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Multidimensional Clustering of Regenerative Alveolar Duct Cells after Murine Pneumonectomy
Author(s) -
Ysasi Alexandra,
Bennett Robert,
Valenzuela Cristian,
Servais Andrew,
Wagner Willi,
Tsuda Akira,
Ackermann Maximilian,
Mentzer Steven
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.867.5
Subject(s) - pneumonectomy , cd31 , biology , laser capture microdissection , cell , microbiology and biotechnology , pathology , lung cancer , angiogenesis , gene expression , genetics , medicine , gene
Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post‐pneumonectomy compensatory lung growth have identified areas of increased growth in subpleural alveolar ducts. To identify transcriptional patterns in these regenerative “hotspots”, we isolated single cells from alveolar ducts at post‐pneumonectomy days 1, 3 and 7. We used laser microdissection, enzymatic digestion and microfluidic isolation to capture individual cells from alveolar ducts, then performed single‐cell transcriptional analysis using the C1 integrated microfluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi‐dimensional data set was analyzed using visualization software based on the tSNE similarity analysis algorithm (viSNE, Cytobank). The similarity analysis identified six clusters of cells. To obtain a provisional cell type identity for these clusters, previously obtained post‐pneumonectomy cell sorted bulk transcriptional data was projected on the viSNE map using a similarity matrix analysis. The viSNE cluster with the highest overall level of gene expression was genotypically similar to myofibroblasts (64.04% of Acta2+ cells), endothelial progenitor cells (66.67% of sorted CD31+, CD34+cell samples), and type II pneumocytes (83.33% of sorted CD45−, MHC Class II+, phosphine+ cell samples). Another cluster genotypically overlapped primarily with sorted endothelial cells (94.74% of sorted CD31+ cell samples), but also some monocytes (50% of sorted CD11b+ cell samples) and alveolar macrophages (61.54% of sorted CD45+, CD11b−, CD11c+, F4/80+, Gr1− cell samples). One cluster was present only after pneumonectomy (99.23% post‐pneumonectomy, 0.77% un‐operated control), suggesting time point dependent gene activation. The transcriptional pattern of the six clusters was analyzed for genes associated with lung repair, matrix production and angiogenesis. The data demonstrated that multiple cell types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .