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Kondo T, Suga H, Takeuchi K, Fuse Y, Sato Y, Hirose T, Hideyuki H, Nagata Y, Saito R. Benchmark for Setting ACTH Cell Dosage in Clinical Regenerative Medicine for Post-Operative Hypopituitarism. Diseases 2025; 13:112. [PMID: 40277822 PMCID: PMC12025586 DOI: 10.3390/diseases13040112] [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: 02/28/2025] [Revised: 04/06/2025] [Accepted: 04/07/2025] [Indexed: 04/26/2025] Open
Abstract
BACKGROUND/OBJECTIVES Our objective is to develop hormone-producing pituitary cells that can function in the same manner as the human body and provide more effective treatments than current hormone replacement therapy. We have already established a technique for generating hypothalamic-pituitary organoids using feeder-free human pluripotent stem cells (hPSCs) and demonstrated their effectiveness in vivo through transplantation into hypopituitary mouse models. To prospectively determine the upper limit of transplanting adenohypophyseal cells into humans, we investigated the human maximum secretion capacity of adrenocorticotropic hormone (ACTH) and growth hormone (GH). METHODS We analyzed data from 28 patients with pituitary adenomas, among whom 16 evinced no abnormality of ACTH secretion and 12 showed no GH secretion on corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone-2 (GHRP-2) stimulation testing. RESULTS The average ACTH peak value after CRH stimulation tests was 97.2 pg/mL, and the average GH peak value after GHRP-2 stimulation tests was 25.1 ng/mL. CONCLUSIONS These data will likely serve as benchmarks of ACTH and GH secretion when transplanting cultured cells into humans.
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Affiliation(s)
- Tatsuma Kondo
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
- Department of Endocrinology and Diabetes, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan
| | - Kazuhito Takeuchi
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Yutaro Fuse
- Department of Artificial Intelligence Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8722, Japan;
| | - Yoshiki Sato
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Toshiaki Hirose
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Harada Hideyuki
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Yuichi Nagata
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Ryuta Saito
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
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Wang S, Jiang D, Xiao Y, Qin Q, Zhang H, Ye L, Jin J, Jiang X, Guo Q. Human Pituitary Organoids: Transcriptional Landscape Deciphered by scRNA-Seq and Stereo-Seq, with Insights into SOX3's Role in Pituitary Development. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2414230. [PMID: 39951008 PMCID: PMC11984888 DOI: 10.1002/advs.202414230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2024] [Revised: 02/03/2025] [Indexed: 04/12/2025]
Abstract
The 3D human pituitary organoid represents a promising laboratory model for investigating human pituitary diseases. Nonetheless, this technology is still in its nascent stage, with uncertainties regarding the cellular composition, intercellular interactions, and spatial distribution of the human pituitary organoids. To address these gaps, the culture conditions are systematically adjusted and the efficiency of induced pluripotent stem cells' (iPSCs') differentiation into pituitary organoids is successfully improved, achieving results comparable to or exceeding those of previous studies. Additionally, single-cell RNA-sequencing (scRNA-seq) and stereomics sequencing (Stereo-seq) are performed on the pituitary organoids for the first time, and unveil the diverse cell clusters, intricate intercellular interactions, and spatial information within the organoids. Furthermore, the SOX3 gene interference impedes the iPSCs' differentiation into pituitary organoids, thereby highlighting the potential of pituitary organoids as an ideal experimental model. Altogether, the research provides an optimized protocol for the human pituitary organoid culture and a valuable transcriptomic dataset for future explorations, laying the foundation for subsequent research in the field of pituitary organoids or pituitary diseases.
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Affiliation(s)
- Shengjie Wang
- Department of Endocrinologythe First Medical Center of Chinese PLA General HospitalBeijing100853China
| | - Deyue Jiang
- Department of Endocrinologythe First Medical Center of Chinese PLA General HospitalBeijing100853China
| | - Yan Xiao
- Department of Endocrinologythe First Medical Center of Chinese PLA General HospitalBeijing100853China
| | - Qiaozhen Qin
- Beijing Institute of Basic Medical Sciences27 Taiping Road of Haidian DistrictBeijing100850China
| | - Heyang Zhang
- Beijing Institute of Basic Medical Sciences27 Taiping Road of Haidian DistrictBeijing100850China
| | - Lingtong Ye
- Department of Endocrinologythe First Medical Center of Chinese PLA General HospitalBeijing100853China
| | - Jide Jin
- Beijing Institute of Radiation Medicine27 Taiping Road of Haidian DistrictBeijing100850China
| | - Xiaoxia Jiang
- Beijing Institute of Basic Medical Sciences27 Taiping Road of Haidian DistrictBeijing100850China
| | - Qinghua Guo
- Department of Endocrinologythe First Medical Center of Chinese PLA General HospitalBeijing100853China
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Placzek M, Chinnaiya K, Kim DW, Blackshaw S. Control of tuberal hypothalamic development and its implications in metabolic disorders. Nat Rev Endocrinol 2025; 21:118-130. [PMID: 39313573 PMCID: PMC11864813 DOI: 10.1038/s41574-024-01036-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/29/2024] [Indexed: 09/25/2024]
Abstract
The tuberal hypothalamus regulates a range of crucial physiological processes, including energy homeostasis and metabolism. In this Review, we explore the intricate molecular mechanisms and signalling pathways that control the development of the tuberal hypothalamus, focusing on aspects that shape metabolic outcomes. Major developmental events are discussed in the context of their effect on the establishment of both functional hypothalamic neuronal circuits and brain-body interfaces that are pivotal to the control of metabolism. Emerging evidence indicates that aberrations in molecular pathways during tuberal hypothalamic development contribute to metabolic dysregulation. Understanding the molecular underpinnings of tuberal hypothalamic development provides a comprehensive view of neurodevelopmental processes and offers a promising avenue for future targeted interventions to prevent and treat metabolic disorders.
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Affiliation(s)
- Marysia Placzek
- School of Biosciences, University of Sheffield, Sheffield, UK.
- Bateson Centre, University of Sheffield, Sheffield, UK.
- Neuroscience Institute, University of Sheffield, Sheffield, UK.
| | | | - Dong Won Kim
- Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Seth Blackshaw
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
- Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
- Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
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Bosone C, Castaldi D, Burkard TR, Guzman SJ, Wyatt T, Cheroni C, Caporale N, Bajaj S, Bagley JA, Li C, Sorre B, Villa CE, Testa G, Krenn V, Knoblich JA. A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids. Nat Methods 2024; 21:2147-2159. [PMID: 39294368 PMCID: PMC11541204 DOI: 10.1038/s41592-024-02412-5] [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: 09/13/2023] [Accepted: 08/12/2024] [Indexed: 09/20/2024]
Abstract
Organoids generating major cortical cell types in distinct compartments are used to study cortical development, evolution and disorders. However, the lack of morphogen gradients imparting cortical positional information and topography in current systems hinders the investigation of complex phenotypes. Here, we engineer human cortical assembloids by fusing an organizer-like structure expressing fibroblast growth factor 8 (FGF8) with an elongated organoid to enable the controlled modulation of FGF8 signaling along the longitudinal organoid axis. These polarized cortical assembloids mount a position-dependent transcriptional program that in part matches the in vivo rostrocaudal gene expression patterns and that is lost upon mutation in the FGFR3 gene associated with temporal lobe malformations and intellectual disability. By producing spatially oriented cell populations with signatures related to frontal and temporal area identity within individual assembloids, this model recapitulates in part the early transcriptional divergence embedded in the protomap and enables the study of cortical area-relevant alterations underlying human disorders.
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Affiliation(s)
- Camilla Bosone
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
| | - Davide Castaldi
- Human Technopole, Milan, Italy
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
| | - Thomas Rainer Burkard
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
| | - Segundo Jose Guzman
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
| | - Tom Wyatt
- Laboratoire "Matière et Systèmes Complexes" (MSC), UMR 7057 CNRS, University of Paris, Paris, France
| | | | - Nicolò Caporale
- Human Technopole, Milan, Italy
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
| | - Sunanjay Bajaj
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
- Department of Neurology, The University of Texas Health Science Center at Houston, McGovern Medical School, Houston, TX, USA
| | - Joshua Adam Bagley
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
| | - Chong Li
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria
| | - Benoit Sorre
- Laboratoire "Matière et Systèmes Complexes" (MSC), UMR 7057 CNRS, University of Paris, Paris, France
- Physics of Cells and Cancer, Institut Curie, Université PSL, Sorbonne University, CNRS UMR168, Paris, France
| | | | - Giuseppe Testa
- Human Technopole, Milan, Italy.
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.
| | - Veronica Krenn
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.
- Department of Biotechnology and Bioscience, University of Milan-Bicocca, Milan, Italy.
| | - Jürgen Arthur Knoblich
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.
- Department of Neurology, Medical University of Vienna, Vienna, Austria.
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Wang Y, Liu W, Jiao Y, Yang Y, Shan D, Ji X, Zhang R, Zhan Z, Tang Y, Guo D, Yan C, Liu F. Advances in the Differentiation of hiPSCs into Cerebellar Neuronal Cells. Stem Cell Rev Rep 2024; 20:1782-1794. [PMID: 39023738 DOI: 10.1007/s12015-024-10763-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/10/2024] [Indexed: 07/20/2024]
Abstract
The cerebellum has historically been primarily associated with the regulation of precise motor functions. However, recent findings suggest that it also plays a pivotal role in the development of advanced cognitive functions, including learning, memory, and emotion regulation. Pathological changes in the cerebellum, whether congenital hereditary or acquired degenerative, can result in a diverse spectrum of disorders, ranging from genetic spinocerebellar ataxias to psychiatric conditions such as autism, and schizophrenia. While studies in animal models have significantly contributed to our understanding of the genetic networks governing cerebellar development, it is important to note that the human cerebellum follows a protracted developmental timeline compared to the neocortex. Consequently, employing animal models to uncover human-specific molecular events in cerebellar development presents significant challenges. The emergence of human induced pluripotent stem cells (hiPSCs) has provided an invaluable tool for creating human-based culture systems, enabling the modeling and analysis of cerebellar physiology and pathology. hiPSCs and their differentiated progenies can be derived from patients with specific disorders or carrying distinct genetic variants. Importantly, they preserve the unique genetic signatures of the individuals from whom they originate, allowing for the elucidation of human-specific molecular and cellular processes involved in cerebellar development and related disorders. This review focuses on the technical advancements in the utilization of hiPSCs for the generation of both 2D cerebellar neuronal cells and 3D cerebellar organoids.
