390 - A Single Cell RNA-Seq Study of Skeletal Muscle Gene Expression in Spastic Cerebral Palsy

Publication Number: 390.3839

Karyn Robinson, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Qi Zheng, Nemours Children's Hospital, Wilmington, DE, United States; Stephanie Lee, Nemours Children's Hospital, Wilmington, DE, United States; Jonathan R. Hicks, University of Delaware, Newark, DE, United States; Wade Shrader, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Robert Akins, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States

Background: Cerebral palsy (CP) is a leading cause of physical disability in children. CP is a static encephalopathy caused by damage to the developing brain that results in neuromotor dysfunction characterized by muscle spasticity, poor coordination and weakness. Individuals with spastic CP have impaired longitudinal skeletal muscle growth and contractures that may contribute to impaired motor control, muscle atrophy, and increased need for clinical care or surgery. Muscle stem cells (satellite cells; SC) are a mixed population of cells responsible for skeletal muscle growth and repair. Studies demonstrate that CP is associated with decreased numbers of SCs in muscle tissue and that bulk SCs isolated from CP muscle exhibit altered function in vitro. It is unclear if SC heterogeneity contributes to this effect or dysfunction in CP.

Objective: We investigated differences in SC subpopulations in cells isolated from participants with spastic CP and controls using single cell RNA-sequencing (scRNA-seq).

Design/Methods: Surgical explants of skeletal muscle were obtained from subjects undergoing orthopedic surgery at the Nemours Children’s Hospital, Delaware following IRB approved informed consent/assent. Seven subjects with spastic CP and 7 control subjects were enrolled in the study. Satellite cells were isolated by double immunomagnetic selection for CXCR4 and NCAM1. SC phenotype was confirmed by MYF5 immunofluorescence. Single cells were isolated from proliferating satellite cell-derived myoblasts (SC-MBs) and lysed to capture RNA molecules. Barcoded next-generation sequencing (NGS) cDNA libraries were prepared and sequenced on a NextSeq 2000.

Results: scRNA-seq profiles from CP and control cohorts were compared using the R package Seurat. Both cohorts displayed the same overall profile of SC subpopulations with cells separating into 14 clusters (Figure 1). Clusters were identified as representing different phases of the cell cycle and different stages of myogenesis. There were fewer differentiating cells in the CP cohort compared to controls and in the male cohort compared to females.

Conclusion(s): SC-MBs isolated from subjects with CP contain similar SC subpopulations as those isolated from control subjects, but there were fewer differentiating cells in the CP cohort. Differentially expressed genes identified within each subpopulation may account for CP-associated differences seen in skeletal muscle satellite cells.

Figure 1: Clustering of cells by Uniform Manifold Approximation Projection (UMAP) according to transcriptome similarity in 2D space.
Cells isolated from controls (n=7) and CP (n=7) both separated into fourteen clusters with each cluster represented by a different color.