Fig. 2. A brain-dependent circulatory factor increases the osteogenic capacity of ocSSCs.
a, Schematic of fluorescence-activated cell sorting (FACS) isolation from non-haematopoietic, non-endothelial cell fraction and the fate of ocSSCs (left) and pvSSCs (right). b–e, SSC kidney capsule transplants. b, Schematic of WT female ocSSC kidney capsule transplants into Esr1fl/fl and Esr1Nkx2.1-cre females. c, Representative images of the graft region with host-derived haematopoiesis (top, white arrowheads), BV and Movat’s pentachrome staining of bone (yellow), cartilage (blue) and marrow (red). d,e, Fractional areas from stained kidney graft sections (N = 6, 5) (d) and bone mineral density (BMD) from grafts by µCT (N = 4, 4; Esr1fl/fl (black) and Esr1Nkx2.1-cre (red) females) (e). f–h, SSC transplants into the medial basal hypothalamus (mbh). f, Schematic of stereotaxic bilateral delivery of control ocSSCs (about 550 live cells) from Esr1fl/fl-CAG-Luc-GFP into the mbh of Esr1fl/fl and Esr1Nkx2.1-cre females (N = 7, 6). g, Representative images of pentachrome-stained brain sections (top) with ossicles (lower left) and anti-GFP (lower right) 6 weeks after injection. 3V, third ventricle. h, BV in the mbh of Esr1fl/fl (black) and Esr1Nkx2.1-cre (red) females. i, Per cent ocSSCs, pvSSCs and APCs (Methods) in femurs of Esr1fl/fl and Esr1Nkx2.1-cre 3-week-old (N = 3, 3) and 10-week-old females (N = 5, 5) and males (N = 5, 3). j, Quantification (left) of Alizarin Red (AR; osteogenesis) or Alcian Blue (AB; chondrogenesis) staining of differentiated ocSSCs from 3-week-old, 10-week-old and 54-week-old females with representative images (right) including Oil Red O (adipogenesis) staining (technical replicates in cell culture assays (n) = 3–4 per group). k, Uniform manifold approximation and projection (UMAP) plot (Leiden clustering) of Smart-Seq2 scRNA-seq data of high-quality filtered single ocSSCs from 7-week-old females (left) with dot plot of cluster-specific markers (right). l, UMAP (left), distribution of genotypes within cluster populations (middle) and dot plot of anti-inflammatory and pro-osteogenic markers (right) of Esr1fl/fl and Esr1Nkx2.1-cre ocSSCs. One-way ANOVA in d and i (Tukey’s multiple-comparisons test). Mann–Whitney test, two-tailed in h. Unpaired Student’s t-test, two-tailed for e, i (for the 3-week time point) and j. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars ± s.e.m. Graphic in b (kidney) was reproduced from BioRender (https://www.biorender.com). Graphic in b (mouse) was adapted from Mind the Graph (https://mindthegraph.com) under a Creative Commons licence CC BY-SA 4.0.