Extended Data Fig. 1: Analysis of CTSK–mGFP cells in mouse femur. | Nature

Extended Data Fig. 1: Analysis of CTSK–mGFP cells in mouse femur.

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

Extended Data Fig. 1

a, CTSK–mGFP mesenchymal cells (green) were visualized in the mouse long bones at E14.5. Scale bar, 200 µm. Enlarged images of areas marked by the dotted white boxes are provided in i and ii. b, Immunostaining for CD200 (magenta) confirmed co-localization (shown by yellow arrows) with CTSK–mGFP cells (green) in the periosteum. A separate pool of CD200+ cells are detected at the future primary ossification site (marked by dotted orange line). Scale bar, 20 µm. Images in a and b are representative of 3 independent experiments. c, CTSK–mGFP mesenchymal cells in the long bones of mice were detected by FACS at E16.5. d, Visualization of CTSK–mGFP cells (green) in mouse long bones at E16.5. Scale bar, 500 µm. An enlarged view of the areas marked by dotted yellow boxes are shown in i and ii. CTSK–mGFP cells (green) were detected in the mouse periosteum (i and ii). e, CD200 (magenta) immunostaining confirmed co-localization with CTSK–mGFP cells (green) in the periosteum (top panels). CTSK–mGFP cells in the primary spongiosa morphologically consistent with osteoclasts stained negative for CD200 (bottom panels). Scale bar, 20 µm. Images in ce are representative of 3 independent experiments. f, Visualization of CTSK–mGFP cells (green) in the periosteum (dotted white line) of mouse femur at postnatal day 6 (top) and 12 (bottom). Scale bar, 20 µm. g, CTSK–mGFP visualization shows rare mGFP+ osteocytes, an enlarged view of the dotted white box is provided (i). Scale bar, 20 µm. Image representative of 3 independent experiments. Quantification of total CTSK–mGFP-labelled periosteal cells and mGFP-labelled osteocytes in the mouse femur (ii). ***P = 6.95 × 10−16; two-tailed Student’s t-test. Data are mean ± s.e.m., n = 12 distinct areas of periosteum from 3 independent experiments. h, An enlarged view from Fig. 1e. Representative images from 3 independent experiments. i, FACS plots showing expression of CD49f (left) and CD51 (right) in CTSK–mGFP cells isolated from long bones of 7-day-old mice. Representative plot from 5 independent experiments. j, Femurs from 8-week-old Ctskcre mice were immunostained for Runx2 (magenta, top), alkaline phosphatase (ALPL) (magenta, middle) and osteocalcin (magenta, bottom). Co-localization is shown by yellow arrows. Scale bar, 20 µm. Representative images from 3 independent experiments. k, Femurs of 12-day-old Ctskcre mice were immunostained for gremlin 1 (magenta, top) and nestin(magenta,bottom)).Dotted white line indicates periosteum. Scale bar, 20 µm. Representative images from 3 independent experiments.

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