title: Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.
abstract: Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34(+) cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remained CD34(+) - acquired significant DNA-hypermethylation. DNA-hypermethylation occurred particularly in up-stream promoter regions, shore-regions of CpG islands, binding sites for PU.1, HOXA5 and RUNX1, and it was reflected in differential gene expression and variant transcripts of DNMT3A. Low concentrations of DNAm inhibitors slightly increased the frequency of colony-forming unit initiating cells. Our results demonstrate that HPCs acquire DNA-hypermethylation at specific sites in the genome which is relevant for the rapid loss of stemness during in vitro manipulation.
authors.affiliation: Helmholtz-Institute for Biomedical Engineering, RWTH University Medical School, Aachen, Germany.
authors.forename: Carola Ingrid
authors.initials: CI
authors.lastname: Weidner
authors.forename: Thomas
authors.initials: T
authors.lastname: Walenda
authors.forename: Qiong
authors.initials: Q
authors.lastname: Lin
authors.forename: Monika Martina
authors.initials: MM
authors.lastname: Wölfler
authors.forename: Bernd
authors.initials: B
authors.lastname: Denecke
authors.forename: Ivan Gesteira
authors.initials: IG
authors.lastname: Costa
authors.forename: Martin
authors.initials: M
authors.lastname: Zenke
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wagner
mesh_terms: D018952:Antigens, CD34 / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000235:genetics; D013234:Stem Cells / Q000166:cytology*
chemical_list: D018952:Antigens, CD34; D004247:DNA