Design of experiments assisted optimization of induced Pluripotent Stem Cell (iPSC) differentiation into airway progenitors for a cell and gene therapy against primary ciliary dyskinesia - 09/05/26

Primary ciliary dyskinesia (PCD) is a rare 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 employing induced pluripotent stem cell (iPSC) derived NKX2.1+ airway progenitors (AP). A common bottleneck regarding iPSC differentiation is the emergence of unwanted cell types. In a therapeutic context it can reduce the therapy efficiency. iPSC differentiation into AP often leads to the coexistence of hepatic and pulmonary cell lineages. This well described phenomenon was observed in a previous single cell RNA sequencing analysis we performed on iPSC derived AP. To overcome this issue, most of the published protocols require a cell sorting (CS) step to isolate the AP. Nevertheless, airway extracellular markers are poorly described for this differentiation stage. Moreover, CS can damage the cells and represent a severe bottleneck in a GMP-compliant bioproduction. We are thus developing a CS-less iPSC differentiation protocol to obtain a homogenous AP population.

Design of experiments (DOE) is a statistical method allowing optimal value identification for each critical variable of a protocol, with a reduced number of experiments to perform. A DOE approach has been applied to optimize our differentiation protocol by modulating cytokines concentration and duration of exposure. Then, to further purify our AP, we added a single cell passaging step at a critical moment of the optimized protocol so that each cell can be surrounded for a while by our optimized cytokines cocktail without being influenced by neighboring cells signals.

The DOE approach allowed to increase 50 times NKX2.1 (AP marker) expression and to reduce drastically unwanted cell type markers’ expression, neural and hepatic markers expression are respectively 20 and 100 times lower in our resulting cell population compared to our standard protocol. The addition of the single cell passaging step to the optimized protocol allowed an additional increase of 250% of NKX2.1 expression in the resulting cell population. This protocol has been successfully tested on two different iPSC cell lines. The AP's ability to further differentiate into functional ciliated cells has also been confirmed.

The resulting progenitors will be characterized by bulk RNA sequencing. The engraftment ability of the AP also remains to be assessed in vitro on primary human bronchial epithelial cells and on human bronchial explants. Then, it will be tested in vivo in a Yucatan mini pig model.