Fig. 5: Co-culture assay of TBBC and human mesenchymal stem cells (MSC).

a Flow chart illustrating in vitro assay setup. TBBCs were pre-differentiated for 13 days on air-liquid interface prior to co-culture, on the other side, MSCs were pre-induced as pellets in chondrogenic medium or pre-induced as monolayers in osteogenic medium for 3 days before co-culture with TBBCs. RNA of MSC samples were collected every 5 days post-co-culture for PCR and fixed at the endpoint (Day 21) for staining. b Real-time PCR measurement of SOX9/ACAN (Chondrogenic-related) and RUNX2/BGLAP (Osteogenic-related) gene expressions on MSCs at D5, D10, and D15 post co-culture (n = 2 experimental replicates per group). w/nc denoting co-culture with non-TO control, w/TO denoting co-culture with TO-TBBC. Two-tailed Student t-tests were performed and p values are indicated to show statistical significance. c HE and Alcian Blue staining on MSC spheroids which were co-cultured with control or TO cells for 21 days, to reveal the progress of chondrogenesis (n = 2 independent experiments). MSC spheroids cultured alone under basic conditions and spheroids under standard differentiation conditions were examined in parallel as negative and positive controls. Scale bar, 100 μm. d, e Schematic diagrams showing two in vivo approaches to perform TBBC-MSC co-culture assay. Dual-cell transplantation onto immunodeficient NSG mice was performed either by subcutaneous injection of TBBCs (2 × 10⁶ cells/spot): MSC (2 × 10⁶ cells/spot) mixture resuspended with 50% Growth factor-reduced Matrigel in a total volume of 200 μl/spot, or via minor surgery with aggregated MSC (10⁶ cell/spheroid) surrounded by a gel-like layer of TBBCs resuspended in Matrigel. Cartoon mice in this graph were adapted from the online source clipartcraft.com. f HE and Alcian Blue staining on xenografts collected from approach D (n = 2 independent experiments). The human origin of growths found in nodules was confirmed by IHC staining with an anti-human nucleoli antibody. Scale bar, 200 μm. g HE and Alcian Blue staining on xenografts collected from approach E (n = 2 independent experiments). The human origin of growths was confirmed by IHC anti-human nucleoli staining. Scale bar, 200 μm. Source data are provided as a Source Data file.