The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction.

Objective: Ex vivo expansion of primitive hematopoietic cells remains of interest for gene therapy and transplantation. Previous studies reported loss of repopulating activity following culture of cells for more than 4-7 days in the presence of cytokines or stromal cells. In the current study, we investigated whether prolonged culture and transduction in the presence of the carboxy-terminal portion of fibronectin (FN) could maintain or expand retrovirally transduced repopulating hematopoietic stem cells (HSCs).

Effect of chronic cytokine therapy on clonal dynamics in nonhuman primates.

Hematopoietic cytokines such as filgrastim are used extensively to stimulate granulocyte production or to mobilize hematopoietic progenitors into the circulation; however, their effect on more primitive hematopoietic progenitor and stem cells in vivo is unknown, particularly in large animals or humans. In particular, there is concern that chronic therapy with cytokines could result in stem cell exhaustion or clonal dominance; however, direct assessment of the dynamics of individual stem and progenitor cell clones in vivo has not been previously reported. A number of models can be proposed regarding the mechanisms by which the marrow responds to cytokine stimulation, including recruitment of previously quiescent clones, stimulation of proliferation of already active clones, or prevention of apoptosis of more mature progenitors from all clones.

Retrovirally transduced muscle-derived cells contribute to hematopoiesis at very low levels in the nonhuman primate model.

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.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector.

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice.

Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates.

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.

Retroviral transduction and engraftment ability of primate hematopoietic progenitor and stem cells transduced under serum-free versus serum-containing conditions.

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.