Fig. 3: ROR2 is required for HFSC self-renewal and long-term maintenance.

a CFE of Ror2 Ctrl and cKO HFSCs from telogen-phase back skins. Colonies from FACS-purified HFSCs are stained with Rhodamine B (left). Quantification of CFE (right). Numbers of colonies which sizes ≥ 3 mm² were counted. Data are reported as average ± s.d.; n = 3 independent wells; **p = 0.0007. Results are representative of at least two independent experiments. b Ror2 cKO HFSCs could not be passaged and maintained for long-term. Individual Ror2 Ctrl or cKO HFSC colonies were isolated, cultured and passaged. Ror2 depletion was verified for individual colonies. (Left) Ror2 Ctrl HFSCs could be passaged and maintained with feeders as evidenced by YFP expression, whereas YFP+ Ror2 cKO HFSCs got lost along passaging. White dashed lines denote the outline of HFSC colonies. Scale bar represents 200 μm. (Right) Plot graph showing that Ror2 cKO HFSCs could not be long-term maintained in culture. Data are reported as average ± s.d; n = 3 groups, 6 colonies per group were examined. c Cultured Ror2 cKO HFSCs display differentiation-like morphology and reduced proliferation ability. (Left) Differential interference contrast (DIC) images of long-term cultured Ror2 Ctrl and cKO HFSCs. (Right) Proliferation abilities of Ror2 Ctrl and cKO HFSCs were measured based on the daily counts of cell numbers. Data are reported as average ± s.d; n = 3 wells of individual samples. Scale bar represents 50 μm. d Cultured Ror2 cKO HFSCs show reduced expression of HFSC markers, Cd34 and Krt15. qPCR of mRNAs (left) and immunoblotting (right) from higher passaged Ror2 Ctrl and cKO HFSCs. Data are reported as average ± s.d; n = 3 independent HFSC colonies; Ror2, **p < 0.0001; Ccnd1, **p = 0.0001; Cd34, **p = 0.0075; Krt15, **p = 0.0028. All p values were calculated using unpaired two-sided t-test. Source data are provided as a Source Data file.