Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C047

Oral Communications

Identification and characterisation of the senescent phenotype of human primary myogenic precursor cells

T. G. Francis1, O. Jaka1, N. Lazarus1, G. Ellison-Hughes1, S. Harridge1

1. Centre for Human & Applied Physiological Sciences, King's College London, London, LDN, United Kingdom.

Cellular senescence, and the senescence associated secretory phenotype (SASP), are believed to contribute to the physiological ageing process(1). However, their role within skeletal muscle, a highly regenerative tissue which shows a decline in mass and function with age, is less clear. Inducing replicative senescence in cultured human primary muscle precursor cells (MPCs) is challenging due to i) the amount of time in culture and ii) the likelihood that even cultures with initially high myogenic purity can become overrun with fibroblast cells(2). To overcome these limitations, senescence can be induced using the chemotherapy drug, Doxorubicin(3), but its effect on inducing senescence in human MPCs is unknown. The aim of the present study was to investigate the characteristics of human primary MPCs when senescence was induced via two different approaches: i) passaging to replicative senescence (RS) and ii) 24 hours of Doxorubicin (DOX, 0.2 µM) treatment. Both approaches were conducted on the same MPC populations isolated from muscle biopsy samples obtained from young healthy male volunteers (n=6, 22±1 years). Cumulative population doublings (CPD), the presence of senescence-associated β-galactosidase (SA β-gal) and the expression of p16 together with the absence of proliferation marker, Ki67, were monitored to determine cell senescence. Additionally, mRNA levels of established SASP factors (PAI-1, TGF-β1, IL-8 and IGFBP-3) were measured. Three of the six MPC populations maintained myogenic purity through to RS, which was reached at 25.8±1.9 CPD. In RS there was a trend for increased p16 expression (n=3, fold change=2.2±1.0, ns), decreased Ki67 expression (n=3, fold change=0.4±0.1, p=0.01, paired t-test) and increased number of SA β-gal-positive MPCs (n=2, 75% increase), compared to early passage. Over 35 days after DOX treatment, MPCs showed 99-100% of cells positive for SA β-gal (n=2). There was increased p16 expression (n=6, fold change=1.6±0.3, p<0.05, Dunnett's test) and decreased Ki67 expression (n=6, fold change=0.1±0.0, p<0.05, Dunnett's test) in DOX-treated MPCs, compared to pre-DOX treatment. MPC populations which reached RS with maintained myogenic purity were used for comparison against paired DOX-treated MPCs for analysis of SASP factors (n=3). Here PAI-1 and IGFBP-3 showed significantly higher mRNA expression levels following RS compared to early passage and DOX treated MPCs (p<0.05, RM ANOVA). DOX-treated MPCs were not significantly different to early passage for any SASP factor. In conclusion, we have characterised the replicative senescence and DOX-induced senescence phenotype in MPCs. Replicative senescent MPCs showed increased SASP factor expression, whereas DOX-treated senescent MPCs did not. Further analysis should determine the similarities of these two models as regards to their senescent phenotype, as well as whether these models reflect in vivo ageing in skeletal muscle.

Where applicable, experiments conform with Society ethical requirements