FIG. 2.

In vivo bioluminescent imaging (BLI) of transplanted mESCs. (A) Representative BLI images of animals following intramuscular (gastrocnemius muscle) transplantation of mESCs show a decrease in BLI signal in allogeneic animals (BALB/c), as opposed to syngeneic (129/Sv) mice, reaching background levels between post-transplant Days 21 and 28. Color scale bar values are in photons/sec/cm²/steridian (p/s/cm²/sr). (B) Graphical representation of longitudinal BLI after mESC transplantation into syngeneic (129/Sv, n = 8) and allogeneic (BALB/c, n = 8) mouse strains. Note that in allogeneic recipients, BLI signals decreases to background levels over the course of 28 days, whereas BLI signals in the syngeneic recipients increase over time, suggesting mESC proliferation. Data points represent mean ± SD; *p < 0.01, **p < 0.001 (by t-test). Dotted line = background BLI signal. (C) Ex vivo images of harvested muscles (left panels) show intramuscular tumors in syngeneic, as opposed to allogeneic muscles. Corresponding H&E-stained tissue sections (right panels) show presence of teratoma in syngeneic muscles, whereas no signs of teratoma formation are observed in allogeneic muscles. Black arrow: Osteoid differentiation (mesoderm); White arrow: glandular differentiation (endoderm); Black arrow head: neural differentiation (ectoderm). Scale bars: 100 μm. (D) Immunofluorescent staining of allogeneic transplants reveals severe infiltration of CD45⁺ cells (red) surrounding eGFP⁺ mESCs (green). Nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars: 100 μm. (E) To quantify subpopulations of graft infiltrating inflammatory cells, flow cytometry analysis of enzymatically digested muscles was performed at Day 10 following transplant. Graft infiltration of CD3⁺ (T-cells), CD4⁺ (T-helper cells), CD8⁺ (cytotoxic T-cells), Mac-1⁺Gr-1⁻ (macrophages), and Mac-1⁺Gr-1⁺ (neutrophils) was observed. Note that CD3⁺ and CD8⁺ cells infiltrated the allogeneic grafts at a significantly higher rate than the syngeneic grafts (n = 8 per group). Bars represent mean ± SD; *p < 0.05 (by t-test).