Figure 2.

Runx2-Dlx5-Osx-ATF4 rapidly and stably induce osteoblast reprogramming. (A) Runx2-Dlx5-Osx-ATF4 induced iOBs exhibit osteoblast cell morphology. After 25 days of direct reprogramming, iOBs exhibit compact cell clusters (b), cuboidal (c) and nodular (d) osteoblast-like cell morphology and GFP expression (f-h) (live GFP images). No cell clusters or GFP⁺ cells are visible in the mock transduction group (a and e). (B) Immunofluorescence staining for GFP and the mature osteoblast marker osteocalcin. Nuclei were stained with DAPI. Scale bars: 1000 μm. (C) Flow cytometry analysis shows that the percentage of Col2.3GFP⁺ cells increased over time course during reprogramming (left panel). All data represent mean ± SD (n=3; One-way ANOVA; *, P=0.018; #, P<0.001; relative to Day 5). At day 25 of reprogramming, RDOA induced around 3.46 % Col2.3GFP⁺ iOB cells (right panel). (D) qRT-PCR analysis shows the expression of osteogenic specific markers increased over time course during reprogramming. All data represent mean ± SD (n=3; Multiple t test; For Col1a1: #, P <0.001; *, P = 0.019; + P = 0.011; For Bglap: ++, P = 0.009; **, P = 0.007; *, P = 0.040; +, P = 0.038; ^, P = 0.045; For Ibsp: *, P = 0.011; +, P = 0.006; #, P <0.001; relative to Fibroblast). (E) qRT-PCR analysis shows that expression of endogenous reprogramming factors increased over time course during reprogramming. All data represent mean ± SD (n=3; Multiple t test; For Runx2 (endogenous): *, P = 0.028; +, P = 0.012; ^, P = 0.046; **, P = 0.001; For Sp7 (endogenous): #, P <0.001; **, P = 0.001; *, P = 0.012; +, P = 0.003; ^, P = 0.004; For Dlx5 (endogenous): #, P <0.001; *, P = 0.020; +, P = 0.039; ^, P = 0.042; **, P = 0.015; ++, P = 0.044; relative to Fibroblast). See also Figure S5 - S7.