|
1
|
Zhu Y, Warmflash A. Dependence of cell fate potential and cadherin switching on primitive streak coordinate during differentiation of human pluripotent stem cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.31.635963. [PMID: 39975234 PMCID: PMC11838492 DOI: 10.1101/2025.01.31.635963] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 02/21/2025]
Abstract
During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT), which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated. To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall, cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation.
Collapse
Affiliation(s)
- Ye Zhu
- Department of Bioengineering, Rice University, Houston, TX 77005
| | - Aryeh Warmflash
- Department of Bioengineering, Rice University, Houston, TX 77005
- Department of Biosciences, Rice University, Houston, TX 77005
| |
Collapse
|
|
2
|
Xiong J, Ma R, Xie K, Shan C, Chen H, Wang Y, Liao Y, Deng Y, Ye G, Wang Y, Zhu Q, Zhang Y, Cai H, Guo W, Yin Y, Li Z. Recapitulation of endochondral ossification by hPSC-derived SOX9 + sclerotomal progenitors. Nat Commun 2025; 16:2781. [PMID: 40118845 PMCID: PMC11928506 DOI: 10.1038/s41467-025-58122-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Accepted: 03/11/2025] [Indexed: 03/24/2025] Open
Abstract
Endochondral ossification generates most of the load-bearing bones, recapitulating it in human cells remains a challenge. Here, we report generation of SOX9+ sclerotomal progenitors (scl-progenitors), a mesenchymal precursor at the pre-condensation stage, from human pluripotent stem cells and development of osteochondral induction methods for these cells. Upon lineage-specific induction, SOX9+ scl-progenitors have not only generated articular cartilage but have also undergone spontaneous condensation, cartilaginous anlagen formation, chondrocyte hypertrophy, vascular invasion, and finally bone formation with stroma, thereby recapitulating key stages during endochondral ossification. Moreover, self-organized growth plate-like structures have also been induced using SOX9+ scl-progenitor-derived fusion constructs with chondro- and osteo-spheroids, exhibiting molecular and cellular similarities to the primary growth plates. Furthermore, we have identified ITGA9 as a specific surface marker for reporter-independent isolation of SOX9+ scl-progenitors and established a culture system to support their expansion. Our work highlights SOX9+ scl-progenitors as a promising tool for modeling human skeletal development and bone/cartilage bioengineering.
Collapse
Affiliation(s)
- Jingfei Xiong
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Runxin Ma
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Kun Xie
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Ce Shan
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Hanyi Chen
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Yuqing Wang
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Yuansong Liao
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Yanhui Deng
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Guogen Ye
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Yifu Wang
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Qing Zhu
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
- Department of Anesthesiology, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education, Sichuan University, Chengdu, China
| | - Yunqiu Zhang
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Haoyang Cai
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China
| | - Weihua Guo
- Yunnan Key Laboratory of Stomatology, Department of Pediatric Dentistry, The Affiliated Stomatology Hospital of Kunming Medical University, Kunming Medical University, Kunming, China
| | - Yike Yin
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China.
| | - Zhonghan Li
- Center of Growth Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, China.
- Yunnan Key Laboratory of Stomatology, Department of Pediatric Dentistry, The Affiliated Stomatology Hospital of Kunming Medical University, Kunming Medical University, Kunming, China.
| |
Collapse
|
|
3
|
Kobatake T, Miyamoto Y, Fujihara Y, Saijo H, Hoshi K, Hikita A. Small extracellular vesicles derived from auricular chondrocytes promote secretion of interleukin 10 in bone marrow M1-like macrophages. Regen Ther 2025; 28:421-430. [PMID: 39925964 PMCID: PMC11804269 DOI: 10.1016/j.reth.2025.01.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Revised: 12/27/2024] [Accepted: 01/10/2025] [Indexed: 02/11/2025] Open
Abstract
Introduction Elucidation of the paracrine interaction between chondrocytes and macrophages is useful for understanding the mechanisms of cartilage regeneration. Extracellular vesicles are granular substances with a diameter of approximately 150 nm, surrounded by a phospholipid bilayer membrane. In recent years, research has been conducted on clinical applications of extracellular vesicles. It has been shown that macrophages promote cartilage maturation, and macrophages acquire anti-inflammatory properties through cartilage, but the detailed mechanism of paracrine action involving extracellular vesicles remains unclear. Therefore, we focused on the effect of chondrocyte-derived extracellular vesicles on changes in macrophage characteristics. Methods Macrophages induced with granulocyte-macrophage colony stimulating factor (M1-like macrophages) and auricular chondrocytes were co-cultured using cell culture inserts and exosome inhibitors, and the expression of macrophage markers were analyzed. Next, extracellular vesicles separated from auricular chondrocytes were added to in vitro macrophage culture medium, and time-lapse observations of macrophage uptake of auricular chondrocyte-derived extracellular vesicles were performed. In addition, the effects of extracellular vesicles on the expression of macrophage markers were also analyzed. Results The expression of CD206, an M2 macrophage marker, was increased in macrophages due to the paracrine effect of chondrocytes, and CD206 expression was further increased by pharmacological inhibition of chondrocyte-derived exosomes. It was shown that chondrocyte-derived extracellular vesicles were taken up by macrophages and promoted the production of interleukin-10, an anti-inflammatory cytokine while reducing CD206 expression. Conclusions Auricular chondrocyte-derived extracellular vesicles promoted the production of interleukin-10 in bone marrow M1-like macrophages but reduced CD206 expression.
Collapse
Affiliation(s)
- Tetsuya Kobatake
- Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
| | - Yoshiyuki Miyamoto
- Division of Dentistry and Oral Surgery, Mitsui Memorial Hospital, Kanda-Izumi-cho 1, Chiyoda-ku, Tokyo, 101-8643, Japan
- Department of Oral-Maxillofacial Surgery and Orthodontics, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
| | - Yuko Fujihara
- Department of Dentistry and Oral Surgery, Tokyo Teishin Hospital, 2-14-23 Fujimi, Chiyoda-ku, Tokyo 102-8798, Japan
| | - Hideto Saijo
- Department of Oral-Maxillofacial Surgery and Orthodontics, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
- Department of Oral and Maxillofacial Surgery, Field of Oral and Maxillofacial Rehabilitation, Advanced Therapeutics Course, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima 890-8544, Japan
| | - Kazuto Hoshi
- Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
- Department of Oral-Maxillofacial Surgery and Orthodontics, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
- Division of Tissue Engineering, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
| | - Atsuhiko Hikita
- Division of Tissue Engineering, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
- Clinical Stem Cell Biology, The University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
| |
Collapse
|
|
4
|
Mizushina Y, Sun L, Nishio M, Nagata S, Kamakura T, Fukuda M, Tanaka K, Toguchida J, Jin Y. Hydroxycitric acid reconstructs damaged articular cartilages by modifying the metabolic cascade in chondrogenic cells. OSTEOARTHRITIS AND CARTILAGE OPEN 2025; 7:100564. [PMID: 39835169 PMCID: PMC11743121 DOI: 10.1016/j.ocarto.2024.100564] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2024] [Accepted: 12/19/2024] [Indexed: 01/22/2025] Open
Abstract
Objective Osteoarthritis, a degenerative joint disease, requires innovative therapies due to the limited ability of cartilage to regenerate. Since mesenchymal stem cells (MSCs) provide a cell source for chondrogenic cells, we hypothesize that chemicals capable of enhancing the chondrogenic potential of MSCs with transforming growth factor-beta (TGFβ) in vitro may similarly promote chondrogenesis in articular cartilage in vivo. Design Chemical compounds that enhance the TGFβ signaling for chondrogenesis were investigated utilizing mesenchymal stem cells derived from human induced pluripotent stem cells. The mechanisms of action underlying the identified compound were explored in vitro, and its therapeutic effects were validated in vivo using a mouse model of exercise-induced osteoarthritis. Results Hydroxycitric acid (HCA) emerged as the lead compound. In vitro, HCA effectively enhanced chondrogenesis by inhibiting ATP citrate lyase, inducing citrate and alpha-ketoglutarate (α-KG), while reducing cytosolic acetyl coenzyme A (Ac-CoA). This induction of α-KG promoted collagen prolyl-4-hydroxylase activity, boosting hydroxyproline production and matrix formation. The reduction of Ac-CoA attenuated the inhibitory effect of β-catenin on mitochondrial activity by diminishing its acetylation. In vivo, orally administered HCA accumulated in joint tissues of mice and histological examination demonstrated newly synthesized cartilage tissues in damaged area. Analysis of joint tissue extracts revealed a downregulation of Ac-CoA and an upregulation of citrate and α-KG, accompanied by a systemic increase in an anabolic biomarker. Conclusions HCA demonstrates promise as an osteoarthritis therapy by enhancing chondrogenic differentiation. Its ability to modulate crucial metabolic pathways and facilitate cartilage repair suggests potential for clinical translation, addressing a critical need in the treatment of osteoarthritis.
Collapse
Affiliation(s)
- Yoshiyuki Mizushina
- Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
- Central R & D Laboratory, Kobayashi Pharmaceutical Co., Ltd., 1-30-3 Toyokawa, Ibaraki, 567-0057, Japan
| | - Liping Sun
- Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Megumi Nishio
- Department of Fundamental Cell Technology, Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Sanae Nagata
- Department of Fundamental Cell Technology, Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Takeshi Kamakura
- Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Masayuki Fukuda
- Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Kousuke Tanaka
- Central R & D Laboratory, Kobayashi Pharmaceutical Co., Ltd., 1-30-3 Toyokawa, Ibaraki, 567-0057, Japan
| | - Junya Toguchida
- Department of Fundamental Cell Technology, Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
- Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| | - Yonghui Jin
- Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-Ku, Kyoto, 606-8507, Japan
| |
Collapse
|
|
5
|
Hojo H, Tani S, Ohba S. Modeling of skeletal development and diseases using human pluripotent stem cells. J Bone Miner Res 2024; 40:5-19. [PMID: 39498496 DOI: 10.1093/jbmr/zjae178] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 09/28/2024] [Accepted: 11/02/2024] [Indexed: 01/07/2025]
Abstract
Human skeletal elements are formed from distinct origins at distinct positions of the embryo. For example, the neural crest produces the facial bones, the paraxial mesoderm produces the axial skeleton, and the lateral plate mesoderm produces the appendicular skeleton. During skeletal development, different combinations of signaling pathways are coordinated from distinct origins during the sequential developmental stages. Models for human skeletal development have been established using human pluripotent stem cells (hPSCs) and by exploiting our understanding of skeletal development. Stepwise protocols for generating skeletal cells from different origins have been designed to mimic developmental trails. Recently, organoid methods have allowed the multicellular organization of skeletal cell types to recapitulate complicated skeletal development and metabolism. Similarly, several genetic diseases of the skeleton have been modeled using patient-derived induced pluripotent stem cells and genome-editing technologies. Model-based drug screening is a powerful tool for identifying drug candidates. This review briefly summarizes our current understanding of the embryonic development of skeletal tissues and introduces the current state-of-the-art hPSC methods for recapitulating skeletal development, metabolism, and diseases. We also discuss the current limitations and future perspectives for applications of the hPSC-based modeling system in precision medicine in this research field.
