In vitro retroviral transfer of ras genes to single hemopoietic progenitors

Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.     

Culture studies of human pluripotent hemopoietic progenitors

Conclusions Culture conditions are described that promote the growth of human pluripotent hemopoietic progenitors and facilitate their quantitation. These primitive cells form mixed colonies that may contain all elements of myeloid differentiation, including granulocytes, erythroblasts, megakaryocytes, and macrophages. Some mixed colonies contain, in addition to mature progeny, early progenitors that can be identified by their ability to form secondary hemopoietic colonies. The production of secondary mixed hemopoietic colonies by cells present in redispersed primary mixed hemopoietic colonies supports the view that some CFU-GEMM may self-replicate in culture, thus fulfilling one of the major operational requirements for pluripotent hemopoietic stem cells. Assessment of the proliferative state of CFU-GEMM under steady state conditions and in various clinical disorders suggests that the proliferative activity may reflect conditions that are associated with perturbations at the level of pluripotent progenitors.The recently observed increased plating efficiency of approximately 10 mixed colonies per 10⁵ plated mononuclear cells will facilitate investigation of regulatory events at the level of pluripotent progenitors. With the use of putative stimulators, it might be feasible to modulate the cellular composition of mixed colonies and thus, identify mechanisms that determine the choice of pluripotent cells to self-replicate or to differentiate and mature into cells of specific phenotype.Supported by the Medical Research Council of Canada, University of Toronto and the Banting Research FoundationFellow of the Medical Research Council of Canada and financially assisted by Deutsche Forschungsgemeinschaft     

Sensitivity of chronic myeloid leukemia hemopoietic progenitors to PTT-119 in combination with human recombinant interferon alpha and gamma

Summary PTT-119, a new synthetic alkylating compound, has shown a marked in vitro inhibitory effect on chronic myeloid leukemia (CML) granulo-monocytic precursors (CFU-GM) at doses greater than 5µg/ml. Based on previous experiences of synergistic associations between alkylating drugs and biological modifiers, we tested the effects of low doses of PTT-119 (from 0.1 to 1µg/ml) in concert with alpha, gamma, or alpha + gamma interferons and compared to IFNs alone, in order to investigate an alternative choice for treatment of CML patients in chronic phase. Our results showed a significantly higher CFU-GM cloning inhibition after addition of 100 or 1,000 U/ml of alpha IFN to 0.1µg/ml PTT-119 (from 39.6% ± 26.6 SD to 80.7% ± 10 SD and 91.5% ± 8 SD, respectively), while gamma IFN resulted in only a slight increase in colony growth inhibition when compared to the drug used alone. The association of alpha plus gamma IFN coupled with PTT-119 treatment did not significantly improve the results observed after exposure of leukemic progenitors to PTT-119 and alpha IFN alone. We conclude that a combined treatment with PTT-119 and IFN is probably worth testing both for purging methods before autologous bone marrow transplantation and for in vivo administration in chronic myeloid leukemia.     

Differential protection of normal and malignant human myeloid progenitors (CFU-GM) from Ara-C toxicity using cycloheximide

Cycloheximide, a reversible protein synthesis inhibitor, is thought to block DNA replication in normal cells by preventing synthesis of a labile protein. In animal systems, cycloheximide protects normal cells from cytotoxic S-phase specific agents, such as cytosine arabinoside (Ara-C). Malignant cells appear not to be susceptible to cycloheximide- induced cycle arrest and, subsequently, may not be protected from Ara-C cytotoxicity. The effect of cycloheximide on granulocyte/macrophage progenitors (CFU-GM) after in vitro Ara-C exposure was examined using normal human bone marrow, malignant progenitors from patients with chronic myelogenous leukemia (CML), and clonogenic cells from the human acute nonlymphocytic leukemia cell lines HL-60 and KG-1. Mononuclear or clonogenic cells were incubated for one hour with cycloheximide, followed by the addition, for three or 17 hours, of Ara-C before being plated in a methylcellulose culture system. CFU-GM survival was significantly increase if normal cells were treated with cycloheximide before Ara-C exposure. Similar cycloheximide pretreatment of CML progenitors and clonogenic HL-60 and KG-1 cells failed to protect CFU- GM from Ara-C-induced cytotoxicity.     

