Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells
Posted on: 20 July 2021
Preprint posted on 30 June 2021
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
Categories: cell biology, developmental biology
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?
doi: https://doi.org/10.1242/prelights.30108
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(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.
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