Epigenetic Classification of Human Mesenchymal Stromal Cells

Abstract

SummaryStandardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures.

Senescence‐associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells

“…We performed additional limiting dilutions of the aforementioned MSC preparations, which were further differentiated toward adipogenic or osteogenic lineages for 2 weeks. The individual subclones revealed very heterogeneous in vitro differentiation potential as described in our previous work (Schellenberg et al ., ; de Almeida et al ., ) (Fig. S5).…”

“…Due to their regenerative and immunomodulatory potential, they are applied for a huge variety of therapeutic approaches, for example, for orthopedic injuries, autoimmune diseases, or cardiovascular disorders (Kim & Cho, ). However, precise molecular markers for MSCs are still elusive (de Almeida et al ., ) and there is a growing perception that these cell preparations are highly heterogeneous and only a small subfraction reflects multilineage differentiation potential (Phinney, ; Schellenberg et al ., ). It has been demonstrated that the number of colony‐forming units and the number of cells with high adipogenic and osteogenic differentiation potential decline rapidly during culture expansion (Schellenberg et al ., , ).…”

Cell type-specific DNA methylation in neonatal cord tissue and cord blood: a 850K-reference panel and comparison of cell types

“…However, these limitations are hard to overcome as in the field there seems to be no general consensus on the molecular markers that can be used to distinguish these sub-populations [22]. The use of an epigenetic score has been previously proposed to distinguish MSCs from fibroblasts, and this is ascertained by the DNA methylation difference on 2 CpGs (cg22286764, C3orf35 , and cg05684195, CIDEC ) [23]. The resulting beta score range from −1 to 1, where a positive value indicates MSCs, and negative indicates fibroblasts.…”

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].…”