(A) Colony forming efficiency assay of BM MSCs isolated from Prrx1-CreERT; Ptch1f/f and control mice. These plates were stained with crystal violet. Right panels, quantitation data, *p<0.05, n = 3. (B) Cell proliferation of MSCs isolated from Prrx1-CreERT; Ptch1f/f and control mice by Ki67 staining. Right panel: quantitation data. *p<0.05, n = 3. (C) Prrx1-CreERT; Ptch1f/f MSCs showed an alteration in tri-lineage differentiation activities. Osteoblast differentiation was judged by Von Kossa and Alizarin Red staining, chondrocyte differentiation was judged by Toluidine Blue staining, and adipocyte differentiation was judged by Oil Red O staining. (D) Quantitative PCR analysis of lineage-specific markers of Ptch1-/- and control MSCs cultured with osteoblast medium, chondrocyte medium or adipocyte medium. All samples were normalized to GAPDH and then to the controls, *p<0.05, **p<0.01, n = 3. (E) H/E staining of histological sections from implanted Ptch1-/- and control MSC-scaffolds. Right panel, quantitative analysis of amount of bone and adipocytes that formed on the HA/TCP particles using Image-Pro Plus software based on H/E staining. HA/TCP, hydroxyapatite/tricalcium phosphate; B, bone; ad, adipocytes. Right panel: quantitation data. *p<0.05, n = 3. (F) Chondrocyte cells pellet images of differentiated Ptch1-/- and control BM MSCs. Right panel, quantitative data of pellet sizes. *p<0.05, n = 4. (G) Chondrocyte cell pellet assays of differentiation of Ptch1-/- and control BM MSCs. The same numbers of mutant and control MSCs were induced to differentiate into chondrocytes and the section were stained with H/E (left), toluidine blue (middle), or Ki67 (right).