Collapse
Affiliation(s)
- Hironori Hojo
- Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8655, Japan
| | - Shoichiro Tani
- Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States
| | - Shinsuke Ohba
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| |
Collapse
|
|
6
|
Suzdaltseva Y, Selezneva A, Sergeev N, Kiselev SL. Initial WNT/β-Catenin or BMP Activation Modulates Inflammatory Response of Mesodermal Progenitors Derived from Human Induced Pluripotent Stem Cells. Cells 2024; 13:1820. [PMID: 39513926 PMCID: PMC11545028 DOI: 10.3390/cells13211820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Revised: 10/29/2024] [Accepted: 11/01/2024] [Indexed: 11/16/2024] Open
Abstract
Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing.
Collapse
Affiliation(s)
- Yulia Suzdaltseva
- Department of Epigenetics, Vavilov Institute of General Genetics of the Russian Academy of Sciences, 119333 Moscow, Russia
| | | | | | | |
Collapse
|
|
7
|
Wang XH, Liu N, Zhang H, Yin ZS, Zha ZG. From cells to organs: progress and potential in cartilaginous organoids research. J Transl Med 2023; 21:926. [PMID: 38129833 PMCID: PMC10740223 DOI: 10.1186/s12967-023-04591-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2023] [Accepted: 10/04/2023] [Indexed: 12/23/2023] Open
Abstract
While cartilage tissue engineering has significantly improved the speed and quality of cartilage regeneration, the underlying metabolic mechanisms are complex, making research in this area lengthy and challenging. In the past decade, organoids have evolved rapidly as valuable research tools. Methods to create these advanced human cell models range from simple tissue culture techniques to complex bioengineering approaches. Cartilaginous organoids in part mimic the microphysiology of human cartilage and fill a gap in high-fidelity cartilage disease models to a certain extent. They hold great promise to elucidate the pathogenic mechanism of a diversity of cartilage diseases and prove crucial in the development of new drugs. This review will focus on the research progress of cartilaginous organoids and propose strategies for cartilaginous organoid construction, study directions, and future perspectives.
Collapse
Affiliation(s)
- Xiao-He Wang
- Department of Bone and Joint Surgery, the First Affliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China
| | - Ning Liu
- Department of Bone and Joint Surgery, the First Affliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China
| | - Hui Zhang
- Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, Anhui, China
| | - Zong-Sheng Yin
- Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, Anhui, China
| | - Zhen-Gang Zha
- Department of Bone and Joint Surgery, the First Affliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China.
| |
Collapse
|
|
8
|
Ikeda Y, Tani S, Moriishi T, Kuroda A, Matsuo Y, Saeki N, Inui-Yamamoto C, Abe M, Maeda T, Rowe DW, Chung UI, Hojo H, Matsushita Y, Sawase T, Ohba S. Modeling of intramembranous ossification using human pluripotent stem cell-derived paraxial mesoderm derivatives. Regen Ther 2023; 24:536-546. [PMID: 37860130 PMCID: PMC10582276 DOI: 10.1016/j.reth.2023.09.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2023] [Revised: 09/25/2023] [Accepted: 09/28/2023] [Indexed: 10/21/2023] Open
Abstract
Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.
Collapse
Affiliation(s)
- Yuki Ikeda
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
- Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Shoichiro Tani
- Laboratory of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo 113-8655, Japan
| | - Takeshi Moriishi
- Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Aiko Kuroda
- Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Yuki Matsuo
- Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Naoya Saeki
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| | - Chizuko Inui-Yamamoto
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| | - Makoto Abe
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| | - Takashi Maeda
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| | - David W. Rowe
- Department of Reconstructive Sciences, University of Connecticut Health Center, CT 06030, USA
| | - Ung-il Chung
- Laboratory of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo 113-8655, Japan
| | - Hironori Hojo
- Laboratory of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo 113-8655, Japan
| | - Yuki Matsushita
- Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Takashi Sawase
- Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
| | - Shinsuke Ohba
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
| |
Collapse
|
|
9
|
Suzdaltseva Y, Kiselev SL. Mesodermal Derivatives of Pluripotent Stem Cells Route to Scarless Healing. Int J Mol Sci 2023; 24:11945. [PMID: 37569321 PMCID: PMC10418846 DOI: 10.3390/ijms241511945] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 07/07/2023] [Accepted: 07/24/2023] [Indexed: 08/13/2023] Open
Abstract
Scar formation during normal tissue regeneration in adults may result in noticeable cosmetic and functional defects and have a significant impact on the quality of life. In contrast, fetal tissues in the mid-gestation period are known to be capable of complete regeneration with the restitution of the initial architecture, organization, and functional activity. Successful treatments that are targeted to minimize scarring can be realized by understanding the cellular and molecular mechanisms of fetal wound regeneration. However, such experiments are limited by the inaccessibility of fetal material for comparable studies. For this reason, the molecular mechanisms of fetal regeneration remain unknown. Mesenchymal stromal cells (MSCs) are central to tissue repair because the molecules they secrete are involved in the regulation of inflammation, angiogenesis, and remodeling of the extracellular matrix. The mesodermal differentiation of human pluripotent stem cells (hPSCs) recapitulates the sequential steps of embryogenesis in vitro and provides the opportunity to generate the isogenic cell models of MSCs corresponding to different stages of human development. Further investigation of the functional activity of cells from stromal differon in a pro-inflammatory microenvironment will procure the molecular tools to better understand the fundamental mechanisms of fetal tissue regeneration. Herein, we review recent advances in the generation of clonal precursors of primitive mesoderm cells and MSCs from hPSCs and discuss critical factors that determine the functional activity of MSCs-like cells in a pro-inflammatory microenvironment in order to identify therapeutic targets for minimizing scarring.
Collapse
Affiliation(s)
- Yulia Suzdaltseva
- Department of Epigenetics, Vavilov Institute of General Genetics of the Russian Academy of Sciences, 119333 Moscow, Russia;
| | | |
Collapse
|
|
10
|
Kidwai FK, Canalis E, Robey PG. Induced pluripotent stem cell technology in bone biology. Bone 2023; 172:116760. [PMID: 37028583 PMCID: PMC10228209 DOI: 10.1016/j.bone.2023.116760] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/19/2023] [Revised: 03/31/2023] [Accepted: 04/04/2023] [Indexed: 04/09/2023]
Abstract
Technologies on the development and differentiation of human induced pluripotent stem cells (hiPSCs) are rapidly improving, and have been applied to create cell types relevant to the bone field. Differentiation protocols to form bona fide bone-forming cells from iPSCs are available, and can be used to probe details of differentiation and function in depth. When applied to iPSCs bearing disease-causing mutations, the pathogenetic mechanisms of diseases of the skeleton can be elucidated, along with the development of novel therapeutics. These cells can also be used for development of cell therapies for cell and tissue replacement.
Collapse
Affiliation(s)
- Fahad K Kidwai
- Skeletal Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, United States of America
| | - Ernesto Canalis
- Center for Skeletal Research, Orthopedic Surgery and Medicine, UConn Musculoskeletal Institute, UConn Health, Farmington, CT 06030-4037, United States of America
| | - Pamela G Robey
- Skeletal Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, United States of America.
| |
Collapse
|
|
11
|
Zujur D, Al-Akashi Z, Nakamura A, Zhao C, Takahashi K, Aritomi S, Theoputra W, Kamiya D, Nakayama K, Ikeya M. Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair. Front Cell Dev Biol 2023; 11:1140717. [PMID: 37234772 PMCID: PMC10206169 DOI: 10.3389/fcell.2023.1140717] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Accepted: 04/24/2023] [Indexed: 05/28/2023] Open
Abstract
Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.
Collapse
Affiliation(s)
- Denise Zujur
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Ziadoon Al-Akashi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Anna Nakamura
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Chengzhu Zhao
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Laboratory of Skeletal Development and Regeneration, Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Kazuma Takahashi
- Research Institute for Bioscience Product and Fine Chemicals, Ajinomoto Co., Inc, Kawasaki, Japan
| | - Shizuka Aritomi
- Research Institute for Bioscience Product and Fine Chemicals, Ajinomoto Co., Inc, Kawasaki, Japan
| | - William Theoputra
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Daisuke Kamiya
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Takeda-CiRA Joint Program (T-CiRA), Kanagawa, Japan
| | - Koichi Nakayama
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Makoto Ikeya
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Takeda-CiRA Joint Program (T-CiRA), Kanagawa, Japan
| |
Collapse
|
|
12
|
Lamandé SR, Ng ES, Cameron TL, Kung LHW, Sampurno L, Rowley L, Lilianty J, Patria YN, Stenta T, Hanssen E, Bell KM, Saxena R, Stok KS, Stanley EG, Elefanty AG, Bateman JF. Modeling human skeletal development using human pluripotent stem cells. Proc Natl Acad Sci U S A 2023; 120:e2211510120. [PMID: 37126720 PMCID: PMC10175848 DOI: 10.1073/pnas.2211510120] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Accepted: 04/04/2023] [Indexed: 05/03/2023] Open
Abstract
Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.
