Figure 4. S1P deficiency causes defective UPR and subsequent cell death, owing to collagen accumulation in the ER of patient chondrocytes.

(A) Hand radiographs. Arrows mark shortening in tubular bones and delayed ossification of epiphyses and carpal bones in the patient compared with a healthy girl. (B) Flow cytometric profiles of iPSCs derived from fibroblasts of the patient and her parents using antibodies against the pluripotent stem cell markers TRA-1-60 and SSEA4. Normal fibroblasts were used as negative controls. (C) Scheme of generating iPSC-derived teratomas in immune-deficient NRG mice. (D) Images of H&E-stained teratomas. Scale bar: 100 μm. (E) Representative images of teratoma tissue sections. Scale bar: 5 μm. Identical chondrocytes in the patient teratoma have enlarged calnexin⁺ ER and enlarged Lamp1⁺ lysosomes compared with maternal chondrocytes. Arrowheads indicate enlarged ER. (F) Immunofluorescence images of cartilage in teratomas. TO-PRO, nuclear staining; yellow, merged. Arrowheads indicate collagen II retained in calnexin⁺ ER in patient-derived cartilage. Scale bar: 10 μm. (G) Images of TUNEL staining of maternal and patient-derived teratomas. Scale bar: 100 μm. TUNEL⁺ apoptotic cells were detected in cartilages but not in noncartilaginous tissues in the patient teratoma. (H) Expression of COP-II vesicle-related genes in maternal and patient-derived teratoma, as measured by qRT-PCR. Each dot represents a teratoma-bearing mouse. (I) Confocal images of cartilage in teratomas. Scale bar: 20 μm; 6.67 μm (insets). Tango1 and Hsp47 were decreased in patient cartilages compared with maternal cartilages. All data are presented as mean ± SEM of at least 3 independent experiments. *P < 0.05; **P < 0.01, Student’s t test.