16.1.3 - Enhanced Cartilage Tissue Yield from Induced Pluripotent Stem Cells by Initial WNT/beta-Catenin Activation
- S. Diederichs (Heidelberg, DE)
- S. Diederichs (Heidelberg, DE)
- U. Kreuser (Heidelberg, DE)
- W. Richter (Heidelberg, DE)
Abstract
Purpose
Induced pluripotent stem cells (iPSCs) are promising for cartilage tissue engineering as they are unlimited in supply. We have established chondrocyte differentiation of iPSCs, but high cell loss currently compromises tissue yield. During embryo development, cell survival and proliferation are regulated by several pathways including WNT/β-catenin. WNT/β-catenin activation initiates mesoderm commitment and hence might be important for chondrocyte differentiation. We here asked, whether short stimulation of WNT/β-catenin signalling at initiation of differentiation would improve cell survival and tissue yield during subsequent iPSC chondrogenesis.
Methods and Materials
Two human iPSC-lines (IMR90,CB) were differentiated towards mesoderm (14days) using bFGF/serum on matrigel/gelatin followed by 42days of chondrogenic 3D-pellet culture with 10ng/ml TGF-β1. One group was stimulated with a WNT/β-catenin pulse (5µM CHIR99021) for 24h at the start of mesoderm differentiation (d0). Effects were analyzed by Western blot, qPCR, cDNA-microarray, histology, aggregation assay and DNA quantification.
Results
Initial CHIR treatment significantly increased the number of PDGFRα-positive cells at d7 compared to controls. Accordingly, mRNA-levels of mesoderm markers were significantly elevated at d14 and common ectodermal markers reduced, demonstrating an enhanced mesoderm commitment of CHIR-treated iPSCs. While controls quickly formed multiple free-floating small aggregates and only few cells attached to the plastic surface, CHIR-treated cells aggregated into a plastic-adherent cell sheet, which subsequently condensed into one large pellet over 2-14days. In line, CHIR-treated cells expressed ECM and adhesion-related genes at higher levels than controls at d14. As a consequence of improved pellet formation, DNA amounts in the CHIR-group remained significantly higher than in controls during chondrogenic culture, resulting in significantly larger pellets.
Conclusion
Initial WNT activation improved mesoderm commitment; and we demonstrate for the first time that, acting via stimulated cell proliferation, ECM-expression and cell-aggregation, WNT-pulsing is key to rescue low tissue yield during chondrogenesis. This advancement can be highly beneficial for clinical cartilage regeneration, disease modelling and drug screening.