Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Background/Purpose: Fibrosis in systemic sclerosis (SSc), as well as a wide variety of other fibrotic diseases, is largely driven by myofibroblasts. Myofibroblasts are known to selectively produce high amounts of collagen and are found in the deep reticular dermis of patients with diffuse cutaneous SSc (dcSSc). However, our understanding of myofibroblast biology has been limited to mixed cultures of myofibroblasts due to the lack of a unique marker for their selection.
Methods: Skin biopsies from a patient with dcSSc and a healthy control were enzymatically digested and cells sorted into individual wells of a 96 well plate. Libraries were prepared according to the SmartSeq2 protocol and RNAseq performed. Data from individual cells was deconvoluted by barcode and aligned using Tophat. Transcripts were quantified using the Cufflinks. Cuffnorm files were mean centered, normalized, clustered by complete linkage using Cluster 3.0, and visualized using Java Treeview. T-distributed stochastic neighbor embedding (t-SNE) was implemented in R using the Sincell Bioconductor package
Results: Clustering showed different cell types in the dcSSc skin that were easily identified through known markers: keratinocytes, endothelial cells, macrophages, T cells and B cells. In addition, a group of cells was found that expressed smooth muscle actin (SMA/ACT2 gene) and type 1 collagen. On the basis of previously described marker genes and clustering, these cells could be divided into three well-defined groups: pericytes, smooth muscle cells and a third cell type. This third cell type uniquely expressed high levels of integrin alpha 8 (ITGA8), an integrin previously associated with myofibroblasts in mice. This cell type also uniquely expressed a series of other genes including Disintegrin and metalloproteinase domain-containing protein 12 (ADAM12). Mice deleted of cells expressing ADAM12 show severely blunted fibrosis in murine models. t-SNE analysis supported the cell groupings, showing a discrete cellular grouping for the ACTA2/ITGA11/ADAM12-expressing cells. Immunohistochemical staining revealed that several of the unique gene markers associated with the third SMA-expressing cell type co-stained with SMA, staining deep reticular myofibroblasts, and confirming the myofibroblast identity of these cells. The unique markers on these cells correlated closely with the modified Rodnan skin score.
Conclusion: Myofibroblasts have a unique transcriptome profile that provides new insights into the function of these cells. The high correlation between transcriptome markers expressed by these cells with the MRSS supports the key importance of dermal myofibroblasts in SSc skin fibrosis.
To cite this abstract in AMA style:Lafyatis RA, Rice L, Stifano G, Browning J, Simms RW. Single Cell Rnaseq Defines a Unique Transcriptome Profile for Myofibroblasts in the Skin of Patients with Systemic Sclerosis [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/single-cell-rnaseq-defines-a-unique-transcriptome-profile-for-myofibroblasts-in-the-skin-of-patients-with-systemic-sclerosis/. Accessed June 7, 2020.
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