Figure 4.

WNT Signaling Modulation In Vitro Prevents the Onset of Endochondral Ossification In Vivo

(A) Collagen type X immunostaining on pellet cultures obtained from WF-MSCs (+WNT+FGF) or F-MSCs (+FGF). Representative images of three MSC donors. The scale bar represents 1 mm.

(B and C) Gene expression analysis of the hypertrophic markers COL10 (B) and MMP13 (C) on pellet cultures after 3 or 5 weeks of chondrogenic induction. Pellets were obtained from MSCs (n = 3 donors and three pellets per donor) expanded in the indicated conditions. Values represent means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.

(D) Schematic overview of the in vivo experimental protocol.

(E) Pre and post in vivo analysis of pellets formed using F-MSCs (+FGF) or WF-MSCs (+WNT+FGF). GAG: thionine staining. The scale bar represents 1 mm. μCT: Quantum FX μCT 3D images of the calcified area of two representative pellets per condition. The scale bar represents 2 mm. H&E: hematoxylin and eosin staining on histological sections of decalcified samples. Black arrowheads indicate blood vessel invasion. The scale bar represents 1 mm. COL2: collagen type II immunohistochemistry on pellet cultures. The scale bars represent 150 μm (high magnification) and 400 μm (low magnification).

(F) Transcript analysis of pellet cultures formed with cells expanded in the indicated conditions and chondrogenically induced without (→ST) or with IWP2 (→IWP2). Values represent means ± SEM of three pellets from three MSC donors. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(G) Pre and post in vivo analysis of pellets formed using WF-MSCs chondrogenically differentiated with IWP2 (+WNT+FGF→IWP2). The in vivo data are representative of samples from two MSC donors, four pellets per donor and condition. Scale bars are as defined in (E).