Design of experiments assisted optimization of induced Pluripotent Stem Cell (iPSC) directed differentiation toward airway progenitors

Abstract

Primary ciliary dyskinesia (PCD) is a rare genetic disease causing ciliary function impairment and bronchial mucus accumulation among other comorbidities. We assume that the development of an autologous cell and gene therapy using iPSC derived airway progenitors (AP) could restore mucociliary function of PCD patients. Common bottlenecks regarding iPSC differentiation are the persistence of undifferentiated cells and the emergence of unwanted cell types. In a therapeutic context, it can cause uncontrolled cell proliferation or differentiation and reduce the therapy efficiency. iPSC differentiation into AP often leads to the coexistence of hepatic and pulmonary cell lineages. The emergence of hepatic progenitors occurs between the definitive endoderm (DE) and the anterior foregut endoderm (AFE) stages. This key differentiation step relies on the inhibition of the TGF-β and BMP pathways. In our standard protocol (STD), this is performed by removing all cytokines from the differentiation medium. Alternatively, a double inhibition (DI) can be realized by using inhibitors of both pathways.

A comparison of those two protocols (STD vs DI) showed that DI reduces pluripotency and liver markers expression, improving pulmonary marker expression. Nevertheless, DI leads to increased cell mortality. We chose to optimize DI protocol employing a design of experiments (DOE) approach. Besides, the effect of cell density at the critical step of the protocol has been investigated. To assess the efficiency of the iPSC differentiation, the relative expression of the main airway progenitor biomarker NKX2-1 has been measured by RT-qPCR as well as the expression unwanted cell-types markers. The viability of the cells in the end of the differentiation process has also been assessed.

The main effects shown by the screening DOE were caused by three variables linked to the transition from DE to AFE stages. Significant effects linked to the last step of differentiation have also been observed. The optimization DOE applied to the three most critical variables allowed significant modeling of the expression of several genes. The best conditions of the optimization DOE allow more than a 2-fold increase of the NKX2-1 expression compared to our STD protocol without compromising cell viability. We further optimize the protocol by passaging cells at a lower density once the AFE stage is reached, allowing a 10-fold increase of the NKX2-1 expression and an even lower expression of unwanted cell type markers.

The optimized protocol remains to be tested to assess the capacity of these NKX2-1+ lung progenitors to differentiate into bronchial epithelium. Furthermore, a single cell RNA sequencing analysis will be performed to fully characterize the homogeneity of the resulting cells.