Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

Abstract

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Figure 1. Characterization of hUC-MSCs. A, hUC-MSCs at passage 1 displayed a spindle-shaped or polygonal morphology in monolayer culture. B, hUC-MSCs at passage 5 became a predominant fibroblast-like morphology and formed a squamous eddy-like structure. Bar = 50 μm. C, Flow cytometric analysis of surface antigen markers. hUC-MSCs expressed CD44, CD105, CD73, and CD90, but not CD34, CD45, CD19, CD14, or MHC II. PE- and FITC-conjugated mouse monoclonal IgG1 were used as isotype controls.

Figure 2. Effects of Sox9 overexpression on morphological changes and proteoglycan disposition in hUC-MSCs. A, Untreated hUC-MSCs and those transfected with empty vector or Sox9-expressing plasmid had a similar fibroblast-like morphology. In contrast, hUC-MSC cells cultured in the chondrogenic medium (differentiation-induced group: DI) for 48 h were polygonal or irregular in shape and prone to detach from the plate. Bar = 50 μm. B, Assessment of proteoglycan disposition by toluidine blue staining. The degree of toluidine blue staining was low in untreated and empty vector-transfected control hUC-MSC cells with basic maintenance medium. After 2-3 weeks of monolayer culture, Sox9-transfected hUC-MSCs and those treated with chondrogenic medium (DI) showed strong toluidine blue staining. Bar = 100 μm.

Figure 3. RT-PCR analysis of the mRNA expression of Sox9, collagen I, collagen II, and aggrecan in untreated hUC-MSCs cells and those transfected with empty vector or Sox9-expressing plasmid or induced with the chondrogenic medium (differentiation-induced group: DI). Representative gel photographs of RT-PCR products from three independent experiments are shown. d: days.

Figure 4. Sox9 overexpression induces chondrogenic differentiation in hUC-MSC cells. For induction of chondrogenic differentiation, cells were treated as described in Material and Methods. After the treatments, cells were subjected to immunocytochemistry for aggrecan and collagen II. Similar to the treatment with chondrogenic medium, genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs. DI: differentiation-induced group. Bar = 50 μm.