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Affiliation(s)
- Yingxin Wang
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Wenzhu Liu
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Yichang Jiao
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Yitong Yang
- School of Nursing, Jining Medical University, Jining, Shandong, 272067, China
| | - Didi Shan
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Xinbo Ji
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Rui Zhang
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Zexin Zhan
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Yao Tang
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Dandan Guo
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China
| | - Chuanzhu Yan
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China.
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China.
- Brain Science Research Institute, Shandong University, Jinan, Shandong, 250012, China.
- Mitochondrial Medicine Laboratory, Qilu Hospital (Qingdao), Shandong University, Qingdao, 266103, China.
| | - Fuchen Liu
- Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China.
- Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 West Wenhua Road, Jinan, Shandong, 250012, China.
- Brain Science Research Institute, Shandong University, Jinan, Shandong, 250012, China.
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Alonso-Olivares H, Marques MM, Prieto-Colomina A, López-Ferreras L, Martínez-García N, Vázquez-Jiménez A, Borrell V, Marin MC, Fernandez-Alonso R. Mouse cortical organoids reveal key functions of p73 isoforms: TAp73 governs the establishment of the archetypical ventricular-like zones while DNp73 is central in the regulation of neural cell fate. Front Cell Dev Biol 2024; 12:1464932. [PMID: 39376628 PMCID: PMC11456701 DOI: 10.3389/fcell.2024.1464932] [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: 07/15/2024] [Accepted: 09/04/2024] [Indexed: 10/09/2024] Open
Abstract
Introduction Neurogenesis is tightly regulated in space and time, ensuring the correct development and organization of the central nervous system. Critical regulators of brain development and morphogenesis in mice include two members of the p53 family: p53 and p73. However, dissecting the in vivo functions of these factors and their various isoforms in brain development is challenging due to their pleiotropic effects. Understanding their role, particularly in neurogenesis and brain morphogenesis, requires innovative experimental approaches. Methods To address these challenges, we developed an efficient and highly reproducible protocol to generate mouse brain organoids from pluripotent stem cells. These organoids contain neural progenitors and neurons that self-organize into rosette-like structures resembling the ventricular zone of the embryonic forebrain. Using this model, we generated organoids from p73-deficient mouse cells to investigate the roles of p73 and its isoforms (TA and DNp73) during brain development. Results and Discussion Organoids derived from p73-deficient cells exhibited increased neuronal apoptosis and reduced neural progenitor proliferation, linked to compensatory activation of p53. This closely mirrors previous in vivo observations, confirming that p73 plays a pivotal role in brain development. Further dissection of p73 isoforms function revealed a dual role of p73 in regulating brain morphogenesis, whereby TAp73 controls transcriptional programs essential for the establishment of the neurogenic niche structure, while DNp73 is responsible for the precise and timely regulation of neural cell fate. These findings highlight the distinct roles of p73 isoforms in maintaining the balance of neural progenitor cell biology, providing a new understanding of how p73 regulates brain morphogenesis.
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Affiliation(s)
- Hugo Alonso-Olivares
- Instituto de Biomedicina and Departamento de Biología Molecular, Universidad de León, León, Spain
| | - Margarita M. Marques
- Instituto de Desarrollo Ganadero y Sanidad Animal and Departamento de Producción Animal, Universidad de León, León, Spain
| | - Anna Prieto-Colomina
- Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Alicante, Spain
| | - Lorena López-Ferreras
- Instituto de Biomedicina and Departamento de Biología Molecular, Universidad de León, León, Spain
| | - Nicole Martínez-García
- Instituto de Biomedicina and Departamento de Producción Animal, Universidad de León, León, Spain
| | - Alberto Vázquez-Jiménez
- Instituto de Biomedicina and Departamento de Biología Molecular, Universidad de León, León, Spain
| | - Victor Borrell
- Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Alicante, Spain
| | - Maria C. Marin
- Instituto de Biomedicina and Departamento de Biología Molecular, Universidad de León, León, Spain
| | - Rosalia Fernandez-Alonso
- Instituto de Biomedicina and Departamento de Biología Molecular, Universidad de León, León, Spain
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Campos RC, Matsunaga K, Reid MW, Fernandez GE, Stepanian K, Bharathan SP, Li M, Thornton ME, Grubbs BH, Nagiel A. Non-canonical Wnt pathway expression in the developing mouse and human retina. Exp Eye Res 2024; 244:109947. [PMID: 38815793 PMCID: PMC11179970 DOI: 10.1016/j.exer.2024.109947] [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: 11/21/2023] [Revised: 05/13/2024] [Accepted: 05/27/2024] [Indexed: 06/01/2024]
Abstract
The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.
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Affiliation(s)
- Rosanna C Campos
- The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA; Department of Development, Stem Cells and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Kate Matsunaga
- Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Mark W Reid
- The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - G Esteban Fernandez
- The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - Kayla Stepanian
- The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - Sumitha P Bharathan
- The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA; The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - Meng Li
- USC Libraries Bioinformatics Services, University of Southern California, Los Angeles, CA, USA
| | - Matthew E Thornton
- Maternal-Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Brendan H Grubbs
- Maternal-Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Aaron Nagiel
- The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA; The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, USA; Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
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8
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Leal H, Carvalhas-Almeida C, Álvaro AR, Cavadas C. Modeling hypothalamic pathophysiology in vitro for metabolic, circadian, and sleep disorders. Trends Endocrinol Metab 2024; 35:505-517. [PMID: 38307813 DOI: 10.1016/j.tem.2024.01.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Revised: 01/09/2024] [Accepted: 01/10/2024] [Indexed: 02/04/2024]
Abstract
The hypothalamus, a small and intricate brain structure, orchestrates numerous neuroendocrine functions through specialized neurons and nuclei. Disruption of this complex circuitry can result in various diseases, including metabolic, circadian, and sleep disorders. Advances in in vitro models and their integration with new technologies have significantly benefited research on hypothalamic function and pathophysiology. We explore existing in vitro hypothalamic models and address their challenges and limitations as well as translational findings. We also highlight how collaborative efforts among multidisciplinary teams are essential to develop relevant and translational experimental models capable of replicating intricate neural circuits and neuroendocrine pathways, thereby advancing our understanding of therapeutic targets and drug discovery in hypothalamus-related disorders.
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Affiliation(s)
- Helena Leal
- Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; Center for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
| | - Catarina Carvalhas-Almeida
- Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; Center for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
| | - Ana Rita Álvaro
- Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; Center for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal
| | - Cláudia Cavadas
- Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; Center for Innovation in Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
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Sanaki-Matsumiya M, Villava C, Rappez L, Haase K, Wu J, Ebisuya M. Self-organization of vascularized skeletal muscle from bovine embryonic stem cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.22.586252. [PMID: 38585777 PMCID: PMC10996461 DOI: 10.1101/2024.03.22.586252] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2024]
Abstract
Cultured beef holds promising potential as an alternative to traditional meat options. While adult stem cells are commonly used as the cell source for cultured beef, their proliferation and differentiation capacities are limited. To produce cultured beef steaks, current manufacturing plans often require the separate preparation of multiple cell types and intricate engineering for assembling them into structured tissues. In this study, we propose and report the co-induction of skeletal muscle, neuronal, and endothelial cells from bovine embryonic stem cells (ESCs) and the self-organization of tissue structures in 2- and 3-dimensional cultures. Bovine myocytes were induced in a stepwise manner through the induction of presomitic mesoderm (PSM) from bovine ESCs. Muscle fibers with sarcomeres appeared within 15 days, displaying calcium oscillations responsive to inputs from co-induced bovine spinal neurons. Bovine endothelial cells were also co-induced via PSM, forming uniform vessel networks inside tissues. Our serum-free, rapid co-induction protocols represent a milestone toward self-organizing beef steaks with integrated vasculature and innervation.
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Affiliation(s)
- Marina Sanaki-Matsumiya
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Casandra Villava
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Luca Rappez
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Kristina Haase
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Miki Ebisuya
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
- Cluster of Excellence Physics of Life, TU Dresden, Dresden, Germany
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10
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Miwata T, Suga H, Mitsumoto K, Zhang J, Hamada Y, Sakakibara M, Soen M, Ozaki H, Asano T, Miyata T, Kawaguchi Y, Yasuda Y, Kobayashi T, Sugiyama M, Onoue T, Hagiwara D, Iwama S, Oyadomari S, Arima H. Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells. Peptides 2024; 173:171151. [PMID: 38215943 DOI: 10.1016/j.peptides.2024.171151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 12/17/2023] [Accepted: 01/08/2024] [Indexed: 01/14/2024]
Abstract
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.
