Figure 2. MTXr-teratoma formation during methotrexate administration.

Four million undifferentiated EFDIG- and CSIIEG-hESCs were injected into the left and right hind limbs of DHFR-BM transplanted NSG mice (n=8), respectively. One month after hESC injection, mice were treated 5–7 days/wk with PBS or MTX for one month and euthanized at day 60 post hESC-transplantation. (a) Endpoint teratoma diameter in PBS and MTX treated mice. CSIIEG teratomas trended toward growth inhibition in the presence of MTX. Teratomas were also evaluated for GFP marking by qPCR (bottom). The fold change in GFP copy number was calculated by dividing the mean number of copies per genome equivalent in the teratomas divided by the copies per genome equivalent in the input undifferentiated cell populations (b) Representative teratoma sections were stained with hematoxylin and eosin (H&E) to show the presence of germ layer cell derivatives including: Mixed (bone, pigmented epithelium, smooth muscle), Ectoderm, Mesoderm (cartilage), and Endoderm (gut-like structure surrounded by pigmented epithelium). Frozen teratoma sections were stained with DAPI and anti-GFP to detect transgene expression after multi-lineage differentiation. Left to Right: PBS/CSIIEG, PBS/EFDIG, MTX/CSIIEG, MTX/EFDIG, all stained with rabbit anti-GFP primary and FITC conjugated donkey anti-rabbit secondary antibodies.