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

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.

Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro

“…The isolated muscle stem cells were cultured in MEM-based growth media. Various studies widely used FBS-containing MEM to culture pig muscle stem cells in vitro ( Jeong et al, 2013 ; Miersch et al, 2018 ). Doumit et al, 1993 , showed that pig muscle stem cells had a higher proliferation rate in MEM-based media compared to other media, including McCoy’s 5A, Ham’s F12, DMEM, and DMEM/F12.…”

“…Cultured meat is an artificial meat that imitates fresh meat using the in vitro production of muscle tissue, which would solve the problems derived from traditional meat production, including animal welfare and environmental pollution ( Post, 2012 ). Various researchers used fetal bovine serum (FBS)-containing minimum essential medium (MEM) to culture pig muscle stem cells for a long time ( Doumit and Merkel, 1992 ; Miersch et al, 2018 ). However, FBS-containing MEM-based culture conditions do not support the stemness of pig muscle stem cells for an extended period.…”

Low birth weight influences the postnatal abundance and characteristics of satellite cell subpopulations in pigs

“…However, we found a significantly reduced MPC yield in muscles of LBW piglets affecting both cell subpopulations but the negative effect is stronger for the SPS population. SPS cells are thought to form the reserve cell compartment from which SPF cells can be supplied during postnatal growth and thus, are normally characterized by a lower ability to differentiate 36,87 . In our study, expression of MyoG, known to be essential for myoblast differentiation 88 , is increased in SPS cells from LBW piglets.…”

Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets

“…A highly pure and vital population of satellite cells can be obtained with the established protocol developed in our lab 35 , 36 . Those cells were used to conduct three independent xCELLigence assays, which demonstrated a high repeatability of the cell growth measurements.…”

“…Previous studies demonstrated that the vast majority of cells isolated with this method are satellite cells that are able to differentiate. Abundance of myogenic markers were demonstrated with flow cytometry and the ability to terminally differentiate and generate myotubes was shown 35 , 36 . For xCELLigence assays (see below), myogenic cells from single animals were seeded in 10 cm collagen-coated culture dishes (Greiner Bio-One, Kremsmünster, Germany) and incubated at 37 °C, 6% CO 2 and 95.5% humidity.…”