Three-axis classification of mouse lung mesenchymal cells reveals two populations of myofibroblasts

Background:

Mesenchymal cells can be defined as the supportive niche in multiple tissues^1–3. However, these cells continue to be poorly understood with respect to their molecular markers and functional capacities. A major obstacle for this has been their heterogeneity and is complicated by the presumed absence of a differentiation hierarchy. Considering their active role in homeostasis, regeneration, and disease progression, it is crucial to improve our understanding of the mesenchyme and its interaction with other cell populations.

The three-dimensional (3D) organization of organs generates specialized microenvironments within them where distinct cell populations can exist. The mammalian lung is an example of these tissue compartments, and can be broadly defined by the airways and alveoli⁴. While previous work has provided information about the lung epithelium, the respiratory field is yet to reach a joint consensus regarding the mesenchyme. Previous studies have identified subsets of mesenchymal cells and implicated them in regeneration and disease^5–7. However, lineage markers identified for these cells are often shared between subsets which complicates their functional characterization. Some of the observed complexity can also be attributed to spatial cues that influence mesenchymal heterogeneity. An integration of molecular profiles with the positional information would lay a robust foundation for understanding the mesenchymal subsets.

In this preprint, the authors employ a multi-pronged approach to characterize the 3D heterogeneity of the pulmonary mesenchyme. Using single cell transcriptomics, lineage tracing and 3D immunostaining they define three distinct mesenchymal trajectories during development and growth of the mouse lung. They further make use of existing literature and imaging data to predict the functions of distinct mesenchymal subsets. Importantly, this study identifies a marker, Meox2, for the population of ‘interstitial fibroblasts’, which can serve as a signalling hub in the lung.

Key Findings:

Identification of a three-axes spatial heterogeneity of the lung mesenchyme:

Cell Population	Markers
Pericytes	Pdgfrb
Vascular Smooth Muscle Cells (VSMC)	Acta2, Pdgfrb, Tagln
Airway Smooth Muscle Cells (ASMC)	Acta2, Actct1
Myofibroblasts	Acta2, Pdgfra, Fgf18
Co113a1 Matrix Fibroblasts	Wnt2, Col13a1
Col14a1 Matrix Fibroblasts	Twist2, Col14a1
Mesothelial Cells	Msln
Proliferating Cells	Mki67

Table 1. Mesenchymal Cluster Markers

Table 1. Mesenchymal Cluster Markers

Mesenchymal cells from mouse lungs were isolated via a negative gating strategy at distinct time points during development (E17, E19; E=embryonic) and growth (P7, P13, P20, P70; P=post-natal). Single cell RNA sequencing of these cells captured several pre-defined populations and identified their growth-associated dynamics. Based on preliminary analysis, specific markers were assigned to subset of the mesenchymal population (Table 1). The authors identified a proliferating cluster which was enriched in the developmental over post-natal stages. They also affirmed how Adrp, a well-noted lipofibroblast marker, exhibits heterogeneous expression trends and may not be the most specific gene for detecting this cell population. Using their single cell data and available literature on stromal cell-epithelium/-endothelium interactions the authors then developed a spatial annotation scheme for the mesenchyme. Subsets located closest to the distal lung epithelium were classified as the ‘epithelial mesenchyme’, cells in proximity of the endothelium were termed as the ‘vascular mesenchyme’ while stromal cells posited between these two compartments were annotated as the ‘interstitial mesenchyme’. Trajectory analysis confirmed the fate commitment of these cells over the course of development and the post-natal emergence of a supportive niche.

Figure 1. Mesenchymal heterogeneity in the mouse lung. Single cell RNA sequencing data represented as UMAPs highlights the mesenchymal populations during development and growth of the mouse lung (Figure adapted from Figure 1b of Narvaez del Pilar and Chen).

