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. 2023 Feb 20;14(1):804.
doi: 10.1038/s41467-023-36408-0.

Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect

Kengo Abe  1   2   3 , Akihiro Yamashita  1   3 , Miho Morioka  1 , Nanao Horike  1   3 , Yoshiaki Takei  3   4 , Saeko Koyamatsu  1 , Keisuke Okita  5 , Shuichi Matsuda  2 , Noriyuki Tsumaki  6   7   8
Affiliations

Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect

Kengo Abe et al. Nat Commun. .

Abstract

Induced pluripotent stem cells (iPSCs) are a promising resource for allogeneic cartilage transplantation to treat articular cartilage defects that do not heal spontaneously and often progress to debilitating conditions, such as osteoarthritis. However, to the best of our knowledge, allogeneic cartilage transplantation into primate models has never been assessed. Here, we show that allogeneic iPSC-derived cartilage organoids survive and integrate as well as are remodeled as articular cartilage in a primate model of chondral defects in the knee joints. Histological analysis revealed that allogeneic iPSC-derived cartilage organoids in chondral defects elicited no immune reaction and directly contributed to tissue repair for at least four months. iPSC-derived cartilage organoids integrated with the host native articular cartilage and prevented degeneration of the surrounding cartilage. Single-cell RNA-sequence analysis indicated that iPSC-derived cartilage organoids differentiated after transplantation, acquiring expression of PRG4 crucial for joint lubrication. Pathway analysis suggested the involvement of SIK3 inactivation. Our study outcomes suggest that allogeneic transplantation of iPSC-derived cartilage organoids may be clinically applicable for the treatment of patients with chondral defects of the articular cartilage; however further assessment of functional recovery long term after load bearing injuries is required.

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Conflict of interest statement

