Kim Heeje, Whartenby Katharine A, Georgantas Robert W, Wingard John, Civin Curt I
Department of Oncology and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Stem Cells. 2002;20(2):174-82. doi: 10.1634/stemcells.20-2-174.
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.
我们试图确定来自人胎盘/脐带血（CB）或成人动员血（PBSC）的淋巴造血干祖细胞（HSC）是否对Fas诱导的凋亡敏感。将来自CB或PBSC的人CD34+细胞在无血清培养基中培养，添加或不添加造血生长因子（FKT：FLT-3配体[FL]、KIT配体[KL]和血小板生成素[TPO]），添加或不添加可溶性Fas配体（sFasL）或激动性抗Fas抗体。培养5-48小时后，评估细胞活力，并用膜联蛋白V和7-氨基放线菌素D染色，通过荧光激活细胞分选进行凋亡分析。还通过体外造血集落形成细胞（CFC）和体内非肥胖糖尿病/严重联合免疫缺陷小鼠植入潜力（SEP）试验评估培养的细胞。通过实时定量聚合酶链反应测定CD34+细胞中Fas、FLICE抑制蛋白（FLIP）和半胱天冬酶8 mRNA的水平。通过蛋白质印迹法确认FLIP的表达。在存在sFasL或激动性抗Fas抗体的情况下培养的CB或PBSC CD34+细胞中，未观察到活力、CFC或SEP的降低。人CB和动员的PBSC CD34+细胞表达高水平的FLIP、低比例的半胱天冬酶8:FLIP和低水平的Fas。因此，人CB和PBSC CD34+ HSC对Fas途径激动剂具有抗性。FLIP的高水平表达可能为CD34+细胞提供了一层免受Fas介导凋亡的保护。
