Purified canine CD34(+)Lin(-) marrow cells transduced with retroviral vectors give rise to long-term multi-lineage hematopoiesis

Human CD34(+) cells have been shown to retain long-term hematopoietic engra fting potential in preclinical and clinical studies. However, recent studie s of human and murine CD34(-) stem cells suggest that these are functionall y important early progenitors. Using autologous transplantation, we investi gated whether canine CD34(+) and CD34(-) marrow cells could be transduced a nd give rise to long-term hematopoiesis. CD34(+)Lin(-) and CD34(-)Lin(-) ce ll populations purified by fluorescence-activated cell sorting were separat ely cocultivatcd with retroviral vectors LN (CD34(+)Lin(-)) and LNY (CD34(- )Lin(-)), which carry the neomycin (neo) gene. After myeloablative total bo dy irradiation (920 cGy), 3 dogs received transplants of both CD34(+)Lin(-) cells and CD34(-)Lin(-) cells and 2 dogs received only CD34(-)Lin(-) cells . Untransduced autologous marrow cells were given to ensure hematopoietic r ecovery. Using CFU-C assays, transduction efficiencies of CD34(+)Lin(-) cel ls ranged from 6% to 18% with no CFU-C formation from CD34(-)Lin(-) cells. PCR-based detection of the neo gene from WBCs was used to detect transduced cells weekly after transplantation. Additional PCR studies in 3 dogs given both CD34(+)Lin(-) and CD34(-)Lin(-) cells were performed on monocytes, gr anulocytes, and T cells (2 dogs, one at 7.5 months and the other at 9 month s) and granulocytes (I dog at 12 months). LN was detected up to 12 months p osttransplantation in WBCs and mono-myeloid and lymphoid populations from 3 dogs receiving transplants of transduced CD34(+)Lin(-) cells. LINN was not detected at any time after transplantation in 5 dogs that received transdu ced CD34(-)Lin(-) cells. Whereas canine CD34(+)Lin(-) marrow cells contribu ted to long-term multilineage hematopoiesis, progeny of CD34(-)Lin(-) proge nitor cells were not detected after transplantation in these experiments.