N.T. is an inventor and Kyoto University is a holder of the patent on “An efficient chondrocyte induction method” (PCT/JP2014/079117). This patent is licensed to Asahi KASEI corporation. Y.T. is an employee of Asahi KASEI. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Transplantation of cyiPS-Cart in the knee joints in a primate model.
a Two categories of articular cartilage defect. Left: chondral defects extending down to but not through the subchondral bone. Right: osteochondral defects extending down through the subchondral bone. b Primate model for cyiPS-Cart transplantation. Chondral defects were created in the femoral trochlear ridge of the right knee joints in cynomolgus monkeys. CyiPS-Cart (transplantation group) or nothing (empty group) were transplanted into the defects. The monkey image is taken from [https://www.flaticon.com/free-icon/monkey_47138] following the Flaticon license guidelines. c Gross appearance of the joint surface 4 weeks (left) and 17 weeks (right) after surgery. Data were representative of three monkeys.
Fig. 2
Fig. 2. Analysis of immune reactions following allogeneic transplantation of cyiPS-Cart into chondral defects in the knee joints 4 weeks after transplantation.
a Semi-serial histological sections were stained with safranin O or HE, or immunostained for CD3. Scale bars, 100 µm. b The number of CD3+ cells per microscopic field was determined. Four fields were used for each monkey. Three monkeys were used in each group. Each mark indicates one field, and different shapes of marks indicate different monkeys. Error bars denote mean ± SE. ****P < 0.0001 by one-way ANOVA with post hoc Tukey HSD test (n = 12 fields). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Qualities of repaired tissue in chondral defects.
a Samples were harvested 4 or 17 weeks after transplantation. Semi-serial sections were stained with Safranin O, HE, or picrosirius red. Sections stained with picrosirius red were observed under a polarized microscope. A magnification of the boxed regions that cover repaired tissue and native articular cartilage in the third row is indicated in the bottom row. Scale bars, 100 µm. b Sections stained with Safranin O were subjected to a modified Wakitani histological scoring system (n = 3 monkeys in each group) and evaluated by two independent assessors in a blinded manner. Error bars denote mean ± SE. Source data are provided as a Source Data file. c Magnifications of remaining cartilage located between the bottom of the defect and bone in (a). Dotted lines indicate the bottom of defects. Safranin O staining. Scale bars, 100 µm.
Fig. 4
Fig. 4. Immunohistochemical staining of repaired chondral defects.
a Samples were harvested 4 or 17 weeks after transplantation. Semi-serial sections were immunostained for GFP, type II collagen (COL2), and type I collagen (COL1). The boxed regions in the second row are magnified in the third row. Data were representative of three monkeys. b Semi-serial histological sections of samples at 17 weeks after transplantation were immunostained for CD3. Data are representative of three monkeys. Scale bars, 100 µm.
Fig. 5
Fig. 5. scRNA-seq analysis and chondrogenic differentiation of cyiPSCs into pre-transplant cyiPS-Cart.
a Schematic representation of samples subjected to scRNA-seq analysis. Undifferentiated cyiPSCs (cyiPSC), cyiPS-Cart (pre-transplant cyiPS-Cart), intact articular cartilage (cyAC), fibrous tissue formed in chondral defects in the empty group (cyFT), and cyiPS-Cart in chondral defects in the transplantation group (post-transplant cyiPS-Cart) 17 weeks after surgery. b Ridgeplot (Seurat) showing the distribution of single-cell gene expression in each sample. The x-axis of each panel represents the expression levels of the indicated genes. The y-axis represents the number of cells. c CyiPSCs and pre-transplant cyiPS-Cart cells were projected onto UMAP plots with a parameter resolution of 0.5. d Marker gene expression levels are indicated in each cell projected on the UMAP plot using the featureplot function.
Fig. 6
Fig. 6. scRNA-seq analysis of cyAC, cyFT, pre-transplant cyiPS-Cart, and post-transplant cyiPS-Cart.
a The VlnPlot (Seurat) shows the distribution of single-cell gene expression in each sample. The y-axis of each panel represents the expression levels of the indicated genes. b After reducing the cell number for each sample to 320, the data from the samples were integrated. The cells were then clustered with a parameter resolution of 0.2 and projected onto the UMAP plots. c UMAP plot in (b) separated by samples. d The ratio of the number of cells in each cell cluster in each sample (c) is plotted. e COL2A1 and COL1A1 expression levels are indicated in each cell projected on the UMAP plot using the featureplot function. f Heatmap revealing the scaled expression of differentially expressed genes for each cluster defined in (b). g Canonical pathways enriched for each cluster based on differentially expressed genes. The results of Clusters #3 and #4 were omitted because there were very few cells in these clusters.
Fig. 7
Fig. 7. Expression of PRG4 in cyAC, pre-transplant cyiPS-Cart, and post-transplant cyiPS-Cart.
a The expression level of each gene in pre-transplant cyiPS-Cart is plotted on the x-axis and the expression level in post-transplant cyiPS-Cart is plotted on the y-axis. b VlnPlot of PRG4 expression for each sample. c PRG4 expression levels indicated in each cell projected on the UMAP plot in Fig. 6b, using the FeaturePlot function. d Histological sections were immunostained for PRG4 expression. A magnification of the boxed regions in the top row is shown in the bottom row. Data were representative of three cyiPS-Cart organoids and three monkeys. Scale bars, 100 µm. e Cells from the pre-transplant cyiPS-Cart were cultured in the presence or absence of TGF-β1 (left) or TGF-β inhibitor, SB431542 (right). PRG4 mRNA expression was analyzed using real-time RT-PCR. Error bars denote means ± SE. **P = 0.0048, **P = 0.0017 by two-tailed Student’s t-test (n = 3 dishes). Data were representative of three independent experiments. Source data are provided as a Source Data file.
Fig. 8
Fig. 8. Involvement of Sik3 function and fluid flow shear stress (FFSS) on Prg4 expression in mouse chondrocytes.
a Immunoblot expression analysis of Sik3 phosphorylated at T411, an inactive form of Sik3, in wild-type mouse primary chondrocytes treated with 10 µM forskolin for 30 min. Left: blots representative of three independent experiments are shown. Right: quantification of pSik3(pT411) to the β-actin ratio in cultured chondrocytes with or without forskolin treatment. Error bars denote mean ± SE. **P = 0.0041 by two-tailed Student’s t-test (n = 3). b Real-time RT-PCR analysis of Prg4 expression in wild-type mouse primary chondrocytes treated with 10 µM forskolin for 6 h. Error bars denote mean ± SE. *P = 0.0108 by two-tailed Student’s t-test (n = 6 dishes). Data were representative of three independent experiments. c Real-time RT-PCR analysis of Prg4 and Col10 expression in primary chondrocytes obtained from Sik3 knockout (Sik3−/−) and Sik3 transgenic (Sik3tg) mice. Error bars denote mean ± SE. ****P < 0.0001, ****P < 0.0001, ***P = 0.0003, **P = 0.0012, n = 3, two-tailed Student’s t-test (n = 3 dishes). Data were representative of two independent experiments. d Immunohistochemical analysis of Prg4 expression in the knee joints of Sik3 conditional knockout (11Enh-Cre; Sik3flox/flox) mice lacking Sik3 expression in chondrocytes 14 days after birth. Yellow arrows indicate the thickness of the area in which Prg4 was expressed. Data were representative of five conditional knockout mice and four Sik3flox/+ mice. Scale bars: 100 μm. e Real-time PCR analysis of Prg4 expression in wild-type (Sik3+/+) and Sik3 knockout (Sik3−/−) primary chondrocytes subjected to FFSS for the indicated period. Data are representative of two independent experiments. Source data are provided as a Source Data file.
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