Weidner Carola Ingrid, Walenda Thomas, Lin Qiong, Wölfler Monika Martina, Denecke Bernd, Costa Ivan Gesteira, Zenke Martin, Wagner Wolfgang
Helmholtz-Institute for Biomedical Engineering, RWTH University Medical School, Aachen, Germany.
Sci Rep. 2013 Nov 28;3:3372. doi: 10.1038/srep03372.
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.
造血干细胞和祖细胞（HPCs）能够在体外维持培养，但它们的绝大多数子代细胞在培养过程中会丧失干性。在本研究中，我们比较了新鲜分离的和培养扩增的HPCs的DNA甲基化（DNAm）图谱。CD34(+)细胞的培养条件——无论有无间充质基质细胞（MSCs）——对DNAm的影响相对较小，尽管基质支持能显著提高细胞增殖。然而，所有培养的HPCs——即使是那些仍保持CD34(+)的细胞——都出现了显著的DNA高甲基化。DNA高甲基化尤其发生在上游启动子区域、CpG岛的岸区、PU.1、HOXA5和RUNX1的结合位点，并且反映在DNMT3A的差异基因表达和可变转录本中。低浓度的DNAm抑制剂略微增加了集落形成单位起始细胞的频率。我们的结果表明，HPCs在基因组的特定位点获得DNA高甲基化，这与体外操作过程中干细胞干性的快速丧失有关。
