Figure 6. Specification and functional characterization of hormone-producing pituitary placode derivatives (see also Figure S7).

A) Schematic illustration of normal pituitary lineage development in vivo (Tabar, 2011). Examples of hormone producing cells generated using our modified PIP are listed in bold (ACTH, GH, FSH). B) Treatment with SHH from day 7–11 of PIP differentiation induced PITX1 and SIX6 expression as assessed by qRT-PCR. PITX1 and SIX6 mark the pituitary anlage. Low SHH: 20ng/ml C25II SHH; high SHH: 100ng/ml C25II SHH + 1μM puromorphamine. C) Immunocytochemical analysis showed expression of SIX6 at the protein level in a subset of clusters in the presence of SHH treatment. D) Immunocytochemical analysis for expression of LHX3 at day 16 (upon SHH treatment). E–G) Induction of defined endocrine precursor lineages: E) TBX19 expression was highly induced by day 20. F) PIT1 expression at day 20 and 32 of differentiation (see Figure S7A for treatment paradigm). G) GATA2 expression at day 20 and 32 of differentiation. H–L) Immunocytochemical evidence of hormone production H) CGA expression was readily detected by day 16 in SHH-treated PIP cultures. I) FSH, was expressed by day 27. J) ACTH expression was most abundant in SHH-treated PIP culture by day 30 of differentiation. K) GH expression at day 30 of differentiation. L) ELISA measurement of in vitro hormone production after 10 min exposure in HBSS. M) Schematic illustration of transplantation paradigm in nude rat host. N) ACTH plasma levels in grafted adult nude rats and sham-grafted controls at 4 and 6 weeks after transplantation. O) GH plasma levels using a human specific ELISA (6 weeks after transplantation). P–R) Histological analysis 6 weeks after transplantation: P) Robust survival of hNCAM⁺ human cells. Q) ACTH expressing and (R) GH expressing cells in vivo. Scale bars are: 100 μm in (C, P), 50 μm in (D, H, I, J, Q, R, S) and 10 μm in (K). Error bar represents SEM (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 compared with controls PIP condition in (B), hESC in (E, F, G; n = 3 independent experiments), HBSS without cells in (L), and plasma samples from matrigel-only injected (Sham) animals in (N, O; n=3 and n=5 animals).