Bidirectional fibrogenic cross-talk revealed in a human iPSC-derived epithelial-mesenchymal co-culture model of pulmonary fibrosis

  • bioRxiv. 2026 Jan 31:2026.01.30.702837. doi: 10.64898/2026.01.30.702837.
Andrea B Alber  1  2 ,  George Kwong  1  2 ,  Vishal K Gupta  1  2 ,  Porter E Dooley  1  2 ,  Jill R Patel  1  2 ,  Pushpinder S Bawa  1 ,  Kasey Minakin  1  2 ,  Dakota Jones  1  2 ,  Diya Gopal  1  2 ,  Huan Souza  3 ,  Maria Yampolskaya  3 ,  Eitan Vilker  1  2 ,  Chandani Sen  4 ,  Ansley S Conchola  5 ,  Pankaj Mehta  3 ,  Brigitte N Gomperts  4 ,  Tristan Frum  5 ,  Jason R Spence  5 ,  Konstantinos-Dionysios Alysandratos  1  2 ,  Darrell N Kotton  1  2
Affiliations
  • 1. Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA.
  • 2. The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
  • 3. Department of Physics, Boston University, Boston, MA 02215, USA.
  • 4. Department of Pediatrics, David Geffen School of Medicine, UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital, University of California, Los Angeles, CA 90095, USA.
  • 5. Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
PMID: 41659605 DOI: 10.64898/2026.01.30.702837
Abstract

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 SFTPCI73T 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 TGFβ, multiple integrins, and additional genes that have not been previously linked to PF. Finally, we show that small molecule-mediated inhibition of TGFβ or integrins αvβ1/αvβ6 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.

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