Generation of mesenchymal stem cell (MSC) clones. (A): MSC clone at day 7 of expansion (bar: 100µm). (B): Cloning efficiency of MSCs. Shown are the results of three different limiting dilution experiments. The red line corresponds to 37% of wells without clone growth. (C): Phenotype of the MSC clones (10 clones). Gray lines are negative controls.

Regeneration of Sca‐1 expression at the population level. (A): Characteristics of cell sorting as applied to clone AC3 at P6. The Sca‐1 profile of the mother population is shown in black and that of the isotype control in gray. The sorting gate for Sca1‐L is indicated in blue, for Sca1‐M in green and for Sca1‐H in red. (B): Results of in silico (left panel) and in vitro (right panel) marker regeneration studies. Compared are the changes of the marker expression state α and the Sca‐1 fluorescence profiles, respectively. Shown are all sorted subpopulations at day 1‐6 (D1‐D6) of subsequent culture. Colors as in A. (C–E): Regeneration after prolonged clonal expansion. (C) Comparison of regenerated Sca‐1 fluorescence profiles (clone AC5). Compared are profiles obtained at P5 with those at P11. Shown are all subpopulations (L, M and H) and the mother population WCP at day 6 after sorting. The median fluorescence channel is indicated by a dotted line. (D) Sca‐1 median fluorescence (related to that of the isotype control) measured in early‐ (P5 and P6), intermediate‐ (P7 and P8) and late passage (P9‐P12) cells. Box plots are shown with first and third quartiles and mean value; error bar is SD. (E) Time span required for successful regeneration for early‐, intermediate‐ and late‐passage (P9‐P12) cells. For late‐passage cells, only 3 points out of 6 are shown since in 3 cases regeneration was not observed within the time frame of the experiments (10 days). Abbreviations: Sca‐1, stem cell antigen 1; WCP, whole cell population.

Molecular characterization of Sca‐1 subpopulations. (A): Sorting characteristics and self‐organizing maps (SOMs)‐portraits of Sca‐1 subpopulations of clone C1 at P12, immediately after sorting. The SOM portraits of each subpopulation show specific over‐ and under‐expression spots in red and blue, respectively. They are different compared to those observed for the mother population WCP. X indicates the over‐expression spot containing the Ly6 cluster. (B): Differential expression of Ly6 genes (Ly6a, Ly6c1, Ly6c2, Ly6e) genes in the WCP and the L, M and H populations. ΔlogE denotes the expression centered with respect to mean gene expression of the gene in all samples. (C): Structure of the Ly6a gene locus and location of the analyzed CpGs (For: forward; Rev: reverse outer primers). (D–F): Epigenetic states of the Ly6a promoter (n = 3, error bars: SEM). (D) The DNA methylation level of the analyzed CpGs is stable. Changes (ΔDNAme) comparing Sca‐1H (H) and Sca‐1L (L) as well as young (Y) and aged (A) cells are small. (E, F) The H3K4me3 modification level of the promoter correlates with the gene expression. (E) It is higher in Sca‐1H compared to Sca‐1L subpopulations and F) higher in young (P3) WCPs compared to aged (P13) WCPs. The H3K27me3 modification level was low regardless of Sca‐1 sorting and age (* = 0.05, t test). Abbreviations: Sca‐1, stem cell antigen 1; WCP, whole cell population.

Model of bistable epigenetic states of the Ly6a promoter. (A): Upper panel: General regulatory principles. For genes with high promoter CpG density age‐related promoter hyper‐methylation results in a reduced transcriptional activation (left). These changes affect also the expression of genes showing no age‐related promoter methylation changes. In case of the Ly6a model gene, we assumed that they induce a reduced transcriptional activation (right). Lower Panel: Ly6a specific regulatory principles. Shown is the expression T of the Ly6a model gene, scaled by its maximum T_max, in dependence to the activation A (scaled by A_max) of the gene by the underlying transcription factor network. In the bistable region switches between high (red) and low (blue) expression states occur due to stochastic H3K4me3 (de‐) modification reactions (thin arrows). The dashed green line represents instable solutions for T. If A falls below the threshold “a” the system shows monostable Sca‐1L behavior. If A increases above the threshold “b” the system shows monostable Sca‐1H behavior. Aging results in a shift to lower activation A. The thin black line represents the solution without the effects of H3K4me3. (B): Simulated Sca‐1 expression profiles applying the epigenetic model. Shown are profiles immediately after sorting (D0) and after 1 and 6 days of regeneration (D1, D6). The example compares regeneration for early and late passages, that is, after 2 and 7 weeks of cell expansion, respectively. Left panel: At early passages a broad distribution of expression levels of the Ly6a model gene is observed within the mother cell population (black). This distribution completely regenerates within 6 days both for low‐expressing (blue) and high‐expressing cells (red). Right panel: In late passages, the high expression state is less stable and accordingly the distribution of expression levels narrows. Regeneration still occurs, but does not reestablish the distribution of early passages. Abbreviation: Sca‐1, stem cell antigen 1.