TY - JOUR
T1 - Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to fas-mediated apoptosis
AU - Kim, Heeje
AU - Whartenby, Katharine A.
AU - Georgantas, Robert W.
AU - Wingard, John
AU - Civin, Curt I.
PY - 2002
Y1 - 2002
N2 - We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.
AB - We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.
KW - Apoptosis
KW - CD34
KW - Caspase
KW - FLIP
KW - Fas
KW - Progenitor cells
KW - Stem cells
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UR - http://www.scopus.com/inward/citedby.url?scp=0036220563&partnerID=8YFLogxK
U2 - 10.1634/stemcells.20-2-174
DO - 10.1634/stemcells.20-2-174
M3 - Article
C2 - 11897874
AN - SCOPUS:0036220563
VL - 20
SP - 174
EP - 182
JO - Stem Cells
JF - Stem Cells
SN - 1066-5099
IS - 2
ER -