Serum-free culture of human hemopoietic progenitors in attenuated culture media

To elucidate the precise mechanisms of molecular and cellular regulation of hemopoiesis, it is necessary to develop a chemically defined culture assay for purified hemopoietic progenitors. To approach this long-term goal, we attempted to develop a serum-free culture system for enriched human progenitors that permits expression of all hemopoietic lineages and stages of development. Preliminary studies indicated that alpha-medium was superior to Iscove's modified Dulbecco's medium (IMDM) and that culture under low (5%) oxygen condition was better than an ambient level of oxygen. We developed an attenuated (modified quarter-strength) alpha-medium and compared the colony-supporting ability of the three media by plating 1,000 bone marrow null cells per dish in the presence of a combination of recombinant human colony-stimulating factors (CSFs). The numbers of colonies supported in alpha-medium and attenuated alpha-medium were approximately 70% of those in serum-containing cultures. IMDM failed to support colony formation. While, in general, the colony sizes were smaller in the serum-free cultures than in the serum-containing cultures, a variety of types of single lineage and multilineage colonies were seen in serum-free culture. A linear relationship between cell number and colony formation was seen in 100-2,000 cells per dish. Serum-free cultures of enriched human progenitors should be an important tool for analysis of the mechanisms of recombinant CSFs.     

Disparate differentiation in hemopoietic colonies derived from human paired progenitors

We analyzed the differentiation of hemopoietic colonies derived from human paired daughter cells. Candidate progenitor cells were isolated by use of a micromanipulation technique from cultures of My-10 antigen- positive cord blood cells. Then nine to 36 hours later, the paired daughter cells were separated with a micromanipulator and allowed to form colonies in methylcellulose medium containing erythropoietin, phytohemagglutinin leukocyte-conditioned medium, and platelet-poor plasma. The cellular composition of the colonies was determined by differentiating all of the cells of the May-Grunwald-Giemsa-stained preparation. Of a total of 75 evaluable pairs of colonies, 35 consisted of 28 types of disparate pairs revealing nonhomologous lineage combinations. Forty pairs were homologous in lineage expression. However, the proportions of the individual cell lineages were significantly different in the members of some of the homologous pairs. Some pairs revealed significant differences in colony size. These observations are similar to those reported for murine paired progenitors and are consistent with the stochastic model of human stem cell differentiation.     

Stimulatory activities for human multilineage hemopoietic progenitors (CFU-GEMMT) derived from patients with hemochromatosis and from normal volunteers

A medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of human multilineage hemopoietic progenitors (CFU-GEMMT) which form mixed hemopoietic colonies in culture containing granulocytes, erythroblasts, megakaryocytes, macrophages, and T-lymphocytes. PHA-LCM derived from six HLA-typed patients with idiopathic hemochromatosis and from six normal individuals were tested for growth-promoting activities for multilineage hemopoietic colony formation. Four out of six conditioned media obtained from patients with hemochromatosis supported mixed hemopoietic colony formation, as did four of six conditioned media from normal HLA-typed volunteers. Active PHA-LCM preparations from patients with hemochromatosis were similar with respect to the number and size of mixed colonies and their cellular composition when compared with conditioned media obtained from volunteers. The study indicates that no link exists between the HLA phenotype of a particular donor and the predictability of obtaining an active PHA-LCM promoting multilineage hemopoietic colony formation. PHA-LCM derived from patients with hemochromatosis had no advantage with respect to stimulatory activity for mixed colony formation when compared with conditioned media obtained from healthy volunteers.     

