Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Mi Ran Choi,Young Gyu Chai,Kyoung Hwa Jung,Jin Choul Chai,Jungsun Park,Mi Kyung Chung,Young Seek Lee,Taesung Park,Yong-Ho In

doi:10.3858/emm.2012.44.8.057

Abstract

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.

Highlights

Mesenchymal stem cells (MSCs) can be isolated, adhere proficiently to plastic and self-renew; they are considered to have great therapeutic potential
The patterns of these three promoters are in agreement with those reported by Weber et al (2007), who demonstrated the successful enrichment of methylated DNA using methylated DNA immunoprecipitation (MeDIP) methodology
It has been reported that various mechanisms such as telomere shortening, histone modification, DNA methylation and miRNA expression affect the proliferation and differentiation of mesenchymal stem cells (MSCs), resulting in cell senescence

Summary

Introduction

Mesenchymal stem cells (MSCs) can be isolated, adhere proficiently to plastic and self-renew; they are considered to have great therapeutic potential. Because they exist at low frequencies (0.01% to 0.001%) in bone marrow (BM) (Choi et al, 2010), it is necessary to expand MSCs ex vivo prior to clinical use. Some studies have reported that long-term MSC cultures displayed the shortening of telomeres and the absence of telomerase expression and activity (Baxter et al, 2004; Bernardo et al, 2007; Choi et al, 2010) and genetic or epigenetic modifications, contributing to cellular senescence (Dahl et al, 2008; Bork et al, 2010)

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Conclusion