Chondrogenic Differentiation of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells for the Regeneration of Defected Articular Cartilage

Introduction: Mesenchymal stem cells (MSCs) are the best candidates for tissue engineering and cell-based therapeutic strategies, such as the treatment of mesenchymal tissue injuries (1). In recent years, investigators have evaluated novel sources of allogeneic MSCs, including bone marrow, umbilical cord blood (UCB) and possible tissues such as synovium, adipose and periosteum. Because UCB is relatively easy to collect and does not produce strong immune rejection, human UCB-derived MSCs (UCB-MSCs) are utilized as an alternative cell source for stem cell-based therapy and transplantation. In this study, we evaluated the multipotency of UCB-MSCs and assessed the three dimensional cultivation and chondrogenic differentiation of UCB-MSCs in atelocollagen gel for the application of tissue-engineered cartilage constructs. Materials and Methods: Isolation and cultivation of UCB-MSCs Mononuclear cells (MNCs) were separated on Ficoll-Hypaque gradient (density 1.077 g/cm3). The isolated MNCs were washed, and then seeded at a concentration of 1~10 X 106 cells/ml in α-minimum essential medium containing 10% FBS. All cultures were maintained at 37°C in 95% humidified air and 5% CO2 with a half change of culture medium twice a week. After one to three weeks, fibroblast-like adherent cells were observed. When the monolayer of UCB-MSCs colonies reached 60% confluence, the cells were subcultured at a concentration of 1000 cells/cm2. Characterization of UCB-MSCs Immunophenotypes of UCB-MSCs were characterized using fluorescence-activated cell sorter (FACS) with specific MSCs surface markers. Mesodermal differentiation and confirmation For in vitro chondrogenic differentiation, UCB-MSCs were embedded into an atelocollagen solution. To induce chondrogenesis, the UCBMSCs were cultured in basal serum-free media with supplements and TGF-β3. Chondrogenic differentiation was confirmed by RT-PCR and immunofluorescence staining for type II collagen protein. Furthermore, osteogenic and adipogenic potential were demonstrated by quantitative real time PCR, alkaline phosphates activity and Oil Red O staining. Results: Immunophenotyping of UCB-MSCs showed positive expression in CD9low, CD13, CD29, CD44, CD49e, CD73, CD90, CD105, CD117low, CD166 and HLA-ABC, but negative expression in CD14, CD31, CD33, CD34, CD38, CD106, CD133, α-smooth muscle actin (ASMA) and HLA-DR (Fig. 1). The UCB-MSCs in atelocollagen, cartilage-like construct showed that type II collagen protein was expressed after 3 weeks induction (Fig. 2). In addition, chondrogenic specific genes such as type II collagen, aggrecan were also expressed (Fig. 3). Discussion: The use of UCB as an alternate source of hematopoietic progenitor cells (HPCs) was first reported by Gluckman et al. in 1989 (2). Since that time, investigators have evaluated novel sources of MSCs as well as hematopoietic stem cells for tissue engineering and cell-based therapeutic strategies. As we mentioned in our previous study (3), whenever the transplantation of MSCs into cartilage defects, a variety of scaffolds have been utilized as successful carriers for cell delivery. One of the various scaffolds, atelocollagen was eliminated telopeptide regions that were the primary cause of immunogenicity. Therefore, it was suitable for our chondrogenic culture system. In conclusion, we have demonstrated that UCB-MSCs can successfully differentiate into chondrocytes in atelocollagen gel. To our knowledge, this is the first report that human UCBderived stem cells have been successfully differentiated into chondrocytes in atelocollagen gel. These results will provide an in vivo model to regenerate defected articular cartilage. UCB-MSCs can therefore serve as an alternative source for stem cell-based therapy and transplantation. References: 1. Horwitz et al. Nat. Med. 5, 309-13, 1999. 2. Gluckman et al. N. Engl. J. Med. 321, 1174-8, 1989. 3. Choi et al. Biotechnol. Lett. 29, 323-9, 2007. Acknowledgements: This work was supported by Korea Science and Engineering Foundation (KOSEF R01-2005-000-10927-0) and also by Boryung Pharmaceutical Co. Ltd., Ansan, Korea