Muscle Microbiopsy to Delineate Stem Cell Involvement in Young Patients: A Novel Approach for Children With Cerebral Palsy

Abstract

Cerebral palsy (CP), the single largest cause of childhood physical disability, is characterized firstly by a lesion in the immature brain, and secondly by musculoskeletal problems that progress with age. Previous research reported altered muscle properties, such as reduced volume and satellite cell (SC) numbers and hypertrophic extracellular matrix compared to typically developing (TD) children (>10 years). Unfortunately, data on younger CP patients are scarce and studies on SCs and other muscle stem cells in CP are insufficient or lacking. Therefore, it remains difficult to understand the early onset and trajectory of altered muscle properties in growing CP children. Because muscle stem cells are responsible for postnatal growth, repair and remodeling, multiple adult stem cell populations from young CP children could play a role in altered muscle development. To this end, new methods for studying muscle samples of young children, valid to delineate the features and to elucidate the regenerative potential of muscle tissue, are necessary. Using minimal invasive muscle microbiopsy, which was applied in young subjects under general anaesthesia for the first time, we aimed to isolate and characterize muscle stem cell-derived progenitors of TD children and patients with CP. Data of 15 CP patients, 3-9 years old, and 5 aged-matched TD children were reported. The muscle microbiopsy technique was tolerated well in all participants. Through the explant technique, we provided muscle stem cell-derived progenitors from the Medial Gastrocnemius. Via fluorescent activated cell sorting, using surface markers CD56, ALP, and PDGFRa, we obtained SC-derived progenitors, mesoangioblasts and fibro-adipogenic progenitors, respectively. Adipogenic, skeletal, and smooth muscle differentiation assays confirmed the cell identity and ability to give rise to different cell types after appropriate stimuli. Myogenic differentiation in CP SC-derived progenitors showed enhanced fusion index and altered myotube formation based on MYOSIN HEAVY CHAIN expression, as well as disorganization of nuclear spreading, which were not observed in TD myotubes.

An Emerging Role for Epigenetics in Cerebral Palsy

“…Satellite cells, stem cells residing between the sarcolemma of muscle fibers and the surrounding basal lamina, are mediators of muscle growth and repair [53]. When activated, they proliferate and either self-renew to maintain the satellite-cell pool or differentiate toward myoblasts and fuse with existing myofibers or other satellite cells to form multinucleated myotubes [14,16]. This process is controlled by sequential expression of transcription factors [54].…”

“…Of these, spastic CP is the most common type, accounting for approximately 80% of all cases [13]. The primary symptoms in spastic CP involve increased muscle stiffness and tone (spasticity), contractures, muscle weakness, and disrupted gait and body positioning [11,14]. These motor impairments are frequently accompanied by additional deficits such as intellectual disability, epilepsy, and sensory impairments [15].…”

“…Alterations in muscle tissue have been observed in children with CP. These include impaired longitudinal growth contributing to contractures [16], decreased muscle crosssectional area [17], increased sarcomere length [13], reduced muscle mass and volume, fibrotic tissue accumulation, increased extracellular matrix, reduced satellite-cell numbers [14], microanatomic disruption of neuromuscular junctions (NMJ) [18], and muscletype-specific alteration of fiber type distributions [19,20]. In most cases, the specific causes and mechanisms contributing to these alterations have remained elusive.…”

Commentary: Muscle Microbiopsy to Delineate Stem Cell Involvement in Young Patients: A Novel Approach for Children With Cerebral Palsy

“…(D) Fusion index (FI) values are represented by boxplots and dots represent individual subjects (TD: n = 5; CP: n = 14; * p < 0.05). Figures from Corvelyn et al ( 2020 ).…”

“…Corvelyn et al ( 2020 ) are to be congratulated for circumventing some of these biases by developing a method to obtain a non-intraoperative but smaller (~10 mg) muscle biopsy by needle (Bergstrom, 1975 ), from less severely affected (GMFCS I–III) and generally younger children (average ~6 years of age). Unfortunately, their study lacked clarity regarding the actual muscle stem cell population being studied.…”