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Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique

Abstract

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

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Figure 1: Marker profile of sequential preplates and the potential use of FACS to further purify the SAC into separate fractions.
Figure 2: Schematic diagram showing the preplate technique.
Figure 3: Morphology of muscle-derived cells at different stages of their isolation utilizing the preplate technique.
Figure 4: Schematic diagram of the replating process used to further purify SACs by eliminating fibroblast and differentiated cell contamination.

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Acknowledgements

This work was supported in part by the National Institutes of Health (R01HL069368-01A1 BRP; R01-49684), the US Department of Defense (W81XWH-05-01-0337) and the Muscular Dystrophy Association (J.H.). The Stem Cell Research Center receives financial support from the William F. and Jean W. Donaldson Endowed Chair at the Children's Hospital of Pittsburgh of UPMC and the Henry J. Mankin Endowed Chair for Orthopaedic Research at the University of Pittsburgh.

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Correspondence to Johnny Huard.

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Senior author (Johnny Huard) receives consultant fee and royalties related to a patent license for this technology to Cook MyoSite, Inc.

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Gharaibeh, B., Lu, A., Tebbets, J. et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3, 1501–1509 (2008). https://doi.org/10.1038/nprot.2008.142

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