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Affiliation(s)
- Tsutomu Miwata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
| | - Kazuki Mitsumoto
- Department of Endocrinology and Diabetes, Gifu Prefectural Tajimi Hospital, Tajimi, Japan
| | - Jun Zhang
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Yoshimasa Hamada
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Mayu Sakakibara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mika Soen
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hajime Ozaki
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoyoshi Asano
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Takashi Miyata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yohei Kawaguchi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yoshinori Yasuda
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoko Kobayashi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mariko Sugiyama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Takeshi Onoue
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Daisuke Hagiwara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Shintaro Iwama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Seiichi Oyadomari
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Hiroshi Arima
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
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11
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Damiani D, Baggiani M, Della Vecchia S, Naef V, Santorelli FM. Pluripotent Stem Cells as a Preclinical Cellular Model for Studying Hereditary Spastic Paraplegias. Int J Mol Sci 2024; 25:2615. [PMID: 38473862 DOI: 10.3390/ijms25052615] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Revised: 02/12/2024] [Accepted: 02/20/2024] [Indexed: 03/14/2024] Open
Abstract
Hereditary spastic paraplegias (HSPs) comprise a family of degenerative diseases mostly hitting descending axons of corticospinal neurons. Depending on the gene and mutation involved, the disease could present as a pure form with limb spasticity, or a complex form associated with cerebellar and/or cortical signs such as ataxia, dysarthria, epilepsy, and intellectual disability. The progressive nature of HSPs invariably leads patients to require walking canes or wheelchairs over time. Despite several attempts to ameliorate the life quality of patients that have been tested, current therapeutical approaches are just symptomatic, as no cure is available. Progress in research in the last two decades has identified a vast number of genes involved in HSP etiology, using cellular and animal models generated on purpose. Although unanimously considered invaluable tools for basic research, those systems are rarely predictive for the establishment of a therapeutic approach. The advent of induced pluripotent stem (iPS) cells allowed instead the direct study of morphological and molecular properties of the patient's affected neurons generated upon in vitro differentiation. In this review, we revisited all the present literature recently published regarding the use of iPS cells to differentiate HSP patient-specific neurons. Most studies have defined patient-derived neurons as a reliable model to faithfully mimic HSP in vitro, discovering original findings through immunological and -omics approaches, and providing a platform to screen novel or repurposed drugs. Thereby, one of the biggest hopes of current HSP research regards the use of patient-derived iPS cells to expand basic knowledge on the disease, while simultaneously establishing new therapeutic treatments for both generalized and personalized approaches in daily medical practice.
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Affiliation(s)
- Devid Damiani
- Molecular Medicine for Neurodegenerative and Neuromuscular Diseases Unit, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56128 Pisa, Italy
| | - Matteo Baggiani
- Molecular Medicine for Neurodegenerative and Neuromuscular Diseases Unit, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56128 Pisa, Italy
| | - Stefania Della Vecchia
- Molecular Medicine for Neurodegenerative and Neuromuscular Diseases Unit, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56128 Pisa, Italy
- Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy
| | - Valentina Naef
- Molecular Medicine for Neurodegenerative and Neuromuscular Diseases Unit, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56128 Pisa, Italy
| | - Filippo Maria Santorelli
- Molecular Medicine for Neurodegenerative and Neuromuscular Diseases Unit, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56128 Pisa, Italy
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12
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Atamian A, Birtele M, Hosseini N, Nguyen T, Seth A, Del Dosso A, Paul S, Tedeschi N, Taylor R, Coba MP, Samarasinghe R, Lois C, Quadrato G. Human cerebellar organoids with functional Purkinje cells. Cell Stem Cell 2024; 31:39-51.e6. [PMID: 38181749 PMCID: PMC11417151 DOI: 10.1016/j.stem.2023.11.013] [Citation(s) in RCA: 30] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Revised: 05/30/2023] [Accepted: 11/30/2023] [Indexed: 01/07/2024]
Abstract
Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.
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Affiliation(s)
- Alexander Atamian
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Marcella Birtele
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Negar Hosseini
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Tuan Nguyen
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Anoothi Seth
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Ashley Del Dosso
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Sandeep Paul
- Spatial Genomics, 145 Vista Avenue Suite 111, Pasadena, CA 91107, USA
| | - Neil Tedeschi
- Spatial Genomics, 145 Vista Avenue Suite 111, Pasadena, CA 91107, USA
| | - Ryan Taylor
- Spatial Genomics, 145 Vista Avenue Suite 111, Pasadena, CA 91107, USA
| | - Marcelo P Coba
- Department of Psychiatry and Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA 90033, USA
| | - Ranmal Samarasinghe
- Department of Clinical Neurophysiology and Neurology, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Carlos Lois
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Giorgia Quadrato
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
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13
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Zhang S, Xu G, Wu J, Liu X, Fan Y, Chen J, Wallace G, Gu Q. Microphysiological Constructs and Systems: Biofabrication Tactics, Biomimetic Evaluation Approaches, and Biomedical Applications. SMALL METHODS 2024; 8:e2300685. [PMID: 37798902 DOI: 10.1002/smtd.202300685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 08/23/2023] [Indexed: 10/07/2023]
Abstract
In recent decades, microphysiological constructs and systems (MPCs and MPSs) have undergone significant development, ranging from self-organized organoids to high-throughput organ-on-a-chip platforms. Advances in biomaterials, bioinks, 3D bioprinting, micro/nanofabrication, and sensor technologies have contributed to diverse and innovative biofabrication tactics. MPCs and MPSs, particularly tissue chips relevant to absorption, distribution, metabolism, excretion, and toxicity, have demonstrated potential as precise, efficient, and economical alternatives to animal models for drug discovery and personalized medicine. However, current approaches mainly focus on the in vitro recapitulation of the human anatomical structure and physiological-biochemical indices at a single or a few simple levels. This review highlights the recent remarkable progress in MPC and MPS models and their applications. The challenges that must be addressed to assess the reliability, quantify the techniques, and utilize the fidelity of the models are also discussed.
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Affiliation(s)
- Shuyu Zhang
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China
| | - Guoshi Xu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Chaoyang District, Beijing, 100101, China
- University of Chinese Academy of Sciences, Huairou District, Beijing, 100049, China
| | - Juan Wu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Chaoyang District, Beijing, 100101, China
- University of Chinese Academy of Sciences, Huairou District, Beijing, 100049, China
| | - Xiao Liu
- Department of Gastroenterology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Yong Fan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine/Department of Fetal Medicine and Prenatal Diagnosis/BioResource Research Center, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China
| | - Jun Chen
- Intelligent Polymer Research Institute, Australian Institute for Innovative Materials, Innovation Campus, University of Wollongong, North Wollongong, NSW, 2500, Australia
| | - Gordon Wallace
- Intelligent Polymer Research Institute, Australian Institute for Innovative Materials, Innovation Campus, University of Wollongong, North Wollongong, NSW, 2500, Australia
| | - Qi Gu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Chaoyang District, Beijing, 100101, China
- University of Chinese Academy of Sciences, Huairou District, Beijing, 100049, China
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14
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Kamei T, Tamada A, Kimura T, Kakizuka A, Asai A, Muguruma K. Survival and process outgrowth of human iPSC-derived cells expressing Purkinje cell markers in a mouse model for spinocerebellar degenerative disease. Exp Neurol 2023; 369:114511. [PMID: 37634697 DOI: 10.1016/j.expneurol.2023.114511] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 08/11/2023] [Accepted: 08/23/2023] [Indexed: 08/29/2023]
Abstract
Purkinje cells are the sole output neurons of the cerebellar cortex and play central roles in the integration of cerebellum-related motor coordination and memory. The loss or dysfunction of Purkinje cells due to cerebellar atrophy leads to severe ataxia. Here we used in vivo transplantation to examine the function of human iPS cell-derived cerebellar progenitors in adult transgenic mice in which Purkinje-specific cell death occurs due to cytotoxicity of polyglutamines. Transplantation using cerebellar organoids (42-48 days in culture), which are rich in neural progenitors, showed a viability of >50% 4 weeks after transplantation. STEM121+ grafted cells extended their processes toward the deep cerebellar nuclei, superior cerebellar peduncle, and vestibulocerebellar nuclei. The transplanted cells were mostly located in the white matter, and they were not found in the Purkinje cell layer. MAP2-positive fibers seen in the molecular layer of cerebellar cortex received VGluT2 inputs from climbing fibers. Transplanted neural progenitors overgrew in the host cerebellum but were suppressed by pretreatment with the γ-secretase inhibitor DAPT. Hyperproliferation was also suppressed by transplantation with more differentiated organoids (86 days in culture) or KIRREL2-positive cells purified by FACS sorting. Transplanted cells expressed Purkinje cell markers, GABA, CALB1 and L7, though they did not show fan-shaped morphology. We attempted to improve neuronal integration of stem cell-derived cerebellar progenitors by transplantation into the adult mouse, but this was not successfully achieved. Our findings in the present study contribute to regenerative medical application for cerebellar degeneration and provide new insights into cerebellar development in future.
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Affiliation(s)
- Takamasa Kamei
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan; Department of Neurosurgery, Kansai Medical University, Osaka, Japan
| | - Atsushi Tamada
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan
| | - Toshiya Kimura
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan
| | - Akira Kakizuka
- Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Akio Asai
- Department of Neurosurgery, Kansai Medical University, Osaka, Japan
| | - Keiko Muguruma
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan; Laboratory for Lung Development and Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe 650-0047, Japan.
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15
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Ong ALC, Kokaji T, Kishi A, Takihara Y, Shinozuka T, Shimamoto R, Isotani A, Shirai M, Sasai N. Acquisition of neural fate by combination of BMP blockade and chromatin modification. iScience 2023; 26:107887. [PMID: 37771660 PMCID: PMC10522999 DOI: 10.1016/j.isci.2023.107887] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 08/07/2023] [Accepted: 09/07/2023] [Indexed: 09/30/2023] Open
Abstract
Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.
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Affiliation(s)
- Agnes Lee Chen Ong
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Toshiya Kokaji
- Data-driven biology, NAIST Data Science Center, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Arisa Kishi
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Yoshihiro Takihara
- Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-0037, Japan
| | - Takuma Shinozuka
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Ren Shimamoto
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Ayako Isotani
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
| | - Manabu Shirai
- Omics Research Center (ORC), National Cerebral and Cardiovascular Center, 6-1 Kishibe Shinmachi, Suita, Osaka 564-8565, Japan
| | - Noriaki Sasai
- Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan
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16
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Arakawa M, Sakamoto Y, Miyagawa Y, Nito C, Takahashi S, Nitahara-Kasahara Y, Suda S, Yamazaki Y, Sakai M, Kimura K, Okada T. iPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress. Mol Ther Methods Clin Dev 2023; 30:333-349. [PMID: 37637385 PMCID: PMC10448333 DOI: 10.1016/j.omtm.2023.07.005] [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: 07/21/2022] [Accepted: 07/11/2023] [Indexed: 08/29/2023]
Abstract
Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.
