Figure 3.
Proliferation and evaluation of HLA-G expression and secretion of cultured BeWo Cells on the HA-MatrixDecidua containing AF/AM-MSCCO-EV with in vivo-like temperature change and circulation conditions. (a) A scheme of verification procedure for induction of immune-tolerized trophoblast cells (itTBCs) and immune-tolerized EVs (itTBC-EVs). (b) Microscope images of cultured BeWo cells on the HA-MatrixDecidua, at the 1st day (left) and 6th day (right) after cultivation. (c) A graph of 24-hour-cyclic vibration condition during BeWo cells cultivation to promote the circulation of various soluble factors between BeWo cells and the HA-MatrixDecidua. (d) Relative cell proliferation assay of BeWo cells cultured on the 2D-culture plate (Control), on the HA-MatrixDecidua without temperature/vibration conditions (Matrix Only), and on the HA-MatrixDecidua under temperature/vibration conditions (Matrix+T/V). Error bars represent SD. *, P < 0.05; Mann Whitney test. (E) Intracellular HLA-G mRNA isoforms expression of BeWo cells cultured on the HA-MatrixDecidua under temperature/vibration conditions (BeWoMatrix+T/V) was shown by RT-PCR analysis, with GAPDH as a positive control, using designed HLA-G primer set listed in Supplemental Table 1. All HLA-G mRNA isoforms (HLA-G1, G2, G5, and G6) were expressed. (F) FACS analysis of membrane-bound HLA-G expressions on the surface of BeWoControl, BeWoMatrix Only, and BeWoMatrix+T/V cells obtained at passage 12 using MEM-G/9 antibody. Two peaks in HLA-G positive BeWoMatrix+T/V cells were generated due to the more colony generation while more active cell proliferation in the ex vivo culture system (Supplemental Figure 4). (g) The concentrations of sHLA-G in the culture supernatant of BeWoMatrix+T/V and BeWoControl cells using MEM-G/9 antibody bound to sHLA-G1 and HLA-G5. (h) Specific proteins quantification contained in the culture supernatant of BeWoControl, BeWoMatrix Only, and BeWoMatrix+T/V by ELISA. Each EVs were obtained at passage 12 of each cultivation.