title: mRNA expression and promoter DNA methylation status of CDKi p21 and p57 genes in ex vivo expanded CD34 (+) cells following co-culture with mesenchymal stromal cells and growth factors.
abstract: BACKGROUND
  Several studies indicate that ex vivo cytokine-based expansion of cord blood (CB) CD34(+) cells can induce cell cycle abnormality in expanded cells. Cycline-dependent kinase inhibitors, p21 and p57, are critical regulators of cell cycle. We investigated  mRNA expression and promoter DNA methylation status of p21 and p57 genes in ex vivo expanded CD34(+) cells in different culture conditions.
  
  
  METHODS
  Human CB CD34(+) cells were cultured either in cytokine-supplemented liquid culture  or in co-culture with mesenchymal stromal cells (co-culture system) in presence and absence of cytokine. After 14 days, expansion fold in total nucleated cells (TNCs), CD34(+) cells, CD34(+)/CD38(-) cells and colony-forming units in culture (CFU-C) count was  determined. Moreover, mRNA expression level and promoter DNA methylation status of p21 and p57 genes in expanded CD34(+) cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR) and methylation-specific PCR technique, respectively.
  
  
  RESULTS
  Maximum expansion of TNC, CD34(+) cells, CD34(+)/CD38(-) cells and CFU-C was seen at day 10 in all three culture conditions. p21 and p57 mRNA expression level decreased in both cytokine liquid culture and co-culture system with cytokine, while in the co-culture  system without cytokine, the mRNA expression of p21 and p57 genes relatively increased. In addition, promoter DNA methylation status of p21 and p57 genes did not change during expansion in all three culture conditions.
  
  
  CONCLUSIONS
  Co-culture of CB CD34(+) cells  with mesenchymal stromal cells, especially in the absence of cytokines, maintained mRNA expression of p21 and p57 genes in ex vivo expanded CD34(+) cells. Moreover, transcriptional repression of p21 and p57 genes in ex vivo expanded CD34(+) cells was not mediated  by promoter DNA methylation.
authors.affiliation: Department of Hematology and Blood Banking, School of Allied Medical Sciences, Tehran University of Medical Science, Tehran, Iran.
authors.forename: Mohammad Soleiman
authors.initials: MS
authors.lastname: Soltanpour
authors.forename: Naser
authors.initials: N
authors.lastname: Amirizadeh
authors.forename: Farhad
authors.initials: F
authors.lastname: Zaker
authors.forename: Arezoo
authors.initials: A
authors.lastname: Oodi
authors.forename: Mahin
authors.initials: M
authors.lastname: Nikougoftar
authors.forename: Ahmad
authors.initials: A
authors.lastname: Kazemi
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis* / Q000378:metabolism; D018920:Coculture Techniques; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000235:genetics*; D019175:DNA Methylation; D005312:Fetal Blood / Q000166:cytology / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018952:Antigens, CD34; C493956:CDKN1A protein, human; C493964:CDKN1C protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger