The Isolation and Characterization of Muscle Derived Stem Cells from Gastrocnemius Muscle of Rats Using the Modified Preplate Method.
- Author:
Ji Youl LEE
1
;
Soon Young PAIK
;
Soon Hong YUK
;
Jin Ho LEE
;
Sung Ho GHIL
;
Sang Sub LEE
Author Information
1. Department of Urology, The Catholic University of Korea, Korea. uroljy@catholic.ac.kr
- Publication Type:Original Article
- Keywords:
Stem cells;
Muscle;
Rats
- MeSH:
Animals;
Desmin;
Extremities;
Fibroblasts;
Muscle Cells;
Muscle, Skeletal*;
Rats*;
Stem Cells*
- From:Korean Journal of Urology
2004;45(12):1279-1284
- CountryRepublic of Korea
- Language:Korean
-
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.