Normal and malignant human myeloid progenitors differ in their sensitivity to hyperthermia

The effect of in vitro hyperthermia on normal human bone marrow granulocyte-macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) was compared to its effect on clonogenic acute nonlymphocytic leukemic (ANLL) cells. Mononuclear normal bone marrow cells, blasts from patients with ANLL, and HL-60 cells were incubated at room temperature (control) and at 42 degrees-44 degrees C for 0-120 min prior to assay in methylcellulose. The heat sensitivity of the leukemic cells was significantly greater than that of normal bone marrow progenitors. Two-h exposure to 43 degrees C, for example, resulted in survival of 52% of normal marrow CFU-GM, whereas only 3% of leukemic CFU-GM survived (p less than 0.001 for HL-60 cells and p less than 0.005 for patient blast cells). To determine the effect of hyperthermia on more primitive progenitors and on marrow stromal cells, long-term cultures of normal bone marrow were established using control and heat-treated cells. Generation of CFU-GM was detected in the nonadherent fraction of hyperthermia-treated samples throughout the 5-week culture period. Although stromal development was slightly delayed, hyperthermia-treated cells were able to establish stromal layers similar to control cells. These results indicate that normal bone marrow committed progenitor cells are more resistant to hyperthermia than are myeloid leukemic cells. Normal stromal cells and primitive cells assayed in long-term culture are also resistant to hyperthermia that is toxic for leukemic cells. Because of this differential sensitivity to heat, ex vivo hyperthermia may be applicable for removing residual leukemic cells from bone marrow harvested for autologous transplantation.     

Expansion of genetically modified primary human hemopoietic cells using chemical inducers of dimerization

The inability to deliver a therapeutic gene to a sufficient percentage of hematopoietic stem cells is the major obstacle to using gene therapy to treat blood disorders. Providing genetically corrected stem cells with a reversible growth advantage could solve this problem. To this end we have employed small synthetic molecules that can reversibly dimerize and activate fusion proteins which contain a growth factor receptor signaling domain. We have shown that the thrombopoietin receptor (mpl) signaling domain can be used in this system to expand transduced multipotential progenitor cells from mouse bone marrow. In the present study we tested a similar retroviral vector in human CD34-selected cord blood cells. Following transduction, cells cultured in the presence of the dimerizing molecule AP1903 expanded 13.8- to 186-fold relative to cells cultured in the absence of AP1903. The cell type that emerged in suspension culture was erythroid. Contrary to our results in the murine system, cell expansion was transient. Activation of mpl caused the disappearance of BFU-E followed by a transient increase in CFU-E. In contrast, mpl activation had no discernable effect on transduced myeloid progenitor cells. AP1903-mediated expansion was restricted to transduced cells, as demonstrated by immunohistochemical staining. These findings indicate that synthetic dimerizing molecules can be used to expand primary human hematopoietic cells. (Blood. 2000;95:430-436)     

Proliferative state of human pluripotent hemopoietic progenitors (CFU-GEMM) in normal individuals and under regenerative conditions after bone marrow transplantation.

The proliferative state of human pluripotent hemopoietic progenitors (CFU-GEMM) was assessed in normal bone marrow transplant donors and in recipients after engraftment using the tritiated thymidine suicide method. In contrast to BFU-E and CFU-C, CFU-GEMM were found to be quiescent. In samples obtained during early regeneration after transplantation, CFU-GEMM were significantly reduced after short-term exposure to 3H TdR when compared to controls. This observation suggests active participation of CFU-GEMM in bone marrow regeneration.     

Clonal origin of human basophil/mast cells from circulating multipotent hemopoietic progenitors

The origin of the human basophil/mast cell lineage from a pluripotent hematopoietic stem cell has been surmised but never demonstrated. By examining individual hemopoietic colonies in methylcellulose under inverted microscopy and using histochemical stains in conjunction with single-colony histamine assays, we have previously identified basophil/mast cell progenitors in human peripheral blood. We now report that a large proportion of normal human peripheral blood mixed granuloerythropoietic (GEMM) colonies contain histamine, in contrast to a significantly lower frequency of histamine positivity among normal neutrophil-macrophage, eosinophil, erythroid, macrophage, or megakaryocyte colonies. Morphological observations confirmed the presence of basophil/mast cells in the majority of GEMM colonies. In our work, the clonal derivation of basophils/mast cells from circulating multipotent (CFU-GEMM) hemopoietic stem cells was formally demonstrated, using combined histamine and G6PD isoenzyme analysis of single colonies grown in methylcellulose from a normal G6PD heterozygote.     

