To assess the in vivo properties of hESC-derived trigeminal placode precursors, PIP-induced neuronal clusters, derived from a constitutively GFP-positive hESC line (Figures S5A and S5B), were injected into the developing chick embryo targeting the early trigeminal anlage at H&H stage 10–12 (Figure S5C). Human cells were identified based on GFP expression and use of human specific antibodies against cytoplasmic antigen (hCA). Two days after in ovo transplantation, surviving GFP+ cells were found dispersed in the area of the endogenous chick trigeminal ganglion (Figure 4P). We observed extensive GFP+ human fiber bundles coexpressing hCA and peripherin (Figures 4Q and 4R). In contrast, no hCA or peripherin expression was detected in the neural tube of the embryo (Figure S5D). The in vivo fiber outgrowth 2 days after transplantation was reminiscent of the extensive in vitro fiber outgrowth of replated trigeminal neuron clusters (Figure S5A). Peripherin expression in vivo (Figure 4S) confirmed the peripheral neuron identity of the grafted cells.
surviving GFP+ cells
dispersed
Two days
days
in vivo fiber outgrowth
2 days
days