FUNCTIONAL CHARACTERIZATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS-DERIVED PITUITARY CELLS AFTER 60 DAYS OF MONOLAYER CULTURE

CHIRINO FELKER, GONZALO T.; ROMANO FLORIT, CAROLINA; PELANDA, MELINA; LAZZATI, JUAN MANUEL; AMIN, GUADALUPE; CASTAÑEDA SHEILA; WAISMAN ARIEL; MIRIUKA SANTIAGO; PEREZ-MILLÁN, MARIA INES; MORO LUCIA; CAMILLETTI, MARIA ANDREA

Mar del Planta

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL 2023; 2023

SOCIEDAD ARGENTINA DE INVESTIGACION CLINICA

Human induced pluripotent stem cells (hiPSCs) are able to differentiate into any cell type of the body. Ongoing research with hiPSCs-derived pituitary cells in 2D and 3D organoids holds promise for the modeling of hormonal deficiencies. In an earlier study, we adjusted the 2D protocol, guiding iPSCs into pituitary progenitors over 40 days. To cut costs, we optimized by switching to SB4, an affordable analog of BMP4, and substituting FGF8 and FGF10 with recombinant FGF2. Both hiPSCs cultures treated according to the original protocol and those following the adapted version exhibited a pattern of gene expression consistent with adenohypophyseal lineage differentiation and loss of pluripotency. Based on these findings, this study aimed to continue characterizing the adapted protocol over the days previously studied and extending differentiation to day 60, testing various cell culture strategies to replating cells on day 15. At the end of the 60-day protocol, RNA samples were collected for qRT-PCRs and secreted media was separated for hormonal determinations. Preliminary results show that day 60 differentiated cells express the hormonal transcripts GH1, PRL, LHB, and POMC in both protocols. Also, human GH and ACTH levels were measured in Immulite 2000 XPi - Siemens by the chemiluminescence immunoassay and found detected in 60-day cultures media. Furthermore, the gene expression of the hypothalamic development marker RAX was studied during cell differentiation using qRT-PCR. The expression of RAX was increased between days 4 and 15 of the conducted differentiation. In summary, while efficiency is moderate, our results suggest that the adapted protocol may enable the generation of functional pituitary cells in vitro. This model has the potential for research in human pituitary development along with the possibility of studying novel disease genes and genetic variants involved in pituitary disorders.