Isolation of muscle stem cells from rat skeletal muscles

Abstract

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Keywords: CD106; Flow cytometry; Muscle stem cells; Pelvic floor muscles; Rat.

Fig. 1.

MuSCs localization. MuSCs identified by Pax7 expression in the rat Tibialis Anterior (TA) muscle. Left: low magnification image of TA (scale bar 50 µm). Center: Zoomed in images of Pax7+ cell (scale bar 25 µm). Right: quantification of Pax7+ cells.

Fig. 2.

Gating approach for cell isolation protocols. (A) Experimental design for panels B–D. (TA: Tibialis Anterior). FACS plots for the gating approach used in protocols 1 (B), 2 (C), and 3 (D).

Fig. 3.

Phenotypic validation of cell populations isolated using three protocols. (A) Experimental design for panels B-D (TA: Tibialis Anterior; IF: immunofluorescence; GM: Growth Media). (B) Left: Pax7 expression in freshly isolated cells (scale bar 50 µm); right: quantification of Pax7+ cells plated for 2 and 12 h after isolation. (C) Left: MyoD expression in cultured cells (scale bar 50 µm); right: quantification of MyoD+ cells in culture for 72 h. (D) Left: myogenin expression in cultured cells (scale bar 100 µm); right: quantification of Myogenin+ cells in culture for 120 h. (E) Experimental design for panel F. (DM: Differentiation Media). (F) Left: myosin heavy chain (MyHC) expression in differentiated cells (scale bar 50 µm); right: quantification of MyHC + nuclei after 72 h in DM. (G) Quantitative real time PCR in freshly isolated cells for myogenic genes (Pax7, MyoD, Myogenin), comparing P1, P2, and P3 populations. All data are normalized to P1 population.

Fig. 4.

Phenotypic validation of cells isolated using Protocol 1 in gastrocnemius, diaphragm, and pelvic floor muscles. (A) FACS plots for the optimized gating for protocol 1. (GAS: Gastrocnemius; DIA: Diaphragm; C: coccygeus; ICa: iliocaudalis; PCa: pubocaudalis; GM: Growth Media; IF: immunofluorescence). (B) FACS plots showing MuSCs (black gate) in GAS, DIA, C, ICa, and PCa. (C) Experimental design for panel D. (D) Left: Pax7 expression in freshly isolated cells (scale bar 50 µm); right: quantification of Pax7+ cells plated for 2 h after isolation. (E) Experimental design for panel F. (DM: Differentiation Media). (F) Left: MyHC expression in differentiated cells (scale bar 50 µm); right: quantification of MyHC + nuclei after 72 h in DM.