Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for success ful gene therapy of diseases affecting the T-cell lineage. However, the T-c ell progenitor potential of HPC, cultured in vitro for cell number expansio n and gene transfer remains largely unknown. Here, we cultured highly purif ied human umbilical cord blood (CB) CD34(+) CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combin ations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/ flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-r epopulating ability of expanded cells in vitro and in vivo. After up to 5 w eeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural kille r cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of cultur e, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragmen t transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) gen erated thymocytes expressing the retroviral encoded marker gene GFP in vivo . Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultur ed for extensive periods, harbor thymus-repopulating cells that retain T-ce ll progenitor potential after expansion and gene transfer. (C) 1999 by The American Society of Hematology.