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Affiliation(s)
- Masafumi Arakawa
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yuki Sakamoto
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yoshitaka Miyagawa
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Chikako Nito
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Laboratory for Clinical Research, Collaborative Research Center, Nippon Medical School, Tokyo, Japan
| | - Shiro Takahashi
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yuko Nitahara-Kasahara
- Division of Molecular and Medical Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Satoshi Suda
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yoshiyuki Yamazaki
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Mashito Sakai
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Kazumi Kimura
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Takashi Okada
- Division of Molecular and Medical Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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17
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Jeon S, Lee YS, Oh SR, Jeong J, Lee DH, So KH, Hwang NS. Recent advances in endocrine organoids for therapeutic application. Adv Drug Deliv Rev 2023; 199:114959. [PMID: 37301512 DOI: 10.1016/j.addr.2023.114959] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2023] [Revised: 05/21/2023] [Accepted: 06/05/2023] [Indexed: 06/12/2023]
Abstract
The endocrine system, consisting of the hypothalamus, pituitary, endocrine glands, and hormones, plays a critical role in hormone metabolic interactions. The complexity of the endocrine system is a significant obstacle to understanding and treating endocrine disorders. Notably, advances in endocrine organoid generation allow a deeper understanding of the endocrine system by providing better comprehension of molecular mechanisms of pathogenesis. Here, we highlight recent advances in endocrine organoids for a wide range of therapeutic applications, from cell transplantation therapy to drug toxicity screening, combined with development in stem cell differentiation and gene editing technologies. In particular, we provide insights into the transplantation of endocrine organoids to reverse endocrine dysfunctions and progress in developing strategies for better engraftments. We also discuss the gap between preclinical and clinical research. Finally, we provide future perspectives for research on endocrine organoids for the development of more effective treatments for endocrine disorders.
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Affiliation(s)
- Suwan Jeon
- Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
| | - Young-Sun Lee
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea
| | - Seh Ri Oh
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea
| | - Jinseong Jeong
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea
| | - Dong-Hyun Lee
- Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
| | - Kyoung-Ha So
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea; Bio-MAX/N-Bio Institute, Institute of Bio-Engineering, Seoul National University, Seoul 08826, Republic of Korea.
| | - Nathaniel S Hwang
- Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 08826, Republic of Korea; School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea; Bio-MAX/N-Bio Institute, Institute of Bio-Engineering, Seoul National University, Seoul 08826, Republic of Korea; Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea.
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18
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Owen LJ, Rainger J, Bengani H, Kilanowski F, FitzPatrick DR, Papanastasiou AS. Characterization of an eye field-like state during optic vesicle organoid development. Development 2023; 150:dev201432. [PMID: 37306293 PMCID: PMC10445745 DOI: 10.1242/dev.201432] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Accepted: 06/02/2023] [Indexed: 06/13/2023]
Abstract
Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors. This crucial event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate. Using optic vesicle organoids to model the onset of the EF, we generate time-course transcriptomic data allowing us to identify dynamic gene expression programmes that characterize this cellular-state transition. Integrating this with chromatin accessibility data suggests a direct role of canonical EF transcription factors in regulating these gene expression changes, and highlights candidate cis-regulatory elements through which these transcription factors act. Finally, we begin to test a subset of these candidate enhancer elements, within the organoid system, by perturbing the underlying DNA sequence and measuring transcriptomic changes during EF activation.
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Affiliation(s)
- Liusaidh J. Owen
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Jacqueline Rainger
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Hemant Bengani
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Fiona Kilanowski
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - David R. FitzPatrick
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Andrew S. Papanastasiou
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
- School of Informatics, University of Edinburgh, Edinburgh EH8 9AB, UK
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19
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Lázaro J, Costanzo M, Sanaki-Matsumiya M, Girardot C, Hayashi M, Hayashi K, Diecke S, Hildebrandt TB, Lazzari G, Wu J, Petkov S, Behr R, Trivedi V, Matsuda M, Ebisuya M. A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals. Cell Stem Cell 2023; 30:938-949.e7. [PMID: 37343565 PMCID: PMC10321541 DOI: 10.1016/j.stem.2023.05.014] [Citation(s) in RCA: 38] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 04/03/2023] [Accepted: 05/25/2023] [Indexed: 06/23/2023]
Abstract
Differential speeds in biochemical reactions have been proposed to be responsible for the differences in developmental tempo between mice and humans. However, the underlying mechanism controlling the species-specific kinetics remains to be determined. Using in vitro differentiation of pluripotent stem cells, we recapitulated the segmentation clocks of diverse mammalian species varying in body weight and taxa: marmoset, rabbit, cattle, and rhinoceros. Together with mouse and human, the segmentation clock periods of the six species did not scale with the animal body weight, but with the embryogenesis length. The biochemical kinetics of the core clock gene HES7 displayed clear scaling with the species-specific segmentation clock period. However, the cellular metabolic rates did not show an evident correlation. Instead, genes involving biochemical reactions showed an expression pattern that scales with the segmentation clock period. Altogether, our stem cell zoo uncovered general scaling laws governing species-specific developmental tempo.
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Affiliation(s)
- Jorge Lázaro
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain; Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany
| | - Maria Costanzo
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Marina Sanaki-Matsumiya
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Charles Girardot
- European Molecular Biology Laboratory, Genome Biology Unit, 69117 Heidelberg, Germany
| | - Masafumi Hayashi
- Department of Genome Biology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Osaka, Japan
| | - Katsuhiko Hayashi
- Department of Genome Biology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Osaka, Japan
| | - Sebastian Diecke
- Technology Platform Pluripotent Stem Cells, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany
| | | | | | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
| | - Stoyan Petkov
- Platform Degenerative Diseases, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Partner site Göttingen, Göttingen, Germany
| | - Rüdiger Behr
- Platform Degenerative Diseases, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Partner site Göttingen, Göttingen, Germany
| | - Vikas Trivedi
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain.
| | - Mitsuhiro Matsuda
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain.
| | - Miki Ebisuya
- European Molecular Biology Laboratory (EMBL) Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain; Cluster of Excellence Physics of Life, TU Dresden, Dresden, Germany.
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20
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D'Antoni C, Mautone L, Sanchini C, Tondo L, Grassmann G, Cidonio G, Bezzi P, Cordella F, Di Angelantonio S. Unlocking Neural Function with 3D In Vitro Models: A Technical Review of Self-Assembled, Guided, and Bioprinted Brain Organoids and Their Applications in the Study of Neurodevelopmental and Neurodegenerative Disorders. Int J Mol Sci 2023; 24:10762. [PMID: 37445940 DOI: 10.3390/ijms241310762] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 06/18/2023] [Accepted: 06/21/2023] [Indexed: 07/15/2023] Open
Abstract
Understanding the complexities of the human brain and its associated disorders poses a significant challenge in neuroscience. Traditional research methods have limitations in replicating its intricacies, necessitating the development of in vitro models that can simulate its structure and function. Three-dimensional in vitro models, including organoids, cerebral organoids, bioprinted brain models, and functionalized brain organoids, offer promising platforms for studying human brain development, physiology, and disease. These models accurately replicate key aspects of human brain anatomy, gene expression, and cellular behavior, enabling drug discovery and toxicology studies while providing insights into human-specific phenomena not easily studied in animal models. The use of human-induced pluripotent stem cells has revolutionized the generation of 3D brain structures, with various techniques developed to generate specific brain regions. These advancements facilitate the study of brain structure development and function, overcoming previous limitations due to the scarcity of human brain samples. This technical review provides an overview of current 3D in vitro models of the human cortex, their development, characterization, and limitations, and explores the state of the art and future directions in the field, with a specific focus on their applications in studying neurodevelopmental and neurodegenerative disorders.
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Affiliation(s)
- Chiara D'Antoni
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Lorenza Mautone
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Caterina Sanchini
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Lucrezia Tondo
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Greta Grassmann
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
- Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, 00185 Rome, Italy
| | - Gianluca Cidonio
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Paola Bezzi
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Department of Fundamental Neurosciences, University of Lausanne, 1011 Lausanne, Switzerland
| | - Federica Cordella
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Silvia Di Angelantonio
- Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- and Neuro-Science of Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
- D-Tails s.r.l., 00165 Rome, Italy
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21
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Yan YW, Qian ES, Woodard LE, Bejoy J. Neural lineage differentiation of human pluripotent stem cells: Advances in disease modeling. World J Stem Cells 2023; 15:530-547. [PMID: 37424945 PMCID: PMC10324500 DOI: 10.4252/wjsc.v15.i6.530] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 03/14/2023] [Accepted: 04/27/2023] [Indexed: 06/20/2023] Open
Abstract
Brain diseases affect 1 in 6 people worldwide. These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease. Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models, tissue culture models, and epidemiologic patient data that are commonly used to study brain disease. One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells (hPSCs) to neural lineages including neurons, astrocytes, and oligodendrocytes. Three-dimensional models such as brain organoids have also been derived from hPSCs, offering more physiological relevance due to their incorporation of various cell types. As such, brain organoids can better model the pathophysiology of neural diseases observed in patients. In this review, we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.
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Affiliation(s)
- Yuan-Wei Yan
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, United States
| | - Eddie S Qian
- Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN 37232, United States
| | - Lauren E Woodard
- Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, United States
- Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37232, United States
- Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, United States
| | - Julie Bejoy
- Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, United States
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22
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Arioka Y, Okumura H, Sakaguchi H, Ozaki N. Shedding light on latent pathogenesis and pathophysiology of mental disorders: The potential of iPS cell technology. Psychiatry Clin Neurosci 2023; 77:308-314. [PMID: 36929185 PMCID: PMC11488641 DOI: 10.1111/pcn.13545] [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: 10/04/2022] [Revised: 03/04/2023] [Accepted: 03/13/2023] [Indexed: 03/18/2023]
Abstract
Mental disorders are considered as one of the major healthcare issues worldwide owing to their significant impact on the quality of life of patients, causing serious social burdens. However, it is hard to examine the living brain-a source of psychiatric symptoms-at the cellular, subcellular, and molecular levels, which poses difficulty in determining the pathogenesis and pathophysiology of mental disorders. Recently, induced pluripotent stem cell (iPSC) technology has been used as a novel tool for research on mental disorders. We believe that the iPSC-based studies will address the limitations of other research approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis. Notably, studies using integrated iPSC technology with genetic information have provided significant novel findings to date. This review aimed to discuss the history, current trends, potential, and future of iPSC technology in the field of mental disorders. Although iPSC technology has several limitations, this technology can be used in combination with the other approaches to facilitate studies on mental disorders.
