Recombinant Canine Stem Cell Factor Research Articles

Cultivation and Characterization of Canine Skin-Derived Mast Cells

It is essential to develop a technique to culture purified skin-derived mast cells (SMCs) to facilitate immunological research on allergic diseases in dogs. This study was performed to develop an efficient culture system for canine SMCs and to characterize the cells in comparison to canine bone marrow-derived mast cells (BMMCs). Enzymatically digested skin biopsy samples were cultivated in serum-free AIM-V medium supplemented with recombinant canine stem cell factor. Three to five weeks after the initiation of culture, mast cells were collected by a magnetic activated cell separation system using anti-c-Kit antibody. The collected cells were composed of a uniform population showing morphological characteristics of mast cells with a round or oval nucleus and abundant toluidine blue-positive metachromatic granules in the cytoplasm. The results of flow cytometric analysis for the presence of cell membrane c-Kit and Fc epsilon receptor I (FcepsilonRI) indicated that approximately 90% of the cells were mast cells. The cytoplasmic granules were positive for both tryptase and chymase. Apparent dose-dependent degranulation was induced by antibody-mediated cross-linking of immunoglobulin E (IgE) bound to the cells. These cytological and immunological characteristics observed in SMCs were mostly similar to those observed in BMMCs; however, IgE-mediated degranulation was significantly lower in SMCs than BMMCs. The culture system for canine SMCs developed in this study would be useful in understanding the pathophysiology and developing anti-allergic therapeutics in canine allergic dermatitis.

Generation and characterization of bone marrow-derived cultured canine mast cells

Disorders of mast cells, particularly mast cell tumors (MCTs), are common in dogs. There now is evidence that many of these disorders exhibit breed predilections, suggesting an underlying heritable component. In comparison to humans and mice, little is known regarding the biology of canine mast cells. To facilitate the study of mast cell biology in other species, bone marrow-derived cultured mast cells (BMCMCs) often are used because these represent a ready source of large numbers of cells. We have developed a protocol to successfully generate canine BMCMCs from purified CD34 + cells. After 5–7 weeks of culture with recombinant canine stem cell factor (rcSCF), greater than 90% of the cell population consisted of mast cells as evidenced by staining with Wright's-Giemsa, as well as production of chymase, tryptase, IL-8 and MCP-1. These cells expressed cell surface markers typical of mast cells including Kit, FcɛRI, CD44, CD45 and CD18/CD11b. The canine BMCMCs were dependent on rcSCF for survival and proliferation, and migrated in response to rcSCF gradients. Cross-linking of cell surface-bound IgE induced the release of histamine and TNFα. Histamine release could also be stimulated by ConA, compound 48/80, and calcium ionophore. In summary, canine BMCMCs possess phenotypic and functional properties similar to mast cells found in vivo. These cells represent a novel, valuable resource for investigating normal canine mast cell biology as well as for identifying factors that lead to mast cell dysregulation in the dog.

IgE enhances FcεRI expression and IgE-dependent TNF-α release from canine skin mast cells

The role of IgE on mast cell (MC) activation is well known. Recent studies have demonstrated that IgE also has the ability to up-regulate the high affinity IgE receptor (FcεRI) on the surface of human and murine MC, leading to an increased production of cytokines and chemokines. In the present study, we have examined the influence of IgE levels on FcεRI expression, and its consequences on TNF-α production from canine skin MC. Mature MC were enzymatically dispersed from the skin biopsies of 6–8 dogs and were cultured for up to 5 days in medium supplemented with recombinant canine stem cell factor (SCF) (6 ng/ml), in the presence of increasing serum IgE concentrations (ranging from 0 to 80 μg/ml). Subsequently, skin MC were activated with anti-IgE, and TNF-α concentration was assessed 5 h post-activation by a cytotoxic bioassay. FcεRI receptors were identified in MC surface by flow cytometry. MC cultured for up to 5 days in the presence of high serum IgE concentration (8 μg/ml) produced twice the quantity of TNF-α than MC cultured in the absence of serum IgE, in response to stimulation with anti-IgE. Moreover, the percentage of FcεRI-positive skin cells was found to be approximately double in cells cultured with serum IgE compared to that cultured in the absence of IgE, following saturation of IgE receptors. These results suggest that, as found in human and murine MC, IgE may induce an up-regulation of the FcεRI density and an enhancement in the secretory activity of canine skin MC. This study could be of great interest in designing new therapeutic strategies for controlling MC activation in inflammatory and allergic processes.

