Induced pluripotent stem cell derived alveolar type 2 cells as a model for testing novel gene therapies for interstitial lung diseases

Objectives:

Advances in protocols for directed differentiation of induced pluripotent stem cells (iPSC) to alveolar type 2 cells (iAT2) provide an important opportunity for researchers to access an unlimited source of cells that recapitulate the key transcripts and functions of primary AT2 cells. To facilitate development of viral vectors for AT2-related lung diseases, we examined adaptation of these 3D cultures to air-liquid interface (ALI).

Methods:

A dual reporter iPSC line (BU3 NKX2-1GFP/SFTPCtdTomato) was differentiated towards distal lung cell fate using published protocols (Jacob et al. 2019; PMID:31732721). On day 29, iAT2 cells were dissociated from 3D spheres to single cell suspension and seeded on transwell inserts. Since modulation of CHIR99021 is known to mature the AT2 cell phenotype, the cultures were either (i) maintained in regular media (CHIR99021, KGF, dexamethasone, cAMP, IBMX) before airlifting, or (ii) CHIR99021 was removed from the media for 5 days followed by add-back for 24 hr, then airlifted. Gene expression was quantified by TaqMan assay.

Results:

Day 29 differentiated iAT2 3D cultures expressed upregulated levels of AT2-associated genes SFTPA2, SFTPB, SFTPC, ABCA3, LAMP3 and NAPSA, relative to undifferentiated iPSC. Significantly higher levels of AT2 genes were expressed in ALI cultures relative to 3D cultures; this was further enhanced by removal of CHIR99021 from the media for a 5 day period pre-airlift.

Conclusions:

iAT2 showed increased expression of key AT2-associated genes when cultured at ALI. Studies are underway to evaluate the transduction of iAT2-ALI cultures with lentiviral vectors to advance gene therapy for genetic lung diseases.