140

Center for Stem Cell Biology and Regenerative Medicine

Division of Regenerative Medicine
再生医学分野Professor
Associate Professor
Assistant Professor
Project Assistant Professor
Project Assistant Professor

Hideki Taniguchi, M.D., Ph.D.
Naoki Tanimizu, Ph.D.
Yun-Zhong Nie, Ph.D.
Yasuharu Ueno, Ph.D.
Takayoshi Oba, M.D., Ph.D.

教 授
准教授
助 教
特任助教
特任助教

博士（医学）
博士（農学）
博士（医学）
博士（医学）
博士（医学）

谷
谷
聶
上
大

口
水
野
場

英
直
運
康
敬

樹
樹
中
晴
義

Currently, organ transplantation is the only effective treatment for patients with endstage organ failure. Unfortunately, the limited number of transplantable organs
hinders the extensive application of this treatment. On the other hand, recent
development of regenerative medicine that aims to generate transplantable organs
on a dish has attracted much attention. Regenerative medicine is a challenging
scientific field that attempts to convert knowledge from developmental biology andstem cell biology into clinical application. Our established novel organoid culture
technologies reconstruct functional human organs derived from human induced
pluripotent stem cells (hiPSCs), and finally aim to develop a substitute for organtransplantation therapy. Currently, we are trying to conduct the transplantation ofhuman liver organoids (LOs) generated from hiPSCs to treat liver diseases, suchas metabolic disorders and liver fibrosis. Moreover, we expand the application of
our technologies to reconstruct artificial cancer tissue (cancer organoid) with arefractory tumor microenvironment for developing a new drug-screening platform to
discover candidate compounds that could prevent cancer relapse and metastasis.

1.  Development of treatment for metabolic liverdisease by transplantation of human iPS cell
derived 3D-organoids.
Yasuharu Ueno1, Naoki Tanimizu1, Yunzhong Nie1,
Satoshi Okamoto1, Yoshiki Kuse1, Takashi Okumura1,
Toshiharu Kasai1, Marina Zushi1, Emi Yajima1, Erika Jinbo1, Kerrigan Kilpatrick1, Tomomi Tadokoro2
and Hideki Taniguchi1,2:
1
Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City University
The liver plays a crucial role in maintaining home-ostasis in the living organism by performing various
metabolic functions such as glucose metabolism, lipid
metabolism, and ammonia metabolism. On the other
hand, abnormalities in these metabolic functions canlead to a variety of diseases of the liver. Liver transplantation is the only curative therapy for end-stage
liver disease, but the absolute shortage and rapid
availability of donor organs is a serious challenge andalternative treatments are clinically highly demanded. We established a technique to produce humanpluripotent stem cell (hiPSC) derived liver organoids(hiPSC-LO) by inducing differentiation of hepatic endodermal cells, vascular endothelial cells, and mesenchymal cells from hiPSCs and co-culturing them in a
3D manner (Nature 2013, Nature 2017, Cell Reports
2017, Sci Rep 2020). Currently, we are developing anovel therapeutic method using hiPSC-LO transplantation for urea cycle disorders, a serious liver disease,

