Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
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Research Article Development Endocrinology

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells

Abstract

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone–producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Authors

Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi

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Figure 4

Decreased expression of hypothalamic FGF10 was responsible for the impairment of LHX3 expression in OTX2mut-iPSCs.

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Decreased expression of hypothalamic FGF10 was responsible for the impai...
(A) Flow cytometry analysis for E-cadherin in control-iPSC–derived aggregates (day 16). FACS-sorted E-cadherin– and E-cadherin+ cells were defined as hypothalamus and oral ectoderm cells, respectively. (B) Quantitative RT-PCR of OTX2 in FACS-sorted hypothalamus and oral ectoderm (day 16–30). OTX2 expression was higher in hypothalamus than in oral ectoderm. Data show mean ± SEM; n = 3 per group. **P < 0.01, Student’s t test (unpaired, 2-tailed). (C) Coculture of GFP-labeled control-iPSCs and OTX2mut-iPSCs produced chimeric aggregates. (D) The chimeric aggregates were induced for pituitary differentiation. We observed 4 combinations of hypothalamus and oral ectoderm according to the origin. The LHX3 expression in oral ectoderm was observed only in the presence of control-iPSC–derived hypothalamus. (E and F) Quantitative RT-PCR and immunostaining of FGF8 and FGF10 in iPSC-derived tissues. The expression of FGF8 and FGF10 was decreased at both the mRNA and the protein level in the hypothalamus of OTX2mut-iPSCs (day 40). Representative data from 2 independent experiments are shown. Data show mean ± SEM; n = 3 per group. *P < 0.05, Student’s t test (unpaired, 2-tailed). (G and H) Quantitative RT-PCR and immunostaining of LHX3 in iPSC-derived tissue treated with or without recombinant FGF8 and FGF10. FGF8 (100 ng/mL) and/or FGF10 (50 ng/mL) were added in culture medium from day 9 to day 40. Not FGF8 but FGF10 treatment restored LHX3 expression in OTX2mut-iPSC–derived oral ectoderm at both the mRNA and the protein level (day 40). Representative data from 2 independent experiments are shown. Data show mean ± SEM; n = 3 per group. *P < 0.05, 1-way ANOVA (F = 7.67, df = 3, P < 0.01) followed by post hoc Dunnett’s test. Post hoc comparison with control-iPSCs no. 1 (far left) is presented. O, oral ectoderm; H, hypothalamus progenitor. Scale bars: 100 μm.