Myelosuppressive Conditioning Improves Autologous Engraftment of Genetically Marked Hematopoietic Repopulating Cells in Dogs

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.

Differentiation of canine bone marrow cells with hemopoietic characteristics from an adherent stromal cell precursor.

Stromal cell lines were established from canine long-term marrow cultures, cloned by limiting dilution, and maintained in stromal cell-conditioned medium. These cells grew adherent, maintained stable growth rate and morphology under standard conditions (in 20-30% conditioned medium; confluency, 70-90%), and supported hemopoiesis in long-term marrow cultures. In the presence of exogenous recombinant canine stem cell factor (rcSCF), round cells developed from the adherent layer, detached, and remained in culture as viable floating cells. Round floating cells also appeared when cultures were grown to > 90% confluency without rcSCF. Round cells were smaller than adherent cells, expressed CD34, showed basophilic plasma, and stained positive for c-kit, MHC-class II markers, and myeloid markers. In standard assays for colony formation, the detached cells produced granulocyte-macrophage colony-forming units (CFU-GM), fibroblast colony-forming units (CFU-F), and less well-defined colony-forming units. In addition, on allogeneic feeder cells in long-term cultures, these cells generated hemopoietic colonies. Strikingly, the differentiation was reversible: when nonadherent cells were resuspended at lower density in serum-containing medium, they reattached and grew to confluence when, once again, round cells detached. Detached cells from this secondary cycle produced mainly CFU-F and few CFU-GM when placed in clonal assays. These results suggest that some fibroblast-like stromal cells have the potential to differentiate into cells with hemopoietic characteristics. These observations provide evidence for the existence of a quiescent precursor of hemopoietic progenitors in the bone marrow stroma of the adult dog.

Allergen-induced changes in bone marrow progenitors and airway responsiveness in dogs.

An increased production of inflammatory cell progenitors (colony-forming cells, CFUs) may contribute to airway inflammation, since CFUs increase after allergen inhalation in asthmatics. We examined the effect of allergen inhalation, with or without budesonide pretreatment, on bone marrow CFU production in dogs with allergen-induced airway hyperresponsiveness. Allergen inhalation increased airway responsiveness (p < 0.001) as well as the number of CFUs induced in vitro by recombinant canine stem cell factor (p < 0.001) and granulocyte-colony-stimulating factor (p = 0.035). Budesonide reduced the allergen-induced increases in airway responsiveness (p = 0.005) and abolished the allergen-induced increases in the numbers of CFUs (p < 0.005). These findings provide the first direct evidence that allergen inhalation increases bone marrow granulocyte progenitor production and suggest that such increases may contribute to the development of airway hyperresponsiveness in asthma. The effectiveness of inhaled corticosteroids in asthma may result, in part, from effect on bone marrow production of inflammatory cells.

Allergen-induced changes in bone marrow progenitors and airway responsiveness in dogs and the effect of inhaled budesonide on these parameters.

Airway inflammation is implicated in the pathogenesis of the airway hyperresponsiveness in asthma. An increased production of inflammatory cell progenitors may contribute to asthmatic airway inflammation. Although the number of circulating inflammatory cell progenitors in asthmatic subjects increases after allergen inhalation, no direct evidence exists for increased bone marrow progenitor production. We examined the effect of allergen inhalation on bone marrow progenitor production in seven dogs that develop allergen-induced airway hyperresponsiveness. The effect of inhaled budesonide, a corticosteroid known to be effective in the treatment of asthma, on allergen-induced bone marrow progenitor production and airway hyperresponsiveness was also examined. Allergen inhalation increased airway responsiveness (P < 0.001) and the number of granulocyte-macrophage colony-forming units (CFU) when cultured with dog serum and either recombinant canine stem cell factor (rcSCF) (P < 0.001) or granulocyte colony-stimulating factor (rcG-CSF) (P = 0.035). Budesonide treatment reduced the allergen-induced increases in airway responsiveness (P = 0.005) and abolished the allergen-induced increases in the numbers of CFU cultured with dog serum and either rcSCF (P < 0.001) or rcG-CSF (P = 0.009). These findings provide the first direct evidence that allergen inhalation increases bone marrow progenitor production and suggest that such increases may contribute to the development of airway hyperresponsiveness in asthma. In addition, the effectiveness of inhaled corticosteroids in asthma may result, in part, from their ability to suppress bone marrow production of inflammatory cells.

