Isolation and characterization of side population stem cells in articular synovial tissue

Abstract

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.

Conclusion: 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.

Figure 1

Flow cytometric characterization of SP cells. (A) Bovine synovial cells were stained with Hoechst 33342, and excited by a UV laser. Populations including SP cells were determined by live/dead evaluation using PI, single/doublet cells evaluation using FSC-A/SSC-A and microscopic evaluation (B). SP patterns were visible after Hoechst 33342 staining in population 3 (P3). P1 and P2 consisted of small, non-adherent cells.

Figure 2

Expansion of SP cells by preculture. (A-a) Synovial tissue-derived SP cells were cultured in the DMEM (solid rectangles) or StemPro-34SFM (SFM; shaded rectangles) with different concentration of FBS. Data represent the mean ± S.D. of 6 experiments. Asterisks indicate statistically significant difference (P < 0.01) between control (tissue) evaluated by Dunnet's test. Different characters indicate statistically significant difference (P < 0.05) determined by ANOVA and Tukey's multiple comparison tests. (A-b) SP region expanded by preculture in 1%FBS supplemented SFM (upper panel). The regions were determined by verapamil treatment (lower panel). (B) Phase-contrast images of precultured cells in SFM supplemented with 1% FBS. In the condition, small cell colonies could be observed (arrow). Phase-contrast images of the sorted cells (right). Right-upper panels show SP cells (scale bar = 200 μm) and high-magnification image of these (scale bar = 100 μm). Right-lower panels show NSP cells and high-magnification images of these.

Figure 3

Analysis of the stem cell marker gene expressions based on quantitative RT-PCR. (A) Expression scores were obtained by the delta-delta Ct calculation method. All values are mean ± S.D. of 5 samples of SP (S) and NSP (N), and 6 samples of precultured SP (cS) and precultured NSP (cN). Significant differences were observed using ANOVA and Tukey's multiple comparison tests. Asterisks on bars indicate significant differences (P < 0.05). (B) Gel electrophoresis pattern recognition of real-time PCR products. Total RNA without reverse transcribed samples (RT-) were used as negative control.

Figure 4

Characteristics of SP cells on ex vivo expansion. (A-a) Phase-contrast images of cultured SP cells. Scale bar = 100 μm. (A-b) Colony-forming assay with the SP cells. SP cells were seeded at a density of 500 cells per a fibronectin coated 35-mm dish. (A-c) Cell proliferation speed was markedly promoted by addition of growth factors and fibronectin addition. Values are the mean and SEM of 3 independent cell lines. (B) Ho-staining and FACS analysis of ex vivo expanded SP and NSP cells. Open triangles indicate the populations possessing Ho-efflux properties. The NSP cells did not represent the Ho-efflux property. All experiments were performed using 6 times passaged cells, and replicated 3 times using 3 independent cell lines. (C) Analysis of the stem cell marker gene expressions based on quantitative RT-PCR in ex vivo expanded SP cells. Y-axis represents the relative values for SP cells of primary culture (correspond to the mean values of cS in figure 3A). Significant differences between primary SP cells and 7 times passaged SP cells were observed by Dunnet's test. Significant differences between passaged SP cells and passaged NSP cells were observed by student t-test.

Figure 5

Evaluation of differentiation properties of SP cells. (A) In vitro chondrogenesis. SP and NSP cells were cultured for 21 days in TGF-β contained chondrocyte differentiation medium. (A-a) Pellets were stained with toluidine blue and alcian blue. (A-b) Expression of chondrogenic genes was also examined. SP and NSP cells were cultured for 21 days, and chondrocyte specific gene expressions were determined by RT-PCR analysis. Representative data were shown with 3 experiments. (B) In vitro osteogenesis. SP and NSP cells were cultured for 21 days in osteocyte differentiation medium. (B-a) ALP activity was determined in both derivatives. (B-b) Calcium deposition was examined by 1% Alizarin red staining, High-magnification image of stained cells derived from SP cells were shown in right panel. Scale bar is 200 μm. (B-c) Expression of osteocyte specific gene Osteocalcin was determined by RT-PCR. (C) In vitro myogenesis. SP and NSP cells were cultured for 7 days in myocyte differentiation medium. Desmin expressions were determined by immunocytochemical staining (C-a; scale bar = 100 μm), and high-magnification image of SP derived myocyte (C-b; scale bar = 50 μm). (C-c) Myocyte specific gene expressions were determined by RT-PCR.