Figure 5.

In Vivo Vasculogenesis Assay for hiPSC-ECs^WT and hiPSC-ECs^{SERPINE1-FOSB (D3)}

(A and C) H&E and human CD31 staining of FFPE tissue from the Matrigel Plug harvested after 4 (A) and 16 (C) weeks. Both hiPSC^WT and hiPSC^{SERPINE-FOSB (D3)} show vessel formation. Scale bar indicates 100 μm.

(B and D) Vessel density was estimated by quantification of the human CD31⁺ area at 4 and 16 weeks. The 4- and 16-week time points showed no significant difference in human CD31⁺ area. Error bars are shown as SDs; ns, not significant.

(E and F) Double immunofluorescent staining with FOSB and human CD31 antibodies counterstained with DAPI on cryosections from Matrigel Plug Assay containing hiPSC-ECs^WT and hiPSC-ECs^{SERPINE-FOSB (D3)}. FOSB is shown in red, CD31 in green, and DAPI in blue. The left panel shows the hiPSC-ECs^WT experiment, and the right panel shows the hiPSC-ECs^{SERPINE-FOSB (D3)} experiment. Cyan-colored objects represent erythrocytes fluorescing in multiple detection channels. Scale bar indicates 100 μm.

(G) FOSB immunohistochemistry (IHC) on FFPE tissue from Matrigel Plug with hiPSC^{SERPINE-FOSB (D30)} ECs. Scale bar, 50 μm. Surrounding mouse muscle (indicated by M) and the Matrigel Plug (indicated by P). (F) and (G) show the invasion of FOSB⁺ hiPSC-ECs^{SERPINE1-FOSB (D3)} into the striated muscle at 16 weeks post-transplantation.

(H and I) PTAH-stained sections from the in vivo vasculogenesis assay. hiPSC-ECs^WT (left panel) and hiPSC-ECs^{SERPINE-FOSB (D3)} (right panel) are shown. Both images show the surrounding mouse muscle (indicated by M) and the Matrigel Plug (indicated by P). Vessels with and without thrombi are indicated by red and green arrows, respectively (H), and red and green stars, respectively (I). The black arrows indicate areas with infiltration in the mouse muscle. The scale bar indicates 50 μm.

(J) Quantification of vessels containing PTAH⁺ thrombi in hiPSC-ECs^WT and hiPSC-ECs^{SERPINE-FOSB (D3)}, in an area of 5.7 mm². Error bars are shown as SDs; n = 3 and p = 0.1.