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and metabolic insufficiency steatohepatitis (MASH),
for which there is a huge number of patients.
Urea cycle disorders is a severe metabolic liverdisease resulting from the dysfunction of ornithine
carbamoylase (OTC) and other enzymes, which is associated with hyperammonemia. We have optimized
the ECM coating on the culture dishes and further optimized the organoid culture method to establish arobust production method for hiPSC-LO with appropriate ammonia metabolic capacity (Biol Methods
Protoc 2022). In addition, to evaluate the safety of thehiPSC-LOs, we established a highly sensitive method
detecting undifferentiated cells after induction of LO
component cells from hiPSCs (Stem Cell Rev Rep2022). Next, we evaluated the efficacy of hiPSC-LO
transplantation in a severe OTCD animal model witha background of immunodeficiency. Subadrenocortical transplantation of hiPSC-LOs into immunocompromised OTCD mice significantly improved their
hyperammonemic state, suggesting that iPSC-LO
transplantation is effective as the treatment of OTCD.
Liver cirrhosis is the end stage pathological condition of chronic liver diseases such MASH. MASH ischaracterized by reduced liver function and regenerative capacity and is expected to explode in the number of patients worldwide. With the support of
AMED, we are currently developing a novel treatment for MASH cirrhosis by transplantation of a newly developed fused-type hiPSC-LO based on hiPSC-LO production technology. To this end, we haveestablished a stable method for creating fused-type
hiPSC-LOs and now we are examining its efficacy as
MASH treatment by transplanting them into MASH
liver cirrhosis model animals. Given that no effective
treatment has been developed for MASH cirrhosis,
there is great hope for fused-type hiPSC-LOs transplantation.
2.  Liver repopulation with hiPSC derived proliferative progenitors
Yun-Zhong Nie1, Yoshihito Hayashi1, Qing-Lin LI1,
Xiao-Shan Deng1, Luo Na1, Yang Li1, Xia Yang1, Riana Plummer1, Naoki Tanimizu1, Yasuharu Ueno1,
Hideki Taniguchi1,21Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City University
Liver transplantation is a proven therapy for genetic liver disorders. Yet, its clinical application faces
the persistent shortage of transplantable livers. hiPSCs have shown immense potential for cell replacement therapy for disease treatment. However, hiPSC-derived cells that can effectively repopulate in thedamaged tissues such as liver have not been reported.
Here, we present the generation of expandable hiPSC-derived hepatoblast (hiPSC-HB) with robust repopulation capacity after transplantation. These hiPSC-HB exhibited an impressive expansion capability
and displayed bipotential differentiation abilities
both in vitro and in vivo. Moreover, we noticed thatthe repopulation capacity of hiPSC-HB could be enhanced with specific conditions, leading to engraftment levels comparable to primary human hepatocytes (PHHs) in liver failure mice. Further, theengrafted hiPSC-HB matured into functional humanhepatocytes with tissue-specific structural features.
This study marks a breakthrough as the first successful generation of lineages from pluripotent stem cells
capable of in vivo repopulating and restoring tissue
function, offering promising prospects for innovative
disease treatment through regenerative medicine.
3.  Modeling liver diseases with hiPSC-derived organoid
Yun-Zhong Nie1, Yang Li1, Xia Yang1, Riana Plummer1, Xiao-Shan Deng1, Yoshihito Hayashi1, QingLin LI1, Luo Na1, Toshiharu Kasai1, Takashi Okumura1, Naoki Tanimizu1, Yasuharu Ueno1, Hideki
Taniguchi1,2
The developed human cell-based liver ex vivo
models still could not accurately recapitulate liver
physiology and disease progression due to lack of
non-parenchymal cell, including Kupffer cell, hepatic
stellate cells and sinusoidal endothelial cells. Currently, we generated LOs containing Kupffer cells (KuLOs) by recapitulating fetal liver hematopoiesis using
hiPSC-derived erythro-myeloid progenitors (EMPs),
origin of tissue-resident macrophages, and hiPSC-derived LOs. Exposing KuLOs to sepsis-like endotoxins
led to significant organoid dysfunction that closely
resembled the pathological characteristics of the human septic liver. Furthermore, we observed a notablefunctional recovery in KuLOs upon endotoxin elimination, which was accelerated by using Toll-like receptor 4-directed endotoxin antagonist. Moreover, weare trying to establish a mini liver with hepatocytes,
Kupffer cells, hepatic stellate cells, and sinusoidal endothelial cells, which will allow a more precise understanding of the cell-to-cell interactions during liverdisease progression, and provide a novel platform to
explore potential targets for alleviating liver diseaseprogression.
4.  hiPSC-liver bud in vitro growth enhanced by
perfusion culture
Yoshiki Kuse1, Naoki Tanimizu1, Yasuharu Ueno1,
Nie Yunzhong1, Shinya Matsumoto1, Takashi Okumura1, Erica Carolina1, Soichiro Yamabe1, Toshiharu Kasai1, Tomomi Tadokoro2, Satoshi Okamoto2,

