title: Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method.
abstract: OBJECTIVE
  The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells.
  
  
  METHODS
  Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry.
  
  
  RESULTS
  A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB.
  
  
  CONCLUSIONS
  The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.
authors.affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Kuljeet
authors.initials: K
authors.lastname: Singh
authors.forename: Ankita
authors.initials: A
authors.lastname: Srivastava
authors.forename: Nitin
authors.initials: N
authors.lastname: Mathur
authors.forename: Sunesh
authors.initials: S
authors.lastname: Kumar
authors.forename: Lalit
authors.initials: L
authors.lastname: Kumar
authors.forename: Asok
authors.initials: A
authors.lastname: Mukhopadhyay
authors.forename: Vinod
authors.initials: V
authors.lastname: Konchupillai
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology*; D006023:Glycoproteins / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D010455:Peptides / Q000378:metabolism
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human