Fig 2: Aorta-associated progenitor cells may partially be yolk sac-independent.Confocal image of a transversal section from an NCX1−/− mouse embryo (E9.5) immunostained for CD34 and Sca-1. The presence of CD34+/Sca-1+ cells surrounding the aortic region of these embryos that lack a functional vascular network to the yolk sac indicates a yolk sac-independent origin of these progenitor cells. Scale bars: lower magnification 50 µm, higher magnification 10 µm; NT neural tube, A aorta.

Fig 3: Chronic stress augmented colony‐forming capacity and Adrβ3 activity in BM sca‐1+ cells. A, First, 2×104 BM sca‐1+ cells were seeded on a 3‐mm dish in duplicate and incubated for 7 days. Colonies were counted using a low‐magnification inverted microscope. Colony‐forming unit (CFU) assay showed an increased colony‐forming capacity of the sac‐1+ cells from stressed BM. B, The extracts of the sac‐1+ cells (100 μg/well) from the BM of nonstressed and stressed mice were subjected to an Adrβ3 ELISA. Stress had increased levels of Adrβ3 activity in the BM‐derived sac‐1+ cells. C, Following culturing in serum‐free DMEM overnight, the sac‐1+ cells were cultured in the presence and absence of the GLP‐1R agonist exenatide (5 nmol/L) for 24 h, and the levels of CXCL12 mRNA were analyzed by quantitative real‐time PCR. GLP‐1 had no effect on CXCL12 mRNA. Values are mean±SEM (n=5–8, Student t test). BM indicates bone marrow; CXCL12, C‐X‐C motif chemokine 12; GLP‐1, glucagon‐like peptide‐1; PCR, polymerase chain reaction.

Fig 4: DPP4 inhibition rectified the stress‐induced BM CD150+ cell hyperproliferation. A, Quantitative data for the percentage of BrdU+ proliferating cells (per 2×102 BM cells) in the BM niches of 2 experimental group mice (n=5, Student t test). B, Double immunofluorescence of BM niches for proliferating CD150+ cells (PCNA + CD150+). Bar graphs: The numbers of double‐positive PCNA + CD150+ cells (n=6, Student t test). C, CXCL12 mRNA in BM sca‐1+ cells of the experimental group mice (n=6, Student t test). D, Representative Western blotting images and quantitative data for brain GLP‐1R protein and Adrβ3 protein of BM sca‐1+ cells (n=3, Student t test). E, Representative gelatin zymography images and quantitative data for MMP9 activity in BM sca‐1+ cells (n=3, Student t test). Data are mean±SEM. *P<0.01 vs stress‐alone control mice. Adrβ3 indicates adrenergic receptor β3; BM, bone marrow; BrdU, bromodeoxyuridine; CXCL12, C‐X‐C motif chemokine 12; DPP4, dipeptidyl peptidase‐4; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GLP‐1R, glucagon‐like peptide‐1 receptor; MMP9, matrix metalloproteinase 9; PCNA, proliferating cell nuclear antigen; S‐Ana, stressed‐anagliptin treatment.

Fig 5: In healing bones, Sca-1+ cells spontaneously form osteoblasts and osteocytes. A) 2.5 × 104 fresh prospectively isolated, UBC-GFP mice-derived Sca-1+ and Sca-1- cells were encapsulated in TG-PEG hydrogels and implanted for 12 weeks into 4 mm calvarial bone defects in C57BL/6 receiver mice. B) Representative top views of 3D surface rendered micro-CT reconstructions of treated defects (scale bar = 3 mm) and C) quantitative assessment of bone formation within defects, shown as individual as individual data points and means (n = 4, Student's t-test, n.s. not significant). D) Contribution of Sca-1+ cells to newly formed bone structures by sequential micro-CT (upper panel) and fluorescence microscopy evaluation (lower panel; scale bar = 500 µm). E) Representative images of anti-GFP stain-based localization of GFP positive cells within the bone wound (black triangle, osteocytes; white triangle, bone lining cells (osteoblasts); scale bar = 20 µm).