GENE EXPRESSION PROFILE DURING PITUITARY DEVELOPMENT IN A TWO-DIMENSIONAL MONOLAYER DIFFERENTIATION PROTOCOL FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

CHIRINO FELKER GONZALO; MORO LUCIA; AMIN GUADALUPE; CASTAÑEDA SHEILA; MIRIUKA SANTIAGO; PEREZ-MILLÁN, MARIA INES; CAMILLETTI, MARIA ANDREA

Congreso; Reunion de Sociedades de Biociencias 2021; 2021

Multiple pituitary hormone deficiencies (MPHD) can be caused by mutations in several transcription factor genes in mouse and human, including Prop1, Pou1f1, Hesx1, Sox2 and other genes involved in early pituitary embryogenesis. In order to study this process of disease development, we aimed to generate a human in vitro model of embryonic pituitary as a robust tool for functional testing of genetic variants found in MPHD patients. We hypothesized that combining different stimuli and differentiation protocols in vitro [1], [2], it is possible to induce fully differentiated hormone-pituitary cells from human induced pluripotent stem cells (iPSCs). We cultured the iPSCs in the presence of signaling factors involved in pituitary development including Bone Morphogenetic Protein 4 (BMP4), the Smoothened Agonist activator of Sonic Hedgehog pathway (SAG), and Fibroblast Growth Factor 2 (FGF2), for 15 days. During the entire protocol, cell morphology was observed and registered under the microscope, and cellular extracts corresponding to days 0, 4, 7, and 15 of the protocol were collected to assess gene expression by qRT-PCR. We observed an increase in the expression of representative markers of pituitary differentiation (PITX2, SIX1, HESX1, OTX2) and a decrease in the expression of pluripotency markers (NANOG, OCT4) over the days of treatment compared to control iPSCs (cultured with media only), suggesting that the protocols were effective in their cell specification. An interesting finding was the increase in mRNA levels of FOXA2 at days 7 and 15 of the protocol, a novel gene in the etiology of MPHD, poorly characterized in human pituitary development and found mutated in one case of MPHD from our Argentinean cohort [3]. This study establishes a new approach to study protein?s role in pituitary progenitor cell regulation and offers new candidate genes for MPHD that remain unexplained.