Effects of granulocyte colony-stimulating factor and stem cell factor, alone and in combination, on the mobilization of peripheral blood cells that engraft lethally irradiated dogs

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) and recombinant canine stem cell factor (rcSCF), a c-kit ligand, on the circulation of hematopoietic progenitor and stem cells were studied in a canine model. Administration of rcG-CSF (10 micrograms/kg) for 7 days led to a 5.4-fold increase in CFU-GM/mL of blood, while 7 days of rcSCF (200 micrograms/kg) led to an 8.2-fold increase. Although treatment with low-dose rcSCF (25 micrograms/kg) had no effect on the level of peripheral blood progenitors, 7-day exposure to a combination of G-CSF plus low dose SCF led to a 21.6-fold increase (P = .03). To assess the ability of these factors to increase the circulation of cells capable of rescuing animals after lethal total body irradiation (TBI), 1 x 10(8) peripheral blood mononuclear cells (PBMC)/kg were collected and cryopreserved from animals after 7 days of treatment with G-CSF, SCF or a combination of the two. One month later, animals were exposed to 9.2 Gy TBI and transplanted with the previously collected cells. Control animals transplanted with 1 x 10(8) PBMC/kg collected without pretreatment died with marrow aplasia 11 to 29 days after TBI as did animals treated with only low-dose SCF before cell collection. In contrast, all animals given PBMC collected after G-CSF, high-dose SCF, or a combination of G-CSF plus low-dose SCF recovered granulocyte function. Recovery to 500 granulocytes/microL after transplant took 17, 18.8, and 13.6 days, respectively, (P = .056 for the difference between the combination G-CSF-SCF group and the other two groups). In both the G-CSF and SCF groups, 4 of 5 animals completely recovered while 1 of 5 in each group died with prolonged thrombocytopenia. In the combination group, all 5 animals became long- term survivors. These studies demonstrate that both G-CSF and SCF dramatically increase the level of peripheral blood hematopoietic progenitor and stem cells and support the view that these factors can act synergistically.

Effects of Granulocyte Colony-Stimulating Factor and Stem Cell Factor, Alone and in Combination, on the Mobilization of Peripheral Blood Cells That Engraft Lethally Irradiated Dogs

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) and recombinant canine stem cell factor (rcSCF), a c-kit ligand, on the circulation of hematopoietic progenitor and stem cells were studied in a canine model. Administration of rcG-CSF (10 micrograms/kg) for 7 days led to a 5.4-fold increase in CFU-GM/mL of blood, while 7 days of rcSCF (200 micrograms/kg) led to an 8.2-fold increase. Although treatment with low-dose rcSCF (25 micrograms/kg) had no effect on the level of peripheral blood progenitors, 7-day exposure to a combination of G-CSF plus low dose SCF led to a 21.6-fold increase (P = .03). To assess the ability of these factors to increase the circulation of cells capable of rescuing animals after lethal total body irradiation (TBI), 1 x 10(8) peripheral blood mononuclear cells (PBMC)/kg were collected and cryopreserved from animals after 7 days of treatment with G-CSF, SCF or a combination of the two. One month later, animals were exposed to 9.2 Gy TBI and transplanted with the previously collected cells. Control animals transplanted with 1 x 10(8) PBMC/kg collected without pretreatment died with marrow aplasia 11 to 29 days after TBI as did animals treated with only low-dose SCF before cell collection. In contrast, all animals given PBMC collected after G-CSF, high-dose SCF, or a combination of G-CSF plus low-dose SCF recovered granulocyte function. Recovery to 500 granulocytes/microL after transplant took 17, 18.8, and 13.6 days, respectively, (P = .056 for the difference between the combination G-CSF-SCF group and the other two groups). In both the G-CSF and SCF groups, 4 of 5 animals completely recovered while 1 of 5 in each group died with prolonged thrombocytopenia. In the combination group, all 5 animals became long-term survivors. These studies demonstrate that both G-CSF and SCF dramatically increase the level of peripheral blood hematopoietic progenitor and stem cells and support the view that these factors can act synergistically.