Stromal cell dynamics along the spatial axes:

Characterization of individual mesenchymal axes involved the re-clustering of the single cell transcriptomic data to predict growth associated dynamics of each subset, and its eventual validation with appropriate mouse models.

Vascular Axis: Additional analysis of this population identified four cell clusters which included vascular smooth muscle cells (VSMCs) and three subsets of pericytes viz., immature, proliferative and mature. The data also indicated that pericyte maturation, unlike VSMCs, was a post-natal event. Their spatial distribution and morphological differences were further validated with the Pdgfrb^CreER mouse line which labels the vascular mesenchyme.

Epithelial Axis: The three main stromal subsets identified in the epithelial axis comprised of the Pdgfra⁺ myofibroblasts, Cdh4/Hhip/Lgr6⁺ myofibroblasts and ASMCs. The authors identified two distinct subsets of Pdgfra⁺ myofibroblasts, which exhibited differences in overall expression (high or low) and subcellular localisation of this marker. Pdgfra-high cells associated with perinuclear localisation of the receptor and were situated around alveolar pockets, potentially involved in constriction during neonatal maturation. Interestingly, an onset of cell death pathways resulted in a loss of these cells in adult mice, thus highlighting their specific developmental role. On the other hand, Cdh4/Hhip/Lgr6⁺ myofibroblasts were situated proximal to the alveolar ducts and persisted in adult lungs despite their early developmental origin. These cells formed a continuum with the ASMCs and could wrap around alveolar ducts by virtue of their extended projections.

Interstitial Axis: In depth analysis identified Meox2 as a specific marker of the interstitial mesenchyme and localised them proximally within bronchovascular bundles. These cells also expressed low levels of non-perinuclear Pdgfra and could be labelled with the Pdgfra^CreER mouse model. Lineage labelling with the Myh11^CreER mouse line, which labels alveolar myofibroblasts during embryonic development and neonatal growth did not label Meox2⁺ cells. These experiments affirmed that interstitial mesenchymal cells exist as an independent population and do not arise from the alveolar myofibroblasts.

Why I Chose This preprint?

Despite multiple studies focusing on it, there has been a lot of debate regarding the properties and functions of the lung mesenchyme. Several well-established markers often exhibit inconsistency across studies due to the limited understanding of how this cellular compartment changes in a spatial context. The multipronged approach employed in this study addresses this issue and provides a spatio-temporal map of distinct mesenchymal subsets. The functional capabilities predicted by the authors, in addition to their molecular signatures provide a roadmap for future studies involving the lung and its associated pathologies.

Questions For the Authors:

1. How were the morphologies of distinct mesenchymal compartments defined? Will it be beneficial for future studies, to identify quantifiable features for them?
2. Do you know if the abundance or distribution of these stromal compartments changes with age (more than 1 year old mice)?
3. Do you intend to develop in vitro functional assays to further characterize these stromal cells and identify their potential to support different epithelial cell populations in co-cultures?

References:

1. El Agha, E. et al. Mesenchymal Stem Cells in Fibrotic Disease. Cell Stem Cell 21, 166–177 (2017).
2. Jiang, D. & Rinkevich, Y. Defining Skin Fibroblastic Cell Types Beyond CD90. Front. Cell Dev. Biol. 6, 1–3 (2018).
3. McCarthy, N. et al. Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient. Cell Stem Cell 26, 391-402.e5 (2020).
4. Basil, M. C. & Morrisey, E. E. Lung regeneration: a tale of mice and men. Semin. Cell Dev. Biol. 100, 88–100 (2020).
5. El Agha, E. et al. Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis. Cell Stem Cell 20, 261-273.e3 (2017).
6. Biasin, V. et al. PDGFRα and αSMA mark two distinct mesenchymal cell populations involved in parenchymal and vascular remodeling in pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 318, L684–L697 (2020).
7. Zepp, J. A. et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell 170, 1134-1148.e10 (2017).

 

doi: https://doi.org/10.1242/prelights.30537

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