Human iPSC-derived lung mesenchyme and primary developing human lung mesenchyme

Darrell N. Kotton1, Konstantinos-Dionysios Alysandratos1, Andrea B. Alber1, Pushpinder Singh Bawa1
1Boston University, *Corresponding author
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Description Single-cell RNA sequencing of the directed differentiation of human iPSCs into lung mesenchyme, with primary fetal lung mesenchyme at four gestational days for comparison. Seven libraries, 14,494 cells, 12 Seurat clusters. Pulmonary fibrosis (PF) can arise from mutations in alveolar epithelial type 2 (AT2) cell-specific genes, but manifests in fibrotic activation of mesenchymal cells, thus involving fibrogenic epithelial-mesenchymal crosstalk. The ligand-receptor interactions underlying the onset and early progression of PF remain poorly understood. Induced pluripotent stem cell (iPSC)-derived models are powerful tools to study respiratory diseases, yet are currently limited to reductionist single lineage epithelial models or multi-lineage systems that lack purity and lung-specificity of the mesenchyme. Here we generate a human iPSC line carrying both a lung mesenchyme-specific reporter (TBX4-LERtdTomato) and a reporter for mesenchymal activation/differentiation (ACTA2GFP). Applying this line, we develop a directed differentiation protocol capable of generating cells that express key molecular and functional features of primary human developing lung mesenchyme across multiple iPSC genetic backgrounds. We then establish co-cultures of these iPSC-derived lung mesenchymal cells (iLM) with patient-specific iPSC-derived alveolar epithelial type 2 cells (iAT2s) carrying an SFTPC I73T mutation as a model for PF. We find increased expression of fibrotic markers in co-cultures with mutant iAT2s as compared to co-cultures with gene-corrected iAT2s. Moreover, mutant iAT2s express markers of alveolar-basal intermediate (ABI) cells only in the presence of iLM, suggesting that bidirectional crosstalk promotes this aberrant cell state. We identify ligand-receptor pairs enriched in co-cultures with mutant iAT2s, including TGFb, multiple integrins, and additional genes that have not been previously linked to PF. Finally, we show that small molecule-mediated inhibition of TGFb or integrins aVb1/aVb6 attenuates both fibrotic mesenchymal activation and the presence of ABI cells in iLM/iAT2 co-cultures. Thus, we have established a human iPSC-derived co-culture system that recapitulates key molecular hallmarks of bidirectional fibrogenic epithelial-mesenchymal crosstalk in pulmonary fibrosis, and enables the identification and study of potentially druggable pathways involved in disease initiation and progression.
Dataset ID LMEX0000090004
Assay Type Single-cell RNA-seq
Organism Human
Cell Count 14494
Technology 10x Genomics Chromium Single Cell Gene Expression
Reference Alber et al. (2023) | Alber et al. (2026)
FileDescriptionSize
sc.set2.RdsSeurat object for this study, the submitter's own file, 3,676,938,498 bytes. The RNA assay counts slot holds the raw integer counts every downstream artifact derives from. The default assay is SCT, whose corrected counts differ from the raw counts. Cell metadata carries orig.ident, the Seurat clusters and the module scores.3.42 GB
LungMAP IDAlt IDSpeciesSexAge Group
LMSPILM0000007D122PLMHomo sapiensUnknownUnknown
LMSPILM0000001D17TplusHomo sapiensUnknownUnknown
LMSPILM0000002D26TminusHomo sapiensUnknownUnknown
LMSPILM0000003D26TplusHomo sapiensUnknownUnknown
LMSPILM0000004D57PLMHomo sapiensUnknownUnknown
LMSPILM0000005D76PLMHomo sapiensUnknownUnknown
LMSPILM0000006D94PLMHomo sapiensUnknownUnknown