Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells

Abstract

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1+c-kit+Lin− cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G0/G1 (72% ± 3%) or S/G2/M (27% ± 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G0/G1 at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest.