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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.

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Distribution of differentially methylated CpG sites.(a) Overlap of hypermethylated and hypomethylated CpG sites in the expanded cell fractions. (b) For the genes WT1, HOXA5 and NOTCH1 site specific DNAm changes (arrowheads indicate significant CpG sites in comparison to either CD34+ (d0) [green] or CD34− w/o MSC [blue]) and gene expression changes are exemplarily depicted (*p ≤ 0.05; **p ≤ 0.01). (c) Gene Ontology analysis revealed most significant enrichment of DNAm changes in genes related to categories of hematopoietic activation or immune processes (same colour code as in B). (d) Enrichment of DNAm changes in relation to gene regions or CpG islands. Hypermethylation was enriched in TSS1500 and shore regions, whereas hypomethylation was enriched in 3′UTR, intergenic areas and shelf regions. (hypergeometric distribution: +p < 10−5; #p < 10−10; Δp < 10−15).
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f2: Distribution of differentially methylated CpG sites.(a) Overlap of hypermethylated and hypomethylated CpG sites in the expanded cell fractions. (b) For the genes WT1, HOXA5 and NOTCH1 site specific DNAm changes (arrowheads indicate significant CpG sites in comparison to either CD34+ (d0) [green] or CD34− w/o MSC [blue]) and gene expression changes are exemplarily depicted (*p ≤ 0.05; **p ≤ 0.01). (c) Gene Ontology analysis revealed most significant enrichment of DNAm changes in genes related to categories of hematopoietic activation or immune processes (same colour code as in B). (d) Enrichment of DNAm changes in relation to gene regions or CpG islands. Hypermethylation was enriched in TSS1500 and shore regions, whereas hypomethylation was enriched in 3′UTR, intergenic areas and shelf regions. (hypergeometric distribution: +p < 10−5; #p < 10−10; Δp < 10−15).

Mentions: All expanded cell fractions (CD34+ w/o MSC, CD34− w/o MSC and CD34+ w/MSC) revealed a remarkable overlap in hypermethylation (Fig. 2a). Among these was the Wilms tumor 1 gene (WT1; P < 10−123); NOTCH1 (p < 10−25), a known modulator of lineage-specific events in hematopoiesis16; and various genes of the homeobox gene cluster A (particularly HOXA5: p < 10−35; Fig. 2b, Supplementary Fig. S4a). Hypermethylation was also observed in the HOXB cluster (particularly HOXB3; p < 10−17), but not in HOXB4 which has previously been implicated in in vitro expansion of HPCs (Supplementary Fig. S4b)1718. Other relevant genes with hypermethylated CpG sites include the myeloid translocation gene 16 (MTG16; also known as CBFA2T3; p < 10−150) that has been implicated in the maintenance of stem cell quiescence; the retinoid X receptor alpha (RXRA; p < 10−91), whose down-regulation is essential for neutrophil development and the adenosine a2a receptor (ADORA2A; p < 10−109), which inhibits neutrophil degranulation; and the epigenetic regulators histone deacetylase 9 (HDAC9; p < 10−4; Supplementary Fig. S4c) and DNMT3A (p < 10−46). The highly significant hypermethylation within DNMT3A was further validated by bisulfite pyrosequencing in independent samples (Supplementary Fig. S3b). On the other hand, hypomethylated CpG sites were related to the transcription factor PAX5 (p < 10−5) and recombination activating gene 2 (RAG2; p < 10−8), which are involved in lymphoid differentiation (Supplementary Fig. S4d). Gene Ontology (GO) analysis of hypermethylated genes revealed highly significant enrichment in functional categories of the immune system, hematopoietic development and activation (Fig. 2c), whereas classification of hypomethylated CpG sites was hardly significant and rather associated with signaling.


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)

Distribution of differentially methylated CpG sites.(a) Overlap of hypermethylated and hypomethylated CpG sites in the expanded cell fractions. (b) For the genes WT1, HOXA5 and NOTCH1 site specific DNAm changes (arrowheads indicate significant CpG sites in comparison to either CD34+ (d0) [green] or CD34− w/o MSC [blue]) and gene expression changes are exemplarily depicted (*p ≤ 0.05; **p ≤ 0.01). (c) Gene Ontology analysis revealed most significant enrichment of DNAm changes in genes related to categories of hematopoietic activation or immune processes (same colour code as in B). (d) Enrichment of DNAm changes in relation to gene regions or CpG islands. Hypermethylation was enriched in TSS1500 and shore regions, whereas hypomethylation was enriched in 3′UTR, intergenic areas and shelf regions. (hypergeometric distribution: +p < 10−5; #p < 10−10; Δp < 10−15).
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Related In: Results  -  Collection

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f2: Distribution of differentially methylated CpG sites.(a) Overlap of hypermethylated and hypomethylated CpG sites in the expanded cell fractions. (b) For the genes WT1, HOXA5 and NOTCH1 site specific DNAm changes (arrowheads indicate significant CpG sites in comparison to either CD34+ (d0) [green] or CD34− w/o MSC [blue]) and gene expression changes are exemplarily depicted (*p ≤ 0.05; **p ≤ 0.01). (c) Gene Ontology analysis revealed most significant enrichment of DNAm changes in genes related to categories of hematopoietic activation or immune processes (same colour code as in B). (d) Enrichment of DNAm changes in relation to gene regions or CpG islands. Hypermethylation was enriched in TSS1500 and shore regions, whereas hypomethylation was enriched in 3′UTR, intergenic areas and shelf regions. (hypergeometric distribution: +p < 10−5; #p < 10−10; Δp < 10−15).
Mentions: All expanded cell fractions (CD34+ w/o MSC, CD34− w/o MSC and CD34+ w/MSC) revealed a remarkable overlap in hypermethylation (Fig. 2a). Among these was the Wilms tumor 1 gene (WT1; P < 10−123); NOTCH1 (p < 10−25), a known modulator of lineage-specific events in hematopoiesis16; and various genes of the homeobox gene cluster A (particularly HOXA5: p < 10−35; Fig. 2b, Supplementary Fig. S4a). Hypermethylation was also observed in the HOXB cluster (particularly HOXB3; p < 10−17), but not in HOXB4 which has previously been implicated in in vitro expansion of HPCs (Supplementary Fig. S4b)1718. Other relevant genes with hypermethylated CpG sites include the myeloid translocation gene 16 (MTG16; also known as CBFA2T3; p < 10−150) that has been implicated in the maintenance of stem cell quiescence; the retinoid X receptor alpha (RXRA; p < 10−91), whose down-regulation is essential for neutrophil development and the adenosine a2a receptor (ADORA2A; p < 10−109), which inhibits neutrophil degranulation; and the epigenetic regulators histone deacetylase 9 (HDAC9; p < 10−4; Supplementary Fig. S4c) and DNMT3A (p < 10−46). The highly significant hypermethylation within DNMT3A was further validated by bisulfite pyrosequencing in independent samples (Supplementary Fig. S3b). On the other hand, hypomethylated CpG sites were related to the transcription factor PAX5 (p < 10−5) and recombination activating gene 2 (RAG2; p < 10−8), which are involved in lymphoid differentiation (Supplementary Fig. S4d). Gene Ontology (GO) analysis of hypermethylated genes revealed highly significant enrichment in functional categories of the immune system, hematopoietic development and activation (Fig. 2c), whereas classification of hypomethylated CpG sites was hardly significant and rather associated with signaling.

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