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and Hideki Taniguchi1,2:
1
Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City University
To overcome the critical shortage of organ donors,
the generation of hiPSC-derived organs with structures and functions is urgently needed. Although hiPSC-organoid is an innovative technology to reconstitute tissue structure and function, an alternative fororgan transplantation. Blood perfusion is a criticalevent for organ growth by supplying nutrients andoxygen. However, blood perfusion is still lacking in
the present organoid culture system. We are developing perfusion culture systems using two approaches;
hiPSC-liver buds (LBs) connected with reconstructed
hiPSC-blood vessel, and the decellularized liver tissue infused with hiPSC-LBs.
From our first approach, we generated the hiPSC-derived macrovessel using collagen gels, hiPSC-derived vascular smooth muscle cells (SMC), andvascular endothelial cells (EC). Although we clarified
that hiPSC-derived macrovessel is histologically similar to the vascular structure of in vivo blood vessels,
EC-seeded macrovessels did not show angiogenesis
after coculturing with hiPSC-LBs. Therefore, we established a new induction method to differentiate
hiPSC into specific EC lineages that exist around thefetal liver. We demonstrated that hiPSC-derived macrovessel containing those specific ECs had higher angiogenic potentials. Under an optimized culture condition, we successfully induced the connection
between the hiPSC-derived macrovessel with the microvessels within hiPSC-LBs. Therefore, we recently
tried to establish the organoid perfusion system. The
established perfusion system enabled us to culture
the hiPSC-LBs by 14 days from co-culture and increase CD31+ blood vessels in LBs. Optimizing theperfusion culture system, we are trying to enhance
hiPSC-LB growth.
As the second approach, we utilize decellularized
liver tissue which retains in vivo vascular structures.
The decellularization technique has been establishedto prepare the scaffold for organ reconstitution. Decellularized organs potentially retain the architectureof the original tissue, including the extracellular matrix. A recent report shows how the recellularized liver using hepatocytes could exert liver-specific functions after transplantation. However, the vascular
structures within this recellularized liver remain unreconstructed, which might explain limited hepatocyte functions in the recellularized liver. Our current
study attempts to generate a more functional recellularized liver by adding oxygen supply into our perfusion culture system of the recellularized liver contain-

ing hiPSC-LBs.
5.  
Generation of 3D cancer tissue using patient-derived pancreatic cancer cells
Kenta Takeuchi1,, Shunsuke Tabe1, Yuya Yamomoto1, Yasuharu Ueno1, Hideki Taniguchi1,2, and Naoki
Tanimizu1
1
Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City University
Pancreatic adenocarcinoma (PDAC) has a poor
prognosis, with a 5-year survival rate of about 10%
due to delayed diagnosis, drug resistance, and recurrence. Organoid technologies have been applied toinvestigate the properties of PDACs. To recapitulate
tumor microenvironment (TME), which is thought tobe crucial for the poor prognosis of PDAC, we generated multicellular spheroids consisting of primary
PDAC cells isolated from Japanese pancreatic cancer
patients with hiPSC-mesenchymal cells (MCs) and-endothelial cells (ECs), and then fused them to construct fused pancreatic cancer organoid (FPCO). Our
FPCO resembles the tissue structure of clinical tissue
including PDAC ductal structures and dense deposition of extracellular matrix components compared toconventional organoids. Single cell RNA sequence
analysis demonstrated that FPCO contains various
types of cancer-related fibroblasts (CAFs) namely immunological CAF (iCAF), myofibroblastic (myCAF),
and antigen presenting ones (apCAFs). Moreover, the
PDAC organoid showed strong resistance to anti-cancer drugs and re-proliferative capacity after drug
treatment indicating its close resemblance to frequent
relapse in PDAC patients. We are currently making
FPCO containing tumor associated macrophages(TAMs) to recapitulate immunosuppressive TME. We
will apply this new cancer organoid in drug screening
and biological analysis to develop effective therapies
against PDAC.
6.  
Space Organogenesis (Development of advanced 3D organ culture system utilizing microgravity environment)
Tomomi Tadokoro2, Tatsuya Kobayashi1, Yoshiki
Kuse1, Yasuharu Ueno1, Takashi Okumura1, andHideki Taniguchi1,2:
1
Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City Universi-

