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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Stem cell defect in aplastic anemia: reduced long term culture-initiating cells (LTC-IC) in CD34+ cells isolated from aplastic anemia patient bone marrow.

Aplastic anemia is associated with quantitative and functional abnormalities in the hematopoietic stem cell compartment. Currently, one of the most primitive human hematopoietic progenitor cells that can be functionally assayed in vitro is the long-term culture-initiating cell (LTC-IC). This assay identifies primitive cells that are capable of producing colonies after five weeks of long-term culture using a limiting dilution method. Previous investigators have demonstrated a significant reduction in the frequency of LTC-IC in bone marrow mononuclear cells (BMMC) isolated from aplastic anemia patients when compared to normal donors. However, immunosuppression of hematopoiesis is a prevalent feature of aplastic anemia. Therefore, we assayed the frequency of LTC-IC in cells expressing the CD34 antigen isolated from bone marrow, a population highly enriched for LTC-IC, of both aplastic anemia patients that had received immunosuppressive therapies (IST, n=13) and normal donors (n=28), thereby minimizing continued immunosuppression by T-cells and regulation by other autologous CD34- cells. We describe a significant reduction of LTC-IC frequency in purified CD34+ populations from aplastic patients (P>0.0001), thus demonstrating a functional difference between this phenotypically defined cell population from patients and normal donors.