Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Summary

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Abstract

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56⁺ and later as CD56⁺/desmin⁺ cells and (ii) muscle-derived fibroblasts, identified as CD56^– and TE-7⁺. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 10⁶ ± 8.87 x 10⁵ cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56⁺ cells bound to microbeads are retained by the field whereas CD56^– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

Introduction

The repair and regeneration of skeletal muscle requires the action of satellite cells¹, the myogenic stem cells^2,3. In vivo these cells exist in a reversibly quiescent state located between the sarcolemma and basal lamina of every myofibre, but become activated to proliferate, fuse and differentiate as muscle tissue is damaged, repaired and regenerated³. Satellite cells can be isolated from young and elderly human muscle biopsy samples using enzymatic digestion⁴ and their myogenic properties can subsequently be studied in primary culture⁵. The efficiency of this isolation process in regard to both yield a....

Protocol

NOTE: For the studies performed in our lab all subjects gave their written, informed consent to participate and all experiments were performed with UK National Health Service Ethics Committee approval (London Research Ethics Committee; reference: 10/H0718/10) and in accordance with the Human Tissue Act and Declaration of Helsinki.

1. Initial Preparation Prior to Muscle Biopsy (15 min)

1. Make human skeletal muscle growth medium. To a graduated sterile 50 ml conical tube add 2.5 ml of the supplement mix, 10 ml fetal calf serum, antibiotics (penicillin/streptomycin) and glutamine to the correct final concentrations (Table 1

Results

Purified myogenic cells and fibroblasts can be cultured in adipogenic differentiation medium for three days followed by adipogenic nutrition medium from anywhere between 7-30 days to assess their potential for adipogenesis. Using the purified cell populations, Oil Red O staining in combination with immunostaining for adipogenic and myogenic lineage markers showed that only the fibroblast fraction was capable of adipogenic differentiation (Figure 2). The massive accumulation of fat by the fibroblasts is v.......

Discussion

We have described an immunomagentic sorting procedure for the selective enrichment of human muscle-derived precursors from small samples of muscle biopsy material. This technique has been invaluable in our lab for overcoming the loss of human muscle-derived cultures to fibroblasts, but also for understanding the unique behavior of distinct populations of muscle-derived progenitors. Once purified myogenic cells can be investigated for changes in protein and/or gene expression, or used for downstream experiments.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase  D	Roche	11088866001
Dispase II	Sigma	D4693-1G	Must be filter sterilized before use
Trypsin/EDTA	(Gibco) Invitrogen	15400-054
100 μm cell strainer	BD Biosciences	352360
Collagen solution ( 3 mg/ml in 0.1% acetic acid)	Sigma, Dorset, UK	C8919
Minisart SRP15 syringe filter (0.2 μm)	Sartorius	17573ACK	Polytetrafluorethylene (PTFE) membrane
CD56 human Microbeads	Miltenyi Biotech	130-050-401	Be aware of the limited shelf life of microbeads
Anti- fibroblast Microbeads, human	Miltenyi Biotech	130-050-601
40 μm Pre-separation filters	Miltenyi Biotech	130-041-407
Large Cell Collumns	Miltenyi Biotech	130-042-202	These columns come with a flow resistor. Use of the flow resistor is not necessary to obtain the high myogenic purities described here.
LS columns	Miltenyi Biotech	130-042-401
MiniMacs Seperator	Miltenyi Biotech	130-042-102	This separator fits the large cell column but not the LS column.
MidiMACS	Miltenyi Biotech	130-042-302
MACS multistand	Miltenyi Biotech	130-042-303
BSA	Sigma		Must be filter sterilized before use
Oil Red O	Sigma	O0625
Triethyl phosphate	Sigma	538728
Whatman Paper	Sigma	Z241121-1PAK	No. 42, Ashless. To prepare the filter fold the circular filter paper to make a semi circle, then fold the semi-circle in half again to form a cone shape. Fit the cone into a funnel for filtering.
ProLong Gold Antifade Reagent	Molecular Probes, Invitrogen	P36930	This can be purchased with or without DAPI and does not quench initial fluorescence.
AxioVision	Carl Zeiss	Contact Zeiss
Adobe Photoshop CS5 Extended	Adobe (purchased from Pugh Computers)	ADPH16982*