Retrovirus-Mediated Gene Transduction Into Canine Peripheral Blood Repopulating Cells

AbstractGenetically marked peripheral blood progenitor cells were used to investigate their contribution to long-term hematopoietic reconstitution after autologous marrow and peripheral blood cell transplantation. After autologous marrow harvest and cryopreservation, canine peripheral blood progenitor cells were mobilized in three dogs by treatment with recombinant canine stem cell factor for 8 days. Peripheral blood mononuclear cells were collected and enriched for major histocompatibility complex (MHC) class II antigen-positive cells by avidin-biotin immunoadsorption, thereby enriching for repopulating cells. Subsequently, the cells were cocultivated for 24 hours on irradiated vector-producing packaging cells (PA317/LN), followed by an 11-day incubation in a vector containing long-term marrow culture system. On the day of transplantation, the animals were irradiated with 9.2 Gy total body irradiation (TBI), and transduced peripheral blood cells and untransduced cryopreserved marrow cells were infused within 2 hours of TBI. All three dogs engrafted. Two dogs are long-term survivors showing intermittently G418-resistant marrow-derived colony-forming unit granulocyte-macrophage colonies at a median of 1 % and 2%, respectively (range, 1 % to 10%), for now up to 48 weeks after transplantation. Neo-specific sequences were detected by polymerase chain reaction in peripheral blood granulocytes for now up to 65 weeks and in peripheral blood lymphocytes for up to 75 weeks after transplantation. Peripheral blood samples of the dogs were free of helper virus and no side effects from the transduction were observed. One of the three dogs died from chronic canine distemper sclerosing encephalitis on day 84, whereas the other two dogs are alive at 15 and 17 months. Our data show successful retroviral transduction of canine peripheral blood repopulating cells. Long-term persistence of marked myeloid and lymphoid cells after transplantation suggests that peripheral blood contains repopulating cells that contribute to long-term hematopoietic reconstitution after otherwise lethal TBI.

Retrovirus-mediated gene transduction into canine peripheral blood repopulating cells

Genetically marked peripheral blood progenitor cells were used to investigate their contribution to long-term hematopoietic reconstitution after autologous marrow and peripheral blood cell transplantation. After autologous marrow harvest and cryopreservation, canine peripheral blood progenitor cells were mobilized in three dogs by treatment with recombinant canine stem cell factor for 8 days. Peripheral blood mononuclear cells were collected and enriched for major histocompatibility complex (MHC) class II antigen-positive cells by avidin-biotin immunoadsorption, thereby enriching for repopulating cells. Subsequently, the cells were cocultivated for 24 hours on irradiated vector-producing packaging cells (PA317/LN), followed by an 11-day incubation in a vector containing long-term marrow culture system. On the day of transplantation, the animals were irradiated with 9.2 Gy total body irradiation (TBI), and transduced peripheral blood cells and untransduced cryopreserved marrow cells were infused within 2 hours of TBI. All three dogs engrafted. Two dogs are long-term survivors showing intermittently G418-resistant marrow-derived colony- forming unit granulocyte-macrophage colonies at a median of 1% and 2%, respectively (range, 1% to 10%), for now up to 48 weeks after transplantation. Neo-specific sequences were detected by polymerase chain reaction in peripheral blood granulocytes for now up to 65 weeks and in peripheral blood lymphocytes for up to 75 weeks after transplantation. Peripheral blood samples of the dogs were free of helper virus and no side effects from the transduction were observed. One of the three dogs died from chronic canine distemper sclerosing encephalitis on day 84, whereas the other two dogs are alive at 15 and 17 months. Our data show successful retroviral transduction of canine peripheral blood repopulating cells. Long-term persistence of marked myeloid and lymphoid cells after transplantation suggests that peripheral blood contains repopulating cells that contribute to long- term hematopoietic reconstitution after otherwise lethal TBI.

