Differentially expressed mRNA upon <i>in vitro</i> senescence.

<p>36 Genes that were significantly differentially expressed between early and senescent passage of three independent donor samples (SAM, FDR = 3).</p

<i>In vitro</i> differentiation.

<p>MSC of different passages were simultaneously differentiated along adipogenic or osteogenic line. Fat accumulation was visualized by Oil Red-O staining. Adipogenic differentiation potential decreased in higher passages (A, B, C). In negative controls without differentiation (grey triangles) no fat accumulation was observed but the cells grew to a higher density which also resulted in higher OD. Osteogenic differentiation was visualized by van Kossa staining (not demonstrated) or Alizarin red staining. There was a higher propensity for osteogenic differentiation in higher cell passages (D, E, F). Senescence associated β-galactosidase staining increases in the later passages (G, H, I). Representative results of three independent MSC preparations are demonstrated (±SD).</p

miRNA expression changes upon <i>in vitro</i> senescence.

<p>miRNA expression in early and senescent passages of three MSC preparations was determined by microarray analysis (miCHIP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002213#pone.0002213-Castoldi1" target="_blank">[29]</a>. Five miRNAs that are up-regulated during senescence are depicted (*  =  significant by SAM analysis). miRNA expression was also analyzed in the sequential passages of donor 1 and hierarchical cluster analysis revealed that expression of these miRNAs was overall increased during senescence (A). Furthermore, differential miRNA expression was validated by QRT-PCR for hsa-mir-29c, hsa-mir-369-5p and hsa-let-7f in the three MSC preparations that were used for microarray analysis as well as in three additional samples (B).</p

Primer sequences.

<p>Primer sequences.</p

QRT-PCR validation of mRNA expression.

<p>Differential expression of senescent passage (PX) <i>versus</i> P2 was validated by using QRT-PCR for 10 genes (A). Results were in line with microarray data for all tested genes, investigating either the same three MSC preparations (donor 1–3) or three independent donor samples that were isolated in the same culture medium M1 (donor 4–6). Furthermore, differential gene expression was also observed in three MSC preparations isolated under different culture conditions (M2). Differential mRNA expression was not restricted to senescent passages but increased during the course of replicative senescence (B,C).</p

Simulation of MDS development.

<p>(A) Simulated MDS development under the assumptions depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003599#pcbi-1003599-g001" target="_blank">Figure 1</a>. Initially, a single MDS-LT-HSC is present. Evolution of cell numbers in the different compartments is simulated over 55 years. (B) A sharp decline of mature cells is achieved after about 17 years – this would correspond to clinical manifestation of MDS. (C) Simulated cellular composition in the BM at the relevant time frame. (D) Corresponding signal intensities for self-renewal and proliferation are presented. Self-renewal decays due to the accumulation of malignant cells in the BM-niche; proliferation is activated due to ineffective hematopoiesis. (E) The percentage of apoptotic cells in the bone marrow is presented.</p

Morphologic changes and immunophenotype upon senescence.

<p>Replicative senescence is reflected by dramatic changes in morphology. Cells enlarge, generate more vacuoles and cellular debris and ultimately stop proliferation. Representative morphology of MSC in early (P3) and senescent passage (P12) is presented (A, B). The continuous increase in cell size and granularity is reflected by the increasing forward-scatter signal in flow cytometry (FSC, ±SD; C). Immunophenotypic analysis of all MSC preparations was in accordance with the literature whereby the detection level for positive markers was much higher in early passages compared to late passages (black line  =  autofluorescence; D). A representative analysis of three preparations is demonstrated.</p

mRNA expression profile of MSC changes extensively with higher passage.

<p>Differential gene expression of senescent passages <i>versus</i> P2 was analyzed by Affymetix GeneChip technology in three independent MSC preparations. 19,448 ESTs that were detected as present in at least 10 of 13 hybridizations were ordered according to their log₂ratio. 1033 ESTs were more than 2-fold up-regulated (red) and 545 were more than 2-fold down-regulated (green). Analysis of different passages of donor1 demonstrated increasing changes in the global gene expression pattern during <i>in vitro</i> senescence (A). GeneOnthology analysis was performed for the subsets of genes that were >2-fold up-regulated or >2-fold down-regulated in comparison to all genes detected as present on the microarray. The percentages of genes that contributed to representative categories are depicted (B,C; P<0.0001). Probabilities of co-localization of regulated genes plotted onto a human karyogram. The probability of representation of 2-fold up-regulated genes (D) and 2-fold down-regulated genes (E) on chromosomal regions is indicated by color coding.</p

CFU-Frequency upon stimulation with MDS serum.

<p>(A) CD34⁺ HPCs from cord blood were cultured with 10% serum supplements of individual MDS patients or controls. After seven days, cells were re-seeded in methylcellulose medium and after two weeks, the numbers of erythrocyte (BFU-E and CFU-E), granulocyte (CFU-G), macrophage (CFU-M) and combined (CFU-GM and CFU-GEMM) colonies were counted. (B) Erythropoietin (EPO) concentration was significantly higher in MDS-derived serum samples. (C) The growth promoting effect on CD34⁺ cells was then plotted against the EPO concentration. CFSE intensity was normalized to healthy controls of the corresponding experiment. Error bars represent SD (*p<0.05, **p<0.01, ***p<0.001).</p

Serum of MDS patients stimulates proliferation of CD34⁺ cells.

<p>(A) CD34⁺ HPCs from umbilical cord blood were stained with CFSE and subsequently cultivated for five days <i>in vitro</i> in culture medium supplemented with 10% serum of individual patients or of healthy controls. Cell division history was monitored by residual CFSE-staining and dotted lines indicate five cell divisions. (B) Mean fluorescence intensities after five cell divisions were normalized to control samples. In each experiment, 57 MDS serum supplements and 17 control serum supplements were tested in parallel. Mean and standard deviation were calculated over measurements with three different cord blood samples (*p<0.05, **p<0.01, ***p<0.001).</p