FROM INDUCED PLURIPOTENT STEM CELLS TO LHX3+ PITUITARY CELLS: A PROMISING DIFFERENTIATION PROTOCOL FOR THE STUDY OF HORMONAL DEFICIENCIES OF GENETIC ORIGIN

CHIRINO FELKER GONZALO TOMAS; ROMANO FLORIT CAROLINA; AMIN GUADALUPE; CASTAÑEDA SHEILA; WAISMAN ARIEL; MIRIUKA SANTIAGO; PÉREZ MILLAN MARÍA INÉS; MORO LUCÍA; CAMILETTI MARÍA ANDREA

Mar del Plata

Congreso; LXVII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2022

Generation of patient-derived pituitary cells is a promising approachin modeling hormonal deficiencies. Since their discovery,human inducedpluripotent stem cells (iPSCs) obtained by cell reprogrammingof adult somatic cells,have revolutionized biomedical research.To date,only two approaches (two and three-dimensional) havebeen published for obtaining functional secretory pituitary cells fromiPSCs with an efficiency of about 20-30%. In this study,we adaptedthe two-dimensional protocol for differentiation of iPSCs to pituitarycells. Our previous results showed that culturing iPSCs in a cocktailof morphogens (BMP4, SAG and bFGF) induces increased expressionof pituitary-specific genes and loss of pluripotency markers after15 days compared to control iPSCs (without stimulation). Basedon these findings, iPSCs cultures were incubated for additionaldays (up to 40 days) and samples corresponding to several days(0,4,15,30 and 40) were collected for their characterization by qRTPCRand immunostaining. We observed a robust up-regulation of pituitary-like progenitor markers (PITX1,PITX2 and SIX6) and pituitaryplacode marker SIX1,accompanied by a downregulation of pluripotencymarkers OCT4 and NANOG, in treated cultures compared tocontrol iPSCs,from day 4 to 40 (N=4). Transcript levels of early pituitarymarkers HESX1 and OTX2 were found to increase at day 4 andthen decreased by day 15. Induction of other cell lineages was evaluated,however,no differences were found among the cultures,asdetermined by mesoderm (TBXT,TBX6),ectoderm (NESS,TUBB3)and endoderm (SOX17) lineage markers expression. Finally,immunodetectionof the transcription factor LIM Homeobox 3 (LHX3),anearly marker of pituitary lineage committed cells,revealed nuclearLHX3 expression in clusters in treated iPSCs at day 40. In summary,our findings suggest this methodology can be used to obtainpituitary progenitor LHX3+ cells in vitro from iPSCs,and has the potentialto,in the near future,serve as a model for human pituitary development.This is particularly relevant for hormonal deficiencies ofgenetic origin,as it will provide a possible tool to evaluate variants inearly genes involved in the formation of the anterior pituitary gland.