Development of an in vitro potency assay for human skeletal muscle derived cells

• Published in: PloS one 2018-03, Vol.13 (3), p.e0194561-e0194561
• Main Authors: Thurner, Marco, Asim, Faheem, Garczarczyk-Asim, Dorota, Janke, Katrin, Deutsch, Martin, Margreiter, Eva, Troppmair, Jakob, Marksteiner, Rainer
• Format: Article
• Language: English
• Subjects: Acetylcholinesterase; Acetylcholinesterase - metabolism; Anal Canal - pathology; Analysis; Biology and Life Sciences; Biopsy; CD56 antigen; CD56 Antigen - metabolism; Cell Count; Cell culture; Cell differentiation; Cell Differentiation - physiology; Cell fusion; Cell number; Cell Separation - methods; Cell Transplantation - legislation & jurisprudence; Cell Transplantation - methods; Cells, Cultured; Clinical trials; Consumer Product Safety; Differentiation; Double-Blind Method; Enzyme Assays - methods; Enzymes; Fecal incontinence; Fecal Incontinence - etiology; Fecal Incontinence - pathology; Fecal Incontinence - therapy; Female; Flow Cytometry; GPI-Linked Proteins - metabolism; Humans; Internet; Laboratories; Male; Mammals; Markers; Markets; Medical research; Medicine and Health Sciences; Mode of action; Muscle Fibers, Skeletal - pathology; Muscle Fibers, Skeletal - physiology; Muscle Fibers, Skeletal - transplantation; Muscles; Muscular Diseases - complications; Muscular Diseases - pathology; Muscular Diseases - therapy; Musculoskeletal system; Myotubes; Obstetrics; Patients; Placebos; Psychological aspects; Public health; Quality; Regeneration; Research and Analysis Methods; Skeletal muscle; Spinal cord; Surface markers; Thoracic surgery; Trauma; Treatment Outcome
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0194561

Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals. CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence. CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected. AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of in vitro differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).