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Microgravity in orbit does not cause subsidence orconvection and is considered advantageous in expanding cells in three-dimension. By utilizing this microgravity environment, we aim to develop a novel
method for generating human iPSCs-derived livertissue in collaboration with Japan Aerospace Exploration Agency (JAXA). In particular, we attempt to establish a new technique for generating three-dimensional organs containing large blood vessels. After we
prepared hiPSC-LBs and artificial vessels on the earth,
we placed those organoids into the culture container
and launched to the International Space Station“KIBO”. We confirmed that hiPSC-LBs were successfully assembled around the artificial vessel under microgravity, as how in silico simulation suggested. After culturing hiPSC-LBs for a predetermined period
in the incubator installed in “KIBO”, the samples
were then transported back to the earth. Adherence
and fusion of hiPSC-LBs to the artificial vessels wereobserved in the post-flight samples cultured in orbit.
Moreover, endothelial cells started to extend their filopodia-like structure. Using qRT-PCR analysis ofground controls and post-flight samples, comparable
expressions of hepatic, endothelial cell-related, andmesenchymal cell-related genes were observed inboth samples. In addition, gene ontology analysis ofRNA-seq data revealed that genes related to triglyceride homeostasis, cholesterol biosynthetic process,
MAPK pathway, and angiogenesis were enriched in
the post-flight samples, indicating how the space environment could provide an optimal condition for tissue reconstruction. Next, we upgraded our system byusing a large blood vessel composed of hiPSC-derived smooth muscle cells and endothelial cells which
resulted in the improvement of hiPSC-LB functions.
The second space experiment is planned to be conducted in March 2024. These findings will uncover
the effects of gravity on cell growth and differentiation. We hope these space experiment results willcontribute to the subsequent development and understanding of (1) The development of a new technique in human three-dimensional tissue preservation and transportation, which is crucial to thepractical use of regenerative medicine products. (2)The establishment of a novel technique for generating
human organs joined with large blood vessels. (3) The
development of a new three-dimensional culture device simulating the microgravity environment on
Earth.
7.  Generation of bile duct tubules in hiPSC-liver
buds
Ayumu Okumura1, Erica Carolina1, Kenji Aoshima1,
Taichi Tsuyuki1, Minjia Zhong1, Kazuki Yanagisawa1, Yoshiki Kuse1, Takashi Okumura1, Toshiharu
Kasai1, Tomomi Tadokoro2, and Hideki Taniguchi1,2,

and Naoki Tanimizu1
1
Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate
School of Medical Science, Yokohama City University
The biliary system consisting of intrahepatic bile
duct (IHBD), extrahepatic bile ducts (EHBDs), andgallbladder, is a crucial tissue structure for maintaining liver homeostasis by providing the excretion routefor the bile secreted from hepatocytes. Although various types of liver organoids have been established,
the generation of hiPSC-liver organoids associatedwith the bile drainage system consisting of IHBD and
EHBD has not been reported so far.
First, to generate liver organoid containing IHBDlike structures, we developed a new co-culture system in which the hiPSC-liver progenitors are locatedin next to the hiPSC-blood vessel (BV) to recapitulate
the fetal portal vein-IHBD tissue interaction. In thiscondition, hiPSC-liver progenitors differentiated into
cholangiocytes and formed duct structures. We
named this organoid as blood vessel incorporated liver organoid (BVLO). hiPSC-cholangiocytes in BVLO
showed secretory functions in vitro and formed duct
structures within the recipient liver after organoidtransplantation to immunodeficient mice. We applied
BLVO technology to modelling a human congenital
biliary disease for understating underlying mechanisms.
Second, to generate EHBDs, we induced EHBD
progenitor cells from hiPSC- definitive endoderm
cells and generated 3D cystic structures. We are currently analyzing transcriptome data of mouse fetal
EHBD epithelial cells to acquire information aboutproliferation signal for EHBD progenitors and theirfunctional maturation process. Our final goal is toeventually connect these two tubular structures with
hiPSC-derived hepatocytes on a dish to generate
Hepatobiliary Tubular Organoids (HBTO) that possess a long-term hepatic function in vitro as well as invivo.
8.  Generation of a novel treatment for pediatriccraniofacial deformity using human auricular
perichondrium-derived elastic cartilage devices
Takayoshi Oba1, Satoshi Okamoto1, Yasuharu
Ueno1, Chie Ikezuki1, Takuya Ohkuma1, Tenta
Kubo2, and Hideki Taniguchi1,21Division of Regenerative Medicine, Center for
Stem Cell Biology and Regenerative Medicine, The
Institute of Medical Science, The University of Tokyo
2
Department of Regenerative Medicine, Graduate

