title: Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia.
abstract: OBJECTIVE
  To investigate further the cellular defect responsible for impaired granulopoiesis in severe congenital neutropenia (SCN), we have evaluated bone marrow (BM) stem cell reserve and function and BM stromal cell myelopoiesis supporting capacity in two patients with SCN.
  
  
  METHODS
  BM primitive stem cells and myeloid progenitor cells were assessed using flow cytometry, limiting dilution assay, clonogenic assays, and long-term BM cultures (LTBMC). BM stroma function was assessed by evaluating the ability of irradiated stromal layers from the patients to induce granulocyte-macrophage colony formation (CFU-GM) by normal CD34+ cells.
  
  
  RESULTS
  Compared to the normal controls (n = 37), SCN patients displayed a low percentage of CD34+/CD38+ cells (P < 0.05), low CFU-GM colony formation by highly purified CD34+ cells (P < 0.05), low CFU-GM recovery in LTBMC (P < 0.05), and normal primitive stem cells as indicated by the frequency of CD34+/CD38- cells and the number of long-term culture initiating cells. Patient BM stromal layers exhibited normal myelopoiesis supporting capacity as shown by the CFU-GM content of irradiated LTBMC recharged with normal CD34+ cells. In addition, patient LTBMC supernatants displayed 20-fold normal granulocyte colony stimulating factor and 2-fold normal granulocyte-macrophage colony stimulating factor levels.
  
  
  CONCLUSIONS
  These data show that primitive BM stem cells and stromal cells are not affected in SCN patients, while they support further the concept of a primary defect at the myeloid progenitor cell level. To know the differentiation stage at which the underlying defect causes the malfunction will be relevant for further elucidation of its nature at the molecular level.
authors.affiliation: Department of Haematology of the University Hospital of Crete School of Medicine, Heraklion, Greece. epapadak@med.uoc.gr
authors.forename: H A
authors.initials: HA
authors.lastname: Papadaki
authors.forename: F M
authors.initials: FM
authors.lastname: Gibson
authors.forename: M
authors.initials: M
authors.lastname: Psyllaki
authors.forename: E C
authors.initials: EC
authors.lastname: Gordon-Smith
authors.forename: J C
authors.initials: JC
authors.lastname: Marsh
authors.forename: G D
authors.initials: GD
authors.lastname: Eliopoulos
mesh_terms: D000328:Adult; D001853:Bone Marrow / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D006801:Humans; D008297:Male; D009503:Neutropenia / Q000151:congenital* / Q000473:pathology; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology*
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D016298:Hematopoietic Cell Growth Factors