(599) iPSC-derived airway basal cells that exhibit increased competence for multipotent differentiation

Background: Airway basal stem cells are capable of self-renewal and differentiation into specialized airway cell types, primarily ciliated and secretory cells, but also rarer cell types such as ionocytes (of great interest due to their high level expression of CFTR) and pulmonary neuroendocrine cells (PNECs). Deriving airway basal cells of such multipotency from induced pluripotent stem cells (iPSCs) has therapeutic potential, especially for CF patients, whose iPSCs, corrected of CFTR mutation(s), could be used to generate corrected airway basal cells for autologous transplantation and regeneration of functional airway epithelium. We have previously reported a protocol for the generation of iPSC-derived airway basal cells (iBCs) [1] and are using CF patient iBC-derived airway epithelium (generated in air-liquid interface [ALI] cultures) to examine patient specific responses to existing CFTR modulators. Despite similarities between iBCs and primary basal cells, crucial differences exist at the transcriptional and functional levels. For example, iBCs from our published protocol show limited multipotency, giving rise to ciliated and secretory cells, but few ionocytes and PNECs. Additionally, iBCs have low expression of KRT5, basal cell marker. Finally, iBC-derived ALI cultures are less stable than those derived from primary airway basal cells and lose basal cells over time. In the current study, we develop a novel iPSC directed differentiation protocol capable of generating iBCs that not only exhibit full competency for multipotential differentiation, but also address the aforementioned deficiencies of the published protocol.

Methods: We modified published protocols with the objective to fully recapitulate normal lung and airway development by modulating airway signaling pathways at specific timing and concentration. Using iPSCs carrying NKX2-1 and TP63 fluorescent reporters, we first derived NKX2-1+ lung progenitors and then used a revised step-wise protocol employing chemically defined media to derive airway progenitors and subsequentially basal cells expressing their characteristic markers (NKX2-1+, TP63+ and NGFR+). The resulting iBCs were further differentiated into airway epithelium through ALI culture. Immunostaining and single cell RNA sequencing were used to profile the airway epithelial cell types from the new ALI culture.

Results: In ALI culture, iBCs from our new protocol not only developed into ciliated and secretory cells, but also gave rise to abundant FOXI1+ BSDN+ ionocytes, as well as ASCL1+ PNECs. Interestingly, KRT5 expression, seen at a very low level in our published protocol, was high in both proliferating iBCs in 3D culture and in ALI. iBC-derived ALI cultures exhibited increased stability and retention of TP63+ cells, comparable to adult basal cell-derived ALI.

Conclusions: Here we show a strategy to derive higher competency iBCs that can give rise to multiple airway cell types including ionocytes. This should allow more accurate modeling of CF airway epithelium ex vivo, as well as development of more physiologically relevant cells for therapeutic transplantation in vivo. Future studies will focus on further molecular and functional analyses of iBCs generated with this protocol in comparison to basal cells freshly isolated from human lung tissues.

Acknowledgements: Supported by the NIH [R01 HL139876]; CFF [DAVIS17XX0, DAVIS20XX2].

References: [1] Hawkins, F. J., et al. Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells. CSC 2021; 28, 79–95. doi:10.1016/j.stem.2020.09.017