Fig. 1.

Generating 3D-cultured organoids for pre-clinical modeling and treatment of degenerative joint disease. a 3D-cultured SMSC organoids were generated and phenotypically analyzed with miRNA and mRNA sequencing for potential applications in chondrogenesis and further OA modeling and treatment. b SMSCs self‐assembled to attain a spheroid shape in the 4-week cultivation in the SMSC organoids. Filamentous‐actin (F‐actin, green) and chondrogenic marker SOX9 (red) staining demonstrated chondrogenesis in the confined actin cytoskeleton network of SMSC organoids after 4 weeks. c Heatmap of clustering dysregulated miRNA expression profiles with microarray in SMSC organoids compared to 2D cultured control. d Volcano plot of miRNA expression profiles and miR-138 was most significantly downregulated in SMSC organoids. e–f Downregulation of miR-138 was further validated with (e) qRT-PCR and (f) fluorescence in situ hybridization (FISH) in SMSC organoids. (miR-138: red; nucleus: blue). g Sequence of wild type (WT) and mutant (Mut) FOXC1 binding sites for miRNA-138 (left) and conservation level of miR-138 sequence among species (right). h Luciferase reporter assay analysis results to confirm direct interaction between miR-138 and FOXC1 binding sites. Relative luciferase reporter activity was assessed for co-transfected FOXC1 WT (or Mut) with miR-138 mimics or inhibitor in cultured primary human SMSC cells. miRNA-138 mimics control and inhibitor control served as negative controls. i–l FOXC1 expression(red) with immunostaining (i) in cultured SMSC organoids (green for cytoskeleton) and (j) OA cartilage tissues. Sections were counterstained with DAPI for nucleus (blue). k Schematic representation of how miR-138/FOXC1/HIF signaling pathway might mediate chondrogenesis and the therapeutic effects of SMSC organoids in OA. On the basis of described findings, hypoxia microenvironment of the SMSC organoids could downregulate miR-138 expression. miR-138 inhibition upregulated FOXC1 expression, and FOXC1 further suppressed HIF1α transcription and upregulated HIF3α expression, which attenuates the binding of HIF1α to its target genes and hence inhibits HIF1α transcription. l Histological assessments of joint cartilage with HE (1st row), safranin-O staining O (2nd row) and immunostaining for ACAN, MMP13, miR-138, FOXC1, HIF1α and HIF3α in different groups. Sham: no surgery group; ACLT: OA model group with anterior cruciate ligament transection; ACLT + MSC: only SMSC was injected for OA treatment; ACLT + organoid: SMSC organoids were injected for OA treatment