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

GONZALO TOMÁS CHIRINO FELKER; CAROLINA ROMANO FLORIT; MELINA PELANDA; JUAN MANUEL LAZZATI; GUADALUPE AMIN; SHEILA CASTAÑEDA; ARIEL WAISMAN; SANTIAGO MIRIUKA; MARÍA INÉS PÉREZ MILLÁN; LUCÍA MORO; MARÍA ANDREA CAMILETTI

Congreso; LXVIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2023

Human induced pluripotent stem cells (hiPSCs) are able to differentiateinto any cell type of the body. Ongoing research with hiPSCs-derived pituitary cells in 2D and 3D organoids holds promisefor the modeling of hormonal deficiencies. In an earlier study, weadjusted the 2D protocol, guiding iPSCs into pituitary progenitorsover 40 days. To cut costs, we optimized by switching to SB4, anaffordable analog of BMP4, and substituting FGF8 and FGF10 withrecombinant FGF2. Both hiPSCs cultures treated according to theoriginal protocol and those following the adapted version exhibiteda pattern of gene expression consistent with adenohypophyseal lineagedifferentiation and loss of pluripotency. Based on these findings,this study aimed to continue characterizing the adapted protocolover the days previously studied and extending differentiationto day 60, testing various cell culture strategies to replating cellson day 15. At the end of the 60-day protocol, RNA samples werecollected for qRT-PCRs and secreted media was separated for hormonaldeterminations. Preliminary results show that day 60 differentiatedcells express the hormonal transcripts GH1, PRL, LHB, andPOMC in both protocols. Also, human GH and ACTH levels weremeasured in Immulite 2000 XPi - Siemens by the chemiluminescenceimmunoassay and found detected in 60-day cultures media.Furthermore, the gene expression of the hypothalamic developmentmarker RAX was studied during cell differentiation using qRT-PCR.The expression of RAX was increased between days 4 and 15 of theconducted differentiation. In summary, while efficiency is moderate,our results suggest that the adapted protocol may enable the generationof functional pituitary cells in vitro. This model has potential forresearch in human pituitary development along with the possibilityof studying novel disease genes and genetic variants involved inpituitary disorders.