Fibroblast and Myofibroblast Subtypes: Single Cell Sequencing

Methods Mol Biol. 2021:2299:49-84. doi: 10.1007/978-1-0716-1382-5_4.


The stroma constitutes the structural framework of an organ and plays crucial roles in health and following organ damage. The major player of the stroma with respect to extracellular matrix deposition, maintenance, and remodeling is the fibroblast and its activated derivative, the myofibroblast. It has long been recognized that there is considerable variability to the fibroblast phenotype. The recent advent of new single cell "omics" technologies has revolutionized our understanding and appreciation of cellular heterogeneity of fibroblasts been revolutionized. With these tools, the nature and defining characteristics of the cells comprising the stroma is finally being defined not just through a few markers, but by taking a wholistic look at transcriptional programs. It is now apparent that stromal cells are not only transcriptionally diverse, but also functionally, epigenetically, and spatially heterogeneous. Studying populations at single cell resolution has enabled identification of new clusters of cells with unique transcriptional signatures. Whether these clusters truly represent distinct subpopulations or different states of the same population remains to be clarified. In this chapter, we first describe a procedure for purification and preparation of a single cell suspension from tissue samples (in this case the heart) for single cell RNA sequencing. We also describe preparation of high-quality tissue sections for spatial transcriptomics. Secondly, we outline a workflow for computational analysis of single cell RNA sequencing and spatial transcriptomics data, as well as integrating them together, to explore the heterogeneity within fibroblasts/myofibroblasts and identify different subtypes and their locations in the heart.

Keywords: 10× Genomics; Computational analysis; Fibroblasts and myofibroblast subsets; Heart; Single cell RNA sequencing; Spatial transcriptomics; Visium.

MeSH terms

  • Animals
  • Cell Differentiation
  • Cells, Cultured
  • Computational Biology
  • Extracellular Matrix / metabolism
  • Fibroblasts / chemistry
  • Fibroblasts / cytology
  • Gene Expression Profiling / methods*
  • Gene Regulatory Networks*
  • Mice
  • Myofibroblasts / chemistry
  • Myofibroblasts / cytology*
  • Sequence Analysis, RNA
  • Single-Cell Analysis / methods*
  • Workflow