotoxicity and proapoptotic activity of imatinib in conjunction with As2O3 and specific down-regulation of Bcr-Abl protein levels by As2O3 in K562 cells indicate that As2O3 in combination with imatinib might be useful for circumventing resistance to imatinib monotherapy. View Publication

The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model,we have determined whether a lytic CD45 monoclonal antibody can produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration,we found saturating quantities of the antibody on all cells expressing the CD45 antigen,both in marrow and in lymphoid organs. All leukocyte subsets in peripheral blood were markedly diminished during or soon after anti-CD45 treatment,but only the effect on the lymphoid compartment was sustained. In contrast to the prolonged depletion of T and B lymphocytes from the thymus and spleen,peripheral blood neutrophils began to recover within 24 hours after the first anti-CD45 injection and marrow progenitor cells were spared from destruction,despite being coated with saturating quantities of anti-CD45. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis,we asked whether this agent could contribute to donor hematopoietic engraftment following nonmyeloablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment,consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast,the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45(+) lymphocytes. Monoclonal antibodies of this type may therefore have an adjunctive role in nonmyeloablative conditioning regimens for allogeneic stem cell transplantation. View Publication

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures,respectively. In erythrocytes,the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However,the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme,designated IGnT3. The genes for IGnT1 (reported in 1993),IGnT2 (also presented in this study),and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells,IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover,reticulocytes expressed the IGnT3 transcript,but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree,individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006GtextgreaterA; Gly336Arg) and exon 3 (1049GtextgreaterA; Gly350Glu),respectively,of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3,not by IGnT1 or IGnT2. View Publication

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),an endocrine disrupting chemical (EDC),can cause carcinogenesis,immunosuppression,and teratogenesis,through a ligand-activated transcription factor,the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology,the influence of TCDD on hematopoietic stem cells (HSCs),which possess the ability to reconstitute long-term multilineage hematopoiesis,has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34(-),c-kit(+),Sca-1(+),lineage negative (CD34-KSL) cells. The number of the CD34-KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 micro g/kg body weight). Surprisingly,we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR(-/-) mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival. 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

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 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

UNLABELLED: AC133 cells,a subpopulation of CD34+ hematopoietic stem cells,can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected,magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma,and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However,low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature,and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors. 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