The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

Abstract

Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures.

Figure 1

Percentage of total, promoter, and inside CGIs reversing the methylation status. The percentage of CGIs reversing the methylation status (y-axis) is indicated for each chromosome (x axis). Cold colors identify newly methylated CGIs; warm colors identify newly demethylated CGIs. XCI: CGIs associated with genes subjected to X inactivation. No XCI: CGIs not associated with XCI genes.

Figure 2

X chromosome CGIs profiles (promoter versus inside). (a) CGIs associated with gene inside; (b) CGIs associated with gene promoter. Each dot corresponds to a CGI in the X chromosome map; blue dots: CGIs represented in the array; red dots: CGI status after in vitro culture. x axis: dots located at 0 correspond to islands that did not change the methylation status after culture; dots at 1: CGIs that reversed the methylation status from unmethylated to methylated after culture; dots at −1: CGIs that reversed the methylation status from methylated to unmethylated after culture. Light blue area defines a region of 18 Mb at Xp22.33-22.13 characterized by absence of de novo methylation in promoter CGIs.

Figure 3

Percentage representativeness of biological processes: unvaried (inner ring) versus modified imprinted genes (outer ring). The genes with an unvaried methylation status (inner circle) and the modified ones (outer circle) were analyzed by GOstat software to determine their functional involvement in cell biology. The classes, in which a variable number of GO terms were pooled, were arbitrarily determined and represent the main biological processes taking place within a cell, as reported in the legend box.