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

Weidner CI, Walenda T, Lin Q, Wölfler MM, Denecke B, Costa IG, Zenke M, Wagner W - Sci Rep (2013)

Bottom Line: In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs.However, all cultured HPCs - even those which remained CD34(+) - acquired significant DNA-hypermethylation.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.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz-Institute for Biomedical Engineering, RWTH University Medical School, Aachen, Germany.

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.

Show MeSH
Enrichment of TF-binding motifs.(a) The sequences within 124 bp around each of the 9,213 overlappingly hypermethylated CpG sites were used for de novo motif analysis. The six most significant motifs were subsequently compared to known TF-binding sites. (b) Alternatively, these sequences were scanned for known motifs and enrichment and p-values (hypergeometric distribution) were calculated in relation to all CpG sites represented on the array. (c) Proportion of PU.1 binding sites within 124, 200, 500 and 1,000 bp around overlappingly hypermethylated CpG sites (black) was enriched compared to all CpG sites of the array (grey; *p < 0.05; ***p < 0.001; Δp < 10−15).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842544&req=5

f3: Enrichment of TF-binding motifs.(a) The sequences within 124 bp around each of the 9,213 overlappingly hypermethylated CpG sites were used for de novo motif analysis. The six most significant motifs were subsequently compared to known TF-binding sites. (b) Alternatively, these sequences were scanned for known motifs and enrichment and p-values (hypergeometric distribution) were calculated in relation to all CpG sites represented on the array. (c) Proportion of PU.1 binding sites within 124, 200, 500 and 1,000 bp around overlappingly hypermethylated CpG sites (black) was enriched compared to all CpG sites of the array (grey; *p < 0.05; ***p < 0.001; Δp < 10−15).

Mentions: De novo motif discovery in the direct vicinity of hypermethylated CpG sites (124 bp) identified binding sites for forkhead box proteins, HOXA5, PU.1 and RUNX1 amongst several other transcription factors (TFs; Fig. 3a). Alternatively, we scanned for known TF-binding sites and the most significant results were observed for the TFs PU.1 (SPI1), EWSR1-FLI1, EBF1, ELF5, and RUNX1 (Fig. 3b). The importance of PU.1 binding sites is further supported by data of a ChIP-Seq experiment with CD133+ HPCs which were cultured for 10 days under very similar culture conditions (Fig. 3c)20.


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

Weidner CI, Walenda T, Lin Q, Wölfler MM, Denecke B, Costa IG, Zenke M, Wagner W - Sci Rep (2013)

Enrichment of TF-binding motifs.(a) The sequences within 124 bp around each of the 9,213 overlappingly hypermethylated CpG sites were used for de novo motif analysis. The six most significant motifs were subsequently compared to known TF-binding sites. (b) Alternatively, these sequences were scanned for known motifs and enrichment and p-values (hypergeometric distribution) were calculated in relation to all CpG sites represented on the array. (c) Proportion of PU.1 binding sites within 124, 200, 500 and 1,000 bp around overlappingly hypermethylated CpG sites (black) was enriched compared to all CpG sites of the array (grey; *p < 0.05; ***p < 0.001; Δp < 10−15).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842544&req=5

f3: Enrichment of TF-binding motifs.(a) The sequences within 124 bp around each of the 9,213 overlappingly hypermethylated CpG sites were used for de novo motif analysis. The six most significant motifs were subsequently compared to known TF-binding sites. (b) Alternatively, these sequences were scanned for known motifs and enrichment and p-values (hypergeometric distribution) were calculated in relation to all CpG sites represented on the array. (c) Proportion of PU.1 binding sites within 124, 200, 500 and 1,000 bp around overlappingly hypermethylated CpG sites (black) was enriched compared to all CpG sites of the array (grey; *p < 0.05; ***p < 0.001; Δp < 10−15).
Mentions: De novo motif discovery in the direct vicinity of hypermethylated CpG sites (124 bp) identified binding sites for forkhead box proteins, HOXA5, PU.1 and RUNX1 amongst several other transcription factors (TFs; Fig. 3a). Alternatively, we scanned for known TF-binding sites and the most significant results were observed for the TFs PU.1 (SPI1), EWSR1-FLI1, EBF1, ELF5, and RUNX1 (Fig. 3b). The importance of PU.1 binding sites is further supported by data of a ChIP-Seq experiment with CD133+ HPCs which were cultured for 10 days under very similar culture conditions (Fig. 3c)20.

Bottom Line: In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs.However, all cultured HPCs - even those which remained CD34(+) - acquired significant DNA-hypermethylation.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.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz-Institute for Biomedical Engineering, RWTH University Medical School, Aachen, Germany.

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.

Show MeSH