Human CD34-positive Cells Research Articles

Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair

Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remains unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a “proof of concept” animal study. Real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role for this protein in early bone formation and repair.

Adoptive Therapy with T-Cell Precursors for Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract 3525Poster Board III-462Recipients of allogeneic hematopoietic transplantation experience a slow reconstitution of donor-derived B and T cell number and function. This post-transplant period of immunodeficiency is associated with an increased risk of infection and malignant relapse. The developement of these complications notably correlates with the recovery of CD4+T cell subset. We proposed a strategy to enhances in vivo reconstitution by promoting donor-derived T cell development in the recipient's thymus. Recently Notch1-based ex-vivo system have been established to mature cord blood- or bone marrow-derived human HSCs into committed T-cell precursors. We used this system for the generation of T-cell precursors starting from G-CSF mobilized human HSCs. We cultured mobilized human CD34+ hematopoietic stem cells (HSCs) (2.5 × 105) in vitro on OP9 mouse stromal cells expressing the Notch 1 ligand Delta-like-1 (OP9-DL1) in the presence of rhFLT3-ligand (5ng/ml) and rhIL7 (5 ng/ml). After 6 weeks of co-culture we obtained a 3 log increase of human T-linage precursors of CD45RA+CD7high phenotype. Further co-colture (7-9 weeks) leed to the generation of CD4+ and CD8+ double-positive (DP) T cells and even mature CD4+ and CD8+ single positive (SP) ab-TCR lymphocytes.Experiments were designed in order to evaluate whether human CD45RA+CD7high T cell precursors could 1) engraft into NOD-SCID IL2 rg-/− mice 2) leed to in vivo expansion and maturation along T cell developmental pathway.Control mice were irradiated and transplanted with G-CSF-mobilized human CD34+ (dose 5×106 i.v.). 4 weeks after transplant more than 20% human CD45 positive cells engrafted in the bone marrow. Thymic engraftment occured at 8 weeks after transplant, with 80% human CD45 positive cells (thymic cellularity: 2.7×105 cells), mostly with T cell-immature phenotype of CD3-CD4-CD8 triple negative (95%) (TN) and CD4+CD8+double positive (5%) (DP).Co-transplant of CD45RA+CD7high T cell precursors (106 cells i.v.) along with CD34+HSC leed to an accelerated thymic engraftment (95% human CD45 positive cells; thymic cellularity 2.5 × 106 cells) already at 6 wks after transplant. Thymocytes were CD3-CD4-CD8 triple negative (51%) (TN) and CD4+CD8+double positive (DP) (42%) cells and at 8 weeks after transplant matured into CD3+CD4+ and CD3+CD8+ single positive (SP) T cells. Spectratyping analyses revealed a broad diversity of the T-cell receptor (TCR) repertoire. This occured in the complete absence of Graft versus Host Disease (GvHD) suggesting that adoptively transferred ex vivo-generated T-cell precursors developed into host-tolerant mature T cells. Ongonig experiment are needed to clarify the beneficial effect of adoptive immunotherapy with human T cell precursors on peripheral T cell reconstitution and control of infection in the humanized mouse system.We conclude that ex-vivo generation of human T-linage precursors is feasible from the G-CSF-mobilized HSCs and that can be succesfully tranfered in-vivo as a new strategie to enhance T-cell reconstitution after allogeneic HSCT with no risk of GvHD. Disclosures:No relevant conflicts of interest to declare.

Stromal Microenvironment Regulates Progenitor Survival through Nitric Oxide Production.

Abstract 1440Poster Board I-463Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium.These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling. DisclosuresJouni: Région IDF: Employment. Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

Optimization of a retroviral vector for transduction of human CD34 positive cells.

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.

The pediatric preclinical testing program: Description of models and early testing results

The Pediatric Preclinical Testing Program (PPTP) is an initiative supported by the National Cancer Institute (NCI) to identify novel therapeutic agents that may have significant activity against childhood cancers. The PPTP has established panels of childhood cancer xenografts and cell lines to be used for in vivo and in vitro testing. These include panels for Wilms tumor, sarcomas (rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma), neuroblastoma, brain tumors (glioblastoma, ependymoma, and medulloblastoma), rhabdoid tumors (CNS and renal), and acute lymphoblastic leukemia (ALL). Here, we describe the characteristics of the in vivo tumor panels and report results for the in vivo evaluation of two standard agents, vincristine and cyclophosphamide. Solid tumors were grown subcutaneously in immune-deficient mice and tumor dimensions were measured weekly. ALL xenografts were inoculated intravenously and human CD45-positive cells were enumerated weekly. Vincristine-induced objective responses in 6 of 24 (25%) and cyclophosphamide-induced objective responses in 18 of 28 (64%) solid tumor models. Comparable assessments of high levels of activity for these two agents were obtained using a tumor growth delay (TGD) measure. Both agents induced regressions in each of the ALL models evaluated. We have established 51 solid tumor and 10 ALL in vivo models. The models identify vincristine and cyclophosphamide as having broad-spectrum activity. The PPTP tumor panels appear to generally recapitulate the activity of these agents against specific childhood cancers and to have the potential for identifying novel agents having significant clinical activity.

