Background: Intractable pulmonary diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are characterized by irreversible damage to alveolar structures, and there is currently no cure with standard therapies. Stem cell-based regenerative approaches offer promising alternative treatments for these conditions. Human amniotic epithelial cells (hAECs), which exhibit pluripotent-like differentiation potential and are non-tumorigenic, have emerged as a clinically relevant stem cell source.

Objective: This study aimed to induce the differentiation of hAECs into alveolar type 2 (AT2) cells via the forced expression of a single transcription factor.

Methods: We first prepared a lentiviral reporter vector in which green fluorescent protein (GFP) expression is driven by the human surfactant protein C (SFTPC) promoter. Among several candidate transcription factors, NKX2-1 was selected and cloned under the CMV promoter in a lentiviral vector. Primary hAECs were transduced with the SFTPC-GFP reporter and, after four days of culture, subsequently transduced with NKX2-1.

GFP expression began five days post-transduction and reached 10.2 ± 2.3% (mean ± SEM, n = 4) by two weeks. GFP-positive cells were isolated by FACS, and upregulation of NKX2-1 and SFTPC mRNA expression was confirmed by RT-qPCR. SFTPC protein expression was also verified by western blotting.

Results: Comprehensive gene expression analysis using RNA sequencing revealed upregulation of additional surfactant-related genes and those involved in surfactant secretion, indicating differentiation into AT2-like cells. Gene Set Enrichment Analysis (GSEA) demonstrated significant enrichment in surfactant metabolism pathways, and meta-analysis showed gene expression profiles similar to human AT2 cells. Functional assays confirmed the presence of lamellar bodies and uptake of phosphatidylcholine, further supporting the AT2-like function of these cells.

Finally, AT2-like cells derived from hAECs were transplanted into a decellularized rat lung scaffold using a bioreactor system. After one week of culture, successful engraftment into the alveolar structure was observed.

Conclusion: These results demonstrate that forced expression of NKX2-1 in hAECs is sufficient to induce AT2-like cells with molecular and functional characteristics. This strategy lays the foundation for the development of small-scale human lung bioreactors and clinically applicable AT2 cell generation using small molecules.