Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3644-3652
Thymic Repopulation by CD34+ Human Cord Blood Cells After
Expansion in Stroma-Free Culture
Bruno Verhasselt,
Tessa Kerre,
Evelien Naessens,
Dominique Vanhecke,
Magda De Smedt,
Bart Vandekerckhove, and
Jean Plum
From the Department of Clinical Chemistry, Microbiology and
Immunology, University of Ghent, University Hospital of Ghent, Ghent,
Belgium; and the Flanders Interuniversity Institute for Biotechnology,
Ghent, Belgium (Vlaams Interuniversitair Instituut voor Biotechnologie,
VIB).
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.
