FasL is a major physiologic developmental regulator, especially in the immune system, as elucidated by over-abundant lymphopoiesis and autoimmune disorders in mice and humans with inactivating mutations in Fas pathway genes. In addition. Fas has been implicated in the pathogenesis of aplastic anemia[Maciejewski, Br J Haematol 1995]. Since lymphoid cells develop from CD34+ stem/progenitor cells, and since CD34+ cells are deficient in aplastic anemia, the Fas pathway has been studied in normal CD34+ cells. Recent reports[Josefsen, Exp Hematol 1999;Barcena, Exp Hematol 1999] concluded that FasL may stimulate, rather than inhibit, human progenitor cell growth. As a preliminary to investigation of sensitivity of in vivo engrafting cells in normal and aplastic anemia patient bone marrow, we treated human CB CD34+ cells with FasL and then evaluated the cells by viable cell counts. Annexin V-binding/DNA binding assays, and in vitro colonyforming cell(CFC) assays. Cells were pre-cultured in serum-free medium for 5-48 hrs, with or without hematopoietic growth factors(Flt-3 ligand[F], kit ligand[K] and thrombopoietin[T]), and with or without soluble recombinant human FasL. Treatment of the control Jurkat human T leukemia cell line with FasL under these conditions resulted in highly efficient apoptosis, which could be almost completely inhibited by z-VAD-FMK (a caspase inhibitory peptide). The table shows results of 1 of 4 similar experiments with CB CD34+ cells. FasL treatment had no significant effect on CB CD34+ cell viability or progenitor cell development, even with prolonged treatment at high FasL doses. Indeed, confirming the prior results, FasL treatment resulted in no detectable apoptosis of CB CD34+ cells and increased numbers of CFC, by an unknown mechanism(s). FKT FasL Total CFC 1 (control) + 0 1014±156 2 + 1 ng/ml 2184±273 3 + 10 ng/ml 2494±344 4(control) - 0 117±13 5 - 1 ng/ml 462±70 6 - 10 ng/ml 207±36.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology