Figure 1. PDGFRα⁺ Cells Are Tissue-Resident Mesenchymal Cells.

(A) Flow cytometric analysis of PDGFRα⁺ cells from murine skeletal muscle obtained from wild-type (WT) mice at 10–12 weeks of age. Cells were labeled with allophycocyanin (APC)- and phycoerythrin (PE)-conjugated antibodies, then gated as the DAPI⁻ population (P1) and then for PDGFRα⁺-APC antibody (P2).

(B) PDGFRα⁺ cells were analyzed for the expression of endothelial (CD31), mesenchymal (Sca1, CD73, CD105, CD29, CD44), and pericyte markers (NG2 and PDGFRβ) from n = 3 independent experiments. PDGFRα⁺Sca1⁺ and PDGFRα⁺CD105⁺ cells were present as high (hi)- and low (lo)-expression populations. An intermediate (int) population was detected for PDGFRα⁺CD29⁺ cells. As a control, PDGFRα⁺ cells were labeled with immunoglobulin G (IgG) isotype antibodies. Representative histograms (dark peaks) are overlaid with IgG isotype controls (pink peaks).

(C–F) Immunofluorescence staining of PDGFRα⁺ cells with anti-PDGFRα antibody and anti-CD31 (C) or anti-NG2 (E) antibodies in the skeletal muscle of 4-month-old uninjured mice. White arrows show PDGFRα⁺CD31⁻ cells (C); yellow arrows show PDGFRα⁺NG2⁺ cells (E). (D) and (F) represent control IgG immunofluorescence images. Images acquired with a Leica SP5 DM confocal as merged z stacks. Scale bars, 20 μm.

(G) Thoracic aortas from 4-month-old PDGFRαH2B-eGfp mice were isolated, dissected to 1-mm slices (“rings”), and plated in Matrigel. GFP⁺ cell migration was monitored daily for 5 days with an EVOS AMG imaging system (Thermo Fisher Scientific). Vessel-like formation of PDGFRαH2B-eGfp⁺ cells was assessed after 5 days by staining with anti-NG2 antibody (red) and DAPI (blue). Scale bar, 200 μm.

(H) High-power magnification of the dashed rectangle in (G). Scale bar, 30 μm. Images acquired with an EVOS AMG imaging system (Thermo Fisher Scientific). Images are representative of n = 3 independent experiments.

(I) Control immunofluorescence analysis with rabbit IgG (DAKO) as primary antibody. Scale bar, 200 μm.

(J) Aortic sections were treated with serum free-EBM media (Lonza) supplemented with VEGF-A or PDGF-AB for 5 days. Images were acquired with an EVOS AMG cell imaging system and are representative of n = 3 independent experiments. White arrows show scattered (VEGF treatment) versus linear arborizing (PDGF-AB treatment) localization of PDGFRα⁺ cells. Scale bars, 200 μm.

(K) Quantification of PDGFRαH2B-eGfp⁺ cells aligned as linear arborizing tubule-like structures after aortic explant and treatment with PDGF-AB or VEGF-A for 5 days as detected in a field of 0.2 mm². The graph shows the average of n = 4 independent experiments for both groups. Data represent means ± SDs and were calculated by Student’s t test. **p < 0.01.

(L) Quantification of PDGFRαH2B-eGfp⁺ cells migrating outward from explanted aortas. Cells were counted in a field of 0.2 mm² in n = 4 independent experiments for PDGF-AB and n = 3 for VEGF-A. *p < 0.05. Data represent means ± SDs.