Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.


Statistics
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Software and code
Policy information about availability of computer code Data collection Initial processing of 3'-end single-cell RNA-sequencing data was performed using the commercial Cell Ranger pipelines (10X Genomics, versions 2.1.0, as described in the Methods section of the manuscript). For 3'-end single-cell RNA-sequencing, transcripts were mapped to the mm10 reference genome (GRCm38.91) using Cell Ranger (version 2.1.0). For full length single-cell RNA-sequencing, demultiplexed, paired-end FASTQs were aligned to the mouse genome (mm10/gencode.Mv13) using Bowtie (version 1.0.0) and quantified using the RNA-seq by Expectation-Maximization algorithm (RSEM) (version 1.2.31), as described in the Methods section of the manuscript.
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Sample size
No sample size calculations were performed for mouse experiments. Mouse wounds were pooled for single-cell RNA-seq experiments, which involved the analysis of many individual cells. For immunohistochemical analysis, we used 3 mice as biological replicates. For all bone marrow transplantation (BMT) experiments, 3 or more mice were used per experiment. For all Cre recombinase -based lineage tracing and gene deletion experiments, 9 or more mice were used per experiment.
Data exclusions In 3'-end single-cell RNA-seq experiments, cells displaying more than 8,000 UMI/cell, 2,500 genes/cell, and more than 8% mitochondrial gene expression were excluded as low quality cells. Approximately 503 cells were excluded. For full length single-cell RNA-seq, cells with a total number of expressed genes > 11,000, a proportion of counts in mitochondrial genes > 15%, and genes expressed in < 3 cells were excluded as low quality cells. 56 cells were excluded from all replicates.

Replication
Key findings were reproduced throughout the manuscript. For example, immunohistochemical analysis recapitulated the expression profiles of fibroblast subtypes described in the initial 3'-end single-cell RNA-seq experiment. Key functional studies were also independently replicated. These include BMT and Cre recombinase-based lineage tracing assays. In single-cell RNA-seq experiments, individual sequenced cells were considered as replicates. In all in vivo mouse experiments, at least 3 mice were used per experiment. In adipocyte differentiation assays, technical replicates were used.
Randomization Littermate mice were assigned into groups on the basis of genotype.

Blinding
Single-cell RNA-seq analyses were unbiased. All cells were analyzed using computational algorithms that were not biased to recognize any particular cell types. All mouse lineage tracing experiments involved identification of genetically labeled cells and did not require blinding. Adipocyte regeneration phenotype after Pparg deletion was quantified using blinded approach, by investigators who did not know the genotype.

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This study did not involve wild animals.

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All animal experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania and University of California, Irvine.
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Flow Cytometry
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Methodology Sample preparation
Bone marrow cells were flushed from the long bones with Dulbecco's Modified Eagle's Medium supplemented with 5% heatinactivated calf serum (Gibco). To obtain a single cell suspension, cells were filtered through a 45 μm nylon screen. Cells were blocked with anti-rat and anti-mouse IgG (Sigma) for 15 minutes.

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FlowJo software Cell population abundance The purity of sorted cells was >90% as tested by running post-sort samples on flow cytometer machine.
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