Functionally heterogeneous human satellite cells identified by single cell RNA sequencing
Pomerantz, Jason; Barruet, Emilie (2020), Functionally heterogeneous human satellite cells identified by single cell RNA sequencing, Dryad, Dataset, https://doi.org/10.7272/Q65X273X
Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cell population is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (HuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the HuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of HuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ HuSCs are morphologically distinct, and are characterized by resistance to activation compared to CAV1- HuSCs. In vivo, CAV1+ HuSCs demonstrated increased engraftment potential after transplantation in mice. Our findings provide a comprehensive transcriptional view of normal HuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.
Freshly harvested human muscle was stored in DMEM with 30% FBS at 4°C overnight. Muscle was digested, erythrocytes were lysed, and hematopoietic and endothelial cells were depleted with magnetic column depletion. Viable cells were depleted for CD31, CD34, and CD45 expressing cells. Cells that remained after depletion were sorted for CXCR4+/CD29+/CD56+ and collected for single cell rna sequencing (Garcia et al., 2018; Garcia et al., 2017). To capture individual cells, we utilized the Chromium Single Cell 3' Reagent Version 1 or 3 Kit from 10X Genomics (Zheng et al., 2017). For both samples (aged and adult) 18,000 satellite cells isolated as in (Garcia et al., 2018)were loaded onto one well of the 10X chip to produce Gel Bead-in-Emulsions (GEMs). GEMs underwent reverse transcription to barcode RNA before cleanup and cDNA amplification. Libraries were prepared with the Chromium Single Cell 3' Reagent Version 1 or 3 Kit. Each sample was sequenced on 1 lane of the HiSeq2500 (Illumina) run in Rapid Run Mode with paired-end sequencing parameters.
Matrices were obtained via 10x Genomics Cell Ranger pipeline for scRNA seq.
Programming languages such as R or Python should be used to load the matrices