THERMOSENSITIVE NANOPARTICLES CONJUGATING WITH CD34 ANTIBODY AND ITS SOLUTION PEROPERTIES

Poly N-isopropylacrylamide (PNIPAAm) is a well-known temperature-sensitive polymer. When the temperature is higher than the lower critical solution temperature (LCST), PNIPAAm aquous solution is cloudy (phase separation occurred). In contrast, when the temperature is lower than the LCST, PNIPAAm is soluble in water (a homogeneous solution). The lower critical solution temperature (LCST) in aqueous solution of PNIPAAm was about 32~33°C. We prepared nano-scaled PNIPAAm particles containing carboxylic group on their surfaces by introducing acrylic acid monomer. The carboxylic groups were applied to conjugate with the amino group of the CD34 antibody. This immuno-conjugate can be applied on targeting the human CD34 positive cells, peripheral blood progenitor cells included, for cell purification and drug controlled release. In order to the active responding of controlled release of the conjugate in the body influenced by temperature, we hope to estimate the shifting of the gel-collapse temperature or cloud point of the immuno-conjugates by dynamic light scattering (DLS) and UV absorption. The results show that the gel-collapse temperature of the nano-particle was not significantly affected by the content of AA between 1.5~5 mol%. However, cloud point of the solution was elevated by the conjugation of CD34 antibody to 37°C. When CD34-conjugated particle was subsequently incorporated with recombinant FLT3-ligand, which is a smaller molecule compare to CD34 antibody, cloud point of the solution was not affected.

Internal Tandem Duplications of the FLT3 Gene Are Present in Leukemia Stem Cells.

Internal tandem duplication mutations of the FLT3 gene (FLT3/ITD mutations) are the most frequent molecular abnormality in acute myeloid leukemia (AML) and are associated with a worse overall survival. While the normal FLT3 receptor is expressed in early hematopoietic progenitor cells, it has not been determined whether FLT3 mutations are present in the leukemic stem cells. In this study, we sorted primary AML samples into stem cell-enriched CD34+/CD38− fractions and then analyzed the sorted and unsorted cells for FLT3 mutant to wild type ratio. In each case, the FLT3 mutant to wild type ratio was not changed by selection of CD34+/CD38− cells, implying that the mutations are present in the leukemic stem cells. We injected NOD-SCID mice, in parallel groups of 4, with 106 unsorted and 5 x 103 CD34+/CD38− sorted cells from 6 FLT3/ITD AML samples. After 14 weeks, the level of human leukemic engraftment was determined using flow cytometric analysis of human CD45 compared with murine CD45 expression. Three of the 6 samples engrafted in the mice, with human CD45 positive cells ranging from 4 to 97% of total nucleated cells. In all three of the sample groups that successfully engrafted, the FLT3/ITD mutation was present at the same or higher mutant-to-wild type allelic ratio seen in the cells used to inject the mice. We then used a small molecule FLT3 inhibitor, CEP-701, to treat mice injected with CD34+/CD38- FLT3/ITD cells. 14 weeks following injection, 7 of 10 vehicle control mice had an easily detectable (by flow cytometry and colony assay) population of cells that stained for human CD45. Meanwhile, no human CD45+ cells were discernible in the marrows from the CEP-701 treated mice. Colony assays on these marrow samples yielded CFU-Ls from all vehicle-treated animals, with a frequency of 2.1 (range 0.94–3.62) CFU-L/103 human CD45+ cells plated. PCR amplification of genomic DNA obtained from the marrows of engrafted mice revealed the expected FLT3/ITD mutation in 7 out of 10 vehicle-treated mice. The mutation was also detectable by PCR only in 2 of the 8 mice treated with CEP-701. This indicated that these mice were indeed engrafted, but the level of engraftment was too low to be detected by the FACS analysis or colony assays. Quantitative PCR performed on genomic DNA from the marrow samples of the CEP-701 treated mice confirmed that the DNA from the marrows of these two mice contained FLT3/ITD DNA at a level of 0.8% and 0.03%, respectively. The other 6 CEP-701-treated mice had no detectable FLT3/ITD DNA. Finally, as a control experiment, we injected each of 10 mice with 6 x 106 normal donor human CD34+ cells. 5 of the mice were treated with CEP-701, while the other 5 mice were treated with vehicle only. After 14 weeks, analysis of human CD45 expression and CFU-GM/BFU-E colony assays was performed on the marrow of these mice. The assay results show that CEP-701 treatment had no significant effect on normal hematopoietic engraftment. This data provides evidence that 1) Leukemic stem cells harboring FLT3/ITD mutations are dependent on FLT3 signaling for survival and engraftment capability in NOD-SCID mice; 2) FLT3/ITD mutations are present in leukemia stem cells; and 3) FLT3 inhibitors may have activity against leukemia stem cells harboring these mutations.

