161 - Muscle Satellite Cell Subpopulations Exhibit Heterogeneity in Spastic Cerebral Palsy

Poster Number: 161
Publication Number: 161.311

Stephanie K. Lee, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Karyn Robinson, 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 static encephalopathy associated with musculoskeletal dysfunction that often worsens over time. Muscle-resident stem cells (satellite cells, SCs), a heterogenous population that contributes to muscle growth and regeneration, have been implicated in CP pathology. Cultured SC-derived myoblasts from CP muscle exhibit altered proliferation and differentiation, but it is unclear if SC heterogeneity contributes to this effect or dysfunction in CP. 

Objective: To elucidate differences in SC subpopulations associated with CP by analyzing measures of cell cycle activity and proliferation in sub-cloned cells prepared from primary SC populations. 

Design/Methods: An in-vitro, case-control, cell-culture design was used to compare proliferative activity of primary SC-derived myoblast cell populations and cells sub-cloned from those populations. De-identified and cryopreserved cells isolated from muscle biopsies in an IRB-approved study were utilized for experiments. Primary SC isolates from spastic CP (CP, n=3) or control (CN, n=3) muscle were prepared by double immunomagnetic selection for CXCR4 and NCAM1. Sub-clones from each parent population were isolated via low density seeding and validated as SC-derivatives by Pax7 and Myf5 immuno-staining. Sub-clones and parent cells underwent a 4-hour pulse labeling with 5-ethynyl-2’-deoxyuridine (EdU) to mark cells progressing through the DNA-synthesis phase of the cell cycle. Fixed cells were labeled with markers to identify all nuclei (Hoechst 33258), EdUpos nuclei (fluorescent azide), and nuclei in actively cycling cells (anti-Ki67). Proliferation rate (the ratio of EdUpos:Ki67pos nuclei) and cell cycle activity (Ki67pos:total nuclei) were evaluated. 

Results: Among parent cell populations, CP cells had a higher proportion of cycling cells (p=0.01) but slower proliferation rates (p=0.002). Sub-clones exhibited significant differences in proliferation and/or cell cycle relative to parent cells in both CP and control groups with either proliferation rate or cell cycle activity (p < 0.05) but not both differing in all cases but one control, which exhibited significant differences between the parent cells and sub-clones in both cell-cycling (p=0.008) and proliferation (p=0.041). Conclusion(s): Satellite cell subpopulations exhibit heterogeneity in cell cycle activity and proliferation that may be associated with spastic CP. Further research is needed to elucidate the mechanisms responsible for differences observed in satellite cell sub-populations and the degree to which these play roles in the musculoskeletal pathophysiology of CP.