Opposing roles for TGFβ- and BMP-signaling during nascent alveolar differentiation in the developing human lung

Abstract

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFβ- and BMP-signaling, where inhibition of TGFβ- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFβ-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFβ- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

TSPYL1 as a Critical Regulator of TGFβ Signaling through Repression of TGFBR1 and TSPYL2

“…We have investigated whether there is perturbed TGFβ signaling in Tspyl1 KO lungs as proper TGFβ and BMP signaling is important in alveolar development. [ 30 ] We found initially up and then down‐regulation of Tgfbr1 and Smad7 transcripts in the mutant lung. This could be due to the presence of heterogeneous cell types and a complex in vivo environment that encompasses various factors such as ligand availability, signaling cross‐talks, and compensatory mechanisms to modulate the cellular response to the absence of Tspyl1 .…”

Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels

“…Thus, we compared the transcriptional and protein expression of common AT2 cell identity markers in Matrigel and HA hydrogels. We first assessed expression of canonical AT2 genes in alveolar organoids in Matrigel and HA hydrogels using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Frum et al, 2023; Herriges and Morrisey, 2014). No significant differences were observed for the expression of the lamellar body marker LAMP3 and surfactant protein B ( SFTPB ) ( Figure 2A ).…”

“…Given that gene and protein expression are not always correlated (Cote et al, 2016), we next sought to confirm maintenance of AT2 identity at the protein level through immunofluorescence staining of selected AT2 proteins (Beers and Moodley, 2017; Frum et al, 2023; Jacob et al, 2017; Sucre et al, 2018; Wang et al, 2007). First, we stained embedded alveolar organoids for SFTPC, SFTPB, and SFTPA and observed the expected punctate staining patterns both in Matrigel and HA hydrogels ( Figure 2D ).…”

“…Several lung organoid models, derived from primary cells or induced pluripotent stem cells (iPSCs), have been developed to study the proximal airways and distal alveoli (Barkauskas et al, 2013(Barkauskas et al, , 2017Dye et al, 2015;Ekanger et al, 2022;Frum et al, 2023;Gotoh et al, 2014;Hein et al, 2022;Miller et al, 2018Miller et al, , 2019Miller et al, , 2020Rock et al, 2009). Recently, iPSC-derived alveolar type II (AT2) organoids have emerged as a powerful tool to study alveolar homeostasis and disease such as SARS-CoV-2 (Huang et al, 2020;Hurley et al, 2020;Jacob et al, 2017;Mirabelli et al, 2021).…”