Background Human embryonic stem cells (hESCs) have high proliferation potential and can generate differentiated progeny of all three embryonic germ layers. However, the derivation of hESCs from early embryos raises technical and ethical limitations for their use in research and the clinic. Engineered stem cells, known as iPSCs, generated from somatic cells by transduction of defined reprogramming transcription factors, typically OCT4, SOX2, KLF4, and c-MYC, open a new avenue to avoid the controversy of using hESCs.
Objectives The purpose of this study was to test the in vitro chondrogenic potential and in vivo capacity for cartilage regeneration of human iPS (hiPS) cells.
Methods Cell culture and differentiation of EB
We used the hiPSC line (SBI, cat# SC802A-1) generated by direct delivery of four proteins fused to a cell penetrating peptide.
Analysis for condrogenic differentiation
After 21 days of in vitro culture, pellets were analyzed for DNA contents, GAG amount, and the expression of chondrogenic markers.
The hiPS-pellets or alginate-hiPSCs constructs were implanted in the osteochondral defect model rat. The rats received daily injections of cyclosporin A to suppress immune responses in rats. After 6 weeks, the rats were sacrificed.
Results Mesodermal markers increased in EB while undifferentiated ES markers disappeared. After 21 days of chondrogenic culture in micromass pellets, GAG analysis showed that proteoglycan production was significantly greater in chondrogenic pellets than in undifferentiated hiPSCs and EBs. Safranin-O staining demonstrated that the cells in chondrogenic pellets took on the appearance of immature chondrocytes and secreted extracellular matrix. The chondrogenic marker gene and protein expression increased after 21days of pellet culture. The chondrogenic pellets derived from hiPS cells have very low expression of hypertrophic or osteogenic markers.
Also, hiPS cells underwent good chondrogenic differentiation in PLGA scaffold or alginate gel as well. When hiPS cells in either pellet state or in alginate hydrogel were implanted in the osteochondral defects created on the patellar groove of immunosuppressed rats, the defects implanted with chondro-induced hiPS cells showed a significantly better quality of cartilage repair than the control defects.
Conclusions In conclusion, this study provides a proof-of-principle strategy for using hiPSCs as a cell source for cartilage tissue engineering. While successful in vitro induction of chondrogenesis with improved biochemical characteristics were obtained from hiPS cells, the working mechanisms in the implantation of hiPS cells and strategies for further improvement of in vivo cartilage repair with hiPS cells should be investigated in future studies.
Wei Y, Zeng W, Wan R, Wang J, Zhou Q, Qiu S, Singh SR. Chondrogenic differentiation of induced pluripotent stem cells from osteoarthritic chondrocytes in alginate matrix. Eur Cell Mater. 2012 Jan 12;23:1-12.
Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005 May;36(5):758-69.
Disclosure of Interest None Declared