Collapse
Affiliation(s)
- Shireen R. Lamandé
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Elizabeth S. Ng
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Trevor L. Cameron
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Louise H. W. Kung
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Lisa Sampurno
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Lynn Rowley
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Jinia Lilianty
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
| | - Yudha Nur Patria
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
- Department of Child Health, Universitas Gadjah Mada, Yogyakarta55281, Indonesia
| | - Tayla Stenta
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Eric Hanssen
- Ian Holmes Imaging Center and Department of Biochemistry and Pharmacology, Bio21 Institute, University of Melbourne, Parkville, VIC 3010, Australia
| | - Katrina M. Bell
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Ritika Saxena
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
| | - Kathryn S. Stok
- Department of Biomedical Engineering, University of Melbourne, Parkville, VIC 3010, Australia
| | - Edouard G. Stanley
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - Andrew G. Elefanty
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
| | - John F. Bateman
- Murdoch Children’s Research Institute, Parkville, VIC 3052, Australia
- Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia
| |
Collapse
|
|
13
|
Grogan SP, Glembotski NE, D'Lima DD. ALK-5 Inhibitors for Efficient Derivation of Mesenchymal Stem Cells from Human Embryonic Stem Cells. Tissue Eng Part A 2023; 29:127-140. [PMID: 36458467 DOI: 10.1089/ten.tea.2022.0164] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
Objectives: Successful tissue regeneration requires a clinically viable source of mesenchymal stem cells (MSCs). We explored activin receptor-like kinase (ALK)-5 inhibitors to rapidly derive an MSC-like phenotype with high cartilage forming capacity from a xeno-free human embryonic cell line. Methods: Embryonic stem cell (ESC) lines (H9 and HADC100) were treated with the ALK-5 inhibitor SB431542; HADC100 cells were additionally treated with ALK-5 inhibitors SB525334 or GW788388. Cells were then seeded upon human fibronectin in the presence of fibroblast growth factor 2 (FGF2) in a serum-free medium. Flow cytometry was used to assess MSC markers (positive for CD73, CD90, and CD105; negative for CD34 and CD45). Differentiation status was assessed through quantitative polymerase chain reaction. Cartilage forming capacity was determined in high-density pellet cultures, in fibrin gels containing extracellular matrix (fibrin-ECM), and after implantation in ex vivo human osteoarthritic cartilage. Gene expression, histology, and immunostaining were used to assess cartilage phenotype, tissue regeneration, and integration. Results: Exposure to all three ALK-5 inhibitors lead to expression of mesodermal gene markers and differentiation into MSC-like cells (embryonic stem cell-derived mesenchymal stem cells [ES-MSCs]) based on surface marker expression. ES-MSC in pellet cultures or in fibrin-ECM gels expressed high levels of chondrogenic genes: COL2A1, ACAN, and COMP; and low levels of COL1A1 and RUNX2. Cell pellets or fibrin constructs implanted into ex vivo human osteoarthritic cartilage defects produced GAG-rich (safranin O positive) and collagen type II-positive neocartilage tissues that integrated well with native diseased tissue. Conclusions: We developed a protocol for rapid differentiation of xeno-free ESC into MSC-like cells with high cartilage forming capacity with potential for clinical applications. Impact statement Osteoarthritis (OA) is a common disease resulting in significant disability and no approved disease modifying treatment (other than total joint replacement). Embryonic stem cell-derived cell therapy has the potential to benefit patients with cartilage lesions leading to OA and may prevent or delay the need for total joint replacement.
Collapse
Affiliation(s)
- Shawn P Grogan
- Shiley Center for Orthopaedic Research and Education at Scripps Clinic, La Jolla, California, USA
| | - Nicholas E Glembotski
- Shiley Center for Orthopaedic Research and Education at Scripps Clinic, La Jolla, California, USA
| | - Darryl D D'Lima
- Shiley Center for Orthopaedic Research and Education at Scripps Clinic, La Jolla, California, USA
| |
Collapse
|
|
14
|
Smith CA, Humphreys PA, Naven MA, Woods S, Mancini FE, O’Flaherty J, Meng QJ, Kimber SJ. Directed differentiation of hPSCs through a simplified lateral plate mesoderm protocol for generation of articular cartilage progenitors. PLoS One 2023; 18:e0280024. [PMID: 36706111 PMCID: PMC9882893 DOI: 10.1371/journal.pone.0280024] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Accepted: 12/20/2022] [Indexed: 01/28/2023] Open
Abstract
Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.
Collapse
Affiliation(s)
- Christopher A. Smith
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Paul A. Humphreys
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Mark A. Naven
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Steven Woods
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Fabrizio E. Mancini
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Julieta O’Flaherty
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Qing-Jun Meng
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Susan J. Kimber
- Faculty of Biology, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Medicine and Health, University of Manchester, Manchester, United Kingdom
| |
Collapse
|
|
15
|
Abstract
During embryo development, cell proliferation, cell fate specification and tissue patterning are coordinated and tightly regulated by a handful of evolutionarily conserved signaling pathways activated by secreted growth factor families including fibroblast growth factor (FGF), Nodal/bone morphogenetic protein (BMP), Hedgehog and Wnt. The spatial and temporal activation of these signaling pathways elicit context-specific cellular responses that ultimately shape the different tissues of the embryo. Extensive efforts have been dedicated to identifying the molecular mechanisms underlying these signaling pathways during embryo development, adult tissue homeostasis and regeneration. In this review, we first describe the role of the Wnt/β-catenin signaling pathway during early embryo development, axis specification and cell differentiation as a prelude to highlight how this knowledge is being leveraged to manipulate Wnt/β-catenin signaling activity with small molecules and biologics for the directed differentiation of pluripotent stem cells into various cell lineages that are physiologically relevant for stem cell therapy and regenerative medicine.
Collapse
|
|
16
|
Dicks AR, Steward N, Guilak F, Wu CL. Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells. Methods Mol Biol 2023; 2598:87-114. [PMID: 36355287 PMCID: PMC9830630 DOI: 10.1007/978-1-0716-2839-3_8] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.
Collapse
Affiliation(s)
- Amanda R Dicks
- Department of Orthopaedic Surgery, Washington University, St. Louis, MO, USA
- Shriners Hospitals for Children - St. Louis, St. Louis, MO, USA
- Department of Biomedical Engineering, Washington University, St. Louis, MO, USA
- Center of Regenerative Medicine, Washington University, St. Louis, MO, USA
| | - Nancy Steward
- Department of Orthopaedic Surgery, Washington University, St. Louis, MO, USA
- Shriners Hospitals for Children - St. Louis, St. Louis, MO, USA
- Center of Regenerative Medicine, Washington University, St. Louis, MO, USA
| | - Farshid Guilak
- Department of Orthopaedic Surgery, Washington University, St. Louis, MO, USA.
- Shriners Hospitals for Children - St. Louis, St. Louis, MO, USA.
- Department of Biomedical Engineering, Washington University, St. Louis, MO, USA.
- Center of Regenerative Medicine, Washington University, St. Louis, MO, USA.
| | - Chia-Lung Wu
- Department of Orthopaedic Surgery and Rehabilitation, Center for Musculoskeletal Research, University of Rochester, Rochester, NY, USA.
| |
Collapse
|
|
17
|
Grogan S, Kopcow J, D’Lima D. Challenges Facing the Translation of Embryonic Stem Cell Therapy for the Treatment of Cartilage Lesions. Stem Cells Transl Med 2022; 11:1186-1195. [PMID: 36493381 PMCID: PMC9801304 DOI: 10.1093/stcltm/szac078] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Accepted: 10/02/2022] [Indexed: 12/13/2022] Open
Abstract
Osteoarthritis is a common disease resulting in significant disability without approved disease-modifying treatment (other than total joint replacement). Stem cell-based therapy is being actively explored for the repair of cartilage lesions in the treatment and prevention of osteoarthritis. Embryonic stem cells are a very attractive source as they address many of the limitations inherent in autologous stem cells, such as variability in function and limited expansion. Over the past 20 years, there has been widespread interest in differentiating ESC into mesenchymal stem cells and chondroprogenitors with successful in vitro, ex vivo, and early animal studies. However, to date, none have progressed to clinical trials. In this review, we compare and contrast the various approaches to differentiating ESC; and discuss the benefits and drawbacks of each approach. Approaches relying on spontaneous differentiation are simpler but not as efficient as more targeted approaches. Methods replicating developmental biology are more efficient and reproducible but involve many steps in a complicated process. The small-molecule approach, arguably, combines the advantages of the above two methods because of the relative efficiency, reproducibility, and simplicity. To better understand the reasons for lack of progression to clinical applications, we explore technical, scientific, clinical, and regulatory challenges that remain to be overcome to achieve success in clinical applications.
Collapse
Affiliation(s)
- Shawn Grogan
- Corresponding author: Darryl D’Lima, MD, PhD, Shiley Center for Orthopaedic Research and Education, Scripps Health, 10666 N. Torrey Pines Road, La Jolla, CA 92037, USA.
| | - Joel Kopcow
- Shiley Center for Orthopaedic Research and Education, Scripps Health, La Jolla, CA, USA
| | - Darryl D’Lima
- Shiley Center for Orthopaedic Research and Education, Scripps Health, La Jolla, CA, USA
| |
Collapse
|
|
18
|
Satake T, Komura S, Aoki H, Hirakawa A, Imai Y, Akiyama H. Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells. BMC Mol Cell Biol 2022; 23:30. [PMID: 35870887 PMCID: PMC9308249 DOI: 10.1186/s12860-022-00431-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Accepted: 07/11/2022] [Indexed: 11/25/2022] Open
Abstract
Background Lubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation. Methods To generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon, surrounding the Achilles tendons and knee joints of severe combined immunodeficient mice and assessed lubricin expression. Result Wnt3a, activin A, TGF-β1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints. Conclusions The present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.
Collapse
|
|
19
|
Ferreira MJS, Mancini FE, Humphreys PA, Ogene L, Buckley M, Domingos MAN, Kimber SJ. Pluripotent stem cells for skeletal tissue engineering. Crit Rev Biotechnol 2022; 42:774-793. [PMID: 34488516 DOI: 10.1080/07388551.2021.1968785] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Here, we review the use of human pluripotent stem cells for skeletal tissue engineering. A number of approaches have been used for generating cartilage and bone from both human embryonic stem cells and induced pluripotent stem cells. These range from protocols relying on intrinsic cell interactions and signals from co-cultured cells to those attempting to recapitulate the series of steps occurring during mammalian skeletal development. The importance of generating authentic tissues rather than just differentiated cells is emphasized and enabling technologies for doing this are reported. We also review the different methods for characterization of skeletal cells and constructs at the tissue and single-cell level, and indicate newer resources not yet fully utilized in this field. There have been many challenges in this research area but the technologies to overcome these are beginning to appear, often adopted from related fields. This makes it more likely that cost-effective and efficacious human pluripotent stem cell-engineered constructs may become available for skeletal repair in the near future.
