- Aanvrager: Dr J.P. Ng-Blichfeldt
ATS 2017 congres van 19 mei 2017 tot 24 mei 2017 te Washington DC, USA Lung Regeneration Research 6.1.14.009CO
A TS 2017 abstract submission Wnt Activity Marks Lung Progenitors Capable Of Forming Spheres In Vitro
John-Poul Ng-Blichfeldt1•2, Chiharu Ota1, Jan Stolk3, Pieter S. Hiemstra3, Reinoud Gosens2, Melanie Königshoff 11. Comprehensive Pneumology Center, Helmholtz-Zentrum Munich, LudwigMaximilians-University, University Hospita! Grosshadem, Memher ofthe Oerman Center ofLung Research (DZL), Munich, Germany
Molecular Pharmacology, Groningen Research Institute for Asthrna and COPD (GRIAC), University of Groningen, Groningen, The Netherlands 3. Department ofPulmonology, Leiden University Medical Center, Leiden, The Netherlands
Rationale Canonical WntsignaHing regulates self-renewal and differentiation of tissue-resident progenitors during lung development and in a variety of adult tissues. The role ofWnt signalling in progenitor self-renewal has been little explored in the adult lung.
Canonical Wnt signalling regulates lung progenitor self-renewal.
Epithelial cells were isolated from lungs of adult wild type mice, or Wnt-GFP reporter mice (TCF/Lef:H2B-GFP fusion) with EpCAM magnetic beads. Cells were additionally isolated from Wnt-GFP mice by F ACS according to EpCAM and GFP level (hi, low, neg). Isolated cells were characterized by qRT-PCR and flow cytometry.
Epithelial cells from wild type (WT-EpCAM+), or Wnt-GFP mice (EpCAM+GFP11i, EpCAM+opptow, EpCAM+oppneg) were co-cultured with lung fibroblasts in Matrigel, and ability to form spheres (organoids) was investigated. Quantitative immunofluorescence and live imaging were used to determine mechanisms of cell
In WT-EpCA~ cells, 91% expressed proSPC and 4% expressed CCIO, markers for alveolar type 2 and airway club cells, respectively. WT-EpCAM+ cells proliferated in culture to form spheres that exhibited immunostaining for alveolar, airway, or both markers at day 21 (proSPC+: 69.8±5.3%, acetylated a-tubulin+: 10.2±3.6%,
proSPC+/acetylated a-tubulin+: 13.8±3.1 %; Figure 1). Quantification ofKi67 immunostaining indicated highest proliferation early in culture (Ki67+ nuclei per sphere were 71.7% at day 3, 14.0% at day 7 [p<0.0001 versus day 3], and 4.7% at day 14 [p<0.0001 versus day 3]). In Wnt-GFP mice, EpCAM+oppncg, EpCAM+opplow,
and EpCAM+opphi comprised 85±2.7%, 4.8±1.7% and 7.5±1.0% oftotal EpCAM+lung cells, respectively. Sphere-formation was predominantly contained in the EpCAM+opptow population (76 fold versus EpCAM+oppneg, p<0.05), with fewer spheres formed by the EpCAM+opphi population (5.2 fold versus EpCAM+GFP"eg,
p<0.05). The EpCAM+opptow population was enriched for airway epithelial markers (CC10, p63, Krt5) and markers associated with lung progenitors (Itga6, Sca-1, CD90), whereas alveolar epithelial markers (SPC, SP A, Nkx2.1) were less abundant in the EpCAM+GFP1ow population compared to EpCAM+oppneg. Quantitative
immunofluorescence on spheres formed by EpCAM+ cells from Wnt-GFP mice ATS 2017 abstract submission demonstrated loss of Wnt activity in progeny of Wnt-active cells, correlating with increased proliferation at day 3 (GFP+ cells were 16.9% Ki67+, GFP" were 84.4% Ki67+, p<O.OOO 1 ). Live imaging confirmed loss of Wnt activity early during sphere formation, and suggests initia! asymmetrie di vision of Wnt-active cells.
Endogenous Wnt activity is a distinguishing feature of lung epithelial progenitors. Our results suggest Wnt-active lung progenitors asymmetrically divide in vitro to yield progeny exhibiting decreased Wnt activity and increased proliferation, generating the cellular mass required for sphere formation.
This project is funded by the Lung Foundation Netherlands (LongFonds).