**3.1. MM-BMMSCs are characterized by high senescence state and cell cycle abnormalities**

Analysis of β-galactosidase activity revealed a significantly higher SAβGalA in MM-BMSC when compared with HD-BMDSC (**Figure 1A**). Since no significant differences in senescent cells between passages 1 and 4 were observed in both MM-BMMSC and HD-BMMSC, we can exclude the effect of cultivation on SAβGalA. These results were confirmed by a histological β-galactosidase staining of HD-BMMSC and MM-BMMSC in passage 4.

**Figure 1.** MM-BMMSC exhibits a higher senescence state and a lower self-renewal capacity than HD-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Mann-Whitney *U* test and unpaired *t*-test (ELISA analysis). (A) Flow cytometric analysis of SAβGalA. ND-MM-BMMSCs and R-MM-BMMSCs displayed higher activity of SAβGalA in passages 1 and 4 of cell cultures compared to HD-BMMSCs. (B) The colony-forming unit fibroblast (CFU-F) assay was used to study the self-renewal capacity of BMMSC. MM-BMMSC showed a lower self-renewal capacity compared to HD-BMMSC. (C) Cell cycle analysis showed a higher amount of cell in S phase and amount in G1 /G0 phase in MM-BMMSC compared to HD-BMMSC. (D) QPCR analysis displayed decreased cyclin E1, increased cyclin D1 and p21 expression in MM-BMMSC compared to HD-BMMSC. (E) Measurement of the protein level in HD-BMMSC and MM-BMMSC. Cyclin E1 was significantly decreased in MM-BMMSC compared to HD-BMMSC, whereas cyclin D1 and p21 were increased. The protein amount of p16 was slightly reduced in MM-BMMSC compared to HD-BMMSCs. ND-MM-BMMSCs, new diagnosed MM patients; R-MM-BMMSCs, MM patients in relapse; HD-BMMSCs, healthy donor control.

The colony-forming unit fibroblast (CFU-F) assay was used to study the self-renewal capacity of BMMSC. MM-BMMSC showed a lower self-renewal capacity compared to HD-BMMSC (**Figure 1B**). Similar to the senescence study, MM-BMMSC obtained from relapsed patients showed a significantly lower self-renewal capacity than MM-BMMSC, which forms newly diagnosed patients.

MM-BMMSCs are characterized by a lower expression of cyclin E1 and an overexpression of cyclin D1 when compared with HD-BMMSC (**Figure 1D**). In addition, the cell cycle inhibitor p21 was upregulated in MM-BMMSC compared to HD-BMMSC (*p <* 0.05). No changes were observed in the mRNA level of p16. Changes in the mRNA levels were also confirmed using protein analysis (*p* < 0.03; **Figure 1E**). Cyclin E1 was decreased in MM-BMMSC compared to HD-BMMSC (*p* = 0.0416). Cyclin D1 and p21 protein levels were 1.5- to 1.8-fold increased. Protein measurement also showed a slightly reduced level of p16 in MM-BMMSCs, but this change was below 1.5-fold. These results correlated with a higher number of cells in S phase and a reduced number of cells in G1 /G0 phase compared to HD-BMMSCs (*p <* 0.008; **Figure 1C**).
