OR14

Design of experiments assisted optimization of induced pluripotent stem cell (iPSC) directed differentiation toward airway progenitors for a cell and gene therapy against primary ciliary dyskinesia

A Coeur(1,2) C Bourdais(1) M Nadaud(2,3) F Foisset(1) C Urena(1) I Vachier(3) S Assou(1) A Bourdin(2,3) J De Vos(1)

1:IRMB, University of Montpellier, INSERM U1183, CHU Montpellier, Montpellier, 34295, France; 2:PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR9214 CHU Montpellier, Montpellier, 34090, France; 3:Department of Respiratory Diseases and Addictology, Hôpital Arnaud de Villeneuve, CHU Montpellier, France

Primary ciliary dyskinesia (PCD) is a genetic disease causing ciliary function impairment and bronchial mucus accumulation. The mucociliary function of PCD patients may be restored using an autologous cell and gene therapy using induced pluripotent stem cell (iPSC) derived NKX2.1+ airway progenitors (AP). Our standard differentiation protocol (STD) avoid cell sorting wich can damage the cells and be a severe bottleneck in a GMP-compliant bioproduction. STD consists in modulating pathways by introducing molecules in the culture medium only until the cells reach the definitive endoderm (DE) stage, followed by four days without cytokines. Some authors have documented that double inhibition of TFG-B and BMP pathways after the DE stage followed by activation of the RA, BMP and canonical Wnt pathways (DI protocol) can improve the differentiation process. Both protocols lead to the emergence of unwanted cell types such as hepatic progenitors. We improved our STD protocol by modulating the same pathways as DI protocol and optimizing molecules concentration and duration of exposure using a design of experiments (DOE) approach. This statistical method allows optimal value identification for each critical variable of a protocol, with a reduced number of experiments to perform. Compared to STD, the resulting protocol shows 50 times more NKX2.1 (AP marker) expression while neural and hepatic markers expression are respectively 20 and 100 times lower. To eliminate remaining contaminants, transcription factor modulation will be performed during the last differentiation step of the optimized protocol. The differentiated progenitors’ homogeneity will be assessed by single cell RNA sequencing.