(590) Airway epithelial stem cell phenotype and function are regulated by culture condition

Background: The Pathway to a Cure initiative includes a cell-based therapy approach. Although advances in CFTR gene editing support the development of autologous airway epithelial stem cell therapy as a curative treatment for Cystic Fibrosis (CF) lung disease, existing data indicate that gene corrected cells will need to be amplified in vitro prior to transplantation. Since development of a Good Manufacturing Practice (GMP) cell amplification protocol is time and labor intensive, and financially demanding, optimal cell culture methods should be identified prior to protocol development. This goal was addressed by amplifying human basal cells using commonly used media and methods and comparing their phenotype, function, and therapeutic potential.

Methods: Human bronchial basal cells were recovered from 3 non-CF donors, cultured using the modified conditional reprograming culture (mCRC) and Pneumacult (PC) methods, and treated with vehicle or dual SMAD inhibition (DSI). Cell phenotype was assayed using flow cytometry and/or single cell RNA sequencing (scRNAseq). Differentiation potential was assayed following culture at the air-liquid-interface (ALI). Therapeutic potential was quantified using orthotopic transplantation of mCRC and PC-DSI amplified cells into the airways of naphthalene-injured NOD/SCID mice.

Results: Cell proliferation was similar under all test conditions. However, cell morphology and cell surface expression of canonical cell surface markers (NGFR, CD66, Factor3, and CD151) varied significantly across culture conditions. These phenotypic changes were reversible when the cells were returned to the original culture condition. Amplification in PC plus DSI significantly increased the rate of differentiation and the frequency of goblet and ciliated cells in ALI cultures. scRNAseq indicated that Factor3 defined multipotential and secretory primed basal cells and that PC plus DSI cultures contained fewer multipotential basal cells. Gene editing and transplantation studies indicated that Factor3 was necessary for successful repopulation of the conducting airway epithelium after transplantation.

Conclusions: Human bronchial basal cell phenotype and function is plastic and can be manipulated by culture condition. Basal cell application using the mCRC produces cells with optimal function as a cellular therapy. Development of GMP protocols for production of therapeutic basal cells that are amplified using the mCRC approach will need to overcome two limitations that are inherent to this method, use of xenobiotic feeder cells and serum.

Acknowledgements: Research Project funding from the CF Foundation, CF Foundation C3 RDP support of the Epithelial, Administrative, and Immune Cell Cores (donor identification, cell procurement and culture, flow cytometry), and the Nationwide Children’s Hospital CRISPR Core (gene editing).

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