Interaction of human herpesvirus 6 with human CD34 positive cells.

We reported previously that human herpesvirus 6 (HHV-6) suppresses hematopoietic colony formation of erythroid (BFU-E), granulocyte/macrophage (CFU-GM), and megakaryocyte (CFU-Meg) lineages in vitro. Here we describe the interaction between HHV-6 and human CD34+ cells, which are a major source of hematopoietic progenitor cells. CD34+ cells were immunomagnetically isolated from cord blood mononuclear cells using anti-CD34+ antibodies coated onto either Dynabeads trade mark or MACS beads. The CD34+ population selected with Dynabeads showed a broad range of fluorescence. The population selected with MACS beads showed a narrow range of fluorescence. After infection with HHV-6, two transcripts of the immediate early genes were detected with both cell populations. HHV-6 suppressed colony formation of BFU-E, CFU-GM, and CFU-Meg. HHV-6 suppressed cell growth after 3 to 7 days culture in the presence of thrombopoietin (TPO). More differentiated CD34+ cells were more susceptible to the effects of HHV-6. These data indicate that the targets for hematopoietic suppression by HHV-6 are the differentiated cells.

Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.

Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells. A bicistronic retrovirus vector, Ha-MDR-IRES-gp91, was constructed for the co-expression of MDR1 and gp91, a gene responsible for X-linked chronic granulomatous disease (X-CGD). Drug-selected co-expression of P-glycoprotein and gp91 was evaluated in transduced cells. Epstein-Barr virus-transformed B cells from X-CGD patients transduced with Ha-MDR-IRES-gp91 co-expressed human P-glycoprotein and gp91, and acquired superoxide-generating activity. Human CD34-positive cells from an X-CGD patient were transduced with Ha-MDR-IRES-gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha-MDR-IRES-gp91-transduced cells were P-glycoprotein- and gp91-positive by FACS analysis. The superoxide-generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha-MDR-IRES-gp91-transduced bone marrow cells showed co-expression of P-glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P-glycoprotein- and gp91-positive cells were increased in all the four mice examined. When mice transplanted with Ha-MDR-IRES-gp91-transduced cells were repeatedly administered paclitaxel, the ratios of P-glycoprotein- and gp91-positive cells were maintained for over 1 year. These results suggest that MDR1-bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo. This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy.

Preferential inactivation of paediatric solid tumour cells by sequential exposure to Merocyanine 540-mediated photodynamic therapy and Edelfosine: implications for the ex vivo purging of autologous haematopoietic stem cell grafts

Paediatric solid tumours exhibit steep dose–response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms’ tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.

Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene.

Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34+ cells. The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFbeta1-EGFP) or EGFP and a downstream (vector SFbeta1-EGFP-IRES) or upstream (vector SFbeta1-IRES-EGFP) internal ribosomal entry site. For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 +/- 0.4), followed by N2A-PGK-EGFP (26.3 +/- 1.8 MFI), and N2A-TK-EGFP (4.8 +/- 0.1 MFI). Expression from the SFbeta1-EGFP vector (82.6 +/- 6.7 MFI) and the SFbeta1-EGFP-IRES vector (102.8 +/- 6.2 MFI) was higher than from SFbeta1-IRES-EGFP vector (15.5 +/- 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFbeta1-EGFP vector still being four- to fivefold more active than the internal promoters tested. The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration.

TRANSFECTION OF HUMAN ISLETS WITH AN INHIBITOR OF TNF PROTECT THEM AGAINST DESTRUCTION BY ACTIVATED HUMAN PERIPHERAL BLOOD LEUKOCYTES.