Collapse
Affiliation(s)
- Miguel J S Ferreira
- Department of Mechanical, Aerospace and Civil Engineering, School of Engineering, Faculty of Science and Engineering & Henry Royce Institute, The University of Manchester, Manchester, UK
| | - Fabrizio E Mancini
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
| | - Paul A Humphreys
- Department of Mechanical, Aerospace and Civil Engineering, School of Engineering, Faculty of Science and Engineering & Henry Royce Institute, The University of Manchester, Manchester, UK
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
| | - Leona Ogene
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
| | - Michael Buckley
- Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
| | - Marco A N Domingos
- Department of Mechanical, Aerospace and Civil Engineering, School of Engineering, Faculty of Science and Engineering & Henry Royce Institute, The University of Manchester, Manchester, UK
| | - Susan J Kimber
- Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
| |
Collapse
|
|
20
|
Humphreys PA, Mancini FE, Ferreira MJS, Woods S, Ogene L, Kimber SJ. Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone. Semin Cell Dev Biol 2022; 127:17-36. [PMID: 34949507 DOI: 10.1016/j.semcdb.2021.11.024] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 11/23/2021] [Accepted: 11/24/2021] [Indexed: 12/14/2022]
Abstract
Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.
Collapse
Affiliation(s)
- Paul A Humphreys
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK; Department of Mechanical, Aerospace and Civil Engineering, School of Engineering, Faculty of Science and Engineering & Henry Royce Institute, University of Manchester, UK
| | - Fabrizio E Mancini
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK
| | - Miguel J S Ferreira
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK; Department of Mechanical, Aerospace and Civil Engineering, School of Engineering, Faculty of Science and Engineering & Henry Royce Institute, University of Manchester, UK
| | - Steven Woods
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK
| | - Leona Ogene
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK
| | - Susan J Kimber
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, UK
| |
Collapse
|
|
21
|
Kim JG, Rim YA, Ju JH. The Role of Transforming Growth Factor Beta in Joint Homeostasis and Cartilage Regeneration. Tissue Eng Part C Methods 2022; 28:570-587. [PMID: 35331016 DOI: 10.1089/ten.tec.2022.0016] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Transforming growth factor-beta (TGF-β) is an important regulator of joint homeostasis, of which dysregulation is closely associated with the development of osteoarthritis (OA). In normal conditions, its biological functions in a joint environment are joint protective, but it can be dramatically altered in different contexts, making its therapeutic application a challenge. However, with the deeper insights into the TGF-β functions, it has been proven that TGF-β augments cartilage regeneration by chondrocytes, and differentiates both the precursor cells of chondrocytes and stem cells into cartilage-generating chondrocytes. Following documentation of the therapeutic efficacy of chondrocytes augmented by TGF-β in the last decade, there is an ongoing phase III clinical trial examining the therapeutic efficacy of a mixture of allogeneic chondrocytes and TGF-β-overexpressing cells. To prepare cartilage-restoring chondrocytes from induced pluripotent stem cells (iPSCs), the stem cells are differentiated mainly using TGF-β with some other growth factors. Of note, clinical trials evaluating the therapeutic efficacy of iPSCs for OA are scheduled this year. Mesenchymal stromal stem cells (MSCs) have inherent limitations in that they differentiate into the osteochondral pathway, resulting in the production of poor-quality cartilage. Despite the established essential role of TGF-β in chondrogenic differentiation of MSCs, whether the coordinated use of TGF-β in MSC-based therapy for degenerated cartilage is effective is unknown. We herein reviewed the general characteristics and mechanism of action of TGF-β in a joint environment. Furthermore, we discussed the core interaction of TGF-β with principal cells of OA cell-based therapies, the chondrocytes, MSCs, and iPSCs. Impact Statement Transforming growth factor-beta (TGF-β) has been widely used as a core regulator to improve or formulate therapeutic regenerative cells for degenerative joints. It differentiates stem cells into chondrocytes and improves the chondrogenic potential of differentiated chondrocytes. Herein, we discussed the overall characteristics of TGF-β and reviewed the comprehension and utilization of TGF-β in cell-based therapy for degenerative joint disease.
Collapse
Affiliation(s)
- Jung Gon Kim
- Division of Rheumatology, Department of Internal Medicine, Inje University Ilsan Paik Hospital, Goyang, Korea
| | - Yeri Alice Rim
- Catholic iPSC Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Ji Hyeon Ju
- Catholic iPSC Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea.,Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
| |
Collapse
|
|
22
|
Pothiawala A, Sahbazoglu BE, Ang BK, Matthias N, Pei G, Yan Q, Davis BR, Huard J, Zhao Z, Nakayama N. GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes. Development 2022; 149:dev196220. [PMID: 35451016 PMCID: PMC9245189 DOI: 10.1242/dev.196220] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 04/07/2022] [Indexed: 12/02/2023]
Abstract
It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.
Collapse
Affiliation(s)
- Azim Pothiawala
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Berke E. Sahbazoglu
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Bryan K. Ang
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Nadine Matthias
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Guangsheng Pei
- Center for Precision Health, School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Qing Yan
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Brian R. Davis
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Johnny Huard
- Department of Orthopedic Surgery, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
- Center for Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Zhongming Zhao
- Center for Precision Health, School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Naoki Nakayama
- Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
- Department of Orthopedic Surgery, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| |
Collapse
|
|
23
|
Shimomura S, Inoue H, Arai Y, Nakagawa S, Fujii Y, Kishida T, Shin-Ya M, Ichimaru S, Tsuchida S, Mazda O, Kubo T. Hypoxia promotes differentiation of pure cartilage from human induced pluripotent stem cells. Mol Med Rep 2022; 26:229. [PMID: 35593322 PMCID: PMC9178684 DOI: 10.3892/mmr.2022.12745] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Accepted: 04/25/2022] [Indexed: 11/28/2022] Open
Abstract
While cartilage can be produced from induced pluripotent stem cells (iPSCs), challenges such as long culture periods and compromised tissue purity continue to prevail. The present study aimed to determine whether cartilaginous tissue could be produced from iPSCs under hypoxia and, if so, to evaluate its effects on cellular metabolism and purity of the produced tissue. Human iPSCs (hiPSCs) were cultured for cartilage differentiation in monolayers under normoxia or hypoxia (5% O2), and chondrocyte differentiation was evaluated using reverse transcription-quantitative PCR and fluorescence-activated cell sorting. Subsequently, cartilage differentiation of hiPSCs was conducted in 3D culture under normoxia or hypoxia (5% O2), and the formed cartilage-like tissues were evaluated on days 28 and 56 using histological analyses. Hypoxia suppressed the expression levels of the immature mesodermal markers brachyury (T) and forkhead box protein F1; however, it promoted the expression of the chondrogenic markers Acan and CD44. The number of sex-determining region Y-box 9-positive cells and the percentages of safranin O-positive and type 2 collagen-positive tissues increased under hypoxic conditions. Moreover, upon hypoxia-inducible factor (HIF)-1α staining, nuclei of tissues cultured under hypoxia stained more deeply compared with those of tissues cultured under normoxia. Overall, these findings indicated that hypoxia not only enhanced cartilage matrix production, but also improved tissue purity by promoting the expression of HIF-1α gene. Potentially, pure cartilage-like tissues could be produced rapidly and conveniently using this method.
Collapse
Affiliation(s)
- Seiji Shimomura
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Hiroaki Inoue
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Yuji Arai
- Department of Sports and Para‑Sports Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Shuji Nakagawa
- Department of Sports and Para‑Sports Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Yuta Fujii
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Tsunao Kishida
- Department of Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Masaharu Shin-Ya
- Department of Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Shohei Ichimaru
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Shinji Tsuchida
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Osam Mazda
- Department of Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| | - Toshikazu Kubo
- Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
| |
Collapse
|
|
24
|
Chien P, Xi H, Pyle AD. Recapitulating human myogenesis ex vivo using human pluripotent stem cells. Exp Cell Res 2021; 411:112990. [PMID: 34973262 DOI: 10.1016/j.yexcr.2021.112990] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 12/15/2021] [Accepted: 12/22/2021] [Indexed: 11/25/2022]
Abstract
Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.
Collapse
Affiliation(s)
- Peggie Chien
- Department of Microbiology, Immunology and Molecular Genetics, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute, University of California, Los Angeles, CA, 90095, USA
| | - Haibin Xi
- Department of Microbiology, Immunology and Molecular Genetics, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute, University of California, Los Angeles, CA, 90095, USA
| | - April D Pyle
- Department of Microbiology, Immunology and Molecular Genetics, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute, University of California, Los Angeles, CA, 90095, USA.
| |
Collapse
|
|
25
|
Liu TM. Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine. World J Stem Cells 2021; 13:1826-1844. [PMID: 35069985 PMCID: PMC8727229 DOI: 10.4252/wjsc.v13.i12.1826] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 06/29/2021] [Accepted: 11/30/2021] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) represent the most clinically used stem cells in regenerative medicine. However, due to the disadvantages with primary MSCs, such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs, gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application, and variation among donors increasing the uncertainty of MSC efficacy, the clinical application of MSCs has been greatly hampered. MSCs derived from human pluripotent stem cells (hPSC-MSCs) can circumvent these problems associated with primary MSCs. Due to the infinite self-renewal of hPSCs and their differentiation potential towards MSCs, hPSC-MSCs are emerging as an attractive alternative for regenerative medicine. This review summarizes the progress on derivation of MSCs from human pluripotent stem cells, disease modelling and drug screening using hPSC-MSCs, and various applications of hPSC-MSCs in regenerative medicine. In the end, the challenges and concerns with hPSC-MSC applications are also discussed.