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Affiliation(s)
- Yuko Arioka
- Pathophysiology of Mental DisordersNagoya University Graduate School of MedicineNagoyaJapan
- Center for Advanced Medicine and Clinical ResearchNagoya University HospitalNagoyaJapan
| | - Hiroki Okumura
- Pathophysiology of Mental DisordersNagoya University Graduate School of MedicineNagoyaJapan
- Hospital PharmacyNagoya University HospitalNagoyaJapan
| | - Hideya Sakaguchi
- BDR‐Otsuka Pharmaceutical Collaboration Center, RIKEN Center for Biosystems Dynamics ResearchKobeJapan
| | - Norio Ozaki
- Pathophysiology of Mental DisordersNagoya University Graduate School of MedicineNagoyaJapan
- Institute for Glyco‐core Research (iGCORE)Nagoya UniversityNagoyaJapan
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23
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Miwata T, Suga H, Kawaguchi Y, Sakakibara M, Kano M, Taga S, Soen M, Ozaki H, Asano T, Sasaki H, Miyata T, Yasuda Y, Kobayashi T, Sugiyama M, Onoue T, Takagi H, Hagiwara D, Iwama S, Arima H. Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells. Stem Cell Reports 2023; 18:869-883. [PMID: 36963388 PMCID: PMC10147555 DOI: 10.1016/j.stemcr.2023.02.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Revised: 02/19/2023] [Accepted: 02/21/2023] [Indexed: 03/26/2023] Open
Abstract
When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.
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Affiliation(s)
- Tsutomu Miwata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
| | - Yohei Kawaguchi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mayu Sakakibara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mayuko Kano
- Division of Metabolism and Endocrinology, Department of Internal Medicine, St. Marianna University School of Medicine, Kanagawa, Japan
| | - Shiori Taga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan; Regenerative & Cellular Medicine Kobe Center, Sumitomo Pharma Co., Ltd., Kobe, Japan
| | - Mika Soen
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hajime Ozaki
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoyoshi Asano
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hiroo Sasaki
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya, Japan
| | - Takashi Miyata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yoshinori Yasuda
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoko Kobayashi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mariko Sugiyama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Takeshi Onoue
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hiroshi Takagi
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Daisuke Hagiwara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Shintaro Iwama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hiroshi Arima
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
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24
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Rouleau N, Murugan NJ, Kaplan DL. Functional bioengineered models of the central nervous system. NATURE REVIEWS BIOENGINEERING 2023; 1:252-270. [PMID: 37064657 PMCID: PMC9903289 DOI: 10.1038/s44222-023-00027-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 01/16/2023] [Indexed: 02/10/2023]
Abstract
The functional complexity of the central nervous system (CNS) is unparalleled in living organisms. Its nested cells, circuits and networks encode memories, move bodies and generate experiences. Neural tissues can be engineered to assemble model systems that recapitulate essential features of the CNS and to investigate neurodevelopment, delineate pathophysiology, improve regeneration and accelerate drug discovery. In this Review, we discuss essential structure-function relationships of the CNS and examine materials and design considerations, including composition, scale, complexity and maturation, of cell biology-based and engineering-based CNS models. We highlight region-specific CNS models that can emulate functions of the cerebral cortex, hippocampus, spinal cord, neural-X interfaces and other regions, and investigate a range of applications for CNS models, including fundamental and clinical research. We conclude with an outlook to future possibilities of CNS models, highlighting the engineering challenges that remain to be overcome.
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Affiliation(s)
- Nicolas Rouleau
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, Ontario Canada
- Department of Biomedical Engineering, Tufts University, Medford, MA USA
| | - Nirosha J. Murugan
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, Ontario Canada
- Department of Biomedical Engineering, Tufts University, Medford, MA USA
| | - David L. Kaplan
- Department of Biomedical Engineering, Tufts University, Medford, MA USA
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25
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Akter M, Ding B. Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons. Cells 2022; 11:3796. [PMID: 36497056 PMCID: PMC9737271 DOI: 10.3390/cells11233796] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Revised: 11/19/2022] [Accepted: 11/24/2022] [Indexed: 11/29/2022] Open
Abstract
Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions.
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Affiliation(s)
| | - Baojin Ding
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
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26
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Kawata M, Kodani Y, Ohkuma M, Miyachi EI, Kaneko YS, Nakashima A, Suga H, Kameyama T, Saito K, Nagasaki H. Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain. PLoS One 2022; 17:e0276694. [PMID: 36356043 PMCID: PMC9648832 DOI: 10.1371/journal.pone.0276694] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Accepted: 10/11/2022] [Indexed: 11/12/2022] Open
Abstract
The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.
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Affiliation(s)
- Miho Kawata
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Yu Kodani
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Mahito Ohkuma
- Department of Physiology II, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Ei-ichi Miyachi
- Department of Physiology II, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
- Department of Health and Nutrition, Faculty of Health and Science, Nagoya Women’s University, Nagoya, Aichi, Japan
| | - Yoko S. Kaneko
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
- Biochemistry and Molecular Cell Biology, Faculty of Pharmacy, Gifu University of Medical Science, Kani, Gifu, Japan
| | - Akira Nakashima
- Department of Physiological Chemistry, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Toshiki Kameyama
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Kanako Saito
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
| | - Hiroshi Nagasaki
- Department of Physiology I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
- * E-mail:
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27
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Cakir B, Kiral FR, Park IH. Advanced in vitro models: Microglia in action. Neuron 2022; 110:3444-3457. [PMID: 36327894 DOI: 10.1016/j.neuron.2022.10.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Revised: 09/28/2022] [Accepted: 10/04/2022] [Indexed: 11/05/2022]
Abstract
In the central nervous system (CNS), microglia carry out multiple tasks related to brain development, maintenance of brain homeostasis, and function of the CNS. Recent advanced in vitro model systems allow us to perform more detailed and specific analyses of microglial functions in the CNS. The development of human pluripotent stem cells (hPSCs)-based 2D and 3D cell culture methods, particularly advancements in brain organoid models, offers a better platform to dissect microglial function in various contexts. Despite the improvement of these methods, there are still definite restrictions. Understanding their drawbacks and benefits ensures their proper use. In this primer, we review current developments regarding in vitro microglial production and characterization and their use to address fundamental questions about microglial function in healthy and diseased states, and we discuss potential future improvements with a particular emphasis on brain organoid models.
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Affiliation(s)
- Bilal Cakir
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, CT 06520, USA.
| | - Ferdi Ridvan Kiral
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, CT 06520, USA
| | - In-Hyun Park
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, CT 06520, USA.
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28
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Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state. Sci Rep 2022; 12:17381. [PMID: 36253431 PMCID: PMC9576732 DOI: 10.1038/s41598-022-22405-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2021] [Accepted: 10/14/2022] [Indexed: 01/10/2023] Open
Abstract
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.
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Medina-Cano D, Corrigan EK, Glenn RA, Islam MT, Lin Y, Kim J, Cho H, Vierbuchen T. Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids. Development 2022; 149:dev200561. [PMID: 35899604 PMCID: PMC10655922 DOI: 10.1242/dev.200561] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Accepted: 07/04/2022] [Indexed: 11/20/2022]
Abstract
Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.
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Affiliation(s)
- Daniel Medina-Cano
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Emily K. Corrigan
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Rachel A. Glenn
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Cell and Developmental Biology Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA
| | - Mohammed T. Islam
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Yuan Lin
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Juliet Kim
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Hyunwoo Cho
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Thomas Vierbuchen
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
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Watanabe M, Buth JE, Haney JR, Vishlaghi N, Turcios F, Elahi LS, Gu W, Pearson CA, Kurdian A, Baliaouri NV, Collier AJ, Miranda OA, Dunn N, Chen D, Sabri S, Torre-Ubieta LDL, Clark AT, Plath K, Christofk HR, Kornblum HI, Gandal MJ, Novitch BG. TGFβ superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids. Stem Cell Reports 2022; 17:2220-2238. [PMID: 36179695 PMCID: PMC9561534 DOI: 10.1016/j.stemcr.2022.08.013] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2022] [Revised: 08/26/2022] [Accepted: 08/27/2022] [Indexed: 12/25/2022] Open
Abstract
Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.