Effects of recombinant canine stem cell factor, a c-kit ligand, and recombinant granulocyte colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

The effects of recombinant canine stem cell factor (rcSCF) on hematopoiesis were studied in normal dogs and in dogs given otherwise lethal total body irradiation (TBI) without marrow transplant. Results were compared with previous and concurrent data with recombinant granulocyte colony-stimulating factor (rG-CSF). Four normal dogs received 200 micrograms rcSCF per kilogram body weight daily either by continuous intravenous infusion for 28 days (n = 2) or by subcutaneous (SC) injection in two divided doses for 20 days (n = 2). All dogs showed at least a twofold increase in peripheral blood neutrophil counts starting approximately 7 days after the initiation of treatment. Hematocrit level and monocyte, lymphocyte, eosinophil, reticulocyte, and platelet counts were not elevated. Marrow sections after rcSCF treatment showed panhyperplasia. The only toxicity was facial edema during the first few days of rcSCF administration, presumably caused by mast cell stimulation. Ten dogs were given 400 cGy TBI at 10 cGy/min from two opposing 60Co sources. They were given no marrow infusion and received 200 micrograms/kg/d rcSCF SC in two divided doses for 21 days starting within 2 hours of TBI. Five of the 10 dogs showed complete and sustained hematopoietic recovery and survived as compared with 1 of 28 control dogs not receiving growth factor (P &lt; .005). RcSCF treatment allowed for hematopoietic recovery in two of seven dogs administered 500 cGy of TBI but in none of five dogs given 600 cGy of TBI. Results with rcSCF are similar to those obtained with rG-CSF. The rate of neutrophil recovery in rcSCF-treated dogs after 400 cGy TBI was not different from that of rG-CSF-treated dogs (P = .65), but the rate of platelet recovery was faster (P = .06) in the rcSCF-treated animals. Combined treatment with rcSCF and rcG-CSF after 500 cGy TBI did not result in strongly improved survival as compared with results obtained with either factor alone.

Effects of Recombinant Canine Stem Cell Factor, a c-kit Ligand, and Recombinant Granulocyte Colony-Stimulating Factor on Hematopoietic Recovery After Otherwise Lethal Total Body Irradiation

The effects of recombinant canine stem cell factor (rcSCF) on hematopoiesis were studied in normal dogs and in dogs given otherwise lethal total body irradiation (TBI) without marrow transplant. Results were compared with previous and concurrent data with recombinant granulocyte colony-stimulating factor (rG-CSF). Four normal dogs received 200 micrograms rcSCF per kilogram body weight daily either by continuous intravenous infusion for 28 days (n = 2) or by subcutaneous (SC) injection in two divided doses for 20 days (n = 2). All dogs showed at least a twofold increase in peripheral blood neutrophil counts starting approximately 7 days after the initiation of treatment. Hematocrit level and monocyte, lymphocyte, eosinophil, reticulocyte, and platelet counts were not elevated. Marrow sections after rcSCF treatment showed panhyperplasia. The only toxicity was facial edema during the first few days of rcSCF administration, presumably caused by mast cell stimulation. Ten dogs were given 400 cGy TBI at 10 cGy/min from two opposing 60Co sources. They were given no marrow infusion and received 200 micrograms/kg/d rcSCF SC in two divided doses for 21 days starting within 2 hours of TBI. Five of the 10 dogs showed complete and sustained hematopoietic recovery and survived as compared with 1 of 28 control dogs not receiving growth factor (P < .005). RcSCF treatment allowed for hematopoietic recovery in two of seven dogs administered 500 cGy of TBI but in none of five dogs given 600 cGy of TBI. Results with rcSCF are similar to those obtained with rG-CSF. The rate of neutrophil recovery in rcSCF-treated dogs after 400 cGy TBI was not different from that of rG-CSF-treated dogs (P = .65), but the rate of platelet recovery was faster (P = .06) in the rcSCF-treated animals. Combined treatment with rcSCF and rcG-CSF after 500 cGy TBI did not result in strongly improved survival as compared with results obtained with either factor alone.