title: Heterogeneity of in vitro-cultured CD34+ cells isolated from peripheral blood.
abstract: OBJECTIVE
  For transplantation, hematopoietic stem cells (HSC) are obtained from bone marrow, cord blood and mobilized adult peripheral blood. HSCs are present in the blood of healthy adults and can be recovered in leuko-reduction system chambers, with a potential yield of 1 to 4 × 10(6) CD34+ cells per unit. Some groups have investigated this valuable source of stem cells; however, investigations are still needed to support their use.
  
  
  METHODS
  CD34+ cells were purified from leuko-reduction system chambers and cultured with a defined custom medium without animal protein and supplemented with interleukin-3, interleukin-6, Fms-like tyrosine kinase 3, stem cell factor and thrombopoietin. Cells were cultured under 8% and 21% oxygen levels. With the use of multiparametric flow cytometry analysis, the phenotypes of emerging populations were compared between oxygen levels and resting CD34+ cells. Both conventional gating and clustering analysis were used to visualize the cellular outcome.
  
  
  RESULTS
  A maximum expansion of 20-fold was obtained without major differences in viability, number of cells or cellular heterogeneity between atmospheric and physiologic oxygen conditions. Worthy of note, phenotype analysis revealed that megakaryocyte and erythrocyte progenitors were favored, albeit more moderately when submitted to 8% O2.
  
  
  CONCLUSIONS
  This study suggests that the bias of cultured blood CD34+ cells toward megakaryocyte and erythrocyte progenitor cells can be reduced by use of 8% pO2. It also shows how clustering software, such as SPADE, can help visualize the complexity of stem cell differentiation.
authors.affiliation: Department of Biochemistry, Microbiology and Bio-Informatics, Laval University, Québec, Canada; Héma-Quebec Department of Research and Development, Québec, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Jobin
authors.affiliation: Héma-Quebec Department of Research and Development, Québec, Canada.
authors.forename: Marc
authors.initials: M
authors.lastname: Cloutier
authors.affiliation: Héma-Quebec Department of Research and Development, Québec, Canada.
authors.forename: Carl
authors.initials: C
authors.lastname: Simard
authors.affiliation: Department of Biochemistry, Microbiology and Bio-Informatics, Laval University, Québec, Canada; Héma-Quebec Department of Research and Development, Québec, Canada. Electronic address: sonia.neron@hema-quebec.qc.ca.
authors.forename: Sonia
authors.initials: S
authors.lastname: Néron
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000276:immunology*; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004912:Erythrocytes / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008533:Megakaryocytes / Q000166:cytology*; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D051941:fms-Like Tyrosine Kinase 3 / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; C508596:IL3 protein, human; C508600:IL6 protein, human; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D013926:Thrombopoietin; D051941:fms-Like Tyrosine Kinase 3
keywords: flow cytometry; heterogeneity; peripheral blood; phenotype; progenitor cells; stem cells