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 successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks 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 killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.
Verhasselt
B
B
Department of Clinical Chemistry, Microbiology and Immunology, University of Ghent, University Hospital of Ghent, Ghent, Belgium. Bruno.Verhasselt@rug.ac.be
Kerre
T
T
Naessens
E
E
Vanhecke
D
D
De Smedt
M
M
Vandekerckhove
B
B
Plum
J
J
D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D019070:Cell Lineage / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology*; D013926:Thrombopoietin / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology*
D016207:Cytokines; D019089:Stem Cell Factor; D013926:Thrombopoietin