Early-Stage Expression of VE-Cadherin During Endothelial Differentiation of Mesenchymal Stem Cells Using SPR Biosensor

Background and Aim: The mesenchymal-to-endothelial transition isan essential phenomenon during tissue regeneration, leading to anincreased vascular density in target tissues. Thus, the development ofhighly sensitive and accurate biosensor approaches for detecting stemcells differentiation, particularly in the earliest steps, is more crucial thanever. In the current study, a label-free SPR (surface plasmon resonance)method was developed for the monitoring of the early-stage differentiationof mesenchymal stem cells into endothelial-like phenotype in term of VEcadherinover a period of 14 days.Methods: Human amniotic mesenchymal stem cells (hAMSCs) line (CatNo: C10680) was obtained from Iranian Biological Resource Center.Low glucose-DMEM (DMEM/LG, Gibco) cell media were used for thecultivation of hAMSCs. To induce endothelial differentiation, hAMSCswere maintained in endothelial cell growth media, M-199, supplementedwith an EGM-2 cocktail (Cat No: C-22010, Promocell) and 2% fetalcalf serum (FCS, Promocell) for 14 days. The medium was replenished every 2 to 3 days. The differentiation of hAMSCs into endothelial-likephenotype was investigated by SPR biosensor method and also with flowcytometry analysis and immunofluorescence microscopy on days 1, 2,3, 5, 7 and 14. The morphological changes in relation to the endothelialacquisition were monitored through the experiment. A multi-parameterSPR device (MP-SPR Navi 210A, BioNavis Ltd, Tampere, Finland) withgold chips (BioNavis Ltd, Finland) was used to examine antibody-cellaffinity interactions.Results: The combination of SPR method and Ab immobilization on-chipresulted in early detection of VE-cadherin as a cell surface marker inthe endothelial differentiation of hAMSCs. Consistent with endothelialdifferentiation of SCs, a high level of VE-cadherin was encoded on the cellsurface resulting in an increased attachment of the cells to the immobilizedantibody on the chip surface. This attachment, in turn, caused profoundchanges in the refractive index values in the detection region which werereflected as increased RU intensities during differentiation stages. Aftersubtracting the nonspecific binding responses, we recorded 0, 80, 120,360, 510 and 610 RUs×10-4 for differentiating hMSCs following 1, 2, 3,5, 7 and 14 days, respectively. The flow cytometric method was unable todiscriminate the hAMSCs expressing VE-cadherin during the first 4 daysof the differentiation, especially on day 0, 1 and 2, toward endotheliallineage.Conclusion: In the present study, SPR technique could sense the earlystage differentiation of hAMSCs on day 3 in real-time and label-freeform without affecting cell viability, but flow cytometry and fluorescentmicroscopy methods were not able to detect the cell differentiation at thesame time. This sensitive method presents hopeful views for monitoringand identification of rare and specific cell populations like tumor cells,cancer stem cells and etc.

کلیدواژه ها:

Human amniotic mesenchymal stem cells ، SPR Biosensor ، Vecadherin