Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Summary

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca²⁺ signaling.

Abstract

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca²⁺ signals are of great importance. These Ca²⁺ signals arise from Ca²⁺ release from internal Ca²⁺ stores, as well as from Ca²⁺ entry from the extracellular space, particularly the store-operated Ca²⁺ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca²⁺ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca²⁺ probe that provides reliable and reproducible measurements of SOCE.

Introduction

Human skeletal muscles are composed of groups of contractile, multinucleated muscle fibers resulting from the fusion of myogenic precursor cells. Skeletal muscles have the capacity to regenerate after injury thanks to the presence of SCs, the skeletal muscle stem cells located between the plasma membrane of myofibers (sarcolemma) and the basal lamina. In uninjured muscle, SCs are mostly present in a quiescent state. In response to mechanical stress or injury, SCs become activated (myoblasts), proliferate, and undergo either differentiation to form new myofibers or self-renewal to replenish the SC pool^1,2. Over....

Protocol

Human muscle samples (tissues obtained from semitendinous muscles) were collected during orthopedic surgery on healthy patients as surgical waste. All methods relating to the human study were performed in accordance with the guidelines and regulations of the Swiss Regulatory Health Authorities and approved by the Commission Cantonale d'Ethique de la Recherche from the Geneva Cantonal Authorities, Switzerland (protocol CER n° 12-259). Informed and written consents were obtained from all adult subjects involved in the study.

1. Isolation of Myoblasts from Human Skeletal Muscle

1. During all procedures, maintain sterile condi....

Results

After the enzymatic dissociation of a human muscle sample, cells were amplified in GM. Human myoblasts, defined as CD56+/CD146+/CD45-/CD34-/CD144- cells, were obtained after FACS. Myoblasts represented more than 60% of the analyzed population (Figure 1). Primary human myoblasts were replated, grown to confluence, and cultured in DM for 48 h. After immunostaining for the expression of the transcription factor MEF2 and the muscle-specific protein myosin heavy chain (MyHC), we observed that a m.......

Discussion

The isolation and culture of human myoblasts from adult skeletal muscle offers an in vitro model to study muscle differentiation and muscle regeneration. In this paper, we provide a protocol that allows for the purification of high yields of human myoblasts in a simple and cost-limited way. In addition, this technique provides reliable and reproducible results in terms of percentage of myoblasts isolated and their myogenic differentiation efficiency. Indeed, we obtained around 60.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ham’s F10	ThermoFisher Scientific	31550-023
FCS	ThermoFisher Scientific	10270-106	Lot 41G5532K
BSA	Sigma	O5482
fetuin	Sigma	F3385
EGF	Corning	354001
Dexamethansone	Sigma	D1756
Insulin	Sigma	I9278
Creatine	Sigma	27900
Pyruvate	Sigma	P4562
Uridine	Sigma	U3003
Gentamycin	ThermoFisher Scientific	15710-049
Trypsin-EDTA 0.05%	ThermoFisher Scientific	25300-062
70 μm cell strainer	Falcon	352350
40 μm cell strainer	Falcon	352340
Falcon 50 mL tube	Falcon	352070
Falcon 15 mL tube	Falcon	352096
Falcon 5 mL tube (FACS)	Falcon	352058
Culture medium: OPTI-MEM	ThermoFisher Scientific	31985-047
Transfectant reagnt: RNAiMax	ThermoFisher Scientific	1756064
PBS	ThermoFisher Scientific	14190-094
Mounting medium	Fluka	10981
Mouse anti-MyHC *	DSHB	MF20	concentrate
Mouse anti-myogenin	BD Pharmigen	556358
Rabbit anti-MEF2	Santa Cruz Biotech	C-21
Goat anti-mouse Alexa488	ThermoFisher Scientific	A11029
Goat anti-rabbit Alexa 546	ThermoFisher Scientific	A11035
Mouse anti human CD56-Alexa488	BD Pharmingen	557699
Mouse anti human CD146-PECy7	BD Pharmingen	562135
Mouse anti human CD45-PE-CF594	BD Pharmingen	562279
Mouse anti human CD34-APC	BD Pharmingen	345804
Mouse anti human CD144-PE	BD Pharmingen	560410
Alexa Fluor 488 mouse IgG1k	BD Pharmingen	557702
PECy7 mouse IgG1k	BD Pharmingen	557872
PE-CF594 mouse IgG1k	BD Pharmingen	562292
APC mouse IgG1k	BD Pharmingen	550854
PE mouse IgG1k	BD Pharmingen	556650
DAPI	Sigma	D9542	CAUTION:  toxic compound
Paraformaldehyde	Fluka	762400
Fura-2 AM	ThermoFisher Scientific	F1201
Pluronic F-127	ThermoFisher Scientific	P3000MP
Thapsigargin	Sigma	T9033	CAUTION : toxic compound
Ratio imaging acquisition software: Metafluor 6.3	Molecular Device
* : MF 20 was deposited to the DSHB by Fischman, D.A.