Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation

Claudia Miersch,Monika Röntgen,Katja Stange

doi:10.1186/s12860-018-0156-1

Abstract

BackgroundSatellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. Several studies have provided evidence that SC and MPC represent a heterogeneous population differing in their biochemical and functional properties. The identification and characterization of functionally divergent SC subpopulations should help to reveal the precise involvement of SC/MPC in these myogenic processes. The aim of the present work was therefore to separate SC subpopulations by using Percoll gradients and to characterize their myogenic marker profiles and their functional properties (adhesion, proliferation, and differentiation).ResultsSC/MPC from muscles of 4-day-old piglets were isolated by trypsin digestion and enriched by Percoll density gradient centrifugation. A mixed myogenic cell population was obtained from the 40/70% interface (termed: mixed P40/70) of a 25/40/70% Percoll gradient. Thereafter, by using a more stepped 25/40/50/70% Percoll gradient, mixed P40/70 was divided into subpopulation 40/50 (SP40/50) collected from the 40/50% interface and subpopulation 50/70 (SP50/70) collected from the 50/70% interface.All three isolated populations proliferated and showed a myogenic phenotype characterized by the ability to express myogenic markers (Pax7, MyoD1, Desmin, and MyoG) and to differentiate into myotubes. However, compared with mixed P40/70, SP40/50 and SP50/70 exhibited distinct functional behavior. Growth kinetic curves over 90 h obtained by the xCELLigence system and proliferation assays revealed that SP40/50 and mixed P40/70 constituted a fast adhering and fast proliferating phenotype. In contrast, SP50/70 showed considerably slower adhesion and proliferation. The fast-proliferating SP40/50 showed the highest myogenic differentiation potential with higher fusion rates and the formation of more middle-sized and large myotubes.ConclusionsThe described Percoll density gradient centrifugation represents a useful tool for subdividing pig SC/MPC populations with divergent myogenic functions. The physiological role of SC subpopulations during myogenesis and the interaction of these populations can now be analyzed to a greater extent, shedding light on postnatal growth variations in pigs and probably in other animals.

Highlights

Satellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity
Separation of SC/MPC subpopulations by various Percoll density gradients SC isolation and enrichment by tissue digestion with 0.25% trypsin followed by Percoll gradient centrifugation were performed according to Mau et al [23] with the exception that the Percoll gradient was changed from 25%, 40%, 90% to 25%, 40%, 70% to remove erythrocytes
Freshly isolated SP40/ 50 and subpopulation 50/70 (SP50/70) accounted for 34% and 66% of the total cell yield, respectively

Summary

Introduction

Satellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. During peri- and postnatal growth, 30–60% of the nuclei in porcine muscle are proliferating SC [5], and a high proportion of their progeny differentiates to fuse with adjacent myofibers thereby providing the main source of new myonuclei [6,7,8] In this rapid growth phase, the number of SC constantly declines representing 2–5% of nuclei in adult muscle [9, 10]. Activated and proliferating SC are characterized by the expression of MyoD1 [16,17,18] Both Myf and MyoD are important in determining muscle cell fate, either by regulating a return to the quiescent state to renew the SC pool or by initiating cellular differentiation accompanied by Pax downregulation and the induction of the early differentiation marker Myogenin (MyoG) [15, 19]. These cells fuse with each other to form nascent myotubes and/or fuse to differentiated muscle fibers [9]

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