598 - Single Cell Transcriptomic Profiling of Skeletal Muscle in Spastic Cerebral Palsy

Publication Number: 4586.598

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

Background: Cerebral palsy (CP) is a static encephalopathy caused by damage to the developing brain that results in neuromotor dysfunction. Individuals with spastic CP have impaired longitudinal skeletal muscle growth and contractures that may contribute to impaired motor control and increased need for clinical care or surgery. Muscle stem cells (satellite cells; SC), which are responsible for skeletal muscle growth and repair, are decreased in muscle tissue from subjects with CP and exhibit altered function in vitro. The gene expression differences contributing to SC dysfunction in CP are not well characterized.

Objective: We investigated differences in SC-derived myoblasts 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 approval and informed consent/assent. Seven subjects with spastic CP (3 males, 4 females, average age=13.9 ± 3.7) and 7 control subjects (4 males, 3 females, average age 14.3 ± 1.6) were enrolled in the study. Satellite cells were isolated by double immunomagnetic selection for CXCR4 and NCAM1 and phenotype was confirmed by MYF5 immunofluorescence. Single cells were isolated from proliferating SC-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 similar overall expression profiles with SC subpopulations separating into 14 clusters. Cluster identities included cells progressing through different phases of the cell cycle (clusters 0, 2, 4, and 6), growing and migrating cells (3, 5, 8, 9, and 13), and those expressing myogenic and functional muscle proteins (1, 7, 10, 11, and 12). Overall, the proportion of myogenic to proliferating cells was lower in the CP cohort compared to controls. Differentially expressed genes were present in seven of the clusters. Cell Ranger analysis revealed three distinct subpopulations, but the distribution of cells among the three subpopulations differed in the control and CP groups.

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

Clustering of satellite cell-derived myoblasts
scRNA-seq was performed and UMAP non-linear dimensional reduction was used to place similar cells together in low-dimensional space. The control cohort (left) and CP cohort (right) displayed the same overall profile of SC-MB subpopulations with cells separating into 14 clusters; however, the CP cohort had far fewer cells in cluster 7.

GO enrichment analysis of clusters
REVIGO treemaps of enriched biological processes are shown. Different colors represent families of related terms. The size of each rectangle reflects the p-value of the GO term. Clusters 0, 2, 4, and 6 (upper left) may represent cells in the M phase (4 and 6) and S phase (0 and 2) of the cell cycle. Clusters 3, 5, 8, 9, and 13 (upper right) may represent cells that are growing and migrating. Clusters 1, 7, and 10 (lower left) may represent cells that are beginning the process of muscle differentiation. Clusters 11 and 12 (lower right) may represent late-stage muscle differentiation.