Fig. 4: Identification of CD antigens for the prospective assessment of the chondrogenic capacity of ExpLBM cells.

a, Effect of high-density culture on the stable maintenance of ExpLBM cells derived from the PRRX1^3′tdTomato-reporter line. ExpLBM cells were cultured until they attained high confluency and then analysed to detect tdTomato expression during serial passage. All cells eventually expressed low levels of tdTomato (PRRX1^low ExpLBM cells). b, Levels of Alcian blue staining after 2DCI with PRRX1^high and PRRX1^low ExpLBM cells (right; n = 3 biologically independent experiments). Representative images are shown (left). c, Histological analysis after 3DCI with PRRX1^high (top) and PRRX1^low (bottom) ExpLBM cells. d, Volcano plot identifying genes that are differentially expressed between PRRX1^high and PRRX1^low ExpLBM cells. The dashed vertical and horizontal lines indicate the threshold. The red dots represent genes that are differentially expressed (n = 2 biologically independent experiments). The gene names of each identified surface antigen are highlighted in blue. e, Representative flow cytometric analysis data for CD90 (THY1), CD140B (PDGFRB) and CD82 expressed by PRRX1^high and PRRX1^low ExpLBM cells. f, Representative flow cytometric data for CD90 (THY1), CD140B (PDGFRB) and CD82 expression of SEES4- and 1383D2-derived ExpLBM cells. g, Histological analysis after 3DCI with SEES4- (left) and 1383D2 (right)-derived ExpLBM cells. h,i, CD90^highCD140B^highCD82^low and CD90^lowCD140B^lowCD82^high populations that were purified from 414C2-derived ExpLBM cells using a cell sorter were treated with the 2DCI protocol, followed by Alcian blue staining (h) and RT–qPCR analysis (i; n = 3 biologically independent experiments). h, Representative images (left) and the levels of Alcian blue staining (right) are shown. b,h,i, *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance was determined using a two-sided unpaired Student’s t-test.