AMPK Methods and Protocols

Chapter 34

Analysis of Muscle Stem Cell Fate Through Modulation

of AMPK Activity

Marine Theret, Linda Gsaier, Sabrina Ben Larbi, Miche`le Weiss-Gayet,

and Re ́mi Mounier

Abstract

In this chapter, we describe the methods to isolate and culture muscle stem cells (MuSCs) from murine
skeletal muscle in order to decipher the intrinsic effect of AMP-activated kinase activity on MuSC fate.
Culture of MuSCs is a powerful model to recapitulate every step of stem cell behavior observed in vivo:
activation, proliferation, differentiation, fusion and also self-renewal. We provide the detailed procedures to
isolate pure MuSCs by a flow cytometry-based method using the selection of a combination of specific
markers and to characterize MuSC fate (quiescence, activation, and differentiation) in response to AMPK
activity modulation by assessing of the expression of stem cell (e.g., Pax7) and myogenic marker
(e.g., MyoD).

Key wordsMetabolism, Stem cell behavior, Myogenesis, Muscle, Quiescence, Differentiation, Pax7,

Flow cytometry

1 Introduction

Skeletal muscle stem cells (MuSCs) are widely recognized for their

contributions to muscle regeneration [1]. At resting state, MuSCs

(also named satellite cells) express the intracellular marker paired-

box domain transcription factors Pax7 and remain quiescent under

the basal lamina attached to the myofiber. During skeletal muscle

regeneration, these cells are able to recapitulate the myogenic

program to repair damaged myofibers [2]. Upon activation and

expression of proliferation and activation markers such as Ki67 and

myogenic differentiation 1 (MyoD), these cells are capable of both

differentiating (leading to the downregulation of Pax7 and upre-

gulation of MyoD and then myogenin expression) to repair muscle

tissue after injury and self-renewing to replenish the stem cell pool

by upregulating Pax7 and downregulating Ki67 and MyoD expres-

sion [3]. In order to maintain the pool of MuSCs for future needs,

controlling the return into quiescence is crucial [2]. We recently

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_34,©Springer Science+Business Media, LLC 2018

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