Synovium-derived stem cell-based chondrogenesis

Abstract

Synovium is considered a candidate source of cells for cartilage tissue engineering. Compared with mesenchymal stem cells (MSCs) from other sources, synovium-derived stem cells (SDSCs) have a higher capacity for chondrogenic differentiation. Our objective was to define cocktails of growth factors that support the growth and chondrogenic differentiation of SDSCs in chemically defined medium. We established a fast and highly selective technique of negative isolation of SDSC populations. The individual and combined effects of three growth factors-transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2)-were evaluated in serum-free pellet cultures of SDSCs for the chondrogenesis of SDSCs using histology, biochemical analysis, and real-time RT-PCR. In vitro studies identified TGF-beta1 as the key factor for both the growth and chondrogenesis of SDSCs. The highest rates of SDSC growth were observed with the synergistic interaction of all three factors. With respect to chondrogenic differentiation of SDSCs, the interaction of TGF-beta1 and IGF-I applied simultaneously was superior to the sequential application of these two factors or any other combination of growth factors studied. Based on these findings, we propose a two-step protocol for the derivation of chondrogenic SDSCs: a cocktail of TGF-beta1, IGF-I, and FGF-2 is applied first to induce cell growth followed by a cocktail of TGF-beta1 and IGF-I applied to induce chondrogenesis.

Figure 1. Isolation and multi-differentiation property of synovium-derived stem cells (SDSCs)

(A) Harvest sites and target tissue (intimal layer of synovium) in a porcine knee joint; anterior-posterior view is shown. (B) Selection of SDSCs from synovial tissue using negative isolation method. Western blot analysis of lysates from three different sources: synovial tissue (lane 1), conjugated cells (lane 2), and negatively isolated cells (lane 3) are shown. Immunoblots were probed with monoclonal antibodies against macrophages (Calprotectin and CD14), fibroblasts (Vimentin and CD90) and MSCs (CD105 and CD90). β-actin served as an internal control. (C) Morphology property (I/II) and multi-differentiation capacity (III/IV) of SDSCs in monolayer culture. (I) Phase contrast photomicrograph of SDSCs. (II) Four SDSC-colonies were stained with crystal violet. (III) Adipogenesis of SDSCs. After 14 days in complete medium to form single cell-derived colonies, SDSCs were incubated in adipogenesis medium for an additional 21 days, and stained with Oil Red O. Representative lipid vacules are shown. (IV) Osteogenesis of SDSCs. After 14 days in complete medium to form single cell-derived colonies, SDSCs were incubated in osteogenesis medium for an additional 21 days, and stained with Alizarin Red S for calcified nodules.

Figure 2. Higher chondrogenic differentiation capacity of negatively isolated SDSCs

(A&B) histology data for glycosaminoglycan (GAG) and collagen II. (A): SDSC-pellets and (B): SC-pellets. All scale bars are 100 μm. (C&D): biochemical analysis. (C): DNA amount (μg/pellet) and (D): GAG amount (μg/pellet). (E&F): real time RT-PCR. (E): relative collagen II mRNA level. (F): relative aggrecan mRNA level. Data are shown as Ave ± SD (n = 4) after 7 days of culture. Statistically significant differences are shown with * for p<0.05, ** for p<0.01, and *** for p<0.001.

Figure 3. Effects of proliferation growth factor cocktails: biochemical data

(A) Experimental groups. (B): DNA contents (μg/pellet). (C): GAG contents (μg/pellet). (D): mRNA ratio of collagen II to collagen I. Data are shown as Ave ± SD (n = 4) after 18 days of culture. Statistically significant differences compared to P5 are shown with * for p<0.05, ** for p<0.01, and *** for p<0.001.

Figure 4. Effects of proliferation growth factor cocktails: histological data

Distributions of sulfated GAG (Safranin-O), type II collagen (antibody), type I collagen (antibody) and type X collagen (antibody) for experimental groups shown in Fig. 2. Knee cartilage and attached synovial tissue from the same animal served as positive controls. All scale bars are 100 μm.

Figure 5. Effects of differentiation growth factor cocktails: biochemical data

(A) Experimental groups. (B): DNA contents (μg/pellet). (C): GAG contents (μg/pellet). (D): mRNA ratio of collagen II to collagen I. (E): mRNA ratio of aggrecan to collagen I. Data are shown as Ave ± SD (n = 4) after 18 days of culture. Statistically significant differences compared to D5 are shown with * for p<0.05, ** for p<0.01, and *** for p<0.001.

Figure 6. Effects of differentiation growth factor cocktails: histological data

Distributions of sulfated GAG (Safranin O), type II collagen (antibody), type I collagen (antibody) and type X collagen (antibody) for the nine experimental groups (nine treatments) shown in Fig. 4. All scale bars are 100 μm.