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KMID : 1377020130100060380
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Tissue Engineering and Regenerative Medicine
2013 Volume.10 No. 6 p.380 ~ p.387
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Induced chondrogenic differentiation of parthenogenetic murine embryonic stem cells by insulin-like growth factor 2 treatment in a three-dimensional culture environment
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Kang Ho-in
Jeong Young-Dan
Paul Verma
Roh Sang-ho
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Abstract
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The generation of cartilaginous tissues using pluripotent stem cells is one possible solution for cartilage regeneration, as the mass production of cartilaginous cells is required to cure defects and diseases of cartilage. Parthenogenetic ESCs (PESCs) are a useful stem cell source. The defects in full-term development of this cell type enable researchers to avoid ethical concerns. Moreover, in female patients, if the PESCs are derived from the patient’s own oocytes, the cells will have that patient’s genetic information. Here we present data demonstrating that PESCs can be differentiated into chondrogenic cells with induction medium containing multiple factors such as ascorbic acid, dexamethasone, bone morphogenetic protein-2 (BMP-2), and transforming growth factor-beta (TGF-β), and the induction can be promoted by exogenous insulin-like growth factor 2 (IGF-2), which is silenced in PESCs. Before chondrogenic induction of the PESCs, floating aggregates called embryoid bodies (EBs) were formed in suspension culture for 5 days. In the first series of experiments, the EBs were transferred to chondrogenic induction medium that contained serum, ascorbic acid, dexamethasone, BMP-2, BMP-4 and TGF-β, and the cells were cultured for an additional 21 days using either a two-dimensional (2D) or three-dimensional (3D) culture system. In the second series of experiments, we investigated the effects of different concentrations of IGF-2 supplementation (0, 1, 10 or 100 ng/mL) in a 3D culture system. After induction, the gene expression of the chondrocyte-specific markers, CHORDIN-LIKE 1, COLLAGEN-2, AGGRECAN, DECORIN, MMP13 and PAX-1, was analyzed real-time PCR. In experiment 1, the cells from all experimental groups after chondrogenic induction exhibited morphology characteristic of murine chondrocytes as confirmed by Alcian blue and Safranin O staining. The 3D culture system showed increased induction of chondrogenic differentiation in comparison to the 2D system. In experiment 2, the group supplemented with 100 ng/mL IGF-2 showed the highest induction of chondrogenic differentiation in comparison to other groups. The expression levels of all genes were 7-to 35-fold higher in the group treated with 100 ng/mL IGF-2 in comparison to the IGF-2-free control. The result showed that supplementation with the IGF-2 improves the efficiency of chondrogenic differentiation of murine PESCs in a 3D culture environment. The addition of IGF-2 may re-activate imprinted genes in PESCs that are only expressed in the paternal genome and are normally silent in PESCs. Our findings suggest that compensative supplementation of imprinting factor(s) may improve the efficiency of differentiation to specific lineages in monogenic stem cells.
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KEYWORD
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parthenogenesis, embryonic stem cells, chondrogenesis
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