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Recent studies have suggested a remarkable potential of adult stem cells from a variety of organs to give rise to cells of disparate organs, but evidence of such potential at a clonal level is lacking in most if not all studies to date. To assess directly the hematopoietic potential of muscle-derived cells in a relevant large animal, we initiated retroviral-tagging studies in the rhesus macaque to allow tracking at the clonal level by integration site analysis. Four rhesus macaques underwent transplantation with transduced muscle-derived cells after lethal irradiation followed by delayed infusion of an autologous hematopoietic graft. The first animal showed no evidence of hematopoietic recovery and, despite infusion of the backup hematopoietic graft, succumbed due to complications of prolonged cytopenias. In the remaining three animals, the overall contribution of retrovirally tagged muscle-derived cells toward hematopoiesis was exceedingly low. Retroviral integration site analysis among clonally derived muscle cells and bone marrow cells in vivo in one animal suggests a common source. These results demonstrate that harvesting disparate organs for cellular therapy is currently highly inefficient at best.

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34(+) cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34(+) cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF-mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

The ability to efficiently transduce hematopoietic stem and progenitor cells under serum-free conditions would be desirable for safety and standardization of clinical gene therapy protocols. Using rhesus macaques, we studied the transduction efficiency and engraftment ability of CD34-enriched SCF/G-CSF mobilized progenitor cells (PBSC) transduced with standard amphotropic marking vectors under serum-free and serum-containing conditions. Supernatants were collected from producer cells 16 hours after serum-free medium or medium containing 10% fetal calf serum was added. Vector titers were approximately two- to threefold higher when producer cells were cultured in serum-containing medium. However, retroviral transduction of rhesus CFU-GM was improved using serum-free vector-containing medium. For analysis of engraftment with transduced cells, three macaques had CD34+ peripheral blood stem cells split into two fractions for transduction. One fraction was transduced using serum-free vector-containing medium, and the other fraction was transduced using standard serum-containing medium. The two fractions were re-infused simultaneously following total body irradiation. In all three animals, there was equivalent marking from both vectors for 7-9 months post-transplantation. These data are encouraging regarding the removal of serum-containing medium from clinical hematopoietic cell transduction protocols, given the lack of a detrimental effect on transduction and engraftment with transduced cells.