970 The purpose of this study was to evaluate if transfection of human islets with an inhibitor of the cytokine tumor necrosis factor alfa (TNF) would affect their in vivo function or protect them against immune mediated destruction. Human islets were separated into the following three groups: group 1 was transfected with an adenoviral vector coding a TNF inhibitor, group 2 was transfected with an adenoviral vector with the LacZ gene coding β-galactosidase (β-gal), and group 3 was not infected. The islets were then placed under the kidney capsule of immunodeficient NOD-scid mice. Islet graft function was determined by measuring human insulin release in response to glucose injections on days 3, 7, and 14 relative to transplantation. On day 15, half of the mice received injections of activated human peripheral blood leukocytes (HuPBL) in order to study the ability of HuPBL to mediate destruction of transfected vs. nontransfected islets. Glucose stimulated human insulin release was measured at weekly intervals after injection of HuPBL or buffer. Animals were sacrificed and histochemical stains were performed on the graft bearing kidneys 36 days after transplantation (21 days after HuPBL injection) to assess graft presence, transgene expression, and HuPBL infiltration. Throughout the 36 day study period there was no significant difference in insulin production in the three groups of mice that did not receive HuPBL (p = 0.83, analysis of variance). In the groups that were injected with HuPBL, graft performances of the islets transfected with the TNF inhibitor were maintained after injection of HuPBL. Expressed as a percentage of function the day before injections, graft function 21 days later was 137±14% (mean±SE) and 120±22% respectively for buffer injected controls and HuPBL injected mice with TNF inhibitor transfected grafts. In contrast islet grafts performance 21 days after HuPBL injection in mice transplanted with islets not transfected or transfected with LacZ declined dramatically to 62±12% and 59±18% of the function before HuPBL injection. Histological examination supported the conclusion that TNF inhibitor transfected grafts were protected from destruction by HuPBL. In contrast to controls and TNF inhibitor transfected grafts, non-tranfected or LacZ transfected grafts were difficult to locate and had few if any insulin staining structures in HuPBL injected mice. Interestingly the presence of human CD45 positive cells in both spleen and islet graft of all HuPBL injected mice was similar. This indicate that the protective effect of the TNF inhibitor may be due to a local neutralization of the destructive effects of HuPBL. This data demonstrates that transfection of human islets with a TNF inhibitor before transplantation can protect them from destruction mediated by human leukocytes.

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.

末梢血ＣＤ３４陽性細胞分離のための大量単球除去用ナイロンウールバッグの開発

A nylon-wool bag for the large-scale depletion of monocytes to obtain high purity and high yeild selection of human CD34 positive cells in mobilized blood was newly developed. The bag was made of a blood transfusion bag containing a nylon-wool which were treated with human gamma grobulin. After double treatment with the bags, 98.4% (n=4) of monocytes were depleted from mobilized peripheral blood. However, 74.9% of the CD34 positive cells were recovered. After the depletion of monocytes, immunomanetic separation system was used to collect CD34 positive cells. CD34 positive cell purities were increased from 12.6% preseparation to 94.0% postseparation with a mean yield of 54.1%. Fourty-one pint five percent of CD34 positive cells could formed blood cell clones in the presence of growth factors by single cell culture method. Fourteen percent of these clones proliferated to over 105 cells, and 9.4% of the clones contained megakaryocytes.

Activation of Wnt signalling in acute myeloid leukemia by induction of Frizzled-4

Wnt signalling regulates proliferation, self renewal and cell fate. Aberrant Wnt signalling is thought to contribute to AML pathogenesis by enhancing self renewal. Herein, we provide evidence for increased expression of Frizzled-4, a receptor for Wnt ligands, in primary AML blasts compared to normal bone marrow on the protein level. In addition, Frizzled-4 is highly expressed in human CD34 positive cells as well as in lineage negative sorted mouse bone marrow cells. Functionally, Frizzled-4 expression modulates apoptosis and enhances Wnt3a induced beta-catenin stability in myeloid progenitor cells. Frizzled-4-dependent beta-catenin stabilization is dkk-1 sensitive, implicating a specific Wnt-ligand/Frizzled-receptor interaction. These findings indicate enhanced sensitivity of AML blasts for Wnt-ligands and suggest an additional mechanism of Wnt signalling activation in the pathogenesis of AML.

Selective Expression of Two Homeobox Genes in CD34-Positive Cells From Human Bone Marrow

Proteins coded by homeobox-containing genes are sequence-specific DNA-binding proteins that have been implicated in the control of gene expression both in developing as well as in adult tissues. Two recently characterized human homeobox genes, HB24 and HB9, were found to be highly expressed in bone marrow cells enriched for CD34-positive cells, present at low levels in unfractionated bone marrow cells, and essentially undetectable in bone marrow cells depleted of CD34 cells. Treatment of CD34-enriched cells with recombinant interleukin-3 (IL-3) and granulocyte macrophage-colony-stimulating factor for 24 hours increased expression of HB24 threefold and HB9 fourfold. Based on studies with actinomycin D, the HB24 and HB9 transcripts in human CD34-positive cells have short half-lives, estimated to be 30 to 45 minutes. Downregulation of HB24 and HB9 expression was found following the treatment of in vitro cultures of CD34-positive cells with IL-3. Thus, the differentiation of CD34-positive cells along a specific cell lineage likely requires downregulation of both HB24 and HB9.