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Affiliation(s)
- Momoko Watanabe
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
| | - Jessie E Buth
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Jillian R Haney
- Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Neda Vishlaghi
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Felix Turcios
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Lubayna S Elahi
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Wen Gu
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Caroline A Pearson
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Arinnae Kurdian
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Natella V Baliaouri
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Amanda J Collier
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Osvaldo A Miranda
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Natassia Dunn
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Di Chen
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Shan Sabri
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Luis de la Torre-Ubieta
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Amander T Clark
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Kathrin Plath
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Heather R Christofk
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Harley I Kornblum
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Michael J Gandal
- Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Bennett G Novitch
- Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
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Disease Modeling of Pituitary Adenoma Using Human Pluripotent Stem Cells. Cancers (Basel) 2022; 14:cancers14153660. [PMID: 35954322 PMCID: PMC9367606 DOI: 10.3390/cancers14153660] [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: 06/25/2022] [Revised: 07/22/2022] [Accepted: 07/23/2022] [Indexed: 11/17/2022] Open
Abstract
Simple Summary Pituitary adenoma pathophysiology has been studied mainly using murine cell lines, animal models, and pituitary tumor samples. However, the lack of human pituitary cell line is a significant limiting factor in studying the molecular mechanisms of human pituitary tumors. Recently, pituitary induction methods from human-induced pluripotent stem cells (hiPSCs) have been established. These methods can induce human pituitary hormone-producing cells that retain physiological properties. hiPSCs in which tumor-causing gene mutations are introduced using genome-editing techniques, such as CRISPR/Cas9 systems, provide great opportunities to establish in vitro human pituitary adenoma disease models. The models will be a novel platform to discover novel drugs and investigate tumorigenesis and pathophysiology. The purpose of this review is to provide an overview of the applications of iPSCs for pituitary and neoplastic disorder research and genome-editing technologies to create strategies for developing pituitary adenoma models using iPSCs. Abstract Pituitary adenomas are characterized by abnormal growth in the pituitary gland. Surgical excision is the first-line treatment for functional (hormone-producing) pituitary adenomas, except for prolactin-producing adenomas; however, complete excision is technically challenging, and many patients require long-term medication after the treatment. In addition, the pathophysiology of pituitary adenomas, such as tumorigenesis, has not been fully understood. Pituitary adenoma pathophysiology has mainly been studied using animal models and animal tumor-derived cell lines. Nevertheless, experimental studies on human pituitary adenomas are difficult because of the significant differences among species and the lack of reliable cell lines. Recently, several methods have been established to differentiate pituitary cells from human pluripotent stem cells (hPSCs). The induced pituitary hormone-producing cells retain the physiological properties already lost in tumor-derived cell lines. Moreover, CRISPR/Cas9 systems have expedited the introduction of causative gene mutations in various malignant tumors into hPSCs. Therefore, hPSC-derived pituitary cells have great potential as a novel platform for studying the pathophysiology of human-specific pituitary adenomas and developing novel drugs. This review presents an overview of the recent progresses in hPSC applications for pituitary research, functional pituitary adenoma pathogenesis, and genome-editing techniques for introducing causative mutations. We also discuss future applications of hPSCs for studying pituitary adenomas.
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Yamamoto M, Ong Lee Chen A, Shinozuka T, Sasai N. The Rx transcription factor is required for determination of the retinal lineage and regulates the timing of neuronal differentiation. Dev Growth Differ 2022; 64:318-324. [PMID: 35700309 DOI: 10.1111/dgd.12796] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2022] [Revised: 04/25/2022] [Accepted: 05/23/2022] [Indexed: 11/30/2022]
Abstract
Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome-wide expression analysis with embryonic stem cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three-dimensional differentiation of wild-type and mutant cells and compared their gene-expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.
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Affiliation(s)
- Maho Yamamoto
- Developmental Biomedical Science, Division of Biological Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, Japan
| | - Agnes Ong Lee Chen
- Developmental Biomedical Science, Division of Biological Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, Japan
| | - Takuma Shinozuka
- Developmental Biomedical Science, Division of Biological Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, Japan
| | - Noriaki Sasai
- Developmental Biomedical Science, Division of Biological Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, Japan
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Croizier S, Bouret SG. Molecular Control of the Development of Hypothalamic Neurons Involved in Metabolic Regulation. J Chem Neuroanat 2022; 123:102117. [DOI: 10.1016/j.jchemneu.2022.102117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 03/03/2022] [Accepted: 06/03/2022] [Indexed: 10/18/2022]
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Tang XY, Wu S, Wang D, Chu C, Hong Y, Tao M, Hu H, Xu M, Guo X, Liu Y. Human organoids in basic research and clinical applications. Signal Transduct Target Ther 2022; 7:168. [PMID: 35610212 PMCID: PMC9127490 DOI: 10.1038/s41392-022-01024-9] [Citation(s) in RCA: 167] [Impact Index Per Article: 55.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2022] [Revised: 04/26/2022] [Accepted: 05/11/2022] [Indexed: 12/12/2022] Open
Abstract
Organoids are three-dimensional (3D) miniature structures cultured in vitro produced from either human pluripotent stem cells (hPSCs) or adult stem cells (AdSCs) derived from healthy individuals or patients that recapitulate the cellular heterogeneity, structure, and functions of human organs. The advent of human 3D organoid systems is now possible to allow remarkably detailed observation of stem cell morphogens, maintenance and differentiation resemble primary tissues, enhancing the potential to study both human physiology and developmental stage. As they are similar to their original organs and carry human genetic information, organoids derived from patient hold great promise for biomedical research and preclinical drug testing and is currently used for personalized, regenerative medicine, gene repair and transplantation therapy. In recent decades, researchers have succeeded in generating various types of organoids mimicking in vivo organs. Herein, we provide an update on current in vitro differentiation technologies of brain, retinal, kidney, liver, lung, gastrointestinal, cardiac, vascularized and multi-lineage organoids, discuss the differences between PSC- and AdSC-derived organoids, summarize the potential applications of stem cell-derived organoids systems in the laboratory and clinic, and outline the current challenges for the application of organoids, which would deepen the understanding of mechanisms of human development and enhance further utility of organoids in basic research and clinical studies.
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Affiliation(s)
- Xiao-Yan Tang
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Shanshan Wu
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Da Wang
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Chu Chu
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Yuan Hong
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Mengdan Tao
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Hao Hu
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Min Xu
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China
| | - Xing Guo
- Department of Neurobiology, School of Basic Medical Sciences; Nanjing Medical University, Nanjing, China.
| | - Yan Liu
- Institute for Stem Cell and Neural Regeneration, School of Pharmacy; State Key Laboratory of Reproductive Medicine; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine; Nanjing Medical University, Nanjing, China.
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NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model. Int J Mol Sci 2022; 23:ijms23063260. [PMID: 35328681 PMCID: PMC8948797 DOI: 10.3390/ijms23063260] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 03/11/2022] [Accepted: 03/14/2022] [Indexed: 12/10/2022] Open
Abstract
Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.
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Miyake N, Nagai T, Suga H, Osuka S, Kasai T, Sakakibara M, Soen M, Ozaki H, Miwata T, Asano T, Kano M, Muraoka A, Nakanishi N, Nakamura T, Goto M, Yasuda Y, Kawaguchi Y, Miyata T, Kobayashi T, Sugiyama M, Onoue T, Hagiwara D, Iwama S, Iwase A, Inoshita N, Arima H, Kajiyama H. Functional Lactotrophs in Induced Adenohypophysis Differentiated From Human iPS Cells. Endocrinology 2022; 163:bqac004. [PMID: 35085394 DOI: 10.1210/endocr/bqac004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Indexed: 11/19/2022]
Abstract
Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.
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Affiliation(s)
- Natsuki Miyake
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Takashi Nagai
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Satoko Osuka
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Takatoshi Kasai
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Mayu Sakakibara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Mika Soen
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Hajime Ozaki
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Tsutomu Miwata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Tomoyoshi Asano
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Mayuko Kano
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Ayako Muraoka
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Natsuki Nakanishi
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Tomoko Nakamura
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Maki Goto
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Yoshinori Yasuda
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Yohei Kawaguchi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Takashi Miyata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Tomoko Kobayashi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Mariko Sugiyama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Takeshi Onoue
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Daisuke Hagiwara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Shintaro Iwama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Akira Iwase
- Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan
| | - Naoko Inoshita
- Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo 173-0015, Japan
| | - Hiroshi Arima
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Hiroaki Kajiyama
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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Characterization of Hypothalamic MCH Neuron Development in a 3D Differentiation System of Mouse Embryonic Stem Cells. eNeuro 2022; 9:ENEURO.0442-21.2022. [PMID: 35437265 PMCID: PMC9047030 DOI: 10.1523/eneuro.0442-21.2022] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 04/05/2022] [Accepted: 04/11/2022] [Indexed: 01/20/2023] Open
Abstract
Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH+CART+ neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH+CART− and MCH+CART+ neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.
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Ou Y, Che M, Peng J, Zhou M, Wu G, Gong H, Li K, Wang X, Niu P, Qi S, Feng Z. An Efficient Method for the Isolation and Cultivation of Hypothalamic Neural Stem/Progenitor Cells From Mouse Embryos. Front Neuroanat 2022; 16:711138. [PMID: 35185481 PMCID: PMC8854184 DOI: 10.3389/fnana.2022.711138] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Accepted: 01/04/2022] [Indexed: 01/01/2023] Open
Abstract
The hypothalamus is the key region that regulates the neuroendocrine system as well as instinct behaviors, and hypothalamic dysfunction causes refractory clinical problems. Recent studies have indicated that neural stem/progenitor cell (NSPC) in the hypothalamus play a crucial role in hypothalamic function. However, specific hypothalamic NSPC culture methods have not been established, especially not detailed or efficient surgical procedures. The present study presented a convenient, detailed and efficient method for the isolation and cultivation of hypothalamic NSPCs from embryonic day 12.5 mice. The procedure includes embryo acquisition, brain microdissection to quickly obtain hypothalamic tissue and hypothalamic NSPC culture. Hypothalamic NSPCs can be quickly harvested and grow well in both neurosphere and adherent cultures through this method. Additionally, we confirmed the cell origin and evaluated the proliferation and differentiation properties of cultured cells. In conclusion, we present a convenient and practical method for the isolation and cultivation of hypothalamic NSPCs that can be used in extensive hypothalamic studies.
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Affiliation(s)
- Yichao Ou
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- The Laboratory for Precision Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Mengjie Che
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Junjie Peng
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Mingfeng Zhou
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Guangsen Wu
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haodong Gong
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- First Medical Institute, Southern Medical University, Guangzhou, China
| | - Kai Li
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xingqin Wang
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Peirong Niu
- First Medical Institute, Southern Medical University, Guangzhou, China
| | - Songtao Qi
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- The Laboratory for Precision Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- *Correspondence: Songtao Qi,
| | - Zhanpeng Feng
- Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- The Laboratory for Precision Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China
- Zhanpeng Feng,
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Zhang H, Bussmann J, Huhnke FH, Devoldere J, Minnaert A, Jiskoot W, Serwane F, Spatz J, Röding M, De Smedt SC, Braeckmans K, Remaut K. Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2102072. [PMID: 34913603 PMCID: PMC8811815 DOI: 10.1002/advs.202102072] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 10/27/2021] [Indexed: 06/14/2023]
Abstract
Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.
