Expansion of Mobilized Peripheral Blood Progenitor Cells under Defined Culture Conditions Using CD34 + CD71 - CD45 - Cells as a Starting Population

Abstract

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions supporting the expansion of transplantable hematopoietic stem cells. We assessed the expansion potential of CD34⁺ CD71^- CD45^- cells derived from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood under recently defined serum-free culture conditions. The CD34⁺ CD71^- CD45^- cells in mobilized peripheral blood were found to contain the majority (92% ± 5.6) of primitive long-term culture initiating cells (LTCIC) and 53.5%± 16.7 of the more committed colony-forming cells (CFC). Furthermore, this population represents 23.3% ± 4.1 of the total CD34⁺ cells and allows reduction of the cell density important for maintenance/expansion of primitive progenitor cells. CD34⁺ CD71^- CD45^- cells were cultured in defined serum-free media supplemented with 300 ng each of Flt-3 ligand and stem cell factor (SCF), 60 ng of interleukin (IL)-3, and 20 ng each of IL-6 and G-CSF. Mononuclear cells (MNC) and CFC were expanded 50-fold and 200-fold, respectively; primitive progenitor cells (LTC-IC) were maintained at input values after a total of 10 days of expansion. The addition of IL-15 to our cytokine cocktail expanded LTC-IC 2- to 3-fold and CFC to >500-fold. The data presented should allow clinical manipulation (purging) and expansion procedures with mobilized PBPC harvests without the loss of primitive progenitor cells and could be made applicable for large-scale clinical expansion.