Culture of MSCs with H2O2 or PS therefore induced a slight increase of apoptosis on the long term. Low AOPP Levels in Duloxetine HCl Patient Serum Induced MSC Senescence We also assessed senescence by quantifying SA–gal activity of MSCs. of MSCs, notably the expression of SOD2 antioxidant gene. By contrast, the osteoblastic/adipogenic potential of MSCs was increased, whereas their immunosuppressive function was slightly reduced. Discussion Although some functional properties of MSCs were affected upon culture with PS, evidence from preclinical studies and the present one suggested that MSCs can adapt to the oxidative environment and exert their therapeutic effect. for 15?min, and patient serum (PS) stored at ?80C. Serum AB (SAB) was a pool of 200 human male AB plasma purchased from Sigma-Aldrich (ref H4522). Human blood was purchased from the Etablissement Fran?ais du Sang (Toulouse). Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (GE Healthcare) according to standard procedures. Table 1 Clinical characteristics of SSc patients. value 0.05 was considered significant. Results High AOPP Levels in SSc Patient Serum Affected the Proliferation Rate of MSC We first evaluated the proliferation rate of MSCs cultured for 10?days in medium containing 5% serum from healthy patients (SAB) or oxidized SAB that has been submitted to H2O2 (SABH2O2) or HOCl treatment to get 400 or 1,000?mol/L of AOPP (SAB400 and SAB1000). In SAB, MSCs rapidly proliferated during the first 3?days and then weakly proliferated till day 10 (Physique ?(Figure1A).1A). By contrast, MSCs cultured in oxidized SAB did not proliferate compared to day 0 and even died when cultured in SABH2O2. By comparison with MSCs cultured in SAB, growth of MSCs in oxidized SAB was significantly inhibited at whatever the time point, indicating that oxidized human serum inhibited MSC proliferation. Open in a separate window Physique 1 HOCl- or H2O2-induced serum AOPPs and systemic Duloxetine HCl sclerosis (SSc) patient serum decreased the proliferation rate of MSCs and increased the number of apoptotic MSCs. (A) Percentage of MSC proliferation depending on the concentration of advanced oxidation protein products (AOPP) induced by HOCl in human serum AB (SAB): 400?mol/L (SAB400) or 1,000?mol/L (SAB10000), H2O2 (SABH2O2) (indicated the Pearsons correlation coefficient. (C) Gene expression fold change of different profibrotic markers (PS 400 and PS 400, of ?0.5971 ( em p /em ?=?0.0054) (Physique ?(Figure1B).1B). This could not be attributed to variability between MSC samples because proliferation rates of different MSC samples cultured with same patient serum were comparable (data not shown). Finally, expression levels of five genes associated with SSc phenotype did not change (Physique ?(Physique1C).1C). Indeed, long-term exposure to high levels of AOPP and oxidative stress inhibited MSC proliferation but did not change the phenotype of MSCs. High AOPP Levels in Patient Serum Induced MSC Apoptosis We also Duloxetine HCl found that the percentage of apoptotic MSCs in control SAB was 5.33??1.60% at day 3, 7.34??1.77% at day 6, and 5.19??1.17% at day 10. By comparison, no MDS1-EVI1 increase of apoptosis was noticed when MSCs were cultured with SAB400, SAB1000, or SABH2O2 at day 3 or 6 (Physique ?(Figure1D).1D). At day 10, the percentage of apoptotic MSCs was significantly increased with SABH2O2, which mirrored the lower proliferation rate of MSCs observed in Physique ?Figure1A.1A. When MSCs were cultured with PS, the percentage of apoptotic cells was significantly increased at days 6 and 10 for MSCs expanded with PS 400 and PSpool (Physique ?(Figure1D).1D). Expression levels of the pro-apoptotic marker Bax was increased with PS 400, and the antiapoptotic marker Bcl2 was significantly decreased (Physique ?(Figure1E).1E). Culture of MSCs with H2O2 or PS therefore induced a slight increase of apoptosis on the long term. Low AOPP Levels in Patient Serum Induced MSC Senescence We also assessed senescence by quantifying SA–gal activity of MSCs. In the SAB culture condition, we measured 143.7??20.4 at day 3, 199.3??23 at day 6, and 258??80 RFU at day 10 that was normalized to 100 at each time point. Compared to SAB, SA–gal activity of MSCs was increased when cultured with SAB1000 or SABH2O2 although significance was reached only at day 3.