220 CpG METHYLATION PROFILE OF ADIPOGENIC AND ENDOTHELIAL GENE PROMOTERS IN HUMAN ADIPOSE STEM CELLS SUGGESTS A RESTRICTIVE ENDOTHELIAL DIFFERENTIATION POTENTIAL

Abstract

Mesenchymal stem cells (MSCs) have received intense research interest due to their perceived potential application in regenerative medicine; nevertheless, MSCs are primarily restricted to form mesodermal cell types. Adipose stem cells (ASCs) with a CD34+ CD105+ CD45– CD31– immunophenotype can be obtained in an uncultured state with high purity from the stromal vascular fraction of human liposuction material (Boquest et al. 2005 Mol. Biol. Cell 16, 1131–1141). While ASCs differentiate readily into adipocytes, their endothelial lineage commitment has been scarcely reported, and controversy remains regarding ASC contribution to vascularization. To address the epigenetic commitment of ASCs to adipogenic and endothelial lineages, we carried out a bisulfite sequencing analysis of CpG methylation in the promoters of adipogenic (LEP, PPARG2, FABP4, LPL), endothelial (CD31, CD144), and myogenic (MYOG) genes in freshly isolated and in clonal ASC cultures in relation to gene expression and differentiation potential. Uncultured ASCs display mosaic hypomethylation of adipogenic promoters, in contrast to MYOG, CD31, or CD144 which are methylated (Noer et al. 2006 Mol. Biol. Cell 17, in press). Nevertheless, CpG methylation does not reflect transcriptional status of these genes in undifferentiated cells. Culture and adipogenic differentiation of ASCs maintains the hypomethylated profile of adipogenic promoters and the hypermethylation of non-adipogenic promoters. Endothelial stimulation of ASCs in methylcellulose elicits tubule-like networks, up-regulation of CD31 and CD144, and restrictive induction of a CD31+ CD144+ immunophenotype. Discrete and lineage-specific changes in CpG methylation in the CD31 and CD144 promoters take place but no global demethylation that marks endothelial cells occurs. Promoters not involved in endothelial differentiation retain a methylation profile characteristic of undifferentiated cells. Hypermethylation of CD31 and CD144 suggests a restricted commitment of ASCs to the endothelial lineage. This contrasts with hypomethylation of adipogenic promoters which reflects a propensity toward adipogenic differentiation. Despite the up-regulation of lineage-specific transcripts, overall maintenance of promoter methylation after adipogenic, osteogenic, and endothelial differentiation suggests the maintenance of an epigenetic signature characteristic of undifferentiated cells. Analysis of CpG methylation at lineage-specific promoters should provide a robust assessment of epigenetic commitment of stem cells to a specific lineage.