Following the initiation of conditioning as noted above, the CD34 cells were thawed and transduced. Cells were plated at 105 cells/cm2 in VEGFR signaling pathway nontissue culture treated 25 cm2 flasks that had been coated with 4 g/cm2 of the RetroNectin. Prestimulation was then performed overnight in X Vivo 15 serum free medium supplemented with 2 mmol/l L glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin, and containing 100 ng/ml each of the recombinant human cytokines: thrombopoietin, Flt3 ligand, and stem cell factor. The next morning, the cells were resuspended in fresh cytokine containing medium and transduced in separate HSP portions with the SIV NoN and SIV GFP vector supernatants at final vector concentrations of 8 × 107 transducing unit/ml, with protamine sulfate at 4 g/ml. After 2 hours, the medium volume was doubled and cells were incubated at 37 with 5% CO2 for 6 8 hours. A second aliquot of each SIV vector supernatant was added to the cells and incubated overnight.
The next morning, aromatase the cells were washed, then resuspended in PBS with 1% autologous serum. Cell counts and viability were determined using trypan blue exclusion prior to each transplant. Small aliquots of cells were used for methylcellulose CFU assays, qPCR analysis, and flow cytometry for GFP expression. Autologous cells were injected i.v. into each animal in an 1 ml volume, 48 hours post busulfan administration as noted above. Post transplant sample collection and analysis. Blood samples were collected for complete blood counts, serum chemistry panels, PBMCs, immunophenotyping, plasma, serum, and indirect ELISA as previously described.37 Blood and bone marrow were collected at monthly intervals beginning at 1 month post transplantation. Mononuclear cells and granulocytes from peripheral blood samples were isolated by density gradient abiraterone centrifugation over Histopaque at 400g for 30 minutes at 25. Hematopoietic CFU assays were performed on mononuclear cells, as previously described.38,40 Briefly, mononuclear cells were resuspended in RPMI and washed. A total of 5 × 104 cells/plate from peripheral blood and 2 × 104 cells/plate from bone marrow were plated in 1 ml MethoCult GF H4435 containing human Epo, G CSF, GM CSF, stem cell factor, interleukin 3, and interleukin 6 in fibroblast free 35 mm culture plates. After a standardized 10 day incubation period, erythroid and myeloid progenitor colonies were counted.
Individual hematopoietic colonies were collected and then lysed and genomic DNA isolated using the QIAamp DNA blood kit, as recommended by the manufacturer for qPCR. Animals were euthanized by an overdose of pentobarbital and tissue harvests were performed according to established protocols.37 Blood and marrow was collected, and all tissues were collected and fixed in 10% buffered formalin then embedded, sectioned, and stained with hematoxylin and eosin for routine histopathology. Specimens were also placed in cryotubes and quick frozen over liquid nitrogen for qPCR. Immunizations. Group 3 animals were immunized with clinical grade tetanus toxoid vaccine before transplantation and at 1 and 2 months postnatal age. Animals were reimmunized with tetanus at 6 months post transplant, as well as with a single dose of recombinant hepatitis B vaccine as a neo antigen.