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.

Blockade of senescence‐associated micro RNA ‐195 in aged skeletal muscle cells facilitates reprogramming to produce induced pluripotent stem cells.

“…SkMs were isolated from C57BL/6 mice (young: 8–10 weeks, The Jackson Laboratory; old: 24–26 months, National Institute on Aging, USA) as previously described (Gharaibeh et al ., 2008). The isolated cells were then suspended in growth medium (DMEM supplemented with 10% FBS, 10% horse serum, 0.5% chick embryo extract, and 2.5% penicillin/streptomycin) and transferred to coated flasks.…”

Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

“…Using a modified preplate technique, 7 different preplate populations (PP1 to PP6) of cells with various adhering characteristics on collagencoated flasks were separated. The early preplate populations (rapid-adhering cells, ie, PP1 and PP2) contain primarily fibroblasts and myoblasts, whereas the late preplate populations (slow-adhering cells, ie, PP5 and PP6) contain primarily muscle stem cells including MDSCs.…”

“…The early preplate populations (rapid-adhering cells, ie, PP1 and PP2) contain primarily fibroblasts and myoblasts, whereas the late preplate populations (slow-adhering cells, ie, PP5 and PP6) contain primarily muscle stem cells including MDSCs. 7 PP6 cells (SASCs that contain MDSCs) were studied, and PP6 cells from both noninjured and injured muscles were cultured in proliferation medium [Dulbecco's modified Eagle's medium (DMEM); Invitrogen Corp., Carlsbad, CA] supplemented with 10% horse serum, 20% fetal bovine serum, 0.5% chicken embryo extract, and 1% penicillin-streptomycin] in 5% CO 2 at 37°C.…”

“…[3][4][5] Among these muscle precursors, muscle-derived stem cells (MDSCs) exist in normal muscle as a small population that usually expresses specific stem cell markers such as Sca-1 and CD34. 6 -9 Isolation of MDSCs has been widely performed using a marker profile-independent preplate technique that takes advantage of their slow adherence to a collagen-coated surface, 6,7,9,10 and MDSCs are included in a population of slow-adhering stem cells (SASCs) isolated with this technique. Using MDSCs for tissue engineering application and cell transplantation into animals and patients has verified that these cells are highly efficient for stem cellbased therapies in various tissues including regeneration and repair of skeletal and cardiac muscle, bone, articular cartilage, and peripheral nerve.…”

“…In the present study, using a modified preplate technique [preplate population 6 (PP6)], 7 a group of SASCs were isolated from injured and healthy noninjured mouse gastrocnemius muscle at 4 days after laceration injury, which is when muscle stem cells undergo activation and proliferation. 23,24 Studies were then performed to directly compare the various characteristics of SASCs isolated from injured muscle and control noninjured muscle including the ratio of cells positive for Sca-1 and those positive for CD34 (MDSCs), and potential for cell migration, proliferation, and multidifferentiation.…”