Interstitial lung disease (ILD) is characterized by a progressive destruction of the lung parenchyma driven by complex interactions between epithelial, stromal and immune cells. To understand the cellular programs and interactions involved in ILD, we performed single nucleus and spatial transcriptomic analyses of lung tissues from ILD patients or comparators. The samples were obtained through the “Transplant Lung” and “Lung Tissue” biorepositories at Mass General Brigham from a total of 28 unique individuals. For single nucleus analyses, frozen samples were dissociated in detergent-salt-Tris buffers and nuclei were loaded on a 10x Chromium controller and libraries were generated with the 3'v3 Chemistry pipeline before sequencing on an Illumina sequencer. The resulting sequence data was demultiplexed and analyzed with the CellRanger pipeline to generate raw fastq files and processed gene-count matrices for each sample. For spatial transcriptomic analyses, samples were snap frozen in OCT (optimal cutting temperature embedding medium). Blocks were cut into 10um sections in a cryostat and transferred to a 10x Genomics Visium slide. Spatially barcoded libraries were prepared according to manufacturer's instructions and sequencing on an Illumina sequencer. The resulting sequence data was processed with the SpaceRanger pipeline to generate fastq files and processed barcode-count matrices. Using the samples deposited here and additional cohorts, we generated a comprehensive cellular network of the distal lung and its alteration in fibrosis. Integration with histopathology revealed that the transformation of normal parenchyma to fibrotic tissue is accompanied by ectopic bronchiolization and decellularization. Areas of active fibrosis were characterized by co-localization of pro-fibrotic <a href="https://www.ncbi.nlm.nih.gov/gene/115908">CTHRC1</a>-hi fibroblasts and aberrant transitional epithelial cells. We modeled both of these maladaptive cell states in reductionist models to identify the pro-inflammatory ligands involved in the differentiation of alveolar epithelial cells and to define <a href="https://www.ncbi.nlm.nih.gov/datasets/gene/4776/">NFATC4</a> as a key transcription factor during myofibroblast differentiation.

Single Nucleus and Spatial Transcriptomic Analyses of Pulmonary Fibrosis from the “Lung Transplant” and “Lung Tissue” Biorepositories