Fig. 3: Axin2^TdTom cells readily expand, are enriched for progenitor/stem cell markers, and undergo multilineage differentiation in vitro.

A–M TAM was given to 3-month-old mice and at 4 months, Axin2^TdTom cells were extracted from the limb tendons and analyzed or plated in cell culture. A–C Images are focused on Axin2^TdTom cells to show changing cell morphology at 4 days, 7 days, and 10 days in culture. D, E Representative flow cytometry plots showing Axin2^TdTom and Axin2^(Neg) cells prior to (0 days) and at 10 days in culture. F Axin2^TdTom cells express higher relative amounts of Scx, Sox9, Col1a2, Axin2, Mkx and mKI67 transcripts by RT-qPCR relative to Axin2^(Neg) cells after 10 days in culture (n = 3 mice; Multiple T-test with two-stage step-up method of Benjamini, Krieger and Yekutieli; *p < 0.05). Flow cytometry histograms show Axin2^TdTom cells are enriched for CD44/CD90.2 double positive (G) vs Axin2^(Neg) cells at 10 days of culture (H). I, J A higher percentage of Axin2^TdTom cells express Sca-1 compared with Axin2^(Neg) cells at day 10 in culture. K Quantification of CD 90.2, CD 44, Sca-1, and double-positive CD90.2/CD44 Axin2^TdTom cells vs Axin2^(Neg) cells shows the cells are significantly enriched for the markers (n > 4 mice per experimental group; Multiple T-test with Welch correction *p < 0.05, ****p < 0.0001). L The Porcupine and Wnt secretion inhibitor Wnt 974 or vehicle (DMSO) were added to a final concentration of 2.5 μM to cultured Axin2^TdTom cells. BrdU was added to both conditions for 5 days and cells were analyzed for TdTom expression and BrdU incorporation (n ≥ 5 with each n representing cells from an individual mouse per condition were analyzed; unpaired T-test with Welch’s correction *p < 0.05). M Axin2^TdTom cells from 4-month-old mice were cultured and expanded separately for 60 days. After obtaining sufficient Axin2^TdTom cell numbers, adipogenic, osteogenic, and chondrogenic assays were performed and cells were stained (examples shown in Supplementary Fig. 4). Stained vs total cells were quantified per 200 × 200 μm area with at least four measurements per well, in at least three wells per treatment, and each well-represented cells from a different mouse (n = 5). Addition of Wnt974 yielded a higher percentile of chondrogenic cells (T-test with Welch’s correction *p < 0.05; each dot represents a 200 × 200 μm quantification of cells).