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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.

SUBMITTER: Weidner CI

PROVIDER: S-EPMC3842544 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.

Weidner Carola Ingrid CI Walenda Thomas T Lin Qiong Q Wölfler Monika Martina MM Denecke Bernd B Costa Ivan Gesteira IG Zenke Martin M Wagner Wolfgang W

Scientific reports 20131128

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 remai ...[more]

PMID: 24284763

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Project description:BackgroundAccumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of aging. Here we tested this hypothesis in healthy individuals of different ages by examining unrepaired DNA double-strand breaks (DSBs) in hematopoietic stem/progenitor cells matured in their physiological microenvironment.Methodology/principal findingsTo asses DNA damage accumulation and repair capacities, γH2AX-foci were examined before and after exposure to ionizing irradiation. Analyzing CD34+ and CD34- stem/progenitor cells we observed an increase of endogenous γH2AX-foci levels with advancing donor age, associated with an age-related decline in telomere length. Using combined immunofluorescence and telomere-fluorescence in-situ hybridization we show that γH2AX-foci co-localize consistently with other repair factors such as pATM, MDC1 and 53BP1, but not significantly with telomeres, strongly supporting the telomere-independent origin for the majority of foci. The highest inter-individual variations for non-telomeric DNA damage were observed in middle-aged donors, whereas the individual DSB repair capacity appears to determine the extent of DNA damage accrual. However, analyzing different stem/progenitor subpopulations obtained from healthy elderly (>70 years), we observed an only modest increase in DNA damage accrual, most pronounced in the primitive CD34+CD38(-)-enriched subfraction, but sustained DNA repair efficiencies, suggesting that healthy lifestyle may slow down the natural aging process.Conclusions/significanceBased on these findings we conclude that age-related non-telomeric DNA damage accrual accompanies physiological stem cell aging in humans. Moreover, aging may alter the functional capacity of human stem cells to repair DSBs, thereby deteriorating an important genome protection mechanism leading to exceeding DNA damage accumulation. However, the great inter-individual variations in middle-aged individuals suggest that additional cell-intrinsic mechanisms and/or extrinsic factors contribute to the age-associated DNA damage accumulation.

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Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.

Publications

Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.

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