Close

Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells

Kyungtae Lim, Walfred Tang, Dawei Sun, Peng He, Sarah A. Teichmann, John C. Marioni, Kerstin B. Meyer, Emma L. Rawlins

Preprint posted on June 30, 2021 https://www.biorxiv.org/content/10.1101/2021.06.30.450501v1

Derivation of self-renewing organoids from the canalicular stage of human lung development enabled mechanistic investigation of alveolar epithelial cell differentiation.

Selected by Rob Hynds

Background
Lung development has been extensively studied in mice, but substantial differences in anatomy and cell biology exist between mice and human lungs. In this context, primary cell-based organoids offer a window into human lung development.

Human epithelial tip progenitor cells are SOX2+/SOX9+ and function as progenitors of both alveolar and airway epithelium during lung development (Figure 1). As alveolar epithelial differentiation is initiated during the canalicular stage, the tip cells lose SOX2 expression, upregulate an alveolar gene expression program and become cuboidal in shape.

Image 1: The stages of human lung development. Figure from Nikolic, Sun and Rawlins (Development, 2018).

Key findings

In their pre-print, Lim and colleagues contrast the distal lung tip epithelium at the pseudoglandular and canalicular stages of development, identifying that co-expression of the cell surface markers CD44 and CD36 identify tip cells during the canalicular stage. When they cultured tip cells as 3D organoids, two morphologies were present. Folded organoids expressed markers associated with progenitor and alveolar fate, whereas cystic organoids contained columnar cells and resembled organoids derived from the earlier pseudoglandular stage. Importantly, by passaging CD44+CD36+ cells from organoids, it was possible for canalicular stage lung tip progenitors to self-renew in culture.

Through manipulation of their cell culture medium, the authors demonstrate roles for both Wnt signalling and FGF signalling in alveolar and airway fate, respectively. Moreover, the canalicular stage tip organoids are highly primed for differentiation, differentiate more readily than organoids from earlier stages of development, and can readily switch between airway and alveolar fates. Data from ATAC-seq experiments suggest that this might be due to increased chromatin accessibility at key lineage defining promoter regions in the canalicular organoids compared to their pseudoglandular counterparts. Overexpression studies suggest that NKX2.1 and TFAP2C are alveolar and airway lineage defining factors, respectively in this context, while overexpression of naturally occurring variants in the NKX2.1 locus showed multiple different phenotypes in alveolar differentiation, establishing organoids as a model system for further mechanistic studies in this area.

Consistent with Wnt signals driving distal epithelial fate, the authors saw more Wnt target expression in tip epithelium than in stalk or airway epithelium and identified fibroblasts as a source of this Wnt signal. Patterning of the epithelium is achieved through the production of Wnt inhibitory protein NOTUM by myofibroblasts that associate with stalks. Interestingly these fibroblast phenotypes appear to be stable in cell culture. Moreover, similar to NKX2.1 overexpression, a differentiation medium allows canalicular tip organoids to be directed to alveolar type 2 cell fate. Tip-derived AT2-like cells contained lamellar bodies and processed surfactant proteins and thus represent an important new source of human AT2 cells for laboratory investigations.

Conclusions
Overall, this pre-print provides insight the emergence of alveolar progenitor cells during development, with tip cells that are exposed to high levels of fibroblast-derived Wnt upregulating NKX2.1 to assign an alveolar fate. Myofibroblast-derived inhibitory signals protect differentiating stalk cells and enable the emergence and persistence of non-tip fates. The organoid model described will provide a useful in vitro tool to study human lung development during the canalicular stage, as well as later stages involving alveolar differentiation and maturation.

Questions for the authors
Q1. How long do the authors think the window of plasticity that they report in tip epithelial cells might span? Similarly, at what stage would they expect to no longer derive organoids with a “pseudoglandular”-like cystic morphology?

Q2. Are AT2 cells that are derived earlier in development phenotypically or functionally distinct from those that arise later and, if so, where do the authors culture-induced AT2 cells would reside on that spectrum?

Tags: alveolar type ii cells, lung development, respiratory biology

Posted on: 20th July 2021

doi: https://doi.org/10.1242/prelights.30108

Read preprint (No Ratings Yet)




Author's response

Kyungtae Lim shared

Q1: This is a very hard question to answer as we cannot investigate primary human lungs beyond 21 post conception weeks (pcw). However, once the tip epithelium acquires the alveolar signature after 14-15 pcw, the CD36+CD44+ cell population can be isolated and cultured (Fig. 1). Experimentally, the lineage positive (LinPOS) folded organoids were reproducibly grown and cultured when the tip cells were freshly isolated from fetal lungs at all ages from 17 to 21 pcw. At the same time, the lineage negative (LinNEG), pseudoglandular-like cystic organoids were easily observed in the culture in a mixture with the LinPOS organoids. What we learned from the experiment using an SFTPC-eGFP reporter system was that, surprisingly, even the LinNEG cystic organoids originate from the SFTPC-expressing, CD44+CD36+ tip populations (Extended data fig. 2G-I; Image 2, below). We hypothesize this could be due to dedifferentiation of the epigenetically unstable canalicular tip epithelium. And therefore that the canalicular tip cells will always produce both LinNEG and LinPOS organoids in culture. Although it remains a possibility that the canalicular tip epithelium contains a mixture of cell states which may change over time.

Image 2. Lung tip organoids derived from fetal lung tips, 17-21 pcw. Freshly isolated tip cells were infected with lentivirus harbouring SFTPC-eGFP and cultured in self-renewing medium. A tiny number of cystic organoids (right panel) were observed to retain the SFTPC-eGFP signal at the early time point; 2 weeks of the culture. However, prolonged culture longer than 3 weeks made them lose the SFTPC-GFP signals and became lineage negative tip organoids (Fig. 2B,C), whereas the SFTPC-GFP positive folded organoids sustain the alveolar features even after multiple passages in the self-renewing medium.

Q2: Very interesting question. We observed future AT2 cells lining the stalk regions of the differentiating epithelial tube and primitive sac of the canalicular stage lungs (Fig. 4J,K) and learned that NOTUM+ myoepithelial cells are likely involved in the patterning of AT2 cells for later developmental stages. However, we know that the acquisition of alveolar features, along with the AT2 pattering, during the canalicular stage does not essentially mean that the cells are mature AT2 cells. Also, we are not sure whether the culture-induced AT2-like cells resemble the future AT2 cells in the developing lung, or whether they represent a more mature cell state. We hypothesize that they are more mature then fetal AT2 cells based on available data. However, to clearly answer that question we have a plan to compare the transcriptome of the culture-induced AT2-like cells with our unpublished single-cell RNA seq data of human fetal lungs which contains the future AT2 cell population. We are hoping to get sequencing data, and an answer, very soon.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the developmental biology category:

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close