Ex vivo expansion of canine dendritic cells from CD34+ bone marrow progenitor cells.

BACKGROUND: The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. METHODS: CD34+-selected cells and CD34+-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-alpha 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. RESULTS: Two main cell populations were identified, DR++(bright)/CD14- and DR+(dim)/CD14+. Ex vivo expanded CD34+-selected cells showed increased allostimulatory activity compared to both cultured CD34+-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34+-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34+-selected bone marrow to enrich for DR++/CD14- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR+/CD14+ cells (P=0.02). CONCLUSIONS: Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34+-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.

Polyclonal hematopoiesis with variable telomere shortening in human long-term allogeneic marrow graft recipients.

Donor-derived hematopoiesis was assessed in 17 patients who received allogeneic marrow grafts from HLA-matched siblings between 1971 and 1980. Complete blood counts were normal or near normal in all patients except one. Chimerism analyses, using either dual-color XY-chromosome fluorescence in situ hybridization (FISH) or analysis of variable number tandem repeat loci, indicated that 15 out of 16 patients had greater than 97% donor-derived hematopoiesis, whereas 1 patient had indeterminate chimerism. All 12 recipients of grafts from female donors exhibited polyclonal hematopoiesis by X-linked clonal analysis with the use of molecular probes. Of the 17 recipients, 9 exhibited a less than 1.0-kilobase shortening of granulocyte telomere length compared with their respective donors, according to terminal restriction fragment analysis or flow-FISH with a fluorescein-labeled peptide nucleic acid probe. These data suggest that under standard transplantation conditions, the stem cell proliferative potential is not compromised during hematopoietic reconstitution. (Blood. 2000;96:3991-3994)

CD34+ selected bone marrow grafts are radioprotective and establish mixed chimerism in dogs given high dose total body irradiation.

BACKGROUND: Canine stem cell transplantation models have provided important preclinical information for human clinical studies. The recent cloning of cDNA for canine CD34 and the production of monoclonal antibodies that recognize canine CD34 have been the basis for the development of techniques for the large-scale enrichment of canine hematopoietic progenitor cells. In this study, we evaluated the in vivo functional properties of canine bone marrow CD34+ cells after a myeloablative conditioning regimen. METHODS: After 920 cGy total body irradiation, three dogs received infusion of autologous CD34+ selected cells from the marrow, three dogs CD34+ depleted autologous marrow cells, and two dogs received CD34+ autologous marrow cells that were immunomagnetically selected and then further purified by cell sorting. In addition, four dogs received allogeneic marrow enriched for CD34+ cells from dog leukocyte antigen-identical littermates to investigate long-term repopulating function of CD34+ cells. Chimerism studies were performed using polymerase chain reaction to detect highly polymorphic microsatellite markers. RESULTS: In three recipients of autologous marrow enriched for CD34+ cells to between 29% and 70% (1.6 x 10(6) to 3.4x10(6) CD34+ cells/kg), prompt and full hematopoietic recovery occurred, whereas in three dogs that received marrow depleted of CD34+ cells (1 x 10(7) cells/kg), no hematopoietic recovery was achieved. In two dogs that received highly purified CD34+ cells (purity: 98% and 96%, 0.79x10(6) to 0.547x 10(6) CD34+ cells/kg), delayed but full hematopoietic recovery was seen. Three of four allograft recipients of 1.75x10(6) to 6.8x10(6) CD34+ cells/kg engrafted and showed full hematopoietic recovery, whereas one dog rejected the graft. The three long-term survivors showed stable mixed hematopoietic chimerism with predominantly donor hematopoiesis. CONCLUSION: Transplantation of canine CD34+ cells after lethal total body irradiation provides radioprotection and gives rise to long-term hematopoietic reconstitution. Stable donor/host mixed chimerism was observed in allograft recipients most likely as a result of T-cell depletion of the grafts. Our findings suggest a future role for canine preclinical transplant studies involving in vitro manipulation of hematopoietic pro.