Early-acting cytokine-driven ex vivo expansion of mobilized peripheral blood CD34(+) cells generates post-mitotic offspring with preserved engraftment ability in non-obese diabetic/severe combined immunodeficient mice

The ability of ex-vivo expanded peripheral blood stem cells (PBSC) to engra ft non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice has n ot been evaluated to date. We investigated the maintenance of primitive SCI D-repopulating cells (SRC) and long-term culture-initiating cells (LTCIC) i n PBSC expanded with early-acting cytokines, thrombopoietin (TPO), stem cel l factor (SCF) and FlT3-ligand (FL) with or without interleukin 3 (IL-3) an d IL-6 in short-term (6 d) stroma-free serum-free cultures. TPO + SCF + FL and TPO + SCF + FL + IL-3 + IL-6 produced 5.9 +/- 1.97 and 18.25 +/- 4.49 ( mean +/- SEM)-fold increase of CD34(+) cells respectively. We tracked cellu lar division with PKH26 and sorted postmitotic CD34(+) PKH26(low) cells to assess their primitive functional properties. After culture with TPO + SCF + FL, LTCICs among post-mitotic cells increased 12.08 +/- 3.4 times, and 4. 3 +/- 1.6 times when IL-3 + IL-6 were added. CD34(+) PKH26(low) cells cultu red with TPO + SCF + FL provided human multilineage (CD34, CD33 and CD19) e ngraftment in NOD/SCID mice, whereas no human cells were detected in mice i njected with cells cultured with TPO + SCF + FL + IL-3 + IL-6. Percentages of CD34(+)/ CD38(-), CD34(+)/CD33(-), CD34(+)/DR- and cells in G(0)/G(1) ph ase were similar among cells cultured with both cytokine combinations, indi cating that the deleterious impact of IL, 3 + IL-6 on the ability to engraf t is not translated into phenotypic or cycling features. In conclusion, TPO + SCF + FL-expanded PBSC maintain multilineage engraftment ability in NOD/ SCID mice, which is abrogated by the addition of IL-3 + IL-6.