Basal cells (BCs) can serve as precursors for essential and specialized epithelial cell types including secretory cells and multiciliated cells. BCs differentiated into pseudostratified airway epithelium in air-liquid interface culture can recapitulate aspects of in vivo airway biology. This can help scientists gain an understanding of acquired and genetic human airway diseases including COVID-19, influenza, asthma, and cystic fibrosis.

iPSCs are the master stem cells that can produce any cell or tissue in the human body. Researchers from the Center for Regenerative Medicine at Boston Medical Center (CREM) and Boston University, in collaboration with the University of Texas Health Science Center at Houston (UTHealth), sought to develop basal cells programmed directly from patient-specific iPSCs that can be used to model airway diseases and find leading candidates for cell-based therapies designed to reconstitute the airway epithelium.

"Simply put, we have developed a way to reproduce patient-specific airway basal cells in the lab, with the ultimate goal of being able to regenerate the airway for patients with airway diseases," said first author Finn Hawkins, MBBCh, physician-scientist at Boston Medical Center and principal investigator in the CREM and the Pulmonary Center, in a statement.

Creating iPSC derived basal cells

The researchers used a dual-fluorescent reporter system to track and purify cells that emerge as developmentally immature lung progenitors and subsequently augmented the cells during proximal airway epithelial patterning to create iPSC-derived basal cells (iBCs or ibasal cells).

The researchers manipulated the iPSCs in a series of steps aimed to simulate what happens during lung development. During the emergence of the earliest detectable lung epithelial program, rare tumor protein 63 (TP63)+ cells are detected. Using single-cell RNA sequencing, the team demonstrated that the cells also express NK2 homeobox 1 (NKX2-1), the earliest transcriptional regulator in lung cells.

Then, TP63 and secretoglobin Family 3A Member 2 (SCGB3A2) are upregulated in the presence of basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10). In response to BC medium and with inhibition of SMAD signaling, the NKX2-1+/TP63+ cells adopt molecular and functional phenotypes similar to adult basal cells, including the capacity for extensive self-renewal for over 150 days in culture and multilineage airway epithelial differentiation in vitro and in vivo.

To visualize the cells, the researchers used a green fluorescent protein reporter targeted to the endogenous NKX2-1 locus. The resulting iBCs express the cell surface marker nerve growth factor receptor (NGFR) that enables their purification by flow cytometry. Once purified based on NGFR, the cells can be subsequently expanded using 3D culture and are cryopreserved for later directed differentiation for a variety of in vitro and in vivo applications.

Modeling airway diseases

The iBCs and differentiated progeny were used to model perturbations that characterize acquired and genetic airway disease, including mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

"These results could lead to a better understanding, and therefore treatments for, a variety of airway diseases," said Shingo Suzuki, PhD, co-first author and postdoctoral researcher at UTHealth. "If we could make pluripotent stem cells using a sample from a patient who has cystic fibrosis, correct the mutation and replace the defective airway cells with corrected airway basal cells that are otherwise genetically identical, we might eventually be able to cure the disease, and other diseases in the future using this same technology."

The researchers said that the study shows the potential use of ibasal cells.

"We demonstrated the potential of these ibasal cells to model both human development and disease, providing evidence of their capacity to regenerate airway epithelium," Hawkins said. "We expect this will be a significant breakthrough and will contribute to new insights and treatment options for airway diseases, as our results have overcome several important hurdles currently limiting progress in the field."

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