The Isolation and Characterization of Muscle Derived Stem Cells from Gastrocnemius Muscle of Rats Using the Modified Preplate Method

Lee, Ji Youl; Paik, Soon Young; Yuk, Soon Hong; Lee, Jin Ho; Ghil, Sung Ho; Lee, Sang Sub

Abstract

PURPOSE: This study attempted to characterize the muscle derived stem cells isolated from the primary cultured skeletal muscle of the rat gastrocnemius muscle; in addition, we modified the preplate method and then compared this to the original preplate method.
MATERIALS AND METHODS
The hind limbs (gastrocnemius muscles) were removed from a 3-6 week olds SD-rat and the bone was dissected away. The muscle mass was finely minced and chopped using razor blades. In an original preplate method, the cells were dissociated using a triple enzyme mixture (collagenase XI, dipase and trypsin) for 1 hour at 37degreesC. The muscle cell extract was preplated on culture flasks as described by Dr. Qu (Qu et al., 1998). The pp1-pp4 cells were referred to as the early plate (EP) cells, and the pp5-pp6 cells were referred to as the late plate (LP) cells. When we modified the preplate method, the pp1-pp2 cells were called the early plate (EP) cells and the pp3-pp4 cells were called to late plate (LP) cells. The phenotypical characteristics of EP and LP cells were compared by immunostaining and FACS.
RESULTS
In the original preplate methods, the early plate (EP) cells were mixed with myogenic cells (mostly fibroblasts, <15% desmin + cells) and the LP cells were highly purified muscle derived cells that contain pure myogenic cells (>90% desmin + cells). Yet in the modified preplate method, the muscle derived stem cells were determined to be CD34 (+ or -), CD45- and desmin- cells by immunohistochemical staining and FACS.
CONCLUSIONS
In original methods, the LP cells exhibited stem cell properties (CD34+, less than 30%), and they were not from a hematogeous origin (CD45-), but rather, they were from a myogenic origin (desmin+). Yet in the modified preplate method, we purified the LP cells much earlier than the original method. The LP cells displayed CD34+(more than 50%), and CD45-; thus, we isolated more primitive (desmin-) cells.