Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids
- University of Cambridge, United Kingdom
- University College London, United Kingdom
Abstract
The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.
Data availability
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Expression data from fresh human embryonic lung epithelial tip and stalk cells and cultured organoids derived from tip and stalkPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE95860).
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Expression data from mouse embryonic lung epithelial tip progenitor cellsPublicly available at the NCBI Gene Expression Omnibus (accession no: GSM1968996).
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Microdissected E11.5 lung epithelial tip biological replicate 2Publicly available at the NCBI Gene Expression Omnibus (accession no: GSM1968997).
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Microdissected E11.5 lung epithelial tip biological replicate 3Publicly available at the NCBI Gene Expression Omnibus (accession no: GSM1968998).
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Microdissected E11.5 lung epithelial tip biological replicate 4Publicly available at the NCBI Gene Expression Omnibus (accession no: GSM1968999).
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Microdissected E11.5 lung epithelial tip biological replicate 5Publicly available at the NCBI Gene Expression Omnibus (accession no: GSM1969000).
Article and author information
Author details
Funding
Medical Research Council (G0900424)
- Emma L Rawlins
Wellcome (PhD programme for clinicians fellowship)
- Marko Z Nikolić
Addenbrooke's Charitable Trust, Cambridge University Hospitals (N/A)
- Marko Z Nikolić
Ikerbasque, Basque Foundation for Science (N/A)
- Usua Laresgoiti
Cancer Research UK (C6946/A14492)
- Emma L Rawlins
Medical Research Council (MR/P009581/1)
- Emma L Rawlins
Wellcome (Clinical PhD Fellowship)
- Jo-Anne Johnson
Wellcome (92096)
- Emma L Rawlins
Fundacio Universitaria Agusti Pedro i Pons (Short term fellowship)
- Oriol Caritg
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge and University College London Animal Welfare and Ethical Review Body (AWERB). All experiments were thereafter conducted according to Home Office project licenses PPL 70/8012 (Emma Rawlins, University of Cambridge) and 70/7607 (Adam Giangreco, UCL).
Human subjects: Human embryonic and foetal lung tissue. Human embryonic and foetal lungs were obtained from terminations of pregnancy from Cambridge University Hospitals NHS Foundation Trust under permission from NHS Research Ethical Committee (96/085) and the Joint MRC/Wellcome Trust Human Developmental Biology Resource (London and Newcastle, grant 099175/Z/12/Z, www.hdbr.org). Informed consent and consent to publish was obtained.Human adult lung tissue. Fresh healthy adult lung tissue (background tissue from lobectomies for lung cancer) was obtained from Papworth Hospital NHS Foundation Trust (Research Tissue Bank Generic REC approval, Tissue Bank Project number T01939). Informed consent and consent to publish was obtained.
Copyright
© 2017, Nikolić et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids
eLife 6:e26575.
https://doi.org/10.7554/eLife.26575
Further reading
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- Stem Cells and Regenerative Medicine
A new source of progenitor cells can now be used to study hidden aspects of human lung development and pediatric lung disease.
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- Stem Cells and Regenerative Medicine
Harnessing the regenerative potential of endogenous stem cells to restore lost neurons is a promising strategy for treating neurodegenerative disorders. Müller glia (MG), the primary glial cell type in the retina, exhibit extraordinary regenerative abilities in zebrafish, proliferating and differentiating into neurons post-injury. However, the regenerative potential of mouse MG is limited by their inherent inability to re-enter the cell cycle, constrained by high levels of the cell cycle inhibitor p27Kip1 and low levels of cyclin D1. Here, we report a method to drive robust MG proliferation by adeno-associated virus (AAV)-mediated cyclin D1 overexpression and p27Kip1 knockdown. MG proliferation induced by this dual targeting vector was self-limiting, as MG re-entered cell cycle only once. As shown by single-cell RNA-sequencing, cell cycle reactivation led to suppression of interferon signaling, activation of reactive gliosis, and downregulation of glial genes in MG. Over time, the majority of the MG daughter cells retained the glial fate, resulting in an expanded MG pool. Interestingly, about 1% MG daughter cells expressed markers for retinal interneurons, suggesting latent neurogenic potential in a small MG subset. By establishing a safe, controlled method to promote MG proliferation in vivo while preserving retinal integrity, this work provides a valuable tool for combinatorial therapies integrating neurogenic stimuli to promote neuron regeneration.
