論文の公開元へEngraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect
概要
Title
Engraftment of allogeneic iPS cell-derived
cartilage organoid in a primate model of
articular cartilage defect
Author(s)
Abe, Kengo; Yamashita, Akihiro; Morioka, Miho et
al.
Citation
Nature Communications. 2023, 14(1), p. 804
Version Type VoR
URL
rights
https://hdl.handle.net/11094/93295
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
Article
https://doi.org/10.1038/s41467-023-36408-0
Engraftment of allogeneic iPS cell-derived
cartilage organoid in a primate model
of articular cartilage defect
Received: 30 May 2022
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Accepted: 31 January 2023
Kengo Abe1,2,3, Akihiro Yamashita1,3, Miho Morioka1, Nanao Horike1,3,
Yoshiaki Takei3,4, Saeko Koyamatsu1, Keisuke Okita 5, Shuichi Matsuda
Noriyuki Tsumaki 1,3,6
2
&
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 RNAsequence 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.
Articular cartilage covers the ends of bones and provides lubrication,
which is vital for smooth joint movement and shock absorption.
Articular cartilage is avascular and consists of chondrocytes embedded in the extracellular matrix (ECM), which enables the mechanical
functions necessary for joint motion and shock absorption. Cartilage
ECM consists of collagen fibrils composed of type II, IX, and XI collagen
molecules and proteoglycans composed of aggrecan, link protein, and
glycosaminoglycans.
As articular cartilage has a limited capacity for repair, and so far,
no drugs are available for cartilage repair, focal damage or erosion of
articular cartilage frequently leads to debilitating conditions, such as
osteoarthritis. Although cell-based therapies have been proposed, only
a limited number of autologous chondrocytes are generated since
expansion culture steers the chondrocyte character toward that of
fibroblastic cells1, as evidenced in autologous chondrocyte implantation (ACI), wherein more than 90% of the repaired tissue is
1
Department of Tissue Biochemistry, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan. 2Department of Orthopaedic
Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 3Department of Clinical Application, Center for iPS Cell Research and Application,
Kyoto University, Kyoto, Japan. 4Regenerative Medicine Technology Department, Healthcare R&D Center, Asahi Kasei Corporation, Kyoto, Japan. 5Department
of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan. 6Premium Research Institute for Human Metaverse
e-mail: ntsumaki@tsu.med.osaka-u.ac.jp
Medicine (WPI-PRIMe), Osaka University, Osaka, Japan.
Nature Communications | (2023)14:804
1
Article
fibrocartilaginous2. Allogeneic cartilage has been transplanted clinically without matching human leukocyte antigen (HLA) types and
without the use of immunosuppressive drugs3–5. However, whether the
transplanted allogeneic cartilage causes an immune reaction remains
controversial. Some reports suggest low immunogenicity of
chondrocytes6,7, whereas others show that chondrocytes are antigenic
and elicit varying degrees of immune reactions8,9. To the best of our
knowledge, the regenerative mechanisms following allogeneic cartilage transplantation have not yet been reported. Whether transplanted
cartilage achieves engraftment (survives and constitutes repaired tissue directly) or only transiently remains and secretes growth factors to
stimulate recipient progenitor cells has not been analyzed.
Induced pluripotent stem (iPS) cells are a promising source for the
regenerative treatment of articular cartilage damage10,11. Cartilage
consisting of chondrocytes and ECM has been successfully created
from iPS cells by differentiating them into chondrocytes, which are
subsequently transferred into a three-dimensional culture to make iPS
cell-derived chondrocytes produce and accumulate ECM around
themselves to form cartilaginous tissue particles12,13. Owing to the selfrenewal activity of iPS cells, allogeneic iPS cell-derived cartilage organoids can theoretically be produced inexhaustibly and transplanted
into an unlimited number of patients, solving the issues associated
with allogenic cartilage, such as scarcity of donors, risk of disease
transmission, and variations in cartilage qualities between donors.
