GraphPad Prism version 5·0 software was used for statistical analyses. Results are expressed as mean ± standard deviation (s.d.). Relationships between different values were examined by Pearson’s correlation coefficient. A proportion of cell subsets were compared using Student’s t-test for normally or non-normally distributed subsets as appropriate. Statistical significance GSK2126458 in vitro was expressed by a P-value of < 0·05. MSCs isolated from

SSc patients were characterized by expressing the surface molecules CD90, CD105 and CD73. They did not express CD45, CD34 and CD14, as assessed by flow cytometry analysis. Moreover, MSCs showed normal ability in differentiating into osteoblast, adipocytes and chondroblast ABT-263 order in vitro (data not shown). The cumulative population doublings for MSCs isolated from SSc patients, as markers of the replication

rate, was consistently lower than that of HC cells (HC–MSCs 3·07 ± 0·38 versus SSc–MSCs 2·42 ± 0·16, P < 0·0070; Fig. 1a). In order to assess whether this reduced proliferation of SSc–MSCs was due to a growth-arrested status and the different cell cycle distributions with respect to HC cells, both SSc and HC–MSCs were analysed by flow cytometry after DNA staining with PI. Of note, no significant differences were observed between HC– and SSc–MSC, as cell cycle analysis revealed that the large percentage of MSCs obtained from both HC and SSc were in G0/G1 phases [HC–MSCs 80·23 ± 1·79 versus SSc–MSCs 83·00 ± 3·33%, P = not significant (n.s.)]; on the contrary, only a small population of cells were engaged in active proliferation (S+G2/M phases: HC–MSCs 18·75 ± 2·09 versus SSc–MSCs 15·65 ± 3·41%, Loperamide P = n.s., Fig. 1b), although not significantly. Because the above method does not distinguish between actively growing (G1) and growth-arrested (G0) cells, to distinguish more effectively between proliferative and resting

cells we assessed Ki67 gene expression by qPCR analysis. We found that MSCs isolated from SSc patients showed a lower expression of Ki67 gene when compared to HC cells (HC–MSCs 3·44 ± 0·20 versus SSc–MSCs 1·57 ± 0·53 mRNA levels, P = 0·019), confirming that the majority of cells was in G0 phase (Fig. 1c). No differences were observed in the proliferative ability of SSc–MSCs between the two disease subsets. Given the functional implications of the in-vitro senescence of MSCs, we employed β-Gal as a senescence marker. We observed that the percentage of β-Gal-positive stained cells was significantly higher in SSc when compared to HC (HC–MSCs 7·67 ± 4·41% versus SSc–MSCs 26·00 ± 4·34%, P = 0·03, Fig. 2a). Furthermore, we cultured both HC and SSc cells for 24 h in the presence of 5 μg/ml of doxorubicin, which represents a well-accepted in-vitro model to recreate the premature ageing of stem cells [29].