Cytokine-dependent regulation of human hematopoietic cell self-renewal and differentiation in suspension cultures
Creators: name: Zandstra, Peter William
Publisher: name: University of British Columbia
Year: 2009

description: This study investigated the processes limiting hematopoietic progenitor proliferation and differentiation in expansion cultures of human hematopoietic cells. A significant net expansion of many types of progenitors, including direct colony-forming cells (CFC) and their more primitive precursors (long-term culture-initiating cells, LTC-IC) could be reproducibly obtained in cytokine-supplemented, stirred suspension cultures. Due to its homogeneous nature, this culture system represents a simple and effective way to approach clinical scale hematopoietic bioreactor design. Factorial design experiments, undertaken to optimize cytokine combinations for the expansion of primitive cell populations, showed that Flt-3 Ligand (FL) was the only factor that, alone, increased marrow-derived LTC-IC above input values. The effects of FL, Steel Factor (SF) and Interleukin-3 (IL-3) on expansion of these LTC-IC were shown to be additive and greater than or equal to those obtained in the presence of combinations of 13 other factors. A dose response analysis undertaken to define the cytokine concentration requirements for marrow LTC-IC self-renewal revealed that the greatest LTC-IC amplification (62-fold) was obtained in cultures containing significantly elevated levels of cytokines (FL and SF at 300 ng/mL plus IL-3 at 60 ng/mL). Subsequent experiments showed that the LTC-IC expansion was primarily and significantly dependent on a high concentration of FL and, as the frequency of clonal growth from individual cells did not change with cytokine concentration, the decrease in LTC-IC expansion observed at lower cytokine concentrations could be attributed to differentiation rather than selective death. Analysis of the average cell specific cytokine depletion rates from both stirred suspension and purified cell cultures revealed proportional increases in these rates at increased cytokine concentrations. In similar investigations analyzing the responses of specific phenotypically defined cell populations, the greatest factor depletion rates were observed in cultures of the most primitive cells. Examination of the kinetics of SF receptor (c-kii) internalization in response to SF stimulation showed that concentration-dependent SF depletion and c-kit internalization were closely associated, suggesting that the extent and/or duration of cytokine signaling may be an important factor in determining cellular responses. Taken together, these results demonstrate the ability to expand hematopoietic cells in a scaleable stirred suspension culture system. Cytokine type, concentration and depletion rates were shown to be parameters important for the development of bioreactor technology. The observations that the hematopoietic cell types whose maximum proliferation in vitro depends on the highest concentration of cytokines also exhibit the greatest capacity to deplete the same cytokines from the medium may explain why the identification of conditions that support the expansion of hematopoietic stem cells has been so elusive. Additionally, these results provide evidence that extrinsically acting cytokines can alter the self-renewal behavior of primary human hematopoietic cells independent of effects on their viability and proliferation.