Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Summary

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Abstract

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream^1-4.

Introduction

Considerable understanding of mammalian myogenesis has been obtained through the recapitulation of this process in both primary mouse (Mus musculus) myoblast cultures and the well-described mouse-derived cell line, C2C12⁵. Beginning in the 1950s⁶, these cultures have led to much advancement in the understanding of the murinemyogenic program and, by extension, myogenesis in other vertebrates. Additionally, single cell myofiber explant techniques have increased out understanding of interactions between satellite cells and surrounding myofibers^7-9.Cell cultures are particularly attractive for investigations of myogenesis due to t....

Protocol

Ethics Statement: All experimentation involving vertebrate animals described herein was approved in advance by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and is consistent with guidelines established by the Office of Laboratory Animal Welfare, National Institutes of Health of the U.S. Department of Health and Human Services.

1. Preparation for Culture

1. Prepare the base medium as follows: 9 mM NaHCO₃ (1.51 g per 2 L), 20 mM HEPES (9.53 g per 2 L) in DMEM (13.48 g/L; 26.96 g per 2 L).
   1. Determine pH and adjust to 7.40 with HCl or NaOH.
   2. Determin....

Results

Twenty-four hours post-seeding, myogenic precursor cells (MPCs) should be visible attached to the laminin substratum (see Figures 1a and 1d). Following seeding, cells (MPCs) adopt a spindle-like shape, indicative of this cell type (Figure 1) and are MyoD1+ (Figure 2). In Danio species, MPCs appear to be more compact with smaller bipolar processes than do MPCs from Oncorhynchus and Salmo species. However, over four days of cultu.......

Discussion

The myogenic program, in whichever species examined, can be most easily studied through an in vitro system. Indeed, upon isolation, myogenic precursor cells (MPCs) in fish or myosatellite cells (MSCs) in mammals readily enter this highly regulated process involving the proliferation, cell cycle withdrawal, and terminal differentiation of myoblasts and the fusion of those myoblasts into nascent myotubes. The general lack of transgenic gene reporter strains of piscine species (with the possible exception of the ze.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table 1. Detailed Reagent Information
Reagent	Company (Preferred v. Alternate)	Catalog Number (Preferred v. Alternate)	Quantity per Culture
γ-irradiated poly-L-lysine	Sigma-Aldrich (MP Biomedicals)	P5899 (ICN19454405)	5 mg
DMEM (high glucose)	Sigma-Aldrich (cellgro)	MT-50-003-PB (D7777)	2 L
Laminin	BD Biosciences (Sigma-Aldrich)	CB-40232 (L2020)	1 mg
Sodium Bicarbonate (NaHCO3)	Fisher Scientific (Sigma-Aldrich)	BP328-500 (S5761)	1.51 g
HEPES (C8H18N2O4S)	Fisher Scientific (Sigma-Aldrich)	BP310-1 (H6147)	9.53 g
Antibiotic/Antimycotic	Thermo Scientific (Sigma-Aldrich)	SV3007901 (A5955)	17-20 mL
Gentamicin Sulfate	Lonza (Sigma-Aldrich)	BW17-519Z (G1397)	2-3 mL
Donor Equine Sera	Thermo Scientific (Sigma-Aldrich)	SH3007403 (H1270)	75 mL
Fetal Bovine Sera	Thermo Scientific (Sigma-Aldrich)	SH3007103 (F2442)	25 mL
Collagenase (Type IV)	Worthington (Sigma-Aldrich)	LS004189 (C9891)	0.44 g
Trypsin (from Pancreas)	MP Biomedicals (Sigma-Aldrich)	ICN15357125 (T5266)	1 g
Table 2. Consumables, Tools and Equipment
Consumable	Tools	Equipment
Cell Culture Plates	Forceps (Coarse)	Serological Pipettor
Sterile 50 mL Conical Tubes	Forceps (Fine)	pH Meter
Laboratory Tape	Scalpel Handles	Chilling Incubator (Echotherm)
0.2 μm Vacuum Sterilization Systems	Scalpel Blades (#10, #11)	Laminar Flow Hood
Water-repellant Autoclave Paper	Surgical Scissors	Vacuum Manifold
Serological Pipettes	Glass Petri Dishes	Microosmolality Meter
	12-16 G Cannulas with Luer Locks
Table 3. Optimized Volumes for Coating Cell Culture Plates
Plate Size	cm^2 per Well	Poly-L-lysine*	Laminin**
6 well	9.5	1.6 mL	1
24 well	1.9	0.32	0.2
48 well	0.95	0.16	0.1
96 well	0.32	0.06	0.03
*0.1 mg/mL concentration	** 0.020 mg/mL concentration
Table 4. Media for Isolation, Dissociation, and Culture
Reagent	Isolation	Wash	Dissociation
Base Medium	419.25 mL	395.40 mL	297.00 mL
Gentamicin Sulfate**	0.75 mL	0.60 mL	-
Donor Equine Serum	75.00 mL	-	-
Fetal Bovine Serum***	-	-	-
* PSF: penicillin/streptomycin/fungizone cocktail (100x); ** 50 mg/mL concentration; *** Characterized
Table 5. Recommended Dilutions and Plating Volumes
Plate Size	cm^2 per Well	Dilution	Plating Volume
6 well	9.5	1.5-2.0x10^6 cells/mL	1 mL
24 well	1.9	1.5-2.0x10^6 cells/mL	250 μL
48 well	0.95	1.5-2.0x10^6 cells/mL	150 μL
96 well	0.32	1.5-2.0x10^6 cells/mL	50-100 μL
Table 6. Average number of cells per g tissue
Species	Average # cells/g tissue
Danio rerio	6,400,000
Danio dangila	1,783,000
Devario aequipinnatus	1,797,000
Oncorhynchus mykiss	66,800
Table 7. Recommended Incubation Temperatures
Species	Temperature
Danio/ Devario spp.	26 - 28 °C
Oncorhynchus/Salmo spp.	10* - 18 °C
Ambystoma mexicanum	18°C
* Lower temperatures support lower proliferation rates.