Fig. 4.

HUVEC proliferation in response to glucose in presence of pMSCs, or after removing glucose and pMSCs, examined after 24, 48, and 72 h in MTS assay. In response to conditioned medium (CMpMSC), pMSCs had no significant effect on HUVEC proliferation in presence of glucose after 24 h as compared to untreated or glucose-treated HUVECs (a). CMpMSC significantly increased HUVEC proliferation in presence of glucose after 48 h (b) and 72 h (c), as compared to glucose-treated but not untreated HUVECs. Cell–cell contact assay showed that, compared to glucose-untreated or treated HUVECs, pMSCs significantly increased HUVEC proliferation in presence of glucose after 24 h (a) and 48 h (b), while after 72 h (c) pMSCs significantly increased HUVEC proliferation in presence of glucose, as compared to glucose-treated but not untreated HUVECs. HUVEC proliferation after removing effects of glucose and pMSCs. HUVECs initially cultured with 100 mM glucose (100(pre)) in presence of different treatments of pMSCs (CMpMSC(PreCM + 100) and ICpMSC(PreCM + 100)) for 72 h, and then used in proliferation assay using xCELLigence real-time cell analyser. After 24 and 48 h (d, e), proliferation of HUVECs pretreated with glucose alone (100(pre)), or with CMpMSC (PreCM + 100) or ICpMSC (PreCM + 100), did not significantly change as compared to glucose-untreated HUVEC (p > 0.05). As compared to glucose-treated HUVECs (100(pre)), proliferation of HUVECs pretreated with glucose and CMpMSC (PreCM + 100), or glucose and ICpMSC (PreIC + 100), did not significantly change after 24 and 48 h (p > 0.05) (d, e). In contrast, proliferation of HUVECs pretreated with 100 mM glucose alone (100(pre)), or with CMpMSC (PreCM + 100), significantly reduced after 72 h as compared to glucose-untreated HUVEC (f). When compared with glucose-treated HUVECs (100(pre)), proliferation of HUVECs pretreated with glucose and CMpMSC (PreCM + 100) did not significantly change after 72 h of culture. In contrast, proliferation of HUVECs pretreated with glucose and ICpMSC (PreIC + 100) increased significantly after 72 h of culture as compared to glucose-treated but not untreated HUVECs (f). Each experiment performed in triplicate using HUVECs (passage 3–5) and pMSCs (passage 2) from five independent umbilical cord tissues and placentae, respectively. *P value is significant p < 0.05. Bars represent standard errors. pMSC placental mesenchymal stem cell