Effects of recombinant human interleukin-3 on CD34-enriched normal hematopoietic progenitors and on myeloblastic leukemia cells

Induction of proliferation and differentiation in response to recombinant human interleukin-3 (hIL-3) was studied in liquid and semisolid cultures of umbilical cord blood and bone marrow cells that were fractionated by "panning" with anti-My10 antibody according to expression of CD34 antigen. Cells from enriched fractions (70% to 90% CD34+) were found to proliferate strongly in response to hIL-3. Phenotypic analysis and morphologic characterization of the proliferating cells demonstrated a rapid decrease in CD34+ cells and an exponential increase in the number of cells belonging to the neutrophilic, eosinophilic, monocyte/macrophage, and thrombocytic lineages. When combined with recombinant human erythropoietin, burst colonies and cells expressing glycophorin-A were detected, thereby demonstrating the effects of hIL-3 on erythroid progenitors. Further, the development of mixed-erythroid colonies indicated that multipotential cells within CD34-enriched fractions responded to hIL-3. In addition, we examined the effect of hIL-3 on the proliferation of primary acute myeloblastic leukemia cells in liquid culture. We found that hIL-3 was able to induce cell proliferation in a proportion of the cases tested. Heterogeneity of the responses to hIL-3 was in part related to French-American-British classification but could not be correlated with CD34 antigen expression by the leukemic cells. These results indicate that, although the effects of hIL-3 on proliferation and differentiation of cells obtained from normal hematopoietic specimens were primarily borne by CD34+ cells, expression of the CD34 molecule per se is an insufficient condition to determine a growth response to this lymphokine.     

In vivo effects of recombinant human erythropoietin on circulating human hemopoietic progenitor cells

Recombinant human erythropoietin (rhEpo), now available, has become increasingly more important for clinical use, e.g., in the treatment of anemia of chronic renal failure, and has been shown to reverse anemia in these patients. When patients with anemia of chronic renal failure were treated with rhEpo at dosages between 40 and 120 U/kg three times per week, the numbers of circulating erythroid burst-forming units (BFU-E) and granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming units (CFU-GEMM) significantly increased during the first week of therapy. In contrast, the incidence of circulating granulocyte-monocyte CFU (CFU-GM) was not significantly altered.     

Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.     

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were trans-fected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.     

In vivo effects of human recombinant interleukin 6 on hemopoietic stem and progenitor cells and circulating blood cells in normal mice

Recombinant human interleukin 6 (rhIL-6) was administered s.c. every 12 h at a daily dose of 10 micrograms/kg body weight to normal healthy mice. After 4 days the numbers of progenitor cells (erythroid burst-forming units, BFU-E and granulocyte-macrophage colony-forming cells, GM-CFC) were significantly increased (p less than 0.01) in the bone marrows and spleens of treated animals. There was no significant change in spleen colony forming unit (CFU-S) number, whereas mixed-lineage colony-forming cell (Mix-CFC) number was elevated only in bone marrow. The number of nucleated cells in peripheral blood was increased in rhIL-6-treated mice, resulting from a significant (p less than 0.01) increase in neutrophil numbers and a decrease in lymphocyte numbers. The number of platelets in these animals was also higher than in controls (p less than 0.05). These results suggest that rhIL-6 is an effective stimulator of unipotent hemopoietic cells of myeloid, erythroid, and thrombocytic lineages when administered in vivo to mice and indicate a possible therapeutic potential of IL-6 in clinical situations.     

Inhibition of normal human macrophage progenitors by retinoic acid

The effects of retinoic acid on the clonal growth in nutrient agar of neutrophil and macrophage progenitors from normal bone marrow and fetal liver were studied. The technique of whole plate fixation and staining with luxol fast blue and haematoxylin was employed to distinguish colonies and clusters into morphological groups. Retinoic acid at a concentration of 3 X 10(-7) M resulted in 97% inhibition of macrophage clonal growth and a mean increase in neutrophil clonal growth from bone marrow of 150 +/- 14%. These effects of retinoic acid were most obvious on bone marrow cluster forming cells. The inhibition of macrophage cluster growth was independent of exogenous colony stimulating factor. This dual action of retinoic acid is likely to be a direct effect on the progenitor cells since it is (1) independent of cell concentration, (2) not mediated by phagocytic cells or other bone marrow cells, and (3) was maximal in the first 2 d of incubation before the appearance of clusters.