A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment

Anna Benedetti,Gianluca Cera,Ciro Villani,Biliana Lozanoska-Ochser,Daniele De Meo,Marina Bouche

doi:10.1186/s13395-021-00261-w

Anna Benedetti, Gianluca Cera + Show 4 more

Open Access

https://doi.org/10.1186/s13395-021-00261-w

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Journal: Skeletal Muscle	Publication Date: Mar 17, 2021
Citations: 15	License type: open-access

Affiliation: Sapienza University of Rome, Policlinico Umberto I

Abstract

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

Highlights

The muscle is endowed with an exceptional regenerative ability primarily due to a resident population of stem cells called satellite cells (SCs) [1,2,3]
As well as the notion that like all stem cells, SCs are sensitive to stress signals and are among the first muscle resident cells to respond to injury [4, 5], we hypothesized that subjecting a heterogeneous culture of muscle cells to a mild stress stimulus such as ice-cold temperature will lead to the detachment of only the SCs
Satellite cells isolated with the ice-cold treatment (ICT) method displayed a similar clonal myogenicity of 40 % and a doubling time of 17 h to SCs isolated using the magnetic beads method (Figure S1A and B)

Summary

Introduction

The muscle is endowed with an exceptional regenerative ability primarily due to a resident population of stem cells called satellite cells (SCs) [1,2,3]. Ensconced between the basal lamina and the plasma membrane of muscle fibers, SCs respond to injury or various stress stimuli by becoming activated, and undergoing proliferation, selfrenewal, and differentiation to form new myofibers [4, 5]. As SCs become activated, a proportion of them identified as Pax7+MyoD- replenish the stem cell pool, while others acquire the expression of MyoD (Pax7+MyoD+), differentiate into myoblasts and enter the myogenic program. After several rounds of division myoblasts give rise to Myogenin+ myocytes, which fuse together to form new myofibers [1, 4, 6,7,8,9]. The success of SC transplantation therapy depends on having an efficient method to isolate (2021) 11:7 and expand these cells in vitro in undifferentiated state and in sufficient numbers [13]

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