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School of Medical Science, Yokohama City University
Auto-transplantation of rib cartilage segments isthe current most popular treatment for patients withcraniofacial deformity. However, major disadvantages such as limited harvestable amounts and post-operative pain of the donor site remain to be solved. To
this end, a none-invasive, morphologically stablescaffold-free elastic cartilage implantation treatment
for patients with craniofacial deformity is essential.
Our previous study showed the world’s first technology of separating and identifying chondroprogenitor cells from the human auricular perichondrium(Kobayashi S et al. PNAS 2011, Patent registration no.
4748222; PCT/JP2008/051327). We succeeded in developing non-scaffold elastic cartilage, which is obtaina-

ble in vitro, by using three-dimensional rotation culture and U–bottomed micropatterned plate culture(Enomura M et al. Int J Mol Sci 2020, Oba T et al. J
Tissue Eng 2022, Patent application no. 2021-141210;
PCT/JP2022/25582). Furthermore, the size and elasticity of the tissue were maintained after craniofacialtransplantation in immunodeficient mice, indicating
the tissue to be morphologically stable.
Our major goal is to establish a non-invasive novel
promising treatment for pediatric patients with nasal
deformity by transplanting morphologically stablenon-scaffold elastic cartilage. To obtain the clinical
POC of the novel treatment,we are currently establishing the manufacturing system, quality control
methods, product specification, evaluation of nonclinical safeness and determination of clinical protocol.

Publications
1.		Takeuchi K, Tabe S, Takahashi K, Aoshima K, Matsuo M, Ueno Y, Furukawa Y, Yamaguchi K, Ohtsuka M, Morinaga S, Miyagi Y, Yamaguchi T, Tanimizu N, Taniguchi H. Incorporation of human
iPSC-derived stromal cells creates a pancreaticcancer organoid with heterogeneous cancer-associated fibroblasts. Cell Rep. 2023 Nov 12;42(11):113420.
2.		Matsuzaki T, Kawano Y, Horikiri M, Shimokawa
Y, Yamazaki T, Okuma N, Koike H, Kimura M,
Kawamura R, Yoneyama Y, Furuichi Y, Hakuno F,
Takahashi SI, Nakabayashi S, Okamoto S, Nakauchi H, Taniguchi H, Takebe T, Yoshikawa HY,
Preparation of mechanically patterned hydrogels
for controlling the self-condensation of cells. STAR
Protoc. 2023 Jul 28;4(3):102471.
3.		Natsumoto B, Shoda H, Nagafuchi Y, Ota M,Okumura T, Horie Y, Okamura T, Yamamoto K, Tsuji
M, Otsu M, Taniguchi H, Fujio K. Functional evaluation of rare OASL variants by analysis of SLE
patient-derived iPSCs. J Autoimmun. 2023 Jun
22;139:103085.doi: 10.1016/j.jaut.2023.103085.
4.		Ichinohe N, Tanimizu N, Ishigami K, Yoshioka Y,
Fujitani N, Ochiya T, Takahashi M, MitakaT,

CINC-2 and miR-199a-5p in EVs secreted by transplanted Thy1+ cells activate hepatocytic progenitor cell growth in rat liver regeneration. Stem Cell
Res Ther. 2023, May 16;14(1):134.
5.		Tanimizu N, Ichinose N, Mitaka T, β-adrenergic
receptor agonist promotes ductular expansion
during3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury. Sci Rep.
2023 May 1;13(1):7084. doi: 10.1038/s41598-02333882-w.
6.		Tsuzuki S, Yamaguchi T, Okumura T, Kasai K,
Ueno Y, Taniguchi H. PDGF receptors and signaling are required for 3D-structure formation anddifferentiation of human iPSC-derived hepatic
spheroids. International Journal of Molecular
Science. 2023, 24(8), 7075.
7.		Aizawa Y, Takada K, Aoyama J, Sano D, Yamanaka S, Seki M, Kuze Y, Ramilowski JA, Okuda R,
Ueno Y, Nojima Y, Inayama Y, Hatakeyama H,
Hatano T, Takahashi H, Nishimura G, Fujii S, Suzuki Y, Taniguchi H, Oridate N. Establishment ofexperimental salivary gland cancer models usingorganoid culture and patient-derived xenografting. Cell Oncol (Dordr). 2023 Apr;46(2):409-421.