Collapse
Affiliation(s)
- Tong-Ming Liu
- Agency for Science, Technology and Research, Institute of Molecular and Cell Biology, Singapore 138648, Singapore.
| |
Collapse
|
|
26
|
Nakamura A, Murata D, Fujimoto R, Tamaki S, Nagata S, Ikeya M, Toguchida J, Nakayama K. Bio-3D printing iPSC-derived human chondrocytes for articular cartilage regeneration. Biofabrication 2021; 13. [PMID: 34380122 DOI: 10.1088/1758-5090/ac1c99] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Accepted: 08/11/2021] [Indexed: 11/12/2022]
Abstract
Osteoarthritis is a leading cause of pain and joint immobility, the incidence of which is increasing worldwide. Currently, total joint replacement is the only treatment for end-stage disease. Scaffold-based tissue engineering is a promising alternative approach for joint repair but is subject to limitations such as poor cytocompatibility and degradation-associated toxicity. To overcome these limitations, a completely scaffold-free Kenzan method for bio-3D printing was used to fabricate cartilage constructs feasible for repairing large chondral defects. Human induced pluripotent stem cell (iPSC)-derived neural crest cells with high potential to undergo chondrogenesis through mesenchymal stem cell differentiation were used to fabricate the cartilage. Unified, self-sufficient, and functional cartilaginous constructs up to 6 cm2in size were assembled by optimizing fabrication time during chondrogenic induction. Maturation for 3 weeks facilitated the self-organisation of the cells, which improved the construct's mechanical strength (compressive and tensile properties) and induced changes in glycosaminoglycan and type II collagen expression, resulting in improved tissue function. The compressive modulus of the construct reached the native cartilage range of 0.88 MPa in the 5th week of maturation. This paper reports the fabrication of anatomically sized and shaped cartilage constructs, achieved by combining novel iPSCs and bio-3D printers using a Kenzan needle array technology, which may facilitate chondral resurfacing of articular cartilage defects.
Collapse
Affiliation(s)
- Anna Nakamura
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Daiki Murata
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Ryota Fujimoto
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Sakura Tamaki
- Institute for Frontier Life and Medical Institute, Kyoto University, Kyoto, Japan
| | - Sanae Nagata
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Makoto Ikeya
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Junya Toguchida
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Institute for Frontier Life and Medical Institute, Kyoto University, Kyoto, Japan
| | - Koichi Nakayama
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| |
Collapse
|
|
27
|
Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture. Nat Commun 2021; 12:5012. [PMID: 34408142 PMCID: PMC8373964 DOI: 10.1038/s41467-021-25328-6] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Accepted: 08/04/2021] [Indexed: 11/09/2022] Open
Abstract
Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.
Collapse
|
|
28
|
Lee MS, Stebbins MJ, Jiao H, Huang HC, Leiferman EM, Walczak BE, Palecek SP, Shusta EV, Li WJ. Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration. SCIENCE ADVANCES 2021; 7:eabf0907. [PMID: 34138734 PMCID: PMC8133756 DOI: 10.1126/sciadv.abf0907] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 03/29/2021] [Indexed: 05/12/2023]
Abstract
Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.
Collapse
Affiliation(s)
- Ming-Song Lee
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Matthew J Stebbins
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Hongli Jiao
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Hui-Ching Huang
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Ellen M Leiferman
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Brian E Walczak
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Sean P Palecek
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Eric V Shusta
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
- Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI 53792, USA
| | - Wan-Ju Li
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA.
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| |
Collapse
|
|
29
|
Rejuvenated Stem/Progenitor Cells for Cartilage Repair Using the Pluripotent Stem Cell Technology. Bioengineering (Basel) 2021; 8:bioengineering8040046. [PMID: 33920285 PMCID: PMC8070387 DOI: 10.3390/bioengineering8040046] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Revised: 04/01/2021] [Accepted: 04/06/2021] [Indexed: 01/19/2023] Open
Abstract
It is widely accepted that chondral defects in articular cartilage of adult joints are never repaired spontaneously, which is considered to be one of the major causes of age-related degenerative joint disorders, such as osteoarthritis. Since mobilization of subchondral bone (marrow) cells and addition of chondrocytes or mesenchymal stromal cells into full-thickness defects show some degrees of repair, the lack of self-repair activity in adult articular cartilage can be attributed to lack of reparative cells in adult joints. In contrast, during a fetal or embryonic stage, joint articular cartilage has a scar-less repair activity, suggesting that embryonic joints may contain cells responsible for such activity, which can be chondrocytes, chondroprogenitors, or other cell types such as skeletal stem cells. In this respect, the tendency of pluripotent stem cells (PSCs) to give rise to cells of embryonic characteristics will provide opportunity, especially for humans, to obtain cells carrying similar cartilage self-repair activity. Making use of PSC-derived cells for cartilage repair is still in a basic or preclinical research phase. This review will provide brief overviews on how human PSCs have been used for cartilage repair studies.
Collapse
|
|
30
|
Shen M, Quertermous T, Fischbein MP, Wu JC. Generation of Vascular Smooth Muscle Cells From Induced Pluripotent Stem Cells: Methods, Applications, and Considerations. Circ Res 2021; 128:670-686. [PMID: 33818124 PMCID: PMC10817206 DOI: 10.1161/circresaha.120.318049] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.
Collapse
Affiliation(s)
- Mengcheng Shen
- Stanford Cardiovascular Institute
- Division of Cardiovascular Medicine, Department of Medicine
| | - Thomas Quertermous
- Stanford Cardiovascular Institute
- Division of Cardiovascular Medicine, Department of Medicine
| | | | - Joseph C. Wu
- Stanford Cardiovascular Institute
- Division of Cardiovascular Medicine, Department of Medicine
- Department of Radiology, Stanford University School of Medicine, Stanford, CA
| |
Collapse
|
|
31
|
Pretemer Y, Kawai S, Nagata S, Nishio M, Watanabe M, Tamaki S, Alev C, Yamanaka Y, Xue JY, Wang Z, Fukiage K, Tsukanaka M, Futami T, Ikegawa S, Toguchida J. Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias. Stem Cell Reports 2021; 16:610-625. [PMID: 33636111 PMCID: PMC7940258 DOI: 10.1016/j.stemcr.2021.01.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 01/23/2021] [Accepted: 01/25/2021] [Indexed: 12/16/2022] Open
Abstract
Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.
Collapse
Affiliation(s)
- Yann Pretemer
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Shunsuke Kawai
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Sanae Nagata
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Megumi Nishio
- Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Makoto Watanabe
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Life Science Research Center, Technology Research Laboratory, Shimadzu Corporation, Kyoto, Japan
| | - Sakura Tamaki
- Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; Institute for Advancement of Clinical and Translational Sciences, Kyoto University Hospital, Kyoto University, Kyoto, Japan
| | - Cantas Alev
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto, Japan
| | - Yoshihiro Yamanaka
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto, Japan
| | - Jing-Yi Xue
- Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan
| | - Zheng Wang
- Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan; McKusick-Zhang Center for Genetic Medicine and State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Kenichi Fukiage
- Department of Pediatric Orthopaedics, Shiga Medical Center for Children, Moriyama, Japan; Department of Orthopaedic Surgery, Bobath Memorial Hospital, Osaka, Japan
| | - Masako Tsukanaka
- Department of Pediatric Orthopaedics, Shiga Medical Center for Children, Moriyama, Japan
| | - Tohru Futami
- Department of Pediatric Orthopaedics, Shiga Medical Center for Children, Moriyama, Japan
| | - Shiro Ikegawa
- Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan
| | - Junya Toguchida
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Institute for Advancement of Clinical and Translational Sciences, Kyoto University Hospital, Kyoto University, Kyoto, Japan.
| |
Collapse
|
|
32
|
Taghiyar L, Jahangir S, Khozaei Ravari M, Shamekhi MA, Eslaminejad MB. Cartilage Repair by Mesenchymal Stem Cell-Derived Exosomes: Preclinical and Clinical Trial Update and Perspectives. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1326:73-93. [PMID: 33629260 DOI: 10.1007/5584_2021_625] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Osteoarthritis (OA) and other degenerative joint diseases are characterized by articular cartilage destruction, synovial inflammation, sclerosis of subchondral bone, and loss of extracellular matrix (ECM). Worldwide, these diseases are major causes of disability. Cell therapies have been considered to be the best therapeutic strategies for long-term treatment of articular cartilage diseases. It has been suggested that the mechanism of stem cell-based therapy is related to paracrine secretion of extracellular vesicles (EVs), which are recognized as the main secretion factors of stem cells. EVs, and in particular the subclass exosomes (Exos), are novel therapeutic approaches for treatment of cartilage lesions and OA. The results of recent studies have shown that EVs isolated from mesenchymal stem cells (MSCs) could inhibit OA progression. EVs isolated from various stem cell sources, such as MSCs, may contribute to tissue regeneration of the limbs, skin, heart, and other tissues. Here, we summarize recent findings of preclinical and clinical studies on different MSC-derived EVs and their effectiveness as a treatment for damaged cartilage. The Exos isolation techniques in OA treatment are also highlighted.
Collapse
Affiliation(s)
- Leila Taghiyar
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Shahrbano Jahangir
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mojtaba Khozaei Ravari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | | | - Mohamadreza Baghaban Eslaminejad
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| |
Collapse
|
|
33
|
Zhao Y, Liu H, Zhao C, Dang P, Li H, Farzaneh M. Paracrine Interactions Involved in Human Induced Pluripotent Stem Cells Differentiation into Chondrocytes. Curr Stem Cell Res Ther 2020; 15:233-242. [PMID: 31889496 DOI: 10.2174/1574888x15666191224122058] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2019] [Revised: 10/24/2019] [Accepted: 10/30/2019] [Indexed: 12/20/2022]
Abstract
Osteoarthritis (OA), as a degenerative joint disease, is the most common form of joint disorder that causes pain, stiffness, and other symptoms associated with OA. Various genetic, biomechanical, and environmental factors have a relevant role in the development of OA. To date, extensive efforts are currently being made to overcome the poor self-healing capacity of articular cartilage. Despite the pivotal role of chondrocytes, their proliferation and repair capacity after tissue injury are limited. Therefore, the development of new strategies to overcome these constraints is urgently needed. Recent advances in regenerative medicine suggest that pluripotent stem cells are promising stem cell sources for cartilage repair. Pluripotent stem cells are undifferentiated cells that have the capacity to differentiate into different types of cells and can self-renew indefinitely. In the past few decades, numerous attempts have been made to regenerate articular cartilage by using induced pluripotent stem cells (iPSCs). The potential applications of patient-specific iPSCs hold great promise for regenerative medicine and OA treatment. However, there are different culture conditions for the preparation and characterization of human iPSCs-derived chondrocytes (hiChondrocytes). Recent biochemical analyses reported that several paracrine factors such as TGFb, BMPs, WNT, Ihh, and Runx have been shown to be involved in cartilage cell proliferation and differentiation from human iPSCs. In this review, we summarize and discuss the paracrine interactions involved in human iPSCs differentiation into chondrocytes in different cell culture media.
Collapse
Affiliation(s)
- Yunchang Zhao
- Department of Orthopedics III, Zhoukou Central Hospital, Zhoukou, Henan 466000, China
| | - Honghao Liu
- Department of Orthopedics III, Zhoukou Central Hospital, Zhoukou, Henan 466000, China
| | - Chunjie Zhao
- Department of Orthopedics III, Zhoukou Central Hospital, Zhoukou, Henan 466000, China
| | - Peng Dang
- Department of Orthopedics III, Zhoukou Central Hospital, Zhoukou, Henan 466000, China
| | - Haijian Li
- Department of Orthopedics III, Zhoukou Central Hospital, Zhoukou, Henan 466000, China
| | - Maryam Farzaneh
- Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| |
Collapse
|
|
34
|
Nakajima T, Ikeya M. Development of pluripotent stem cell-based human tenocytes. Dev Growth Differ 2020; 63:38-46. [PMID: 33270251 DOI: 10.1111/dgd.12702] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Revised: 11/17/2020] [Accepted: 11/18/2020] [Indexed: 12/12/2022]
Abstract
Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.
Collapse
Affiliation(s)
- Taiki Nakajima
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.,Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.,Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| |
Collapse
|
|
35
|
Kreuser U, Buchert J, Haase A, Richter W, Diederichs S. Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells. Front Cell Dev Biol 2020; 8:581331. [PMID: 33195222 PMCID: PMC7661475 DOI: 10.3389/fcell.2020.581331] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Accepted: 10/07/2020] [Indexed: 12/20/2022] Open
Abstract
Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.
Collapse
Affiliation(s)
- Ursula Kreuser
- Research Center for Experimental Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany
| | - Justyna Buchert
- Research Center for Experimental Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany
| | - Alexandra Haase
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Hannover, Germany
| | - Wiltrud Richter
- Research Center for Experimental Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany
| | - Solvig Diederichs
- Research Center for Experimental Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany
| |
Collapse
|
|
36
|
Middendorf JM, Diamantides N, Shortkroff S, Dugopolski C, Kennedy S, Cohen I, Bonassar LJ. Multiscale mechanics of tissue-engineered cartilage grown from human chondrocytes and human-induced pluripotent stem cells. J Orthop Res 2020; 38:1965-1973. [PMID: 32125023 DOI: 10.1002/jor.24643] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2019] [Revised: 02/17/2020] [Accepted: 02/29/2020] [Indexed: 02/04/2023]
Abstract
Tissue-engineered cartilage has shown promising results in the repair of focal cartilage defects. However, current clinical techniques rely on an extra surgical procedure to biopsy healthy cartilage to obtain human chondrocytes. Alternatively, induced pluripotent stem cells (iPSCs) have the ability to differentiate into chondrocytes and produce cartilaginous matrix without the need to biopsy healthy cartilage. However, the mechanical properties of tissue-engineered cartilage with iPSCs are unknown and might be critical to long-term tissue function and health. This study used confined compression, cartilage on glass tribology, and shear testing on a confocal microscope to assess the macroscale and microscale mechanical properties of two constructs seeded with either chondrocyte-derived iPSCs (Ch-iPSCs) or native human chondrocytes. Macroscale properties of Ch-iPSC constructs provided similar or better mechanical properties than chondrocyte constructs. Under compression, Ch-iPSC constructs had an aggregate modulus that was two times larger than chondrocyte constructs and was closer to native tissue. No differences in the shear modulus and friction coefficients were observed between Ch-iPSC and chondrocyte constructs. On the microscale, Ch-iPSC and chondrocyte constructs had different depth-dependent mechanical properties, neither of which matches native tissue. These observed depth-dependent differences may be important to the function of the implant. Overall, this comparison of multiple mechanical properties of Ch-iPSC and chondrocyte constructs shows that using Ch-iPSCs can produce equivalent or better global mechanical properties to chondrocytes. Therefore, iPSC-seeded cartilage constructs could be a promising solution to repair focal cartilage defects. The chondrocyte constructs used in this study have been implanted into humans for clinical trials. Therefore, Ch-iPSC constructs could also be used clinically in place of the current chondrocyte construct.
Collapse
Affiliation(s)
- Jill M Middendorf
- Sibley School of Mechanical Engineering, Cornell University, Ithaca, New York
| | - Nicole Diamantides
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York
| | | | | | | | - Itai Cohen
- Department of Applied Engineering and Physics, Cornell University, Ithaca, New York.,Department of Physics, Cornell University, Ithaca, New York
| | - Lawrence J Bonassar
- Sibley School of Mechanical Engineering, Cornell University, Ithaca, New York.,Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York
| |
Collapse
|
|
37
|
Kidwai F, Mui BWH, Arora D, Iqbal K, Hockaday M, de Castro Diaz LF, Cherman N, Martin D, Myneni VD, Ahmad M, Futrega K, Ali S, Merling RK, Kaufman DS, Lee J, Robey PG. Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors. Stem Cells 2020; 38:1107-1123. [PMID: 32442326 PMCID: PMC7484058 DOI: 10.1002/stem.3206] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Accepted: 05/01/2020] [Indexed: 12/16/2022]
Abstract
Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.
Collapse
Affiliation(s)
- Fahad Kidwai
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Byron W. H. Mui
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Deepika Arora
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
- Biosystems and Biomaterials DivisionNational Institute of Standards and TechnologyGaithersburgMarylandUSA
| | - Kulsum Iqbal
- Department of Health and Human ServicesDental Consult Services, National Institute of Health Dental ClinicBethesdaMarylandUSA
| | - Madison Hockaday
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Luis Fernandez de Castro Diaz
- Department of Health and Human ServicesSkeletal Disorders and Mineral Homeostasis Section, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Natasha Cherman
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Daniel Martin
- Department of Health and Human ServicesGenomics and Computational Biology Core, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Vamsee D. Myneni
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch/Adult Stem Cell Section, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Moaz Ahmad
- Department of Health and Human ServicesMolecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Katarzyna Futrega
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Sania Ali
- Biology of Global Health, Department of BiologyGeorgetown UniversityWashingtonDistrict of ColumbiaUSA
| | - Randall K. Merling
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Dan S. Kaufman
- Department of MedicineUniversity of CaliforniaLa JollaCaliforniaUSA
| | - Janice Lee
- Department of Health and Human ServicesCraniofacial Anomalies and Regeneration Section, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| | - Pamela G. Robey
- Department of Health and Human ServicesCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of HealthBethesdaMarylandUSA
| |
Collapse
|
|
38
|
Sanjurjo-Rodríguez C, Castro-Viñuelas R, Piñeiro-Ramil M, Rodríguez-Fernández S, Fuentes-Boquete I, Blanco FJ, Díaz-Prado S. Versatility of Induced Pluripotent Stem Cells (iPSCs) for Improving the Knowledge on Musculoskeletal Diseases. Int J Mol Sci 2020; 21:ijms21176124. [PMID: 32854405 PMCID: PMC7504376 DOI: 10.3390/ijms21176124] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 08/06/2020] [Accepted: 08/20/2020] [Indexed: 12/13/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) represent an unlimited source of pluripotent cells capable of differentiating into any cell type of the body. Several studies have demonstrated the valuable use of iPSCs as a tool for studying the molecular and cellular mechanisms underlying disorders affecting bone, cartilage and muscle, as well as their potential for tissue repair. Musculoskeletal diseases are one of the major causes of disability worldwide and impose an important socio-economic burden. To date there is neither cure nor proven approach for effectively treating most of these conditions and therefore new strategies involving the use of cells have been increasingly investigated in the recent years. Nevertheless, some limitations related to the safety and differentiation protocols among others remain, which humpers the translational application of these strategies. Nonetheless, the potential is indisputable and iPSCs are likely to be a source of different types of cells useful in the musculoskeletal field, for either disease modeling or regenerative medicine. In this review, we aim to illustrate the great potential of iPSCs by summarizing and discussing the in vitro tissue regeneration preclinical studies that have been carried out in the musculoskeletal field by using iPSCs.
Collapse
Affiliation(s)
- Clara Sanjurjo-Rodríguez
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
- Correspondence: (C.S.-R.); (S.D.-P.)
| | - Rocío Castro-Viñuelas
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - María Piñeiro-Ramil
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Silvia Rodríguez-Fernández
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Isaac Fuentes-Boquete
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
| | - Francisco J. Blanco
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
- Tissular Bioengineering and Cell Therapy Unit (GBTTC-CHUAC), Rheumatology Group, 15006 A Coruña, Galicia, Spain
| | - Silvia Díaz-Prado
- Cell Therapy and Regenerative Medicine Group, Department of Physiotherapy, Medicine and Biomedical Sciences, Faculty of Health Sciences, University of A Coruña (UDC), 15006 A Coruña, Galicia, Spain; (R.C.-V.); (M.P.-R.); (S.R.-F.); (I.F.-B.)
- Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), 15006 A Coruña, Galicia, Spain;
- Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
- Centro de Investigaciones Científicas Avanzadas (CICA), Agrupación estratégica CICA-INIBIC, University of A Coruña, 15008 A Coruña, Galicia, Spain
- Correspondence: (C.S.-R.); (S.D.-P.)
| |
Collapse
|
|
39
|
Abbadessa A, Crecente-Campo J, Alonso MJ. Engineering Anisotropic Meniscus: Zonal Functionality and Spatiotemporal Drug Delivery. TISSUE ENGINEERING PART B-REVIEWS 2020; 27:133-154. [PMID: 32723019 DOI: 10.1089/ten.teb.2020.0096] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Human meniscus is a fibrocartilaginous structure that is crucial for an adequate performance of the human knee joint. Degeneration of the meniscus is often followed by partial or total meniscectomy, which enhances the risk of developing knee osteoarthritis. The lack of a satisfactory treatment for this condition has triggered a major interest in drug delivery (DD) and tissue engineering (TE) strategies intended to restore a bioactive and fully functional meniscal tissue. The aim of this review is to critically discuss the most relevant studies on spatiotemporal DD and TE, aiming for a multizonal meniscal reconstruction. Indeed, the development of meniscal tissue implants should involve a provision for adequate active molecules and scaffold features that take into account the anisotropic ultrastructure of human meniscus. This zonal differentiation is reflected in the meniscus biochemical composition, collagen fiber arrangement, and cell distribution. In this sense, it is expected that a proper combination of advanced DD and zonal TE strategies will play a key role in the future trends in meniscus regeneration. Impact statement Meniscus degeneration is one of the main causes of knee pain, inflammation, and reduced mobility. Currently used suturing procedures and meniscectomy are far from being ideal solutions to the loss of meniscal function. Therefore, drug delivery (DD) and tissue engineering (TE) strategies are currently under investigation. DD systems aim at an in situ controlled release of growth factors, whereas TE strategies aim at mimicking the anisotropy of native meniscus. The goal of this review is to discuss these two main approaches, as well as synergies between them that are expected to lead to a real breakthrough in the field.
Collapse
Affiliation(s)
- Anna Abbadessa
- Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), IDIS Research Institute, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.,Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Campus Vida, Universidade de Santiago de Compostela, Santiago de Compostela, Spain
| | - José Crecente-Campo
- Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), IDIS Research Institute, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.,Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Campus Vida, Universidade de Santiago de Compostela, Santiago de Compostela, Spain
| | - María José Alonso
- Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), IDIS Research Institute, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.,Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Campus Vida, Universidade de Santiago de Compostela, Santiago de Compostela, Spain
| |
Collapse
|
|
40
|
Nakayama N, Pothiawala A, Lee JY, Matthias N, Umeda K, Ang BK, Huard J, Huang Y, Sun D. Human pluripotent stem cell-derived chondroprogenitors for cartilage tissue engineering. Cell Mol Life Sci 2020; 77:2543-2563. [PMID: 31915836 PMCID: PMC11104892 DOI: 10.1007/s00018-019-03445-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 12/24/2019] [Accepted: 12/27/2019] [Indexed: 02/06/2023]
Abstract
The cartilage of joints, such as meniscus and articular cartilage, is normally long lasting (i.e., permanent). However, once damaged, especially in large animals and humans, joint cartilage is not spontaneously repaired. Compensating the lack of repair activity by supplying cartilage-(re)forming cells, such as chondrocytes or mesenchymal stromal cells, or by transplanting a piece of normal cartilage, has been the basis of therapy for biological restoration of damaged joint cartilage. Unfortunately, current biological therapies face problems on a number of fronts. The joint cartilage is generated de novo from a specialized cell type, termed a 'joint progenitor' or 'interzone cell' during embryogenesis. Therefore, embryonic chondroprogenitors that mimic the property of joint progenitors might be the best type of cell for regenerating joint cartilage in the adult. Pluripotent stem cells (PSCs) are expected to differentiate in culture into any somatic cell type through processes that mimic embryogenesis, making human (h)PSCs a promising source of embryonic chondroprogenitors. The major research goals toward the clinical application of PSCs in joint cartilage regeneration are to (1) efficiently generate lineage-specific chondroprogenitors from hPSCs, (2) expand the chondroprogenitors to the number needed for therapy without loss of their chondrogenic activity, and (3) direct the in vivo or in vitro differentiation of the chondroprogenitors to articular or meniscal (i.e., permanent) chondrocytes rather than growth plate (i.e., transient) chondrocytes. This review is aimed at providing the current state of research toward meeting these goals. We also include our recent achievement of successful generation of "permanent-like" cartilage from long-term expandable, hPSC-derived ectomesenchymal chondroprogenitors.
Collapse
Affiliation(s)
- Naoki Nakayama
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA.
- Department of Orthopaedic Surgery, The University of Texas Health Science Center at Houston Medical School, Houston, TX, USA.
| | - Azim Pothiawala
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
| | - John Y Lee
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Miller School of Medicine, University of Miami, Miami, FL, USA
| | - Nadine Matthias
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
| | - Katsutsugu Umeda
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Department of Pediatrics, Kyoto University School of Medicine, Kyoto, Japan
| | - Bryan K Ang
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Weil Cornell Medicine, New York, NY, USA
| | - Johnny Huard
- Department of Orthopaedic Surgery, The University of Texas Health Science Center at Houston Medical School, Houston, TX, USA
- Steadman Philippon Research Institute, Vail, CO, USA
| | - Yun Huang
- Institute of Bioscience and Technology, Texas A&M University, Houston, TX, USA
| | - Deqiang Sun
- Institute of Bioscience and Technology, Texas A&M University, Houston, TX, USA
| |
Collapse
|
|
41
|
Aisenbrey EA, Bilousova G, Payne K, Bryant SJ. Dynamic mechanical loading and growth factors influence chondrogenesis of induced pluripotent mesenchymal progenitor cells in a cartilage-mimetic hydrogel. Biomater Sci 2020; 7:5388-5403. [PMID: 31626251 DOI: 10.1039/c9bm01081e] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Human induced pluripotent stem cells (iPSCs) have emerged as a promising alternative to bone-marrow derived mesenchymal stem/stromal cells for cartilage tissue engineering. However, the effect of biochemical and mechanical cues on iPSC chondrogenesis remains understudied. This study evaluated chondrogenesis of induced pluripotent mesenchymal progenitor cells (iPS-MPs) encapsulated in a cartilage-mimetic hydrogel under different culture conditions: free swelling versus dynamic compressive loading and different growth factors (TGFβ3 and/or BMP2). Human iPSCs were differentiated into iPS-MPs and chondrogenesis was evaluated by gene expression (qPCR) and protein expression (immunohistochemistry) after three weeks. In pellet culture, both TGFβ3 and BMP2 were required to promote chondrogenesis. However, the hydrogel in growth factor-free conditions promoted chondrogenesis, but rapidly progressed to hypertrophy. Dynamic loading in growth factor-free conditions supported chondrogenesis, but delayed the transition to hypertrophy. Findings were similar with TGFβ3, BMP2, and TGFβ3 + BMP2. Dynamic loading with TGFβ3, regardless of BMP2, was the only condition that promoted a stable chondrogenic phenotype (aggrecan + collagen II) accompanied by collagen X down-regulation. Positive TGFβRI expression with load-enhanced Smad2/3 signaling and low SMAD1/5/8 signaling was observed. In summary, this study reports a promising cartilage-mimetic hydrogel for iPS-MPs that when combined with appropriate biochemical and mechanical cues induces a stable chondrogenic phenotype.
Collapse
Affiliation(s)
- Elizabeth A Aisenbrey
- Department of Chemical and Biological Engineering, University of Colorado at Boulder, 3415 Colorado Ave, Boulder, CO 80309, USA.
| | | | | | | |
Collapse
|
|
42
|
Fowler JL, Ang LT, Loh KM. A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids. WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY 2019; 9:e368. [PMID: 31746148 DOI: 10.1002/wdev.368] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2019] [Revised: 09/17/2019] [Accepted: 10/21/2019] [Indexed: 12/11/2022]
Abstract
Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell types; yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations. These are timely issues in view of the fact that hPSC-derived cell populations have or are being transplanted into patients in over 30 ongoing clinical trials. While many in vitro differentiation protocols purport to "mimic development," the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell type in vivo remains largely unknown. Consequently, most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single-cell RNA-sequencing and other analyses. The presence of unwanted cell types in differentiated hPSC populations does not portend well for transplantation therapies. This provides an impetus to precisely control differentiation to desired ends-for instance, by logically blocking the formation of unwanted cell types or by overexpressing lineage-specifying transcription factors-or by harnessing technologies to selectively purify desired cell types. Conversely, approaches to differentiate three-dimensional "organoids" from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as organoids) remains to be fully resolved. This article is categorized under: Adult Stem Cells > Tissue Renewal > Regeneration: Stem Cell Differentiation and Reversion Gene Expression > Transcriptional Hierarchies: Cellular Differentiation Early Embryonic Development: Gastrulation and Neurulation.
Collapse
Affiliation(s)
- Jonas L Fowler
- Stanford Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, Stanford, California.,Department of Developmental Biology, Bio-X, Cancer Institute, Cardiovascular Institute, ChEM-H, Diabetes Research Center, Maternal & Child Health Research Institute, Wu Tsai Neurosciences Institute, Stanford University School of Medicine, Stanford, California
| | - Lay Teng Ang
- Stanford Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, Stanford, California
| | - Kyle M Loh
- Stanford Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, Stanford, California.,Department of Developmental Biology, Bio-X, Cancer Institute, Cardiovascular Institute, ChEM-H, Diabetes Research Center, Maternal & Child Health Research Institute, Wu Tsai Neurosciences Institute, Stanford University School of Medicine, Stanford, California
| |
Collapse
|
|
43
|
Diederichs S, Klampfleuthner FAM, Moradi B, Richter W. Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway. Front Cell Dev Biol 2019; 7:270. [PMID: 31737632 PMCID: PMC6838640 DOI: 10.3389/fcell.2019.00270] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Accepted: 10/22/2019] [Indexed: 12/15/2022] Open
Abstract
A major problem with chondrocytes derived in vitro from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in deriving chondrocytes from human iPSCs without using pro-hypertrophic bone-morphogenetic-proteins. IPSC-chondrocytes had a high cartilage formation capacity and deposited two-fold more proteoglycans per cell than adult ACs. Importantly, cartilage engineered from iPSC-chondrocytes had similar marginal expression of hypertrophic markers (COL10A1, PTH1R, IBSP, ALPL mRNAs) like cartilage from ACs. Collagen X was barely detectable in iPSC-cartilage and 30-fold lower than in hypertrophic cartilage derived from mesenchymal stromal cells (MSCs). Moreover, alkaline phosphatase (ALP) activity remained at basal AC-like levels throughout iPSC chondrogenesis, in contrast to a well-known significant upregulation in hypertrophic MSCs. In line, iPSC-cartilage subjected to mineralizing conditions in vitro showed barely any mineralization, while MSC-derived hypertrophic cartilage mineralized strongly. Low expression of Indian hedgehog (IHH) like in ACs but rising BMP7 expression like in MSCs suggested that phenotype stability was linked to the hedgehog rather than the bone morphogenetic protein (BMP) pathway. Taken together, unlimited amounts of AC-like chondrocytes with a high proteoglycan production reminiscent of juvenile chondrocytes and resistance to hypertrophy and mineralization can now be produced from human iPSCs in vitro. This opens new strategies for cartilage regeneration, disease modeling and pharmacological studies.
Collapse
Affiliation(s)
- Solvig Diederichs
- Research Center for Experimental Orthopaedics, Center for Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Heidelberg, Germany
| | - Felicia A M Klampfleuthner
- Research Center for Experimental Orthopaedics, Center for Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Heidelberg, Germany
| | - Babak Moradi
- Clinic for Orthopaedics and Trauma Surgery, Center for Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Heidelberg, Germany
| | - Wiltrud Richter
- Research Center for Experimental Orthopaedics, Center for Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Heidelberg, Germany
| |
Collapse
|
|
44
|
The Role of Extracellular Matrix Expression, ERK1/2 Signaling and Cell Cohesiveness for Cartilage Yield from iPSCs. Int J Mol Sci 2019; 20:ijms20174295. [PMID: 31480758 PMCID: PMC6747490 DOI: 10.3390/ijms20174295] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Revised: 08/26/2019] [Accepted: 08/29/2019] [Indexed: 02/07/2023] Open
Abstract
Current therapies involving chondrocytes or mesenchymal stromal cells (MSCs) remain inefficient in restoring cartilage properties upon injury. The induced pluripotent stem-cell (iPSC)-derived mesenchymal progenitor cells (iMPCs) have been put forward as a promising alternative cell source due to their high proliferation and differentiation potential. However, the observed cell loss during in vitro chondrogenesis is currently a bottleneck in establishing articular chondrocyte generation from iPSCs. In a search for candidate mechanisms underlying the low iPSC-derived cartilage tissue yield, global transcriptomes were compared between iMPCs and MSCs and the cell properties were analyzed via a condensation assay. The iMPCs had a more juvenile mesenchymal gene signature than MSCs with less myofibroblast-like characteristics, including significantly lower ECM- and integrin-ligand-related as well as lower α-smooth-muscle-actin expression. This correlated with less substrate and more cell-cell adhesion, impaired aggregate formation and consequently inferior cohesive tissue properties of the iMPC-pellets. Along lower expression of pro-survival ECM molecules, like decorin, collagen VI, lumican and laminin, the iMPC populations had significantly less active ERK1/2 compared to MSCs. Overall, this study proposes that this ECM and integrin-ligand shortage, together with insufficient pro-survival ERK1/2-activity, explains the loss of a non-aggregating iMPC sub-fraction during pellet formation and reduced survival of cells in early pellets. Enhancing ECM production and related signaling in iMPCs may be a promising new means to enrich the instructive microenvironment with pro-survival cues allowing to improve the final cartilage tissue yield from iPSCs.
Collapse
|
|
45
|
Kawata M, Mori D, Kanke K, Hojo H, Ohba S, Chung UI, Yano F, Masaki H, Otsu M, Nakauchi H, Tanaka S, Saito T. Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem Cell Reports 2019; 13:530-544. [PMID: 31402337 PMCID: PMC6739881 DOI: 10.1016/j.stemcr.2019.07.012] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2019] [Revised: 07/15/2019] [Accepted: 07/16/2019] [Indexed: 01/08/2023] Open
Abstract
A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5-9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.
Collapse
Affiliation(s)
- Manabu Kawata
- Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Daisuke Mori
- Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Kosuke Kanke
- Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hironori Hojo
- Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Shinsuke Ohba
- Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Ung-Il Chung
- Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Fumiko Yano
- Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hideki Masaki
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Makoto Otsu
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Sakae Tanaka
- Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Taku Saito
- Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
| |
Collapse
|
|
46
|
Nakajima T, Ikeya M. Insights into the biology of fibrodysplasia ossificans progressiva using patient-derived induced pluripotent stem cells. Regen Ther 2019; 11:25-30. [PMID: 31193176 PMCID: PMC6517845 DOI: 10.1016/j.reth.2019.04.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Revised: 03/18/2019] [Accepted: 04/05/2019] [Indexed: 12/12/2022] Open
Abstract
The demand for development of new drugs remains on the upward trend because of the large number of patients suffering from intractable diseases for which effective treatment has not been established yet. Recently, several researchers have attempted to apply induced pluripotent stem cell (iPSC) technology as a powerful tool for studying the mechanisms underlying the onset of various diseases and for new drug screening. This technology has made an enormous breakthrough, since it permits us to recapitulate the disease phenotype in vitro, outside of the patient's body. Here, we discuss the latest findings that uncovered a mechanism underlying the pathology of a rare genetic musculoskeletal disease, fibrodysplasia ossificans progressiva (FOP), by modeling the phenotypes with FOP patient-derived iPSCs, and that discovered promising candidate drugs for FOP treatment. We also discussed future directions of FOP research.
Collapse
Affiliation(s)
- Taiki Nakajima
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan
| |
Collapse
|
|
47
|
Tam WL, Luyten FP, Roberts SJ. From skeletal development to the creation of pluripotent stem cell-derived bone-forming progenitors. Philos Trans R Soc Lond B Biol Sci 2019; 373:rstb.2017.0218. [PMID: 29786553 DOI: 10.1098/rstb.2017.0218] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/18/2017] [Indexed: 02/06/2023] Open
Abstract
Bone has many functions. It is responsible for protecting the underlying soft organs, it allows locomotion, houses the bone marrow and stores minerals such as calcium and phosphate. Upon damage, bone tissue can efficiently repair itself. However, healing is hampered if the defect exceeds a critical size and/or is in compromised conditions. The isolation or generation of bone-forming progenitors has applicability to skeletal repair and may be used in tissue engineering approaches. Traditionally, bone engineering uses osteochondrogenic stem cells, which are combined with scaffold materials and growth factors. Despite promising preclinical data, limited translation towards the clinic has been observed to date. There may be several reasons for this including the lack of robust cell populations with favourable proliferative and differentiation capacities. However, perhaps the most pertinent reason is the failure to produce an implant that can replicate the developmental programme that is observed during skeletal repair. Pluripotent stem cells (PSCs) can potentially offer a solution for bone tissue engineering by providing unlimited cell sources at various stages of differentiation. In this review, we summarize key embryonic signalling pathways in bone formation coupled with PSC differentiation strategies for the derivation of bone-forming progenitors.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.
Collapse
Affiliation(s)
- Wai Long Tam
- Laboratory for Developmental and Stem Cell Biology (DSB), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Herestraat 49 Box 813, 3000 Leuven, Belgium.,Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, O&N 1 Herestraat 49 bus 813, 3000 Leuven, Belgium
| | - Frank P Luyten
- Laboratory for Developmental and Stem Cell Biology (DSB), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Herestraat 49 Box 813, 3000 Leuven, Belgium.,Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, O&N 1 Herestraat 49 bus 813, 3000 Leuven, Belgium
| | - Scott J Roberts
- Laboratory for Developmental and Stem Cell Biology (DSB), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Herestraat 49 Box 813, 3000 Leuven, Belgium .,Bone Therapeutic Area, UCB Pharma, 208 Bath Road, Slough, Berkshire SL1 3WE, UK
| |
Collapse
|
|
48
|
An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva. Stem Cell Reports 2018; 11:1106-1119. [PMID: 30392977 PMCID: PMC6235670 DOI: 10.1016/j.stemcr.2018.10.007] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2018] [Revised: 10/05/2018] [Accepted: 10/05/2018] [Indexed: 12/11/2022] Open
Abstract
Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.
Established a screening system for fibrodysplasia ossificans progressiva (FOP) Identified two hit compounds that are effective in multiple FOP model mice An mTOR signaling modulator opens the door to a therapeutic strategy
Collapse
|
|
49
|
Adkar SS, Wu CL, Willard VP, Dicks A, Ettyreddy A, Steward N, Bhutani N, Gersbach CA, Guilak F. Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing. Stem Cells 2018; 37:65-76. [PMID: 30378731 DOI: 10.1002/stem.2931] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2018] [Revised: 08/26/2018] [Accepted: 09/05/2018] [Indexed: 01/23/2023]
Abstract
The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65-76.
Collapse
Affiliation(s)
- Shaunak S Adkar
- Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.,Shriners Hospitals for Children-St. Louis, St. Louis, Missouri, USA.,Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA
| | - Chia-Lung Wu
- Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.,Shriners Hospitals for Children-St. Louis, St. Louis, Missouri, USA
| | | | - Amanda Dicks
- Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.,Shriners Hospitals for Children-St. Louis, St. Louis, Missouri, USA.,Department of Biomedical Engineering, Washington University, St. Louis, Missouri, USA
| | - Adarsh Ettyreddy
- Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA
| | - Nancy Steward
- Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.,Shriners Hospitals for Children-St. Louis, St. Louis, Missouri, USA
| | - Nidhi Bhutani
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Charles A Gersbach
- Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA
| | - Farshid Guilak
- Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.,Shriners Hospitals for Children-St. Louis, St. Louis, Missouri, USA.,Department of Biomedical Engineering, Washington University, St. Louis, Missouri, USA.,Cytex Therapeutics, Inc., Durham, North Carolina, USA
| |
Collapse
|
|
50
|
Nakajima T, Shibata M, Nishio M, Nagata S, Alev C, Sakurai H, Toguchida J, Ikeya M. Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells. Development 2018; 145:145/16/dev165431. [PMID: 30139810 PMCID: PMC6124548 DOI: 10.1242/dev.165431] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Accepted: 07/24/2018] [Indexed: 12/27/2022]
Abstract
Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies. Summary: Protocols for the differentiation of human iPSCs to somite derivatives (myotome, sclerotome, syndetome and dermatome) are developed and applied to the modeling of the bone disease fibrodysplasia ossificans progressiva.
Collapse
Affiliation(s)
- Taiki Nakajima
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| | - Mitsuaki Shibata
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| | - Megumi Nishio
- Department of Tissue Regeneration, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Sanae Nagata
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| | - Cantas Alev
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| | - Junya Toguchida
- Department of Tissue Regeneration, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.,Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.,Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
| |
Collapse
|