iPSC-Derived Lung Organoids & Airway Epithelium

Definitive Endoderm to NKX2.1+ Lung Progenitors

Following the Hawkins/Kotton-style protocol, iPSCs are committed to definitive endoderm with Activin A, ventralized to anterior foregut by dual-SMAD inhibition, and patterned to NKX2.1+ lung-fated progenitors with FGF7, FGF10, BMP4, and retinoic acid. Sorting on CD47^hi/CD26^lo isolates lung progenitors that further differentiate into distal SFTPC+ alveolospheres (3D Matrigel) or proximal multiciliated airway epithelium (air-liquid interface).

1. Definitive endoderm (Day 0–3)

   High-density iPSCs in RPMI + Activin A + CHIR (Day 1 only); validate >80% SOX17+/FOXA2+.

2. Anterior foregut (Day 3–6)

   Switch to SFD basal medium with dual-SMAD inhibition: Dorsomorphin (2 µM) + SB431542 (10 µM).

3. Lung patterning (Day 6–15)

   Switch to FGF7 + FGF10 + BMP4 + RA in CBFRA medium for ~10 days. Quantify NKX2.1+ progenitors.

4. Progenitor enrichment (Day 15)

   FACS sort on CD47^hi/CD26^lo for high-purity NKX2.1+ lung progenitors (alternatively use NKX2.1^GFP reporter line).

5. Distal: alveolospheres

   Embed sorted progenitors in Matrigel droplets; mature in CHIR + KGF + dexamethasone + cAMP + IBMX (CK+DCI). SFTPC+ alveolospheres by Day 35–45.

6. Proximal: ALI airway

   Plate progenitors on Transwell inserts; expand submerged in PneumaCult-Ex Plus, then ALI for 3+ weeks. Validate beating cilia (FOXJ1+, video) and goblet cells (MUC5AC).

Distal Alveolar Type-2–Like Cells

Alveolospheres self-organize into spherical cysts of pro-SFTPC+/SFTPB+/ABCA3+ AT2-like cells with apical lumens. Lamellar bodies are visible by EM, and surfactant secretion can be quantified by ELISA. These structures permit modeling of surfactant deficiency (SFTPC mutations), IPF (telomere-dysfunction lines), and viral infection.

ACE2 / TMPRSS2 Entry & Antiviral Profiling

iPSC-derived AT2-like cells express ACE2 and TMPRSS2 and are productively infected by SARS-CoV-2 spike-pseudotyped lentiviruses and authentic virus (BSL-3). I have used analogous systems (Calu-3 epithelial cells) for spike-uptake assays and antiviral entry-inhibitor screens. See related work on the endocytosis page.

Surfactant Deficiency & Fibrosis Modeling

SFTPC^I73T mutant alveolospheres show ER stress, impaired surfactant trafficking, and proteostatic collapse, modeling familial IPF. Co-culture with iPSC-derived mesenchyme in cytokine-driven fibrotic medium (TGFβ, IL-13) recapitulates myofibroblast activation and ECM deposition for anti-fibrotic compound screens.

Identity Confirmation — Lung Lineages

NKX2.1+ progenitor purity is monitored by flow cytometry (or via the NKX2.1^GFP reporter line); distal alveolosphere identity is confirmed by SFTPC/SFTPB/ABCA3, and proximal airway identity by FOXJ1 (multiciliated) and MUC5AC (goblet).