Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis.
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.
Kim
Heeje
H
Department of Oncology and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Whartenby
Katharine A
KA
Georgantas
Robert W
RW
Wingard
John
J
Civin
Curt I
CI
D000818:Animals; D000906:Antibodies; D018952:Antigens, CD34 / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D053222:Fas Ligand Protein; D005260:Female; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D010920:Placenta / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D011247:Pregnancy; D019014:fas Receptor / Q000187:drug effects / Q000378:metabolism*
D000906:Antibodies; D018952:Antigens, CD34; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; C506387:Cflar protein, mouse; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases