Isolation and characterization of side population stem cells in articular synovial tissue.
BACKGROUND
Autologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 (Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined.


METHODS
Synovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg-2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes.


RESULTS
SP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1%FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1 -all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte.


CONCLUSIONS
In the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.
Institute of Advanced Clinical Medicine, Kinki University School of Medicine, Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan. teramura@med.kindai.ac.jp
Takeshi
T
Teramura
Kanji
K
Fukuda
Shinji
S
Kurashimo
Yoshihiko
Y
Hosoi
Yoshihisa
Y
Miki
Shigeki
S
Asada
Chiaki
C
Hamanishi
D000818:Animals; D015415:Biomarkers; D002358:Cartilage, Articular / Q000166:cytology; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D005434:Flow Cytometry; D007596:Joints / Q000166:cytology*; D032342:Muscle Cells / Q000166:cytology; D010011:Osteocytes / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D013583:Synovial Membrane / Q000166:cytology*
D015415:Biomarkers