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.

In vitro effects of interferon-gamma and tumor necrosis factor-alpha on CD34+ bone marrow progenitor cells from aplastic anemia patients and normal donors.

Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.