Quiescent (5-fluorouracil-resistant) aplastic anemia hematopoietic cells in vitro

Bone marrow from aplastic anemia (AA) patients shows reduced numbers in long-term culture (LTC)-initiating cell (LTC-IC) assays. The LTC-IC assay is based on assumptions of the culture kinetics of normal hematopoietic stem cells (HSC), which are not necessarily justified in a disease state. We therefore undertook a detailed examination of the kinetics of quiescent HSC from AA patients in LTC. Colony formation by quiescent HSC in LTC was tested by pretreating control (n = 6) and AA bone marrow (n = 7) with 5-fluorouracil. Secondly, we manipulated normal samples to inoculate cultures with proportions of CD34 + cells similar to those from AA samples. We obtained enough CD34 + cells to reconstitute one AA sample to “normal” levels. Patient cells showed altered kinetics with rapid proliferation and premature termination of LTC. In vivo, decreased numbers of HSC may induce rapid proliferation and differentiation; a similar phenomenon could explain the observations in culture. We therefore manipulated normal samples to contain a proportion of CD34 + HSC similar to that in AA samples. Although absolute numbers of secondary colonies in LTC were reduced, the kinetics of culture were not altered. However, when AA CD34 + HSC were reconstituted to “normal” levels, the cultures still demonstrated early termination. The kinetics of LTC are not affected by CD34 + HSC number. However, quiescent HSC derived from patients with AA have qualitative differences from normal cells, as reflected by distinct kinetics in long-term culture. This has implications for the interpretation of the LTC-IC assay with AA samples.