Abstract

Replication and fusion of skeletal muscle satellite cells (SkMSCs) are essential for skeletal muscle maintenance and repair. Advancing age impairs SkMSCs replication potential and fusion capacity, however SkMSCs isolated from older human skeletal muscle do not consistently demonstrate these defects, making it difficult to study SkMSC isolated from aged human muscle. PURPOSE: To investigate if serial passaging of SkMSCs can mimic aging associated defects in replication and fusion. We hypothesized that serial passaging of primary human SkMSCs induces replicative senescence and poor fusion capacity in part through inhibition of the cell cycle regulator cyclin-dependent kinase 4 (CDK4) through the activation of CDK inhibitor p16Ink4a. METHODS: SkMSCs were isolated from vastus lateralis biopsies of young men. SkMSCs were serially passaged every five days and passaging continued until SkMSCs were unable to replicate. Population doubling level (PDL) was calculated using passage specific final and starting SkMSC counts. Expression of the myogenic regulator, myogenin (MyoG) and the regeneration regulator, paired box 7 (Pax7) were analyzed via rt-PCR. Senescence was determined by SA-B-gal staining and fusion capacity determined by immunohistochemical staining. RESULTS: Primary human SkMSCs failed to replicate at passage 16 (Pass16) and Pass16 SkMSCs exhibited decreased fusion. PDL was decreased from passage 4 to 16 (Pass4: 3.9 vs. Pass16: 0.6, AU). CDK4 mRNA decreased (Pass4: 1.0 vs. Pass16: 0.4, AU) and p16Ink4a mRNA was increased (Pass4: 1.0 vs. Pass16: 4.9, AU). Pax7 mRNA was unchanged (Pass4: 1.0 vs. Pass16: 1.3, AU), while myoG mRNA was increased (Pass4: 1.0 vs Pass16: 8.6, AU). CONCLUSIONS: Serially passaging SkMSCs isolated from young humans mimics an aged phenotype evidenced by impaired replication and fusion. Our findings suggest serial passaging of primary human SkMSC may be used to study aged SkMSCs.

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