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Affiliation(s)
- Heyang Zhang
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
| | - Jeroen Bussmann
- Division of BioTherapeuticsLeiden Academic Center for Drug ResearchLeiden UniversityLeiden2333 CCThe Netherlands
| | - Florian H. Huhnke
- Max Planck Institute for Medical ResearchDepartment of Cellular Biophysics70569StuttgartGermany
| | - Joke Devoldere
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
| | - An‐Katrien Minnaert
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
| | - Wim Jiskoot
- Division of BioTherapeuticsLeiden Academic Center for Drug ResearchLeiden UniversityLeiden2333 CCThe Netherlands
| | - Friedhelm Serwane
- Max Planck Institute for Medical ResearchDepartment of Cellular Biophysics70569StuttgartGermany
- Center for NanoScienceLudwig‐Maximilian‐University MunichD‐80333MunichGermany
- Faculty of PhysicsLudwig‐Maximilian‐UniversityD‐80539MunichGermany
- Munich Cluster for Systems Neurology (SyNergy)D‐81377MunichGermany
| | - Joachim Spatz
- Max Planck Institute for Medical ResearchDepartment of Cellular Biophysics70569StuttgartGermany
- Department of Biophysical ChemistryUniversity of Heidelberg69120HeidelbergGermany
| | - Magnus Röding
- RISE Research Institutes of SwedenBioeconomy and Health, Agriculture and FoodGöteborg41276Sweden
- Department of Mathematical SciencesChalmers University of Technology and University of GothenburgGöteborg41296Sweden
| | - Stefaan C. De Smedt
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
- Cancer Research Institute Ghent (CRIG)Ghent UniversityGhent9000Belgium
| | - Kevin Braeckmans
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
- Center for Advanced Light MicroscopyGhent UniversityGhent9000Belgium
| | - Katrien Remaut
- Laboratory of General Biochemistry and Physical PharmacyFaculty of Pharmaceutical SciencesGhent UniversityGhent9000Belgium
- Cancer Research Institute Ghent (CRIG)Ghent UniversityGhent9000Belgium
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Kano M, Sasaki H, Miwata T, Suga H. Recipe for pituitary organoids. Front Endocrinol (Lausanne) 2022; 13:1025825. [PMID: 36743928 PMCID: PMC9892717 DOI: 10.3389/fendo.2022.1025825] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Accepted: 12/15/2022] [Indexed: 01/20/2023] Open
Abstract
Generation of a variety of organs and tissues from human pluripotent stem cells (hPSCs) has been attempted in vitro. We here present a simple and efficient method for induction of hypothalamic and pituitary tissues from hPSCs. On provision of exogenous agents important for early hypothalamus-pituitary organogenesis, including bone morphogenetic protein 4 and activators of sonic hedgehog, in three-dimensional culture, hPSCs spontaneously form spherical organoids with two distinct tissues, hypothalamus and adenohypophysis. The pituitary tissues derived from hPSCs not only secrete adenocorticotropic hormone, but also retain both positive and negative feedback mechanisms, recapitulating mature endocrine organs in vivo. Furthermore, the results of ectopic transplantation with mouse models of hypopituitarism suggest that these hypothalamus-pituitary organoids have potential as engraftment organs. In addition to their use in transplantation for patients with hypopituitarism they will allow establishment of disease models in vitro and enable research impossible in humans. Hypothalamus-pituitary organoids promise to be a powerful tool in regenerative medicine, drug discovery, and basic research into pituitary development.
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Affiliation(s)
- Mayuko Kano
- Division of Metabolism and Endocrinology, Department of Internal Medicine, St. Marianna University School of Medicine, Kanagawa, Japan
- *Correspondence: Mayuko Kano, ; Hidetaka Suga,
| | - Hiroo Sasaki
- Department of Neurosurgery, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tsutomu Miwata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
- *Correspondence: Mayuko Kano, ; Hidetaka Suga,
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41
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Yokoi R, Shibata M, Odawara A, Ishibashi Y, Nagafuku N, Matsuda N, Suzuki I. Analysis of signal components < 500 Hz in brain organoids coupled to microelectrode arrays: A reliable test-bed for preclinical seizure liability assessment of drugs and screening of antiepileptic drugs. Biochem Biophys Rep 2021; 28:101148. [PMID: 34693037 PMCID: PMC8517166 DOI: 10.1016/j.bbrep.2021.101148] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 10/03/2021] [Accepted: 10/04/2021] [Indexed: 12/25/2022] Open
Abstract
Brain organoids with three-dimensional structure and tissue-like function are highly demanded for brain disease research and drug evaluation. However, to our knowledge, methods for measuring and analyzing brain organoid function have not been developed yet. This study focused on the frequency components of an obtained waveform below 500 Hz using planner microelectrode array (MEA) and evaluated the response to the convulsants pentylenetetrazol (PTZ) and strychnine as well as the antiepileptic drugs (AEDs) perampanel and phenytoin. Sudden and persistent seizure-like firing was observed with PTZ administration, displaying a concentration-dependent periodic activity with the frequency component enhanced even in one oscillation characteristic. On the other hand, in the administration of AEDs, the frequency of oscillation decreased in a concentration-dependent manner and the intensity of the frequency component in one oscillation also decreased. Interestingly, at low doses of phenytoin, a group of synchronized bursts was formed, which was different from the response to the perampanel. Frequency components contained information on cerebral organoid function, and MEA was proven useful in predicting the seizure liability of drugs and evaluating the effect of AEDs with a different mechanism of action. In addition, frequency component analysis of brain organoids using MEA is an important analysis method to perform in vitro to in vivo extrapolation in the future, which will help explore the function of the organoid itself, study human brain developments, and treat various brain diseases.
Frequency analysis <500 Hz was performed in brain organoids coupled to planner microelectrode arrays (MEA). Concentration-dependent changes in frequency components were detected in responses to convulsants and antiepileptic drugs (AEDs). Analysis of signal components <500 Hz in brain organoids is a useful method for preclinical seizure liability assessment of drugs and screening of antiepileptic drugs.
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Guy B, Zhang JS, Duncan LH, Johnston RJ. Human neural organoids: Models for developmental neurobiology and disease. Dev Biol 2021; 478:102-121. [PMID: 34181916 PMCID: PMC8364509 DOI: 10.1016/j.ydbio.2021.06.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 06/08/2021] [Accepted: 06/24/2021] [Indexed: 12/25/2022]
Abstract
Human organoids stand at the forefront of basic and translational research, providing experimentally tractable systems to study human development and disease. These stem cell-derived, in vitro cultures can generate a multitude of tissue and organ types, including distinct brain regions and sensory systems. Neural organoid systems have provided fundamental insights into molecular mechanisms governing cell fate specification and neural circuit assembly and serve as promising tools for drug discovery and understanding disease pathogenesis. In this review, we discuss several human neural organoid systems, how they are generated, advances in 3D imaging and bioengineering, and the impact of organoid studies on our understanding of the human nervous system.
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Affiliation(s)
- Brian Guy
- Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD, 21218, USA
| | - Jingliang Simon Zhang
- Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD, 21218, USA
| | - Leighton H Duncan
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA
| | - Robert J Johnston
- Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD, 21218, USA.
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Huang WK, Wong SZH, Pather SR, Nguyen PTT, Zhang F, Zhang DY, Zhang Z, Lu L, Fang W, Chen L, Fernandes A, Su Y, Song H, Ming GL. Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells. Cell Stem Cell 2021; 28:1657-1670.e10. [PMID: 33961804 PMCID: PMC8419002 DOI: 10.1016/j.stem.2021.04.006] [Citation(s) in RCA: 98] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Revised: 02/21/2021] [Accepted: 04/07/2021] [Indexed: 12/12/2022]
Abstract
Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.
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Affiliation(s)
- Wei-Kai Huang
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Program in Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Samuel Zheng Hao Wong
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Sarshan R Pather
- Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Phuong T T Nguyen
- Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Feng Zhang
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Daniel Y Zhang
- Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Zhijian Zhang
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Lu Lu
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Wanqi Fang
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Luyun Chen
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Analiese Fernandes
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Yijing Su
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Hongjun Song
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Guo-Li Ming
- Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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Bi FC, Yang XH, Cheng XY, Deng WB, Guo XL, Yang H, Wang Y, Li J, Yao Y. Optimization of cerebral organoids: a more qualified model for Alzheimer's disease research. Transl Neurodegener 2021; 10:27. [PMID: 34372927 PMCID: PMC8349709 DOI: 10.1186/s40035-021-00252-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Accepted: 07/17/2021] [Indexed: 12/18/2022] Open
Abstract
Alzheimer's disease (AD) is a neurodegenerative disease that currently cannot be cured by any drug or intervention, due to its complicated pathogenesis. Current animal and cellular models of AD are unable to meet research needs for AD. However, recent three-dimensional (3D) cerebral organoid models derived from human stem cells have provided a new tool to study molecular mechanisms and pharmaceutical developments of AD. In this review, we discuss the advantages and key limitations of the AD cerebral organoid system in comparison to the commonly used AD models, and propose possible solutions, in order to improve their application in AD research. Ethical concerns associated with human cerebral organoids are also discussed. We also summarize future directions of studies that will improve the cerebral organoid system to better model the pathological events observed in AD brains.
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Affiliation(s)
- Feng-Chen Bi
- School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, 750004, China
- Key Laboratory of Traditional Chinese Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China
| | - Xin-He Yang
- School of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China
| | - Xiao-Yu Cheng
- Department of Neurology and Suzhou Clinical Research Center of Neurological Disease, The Second Affiliated Hospital, Soochow University, Suzhou, 215004, China
| | - Wen-Bin Deng
- School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, 510275, China
| | - Xiao-Li Guo
- School of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China
| | - Hui Yang
- Research Center of Medical Science and Technology, Ningxia Medical University, Yinchuan, 750004, China
| | - Yin Wang
- School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, 750004, China.
| | - Juan Li
- Key Laboratory of Traditional Chinese Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China.
