title: Proof of principle: quality control of therapeutic cell preparations using senescence-associated DNA-methylation changes.
abstract: BACKGROUND
  Tracking of replicative senescence is of fundamental relevance in cellular therapy. Cell preparations - such as mesenchymal stromal cells (MSCs) - undergo continuous changes during culture expansion, which is reflected by impaired proliferation and loss of differentiation potential. This process is associated with epigenetic modifications: during in vitro culture, cells acquire senescence-associated DNA methylation (SA-DNAm) changes at specific sites in the genome. We have recently described an Epigenetic-Senescence-Signature that facilitates prediction of the state of cellular aging by analysis of DNAm at six CpG sites (associated with the genes GRM7, CASR, PRAMEF2, SELP, CASP14 and KRTAP13-3), but this has not yet been proven over subsequent passages and with MSCs isolated under good manufacturing practice (GMP) conditions.
  
  
  RESULTS
  MSCs were isolated from human bone marrow and GMP-conform expanded for up to 11 passages. Cumulative population doublings (cPDs) and long-term growth curves were calculated based on cell numbers at each passage. Furthermore, 32 cryopreserved aliquots of these cell preparations were retrospectively analyzed using our Epigenetic-Senescence-Signature: DNAm-level was analyzed at six specific CpGs, and the results were used to estimate cPDs, time of culture expansion, and passage numbers. Overall, predicted and real parameters revealed a good correlation, particularly in cPDs. Based on predicted cPDs we could reconstruct long-term growth curves and demonstrated the continuous increase in replicative senescence on molecular level.
  
  
  CONCLUSIONS
  Epigenetic analysis of specific CpG sites in the genome can be used to estimate the state of cellular aging for quality control of therapeutic cell products.
authors.forename: Anne
authors.initials: A
authors.lastname: Schellenberg
authors.forename: Sébastien
authors.initials: S
authors.lastname: Mauen
authors.forename: Carmen Mareike
authors.initials: CM
authors.lastname: Koch
authors.forename: Ralph
authors.initials: R
authors.lastname: Jans
authors.forename: Peter
authors.initials: P
authors.lastname: de Waele
authors.affiliation: Helmholtz-Institute for Biomedical Technology, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074 Aachen, Germany. wwagner@ukaachen.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wagner
mesh_terms: D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000592:standards*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D011786:Quality Control; D013997:Time Factors
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