Extended Data Fig. 3: Functional characterization of non-CTSK MSCs, PSCs and their derivatives. | Nature

Extended Data Fig. 3: Functional characterization of non-CTSK MSCs, PSCs and their derivatives.

From: Discovery of a periosteal stem cell mediating intramembranous bone formation

Extended Data Fig. 3

a, Total numbers of PSCs and non-CTSK MSCs in mouse femurs at postnatal day 7, day 15 and day 32. Significant decreases in number of PSCs are observed at day 15 (**P = 0.006) and day 32 (**P = 0.009) compared to day 7. Significant decreases in non-CTSK MSCs are observed at day 15 (***P = 3.8 × 10−5) and day 32 (***P = 0.0003) compared to day 7. Two-tailed Student’s t-test. Data are mean ± s.d., n = 3 independent experiments; 5 animals per group for day 7, day 15; 3 animals per group for day 32. b, µCT images of the bone formed by non-CTSK MSCs (left) and PSCs (right) 5 weeks after transplantation. Representative images from 5 independent experiments. c, Quantification of bone volume when equal numbers of non-CTSK MSCs and PSCs were transplanted into secondary hosts. Data are mean ± s.e.m., n = 3 independent experiments; two-tailed Student’s t-test. ns, not significant. d, Clonal non-CTSK MSC colonies were split for differentiation into osteoblasts (left, alizarin red staining) and adipocytes (middle, oil red O staining) (scale bar, 20 µm). Separately, chondrocyte differentiation potential was assayed (right, alcian blue staining; scale bar, 100 µm). Representative images from 4 independent experiments. e, Clonal differentiation capacity of 10 colonies isolated from PSCs and non-CTSK MSCs after subsequent culture under osteogenic (left) and adipogenic (right) differentiation conditions. All 10 colonies from each population were equally multipotent. Data are mean ± s.d., n = 3 independent experiments. f, Bright-field images of primary (left), secondary (middle) and tertiary mesenspheres (right) derived from non-CTSK MSCs. Tomato red (red) expression is shown in the insets. Scale bar, 20 µm. Representative images from 3 independent experiments. g, The percentage of PSCs and non-CTSK MSCs able to form mesenspheres. *P = 0.02, one-way ANOVA, Dunnett’s multiple comparison test. Data are mean ± s.d., n = 3 independent experiments. h, FACS analysis of in vitro differentiation of non-CTSK MSCs after 15 days of culture. Red box indicates parent/daughter gate. i, FACS plots of non-CTSK MSC-derived cells after the first round of mammary fat pad transplantation. Colour-coded boxes (red and green) indicate parent/daughter gates. FACS plots in h and i are representative of 3 independent experiments. j, FACS for CD140α (i) and CD146 (ii) in PSCs after transplantation into the mammary fat pad. k, FACS for expression of GFP (i),CD140α (ii), and CD146 (iii) in non-CTSK MSCs after mammary fat pad transplantation. l, PP1 cells were transplanted into the mammary fat pad of primary hosts for 2.5 weeks and then analysed by FACS (i–iii). Colour-coded boxes (green and magenta) indicate parent/daughter gates. m, n, PP2 cells were isolated by FACS and implanted into the mammary fat pad of primary recipients. PP2 derived cells in primary recipients were analysed by FACS (m, i–iv), and PP2 cells were re-isolated for transplantation into secondary recipients. PP2-derived cells in secondary recipients were analysed by FACS (n, i–iv). Colour-coded boxes (green, magenta and orange) indicate parent/daughter gates. Plots in jn are representative of results from 3 independent experiments.

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