Single-cell RNA sequencing for osteochondral fusions

We have developed a protocol to generate hPSC-derived osteochondral fusions that mimick embryonic long bone diaphysis. hPSCs were first differentiated to chondroprogenitors and osteogenesis-committed early chondrocytes. Then they were centrifuged together to genrate a fusion construct. After cultured for 7 days in vitro, the fusion construct was transplanted under the kidney capsule of the NOD/SCID mice to generate a mature osteochondral fusion. The 4-week osteochondral fusion grafts contain full spectrum of cells during endochondral ossification, mimicking the in vivo growth plate tissues of early long bone diaphysis. To exam this tissue in single-cell level for comparison with the published datasets of the true human emrbyo, we conducted single-cell RNA sequencing on the 4-week osteochondral fusion grafts. To collect enough cells for single-cell RNA sequencing, three osteochondral fusions were pooled together and were dissociated to single cells by Liberase after cut into small fragments. Digestion was stopped until no large fragments could be found and the cells were filtered by 40μm strainers. After that, the cells were treated with hemolysis reagents for 10 min at RT. Then 1 mL basal medium composed of DMEM/F12, 10% FBS, 1% pen-strep was added and the cells were centrifuged at 300g for 5 min and were resuspended in the medium mentioned above.