In this study, we analyzed the allogenic transplantation of major
histocompatibility complex (MHC)-mismatched iPS cell-derived cartilage organoids in a primate animal model without the use of immunosuppressive drugs. We differentiated cynomolgus monkey iPS cells
(cyiPSCs) into chondrocytes to create cyiPSC-derived cartilage organoids (cyiPS-Cart). We then transplanted cyiPS-Cart into chondral
defects on the knee joint surface of cynomolgus monkeys in an allogeneic manner. Single-cell RNA-sequencing (scRNA-seq) and molecular analysis of the cyiPS-Cart graft revealed molecular pathways
involved in cell differentiation that remodeled the cyiPS-Cart toward
articular cartilage after transplantation.
Results
Preparation of cyiPS cell-derived cartilage organoid (cyiPS-Cart)
1466A1 cyiPSCs expressing enhanced green fluorescent protein (EGFP)
under a constitutive promoter were used. Cartilage was created from
cyiPSCs14 and human iPSCs12,13 using modified protocols. Briefly,
chondrocytes were induced from cyiPSCs in a chondrogenic medium
for two weeks and transferred to a three-dimensional culture, where
they produced and accumulated ECM to form cartilaginous particles
(Supplementary Fig. 1a). The cyiPSC-derived cartilage organoid (cyiPSCart) particles were 1–3 mm in diameter (Supplementary Fig. 1b).
Histological analysis showed that the particles consisted of cells, and
the ECM was stained positively with safranin O. Immunohistochemical
analysis revealed that the ECM contained type II collagen (Supplementary Fig. 1c). Type I collagen was not detectable except at the
periphery of a particle.
Allogeneic transplantation of cyiPS-Cart in primate chondral
defect model
Cartilage defects can be classified into two categories based on their
depth: chondral defects extending down to but not through the subchondral bone, and osteochondral defects extending down through
the subchondral bone (Fig. 1a). Chondral defects are the most common
in patients with articular cartilage damage or erosion, including during
the early stages of osteoarthritis.
We created chondral defects in the femoral trochlear ridge of the
right knee joints of 12 cynomolgus monkeys and transplanted cyiPSCart (transplantation group) in six monkeys or nothing (empty group)
in the remaining monkeys (Fig. 1b). MHC typing revealed a mismatch
between cyiPSCs and recipient monkeys (Supplementary Table 1).
Nature Communications | (2023)14:804
https://doi.org/10.1038/s41467-023-36408-0
Computed tomography (CT) imaging analysis of the knee joints indicated that bone structures were normal immediately after surgery and
at 4 and 12 weeks after surgery (Supplementary Fig. 2), suggesting that
the defects were chondral and did not extend through the subchondral
bone throughout the experiment.
Three monkeys from each group were sacrificed 4 and 17 weeks
after surgery (n = 3). The gross appearance of the joint surface indicated that chondral defects in the empty group were filled with brown
tissue at 4 and 17 weeks (Fig. 1c). On the other hand, chondral defects in
the transplantation group were filled with transparent tissue at 4 weeks
(Fig. 1c) which later turned white as articular cartilage, making it difficult to distinguish the area where cyiPS-Cart were transplanted from
the surrounding articular cartilage area at 17 weeks after transplantation (Fig. 1c). In each monkey, one transplant site was harvested and
subjected to histological analysis and other two sites were combined
and used for scRNA-seq analysis.
Allogeneic transplantation of cyiPS-Cart did not elicit an
immune reaction in primate chondral defects
We recently reported that allogeneic cyiPS-Cart elicited an immune
reaction when transplanted into osteochondral defects14. However, no
scientific evidence exists on whether allogeneic cartilage elicits an
immune reaction when implanted in chondral defects. To answer this
question, we analyzed histological sections of the transplanted sites.
For control, we created osteochondral defects in additional three
monkeys, transplanted cyiPS-Cart, and sacrificed them 4 weeks later. ...
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