16.1.4 - Differential PI3K/AKT Activity in Endochondral vs. Chondral Development In Vitro
- S. Diederichs (Heidelberg, DE)
- S. Diederichs (Heidelberg, DE)
- F. Klampfleuthner (Heidelberg, DE)
- B. Lotz (Heidelberg, DE)
- W. Richter (Heidelberg, DE)
Abstract
Purpose
A main limitation of cartilage engineering with multipotent stromal cells (MSC) is their inherent endochondral development. Current treatments like WNT inhibition can only reduce chondrocyte hypertrophy, but not induce an articular chondrocyte (AC)-like phenotype in MSC. PI3K/AKT signaling is essential for cartilage neogenesis and chondrocyte hypertrophy in the growth plate. Yet, its role for hypertrophic differentiation of MSC in vitro remains unclear. Aim was to uncover if different PI3K/AKT activity in MSC-derived hypertrophic chondrocytes vs. non-hypertrophic AC might indicate PI3K/AKT inhibition as promising to reduce hypertrophy during MSC chondrogenesis.
Methods and Materials
Human expanded MSC and AC were subjected to chondrogenic 3D culture for 42 days and AKT activity was detected via phospho-AKT Western blot. PI3K/AKT signaling was inhibited by LY294002 (0.25µM - 25µM) from day 21 on. Differentiation was assessed via histology, qPCR, proteoglycan quantification and alkaline phosphatase activity. To evaluate AKT activity under anti-hypertrophic stimulation, MSC pellets were co-treated with the WNT inhibitor IWP-2 (5µM, d14-d42).
Results
Unlike AC, MSC upregulated AKT activity during chondrogenesis in parallel to hypertrophy (figure 1), reaching significantly higher levels than AC from d21 on. LY dose-dependently reduced hypertrophic (IHH, PTHR1, COL10A1 mRNA) along with chondrogenic markers (COL2A1, ACAN) and proteoglycan deposition. In line, LY reduced TGFβ-induced pSMAD2/3 and SOX9 protein. Importantly, no LY dose was capable to selectively target hypertrophic but not chondrocyte markers. This indicated that PI3K/AKT activity played primarily a pro-chondrogenic role and may not regulate hypertrophy. In line with this observation, pAKT protein levels became also upregulated in MSC under anti-hypertrophic treatment with IWP-2.
Conclusion
Although the increasing AKT activity in MSC but not in AC suggested its relevance for endochondral differentiation, we here demonstrated that MSC chondrogenesis strongly depends on PI3K/AKT activation. This indicates that induction and maintenance of PI3K/AKT activity will be crucial for future therapeutic success of MSC-based cartilage regeneration.