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Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts.

ABSTRACT: Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic 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 algorithm. The analysis identified 6 cell clusters; 1 cell cluster was present only after pneumonectomy. This post-pneumonectomy cluster was significantly less transcriptionally active than 3 other clusters and may represent a transitional cell population. A provisional cluster identity for 4 of the 6 cell clusters was obtained by embedding bulk transcriptional data into the tSNE analysis. The transcriptional pattern of the 6 clusters was further 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 cell clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.

SUBMITTER: Ysasi AB

PROVIDER: S-EPMC7186418 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts.

Ysasi Alexandra B AB Bennett Robert D RD Wagner Willi W Valenzuela Cristian D CD Servais Andrew B AB Tsuda Akira A Pyne Saumyadipta S Li Shuqiang S Grimsby Jonna J Pokharel Prapti P Livak Kenneth J KJ Ackermann Maximilian M Blainey Paul C PC Mentzer Steven J SJ

Frontiers in medicine 20200421

Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis o ...[more]

PMID: 32373614

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Project description:Biliary epithelial cells (BEC) are morphologically and functionally heterogeneous. To investigate the molecular mechanism for their diversities, we test the hypothesis that large and small BEC have disparity in their target gene response to their transcriptional regulator, the biliary cell-enriched hepatocyte nuclear factor HNF6. The expression of the major HNF (HNF6, OC2, HNF1b, HNF1a, HNF4a, C/EBPb, and Foxa2) and representative biliary transport target genes that are HNF dependent were compared between SV40-transformed BEC derived from large (SV40LG) and small (SV40SM) ducts, before and after treatment with recombinant adenoviral vectors expressing HNF6 (AdHNF6) or control LacZ cDNA (AdLacZ). Large and small BEC were isolated from mouse liver treated with growth hormone, a known transcriptional activator of HNF6, and the effects on selected target genes were examined. Constitutive Foxa2, HNF1a, and HNF4a gene expression were 2.3-, 12.4-, and 2.6-fold, respectively, higher in SV40SM cells. This was associated with 2.7- and 4-fold higher baseline expression of HNF1a- and HNF4a-regulated ntcp and oatp1 genes, respectively. Following AdHNF6 infection, HNF6 gene expression was 1.4-fold higher (P = 0.02) in AdHNF6 SV40SM relative to AdHNF6 SV40LG cells, with a corresponding higher Foxa2 (4-fold), HNF1a (15-fold), and HNF4a (6-fold) gene expression in AdHNF6-SV40SM over AdHNF6-SV40LG. The net effects were upregulation of HNF6 target gene glucokinase and of Foxa2, HNF1a, and HNF4a target genes oatp1, ntcp, and mrp2 over AdLacZ control in both cells, but with higher levels in AdH6-SV40SM over AdH6-SV40LG of glucokinase, oatp1, ntcp, and mrp2 (by 1.8-, 3.4-, 2.4-, and 2.5-fold, respectively). In vivo, growth hormone-mediated increase in HNF6 expression was associated with similar higher upregulation of glucokinase and mrp2 in cholangiocytes from small vs. large BEC. Small and large BEC have a distinct profile of hepatocyte transcription factor and cognate target gene expression, as well as differential strength of response to transcriptional regulation, thus providing a potential molecular basis for their divergent function.

Dataset Information

Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts.

Publications

Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts.

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