Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells

Abstract

BackgroundFor safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations.ResultsWe see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set.ConclusionsHistone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

Soliciting Strategies for Developing Cell-Based Reference Materials to Advance Mesenchymal Stromal Cell Research and Clinical Translation

“…The PluriTest for pluripotent cells [10] could serve as an important model of analysis of microarray data from MSCs, but will require the identification of a cluster of MSC-specific genes similar in concept to the 450 genes used to identify pluripotent cells. New technologies for epigenetic analyses of cells could provide important information about MSCs [20]. The large amount of data generated are often difficult to interpret given the variability between different MSC preparations, thus underscoring the importance of developing standardized methods to test the reference cells.…”

Bioprocess engineering strategies for autologous human MSC-based therapies: one size does not fit all

“…Ideally these metrics should be also linked to the mechanism of action of the harvested and expanded progenitor cell populations. New technologies for epigenetic analyses of cells could provide important information regarding the classification of types [46] based on their tissue of origin even after extensive culture, and about bioprocessing during differentiation of MSCs and its impact on epigenetic patterns [47]. The generation of specific epigenetic signatures reflects functional properties of MSCs such as their hematopoiesis supportive function [48].…”

Epigenetic regulation of chondrocyte differentiation

“…They also reported that lysinespecific demethylase-1 (Lsd1) and Jmjc-containing histone demethylase-2a (Jhdm2a) are involved in the regulation of lysine methylation. Herlofsen et al have demonstrated that the methylation level of H3K4me3 and H3K36me3 is largely associated with chondrocyte differentiation of human mesenchymal stem cells, assessed by Chip-Seq analysis [64]. Setdb1 (SET domain, bifurcated1) regulates chondrocyte hypertrophy by inhibiting Runx2 activity through H3K9 methylation [65,66].…”