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

Abstract

The ability of ex-vivo expanded peripheral blood stem cells (PBSC) to engraft non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice has not been evaluated to date. We investigated the maintenance of primitive SCID-repopulating cells (SRC) and long-term culture-initiating cells (LTCIC) in PBSC expanded with early-acting cytokines, thrombopoietin (TPO), stem cell factor (SCF) and FlT3-ligand (FL) with or without interleukin 3 (IL-3) and 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 cellular division with PKH26 and sorted post-mitotic CD34+ PKH26low 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+ PKH26low cells cultured with TPO+SCF+FL provided human multilineage (CD34, CD33 and CD19) engraftment in NOD/SCID mice, whereas no human cells were detected in mice injected with cells cultured with TPO+SCF+FL+IL-3+IL-6. Percentages of CD34+/CD38-, CD34+/CD33-, CD34+/DR- and cells in G0/G1 phase were similar among cells cultured with both cytokine combinations, indicating that the deleterious impact of IL-3+IL-6 on the ability to engraft 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.