STABLE INTEGRATION OF RETROVIRALLY TRANSDUCED GENES INTO HUMAN UMBILICAL-CORD BLOOD HIGH-PROLIFERATIVE POTENTIAL COLONY-FORMING CELLS (HPP-CFC) AS ASSESSED AFTER MULTIPLE HPP-CFC COLONY REPLATINGS IN-VITRO

We previously demonstrated stable integration of a transduced thymidin e kinase (TK)-neo gene into immature and replatable stem and progenito r cells, as assessed by the presence of the gene in second-generation colonies, To evaluate whether this integration was still present in th ird- and fourth-generation colonies, nonadherent low-density T-lymphoc yte-depleted (NALT(-)) cells from human umbilical cord blood were pres timulated with recombinant human (rhu) erythropoietin (Epo), steel fac tor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-st imulating factor (CSF), and granulocyte (G)-CSF, Prestimulated NALT(-) cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony for mation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introd uced into hematopoietic progenitor cells without stromal cells as a so urce of virus, As previously reported, proviral integration was detect ed in primary G418(R)-colonies, and in second-generation replated colo nies derived from G418(R) granulocyte erythroid macrophage megakaryocy te colony-forming units and high-proliferative potential colony-formin g cells (HPP-CFCs), Moreover, we now document that proviral integratio n was apparent in cells from colonies derived from third- and fourth-g eneration replated HPP-CFC, suggesting a high degree of stable integra tion of the transduced gene.