- School of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China.
| | - Yao Yao
- School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, 750004, China.
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Takenaka-Ninagawa N, Kim J, Zhao M, Sato M, Jonouchi T, Goto M, Yoshioka CKB, Ikeda R, Harada A, Sato T, Ikeya M, Uezumi A, Nakatani M, Noguchi S, Sakurai H. Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice. Stem Cell Res Ther 2021; 12:446. [PMID: 34372931 PMCID: PMC8351132 DOI: 10.1186/s13287-021-02514-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 07/13/2021] [Indexed: 11/10/2022] Open
Abstract
Background Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. Methods To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). Results All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. Conclusions These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02514-3.
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Affiliation(s)
- Nana Takenaka-Ninagawa
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
| | - Jinsol Kim
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Mingming Zhao
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Masae Sato
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Tatsuya Jonouchi
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Megumi Goto
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Clémence Kiho Bourgeois Yoshioka
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Rukia Ikeda
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Aya Harada
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Takahiko Sato
- Department of Anatomy, Fujita Health University, Toyoake, Aichi, 470-1192, Japan
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Akiyoshi Uezumi
- Muscle Aging and Regenerative Medicine, Research Team for Geriatric Medicine, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Masashi Nakatani
- Division for Therapies against Intractable Diseases, Institute for Comprehensive Medical Science (ICMS), Fujita Health University, Toyoake, Aichi, 470-1192, Japan
| | - Satoru Noguchi
- Department of Neuromuscular Research, National Institute of Neuroscience, Department of Clinical Development, Translational Medical Center, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8551, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
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Jalink P, Caiazzo M. Brain Organoids: Filling the Need for a Human Model of Neurological Disorder. BIOLOGY 2021; 10:740. [PMID: 34439972 PMCID: PMC8389592 DOI: 10.3390/biology10080740] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/05/2021] [Revised: 07/25/2021] [Accepted: 07/26/2021] [Indexed: 02/06/2023]
Abstract
Neurological disorders are among the leading causes of death worldwide, accounting for almost all onsets of dementia in the elderly, and are known to negatively affect motor ability, mental and cognitive performance, as well as overall wellbeing and happiness. Currently, most neurological disorders go untreated due to a lack of viable treatment options. The reason for this lack of options is s poor understanding of the disorders, primarily due to research models that do not translate well into the human in vivo system. Current models for researching neurological disorders, neurodevelopment, and drug interactions in the central nervous system include in vitro monolayer cell cultures, and in vivo animal models. These models have shortcomings when it comes to translating research about disorder pathology, development, and treatment to humans. Brain organoids are three-dimensional (3D) cultures of stem cell-derived neural cells that mimic the development of the in vivo human brain with high degrees of accuracy. Researchers have started developing these miniature brains to model neurodevelopment, and neuropathology. Brain organoids have been used to model a wide range of neurological disorders, including the complex and poorly understood neurodevelopmental and neurodegenerative disorders. In this review, we discuss the brain organoid technology, placing special focus on the different brain organoid models that have been developed, discussing their strengths, weaknesses, and uses in neurological disease modeling.
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Affiliation(s)
- Philip Jalink
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Universiteitsweg 99, CG 3584 Utrecht, The Netherlands;
| | - Massimiliano Caiazzo
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Universiteitsweg 99, CG 3584 Utrecht, The Netherlands;
- Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy
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Lv T, Meng F, Yu M, Huang H, Lin X, Zhao B. Defense of COVID-19 by Human Organoids. PHENOMICS (CHAM, SWITZERLAND) 2021; 1:113-128. [PMID: 35233559 PMCID: PMC8277987 DOI: 10.1007/s43657-021-00015-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/27/2021] [Revised: 05/08/2021] [Accepted: 05/11/2021] [Indexed: 01/08/2023]
Abstract
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has created an immense menace to public health worldwide, exerting huge effects on global economic and political conditions. Understanding the biology and pathogenesis mechanisms of this novel virus, in large parts, relies on optimal physiological models that allow replication and propagation of SARS-CoV-2. Human organoids, derived from stem cells, are three-dimensional cell cultures that recapitulate the cellular organization, transcriptional and epigenetic signatures of their counterpart organs. Recent studies have indicated their great values as experimental virology platforms, making human organoid an ideal tool for investigating host-pathogen interactions. Here, we summarize research developments for SARS-CoV-2 infection of various human organoids involved in multiple systems, including lung, liver, brain, intestine, kidney and blood vessel organoids. These studies help us reveal the pathogenesis mechanism of COVID-19, and facilitate the development of effective vaccines and drugs as well as other therapeutic regimes.
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Affiliation(s)
- Ting Lv
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 200438 China
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, 200040 China
| | - Fanlu Meng
- Shandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, 253023 China
| | - Meng Yu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 200438 China
| | - Haihui Huang
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, 200040 China
| | - Xinhua Lin
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 200438 China
| | - Bing Zhao
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 200438 China
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Davis ES, Voss G, Miesfeld JB, Zarate-Sanchez J, Voss SR, Glaser T. The rax homeobox gene is mutated in the eyeless axolotl, Ambystoma mexicanum. Dev Dyn 2021; 250:807-821. [PMID: 32864847 PMCID: PMC8907009 DOI: 10.1002/dvdy.246] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2020] [Accepted: 08/11/2020] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND Vertebrate eye formation requires coordinated inductive interactions between different embryonic tissue layers, first described in amphibians. A network of transcription factors and signaling molecules controls these steps, with mutations causing severe ocular, neuronal, and craniofacial defects. In eyeless mutant axolotls, eye morphogenesis arrests at the optic vesicle stage, before lens induction, and development of ventral forebrain structures is disrupted. RESULTS We identified a 5-bp deletion in the rax (retina and anterior neural fold homeobox) gene, which was tightly linked to the recessive eyeless (e) axolotl locus in an F2 cross. This frameshift mutation, in exon 2, truncates RAX protein within the homeodomain (P154fs35X). Quantitative RNA analysis shows that mutant and wild-type rax transcripts are equally abundant in E/e embryos. Translation appears to initiate from dual start codons, via leaky ribosome scanning, a conserved feature among gnathostome RAX proteins. Previous data show rax is expressed in the optic vesicle and diencephalon, deeply conserved among metazoans, and required for eye formation in other species. CONCLUSION The eyeless axolotl mutation is a null allele in the rax homeobox gene, with primary defects in neural ectoderm, including the retinal and hypothalamic primordia.
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Affiliation(s)
- Erik S. Davis
- Department of Cell Biology and Human Anatomy, University of California Davis School of Medicine, Davis, California
| | - Gareth Voss
- Department of Neuroscience, Spinal Cord and Brain Injury Research Center, and Ambystoma Genetic Stock Center, University of Kentucky, Lexington, Kentucky
| | - Joel B. Miesfeld
- Department of Cell Biology and Human Anatomy, University of California Davis School of Medicine, Davis, California
| | - Juan Zarate-Sanchez
- Department of Cell Biology and Human Anatomy, University of California Davis School of Medicine, Davis, California
- Davis Senior High School, Davis, California
| | - S. Randal Voss
- Department of Neuroscience, Spinal Cord and Brain Injury Research Center, and Ambystoma Genetic Stock Center, University of Kentucky, Lexington, Kentucky
| | - Tom Glaser
- Department of Cell Biology and Human Anatomy, University of California Davis School of Medicine, Davis, California
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Fukunaga I, Oe Y, Danzaki K, Ohta S, Chen C, Shirai K, Kawano A, Ikeda K, Kamiya K. Modeling gap junction beta 2 gene-related deafness with human iPSC. Hum Mol Genet 2021; 30:1429-1442. [PMID: 33997905 DOI: 10.1093/hmg/ddab097] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Revised: 03/29/2021] [Accepted: 03/31/2021] [Indexed: 02/06/2023] Open
Abstract
There are >120 forms of non-syndromic deafness associated with identified genetic loci. In particular, mutation of the gap junction beta 2 gene (GJB2), which encodes connexin (CX)26 protein, is the most frequent cause of hereditary deafness worldwide. We previously described an induction method to develop functional CX26 gap junction-forming cells from mouse-induced pluripotent stem cells (iPSCs) and generated in vitro models for GJB2-related deafness. However, functional CX26 gap junction-forming cells derived from human iPSCs or embryonic stem cells (ESCs) have not yet been reported. In this study, we generated human iPSC-derived functional CX26 gap junction-forming cells (iCX26GJCs), which have the characteristics of cochlear supporting cells. These iCX26GJCs had gap junction plaque-like formations at cell-cell borders and co-expressed several markers that are expressed in cochlear supporting cells. Furthermore, we generated iCX26GJCs derived from iPSCs from two patients with the most common GJB2 mutation in Asia, and these cells reproduced the pathology of GJB2-related deafness. These in vitro models may be useful for establishing optimal therapies and drug screening for various mutations in GJB2-related deafness.
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Affiliation(s)
- Ichiro Fukunaga
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Yoko Oe
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Keiko Danzaki
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Sayaka Ohta
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Cheng Chen
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Kyoko Shirai
- Department of Otolaryngology, Head and Neck Surgery, Tokyo Medical University, Tokyo 1600023, Japan
| | - Atsushi Kawano
- Department of Otolaryngology, Head and Neck Surgery, Tokyo Medical University, Tokyo 1600023, Japan
| | - Katsuhisa Ikeda
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
| | - Kazusaku Kamiya
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo 1138421, Japan
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Fukunaga I, Oe Y, Chen C, Danzaki K, Ohta S, Koike A, Ikeda K, Kamiya K. Activin/Nodal/TGF-β Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells. Front Cell Dev Biol 2021; 9:602197. [PMID: 33968919 PMCID: PMC8097046 DOI: 10.3389/fcell.2021.602197] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Accepted: 03/30/2021] [Indexed: 11/13/2022] Open
Abstract
Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-β pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.
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Affiliation(s)
| | | | | | | | | | | | | | - Kazusaku Kamiya
- Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo, Japan
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