title: [Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria].
abstract: OBJECTIVE
  To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH.
  
  
  METHODS
  CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted.
  
  
  RESULTS
  The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01).
  
  
  CONCLUSIONS
  The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.
authors: Department of Pediatrics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. xiaoj82108@aliyun.com.
authors: Juan
authors: J
authors: Xiao
authors: Yong-Ji
authors: YJ
authors: Wu
authors: Bing
authors: B
authors: Han
authors: Hong-Yan
authors: HY
authors: Dong
authors: Shi-Ping
authors: SP
authors: Chen
mesh_terms: D000293:Adolescent; D018952:Antigens, CD34 / Q000032:analysis*; D001854:Bone Marrow Cells / Q000166:cytology*; D016026:Bone Marrow Transplantation; D018957:CD59 Antigens / Q000032:analysis*; D002469:Cell Separation; D002648:Child; D005260:Female; D006410:Hematopoiesis; D006457:Hemoglobinuria, Paroxysmal / Q000628:therapy*; D006801:Humans; D008297:Male
chemical_list: D018952:Antigens, CD34; D018957:CD59 Antigens