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Ma L, Thapa BR, Le Suer JA, Tilston-Lünel A, Herriges MJ, Wang F, Bawa PS, Varelas X, Hawkins FJ, Kotton DN. Life-long functional regeneration of in vivo airway epithelium by the engraftment of airway basal stem cells. Nat Protoc 2025; 20:810-842. [PMID: 39501108 DOI: 10.1038/s41596-024-01067-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Accepted: 08/30/2024] [Indexed: 12/11/2024]
Abstract
Durable and functional regeneration of the airway epithelium in vivo with transplanted stem cells has the potential to reconstitute healthy tissue in diseased airways, such as in cystic fibrosis or primary ciliary dyskinesia. Here, we present detailed protocols for the preparation and culture expansion of murine primary and induced pluripotent stem cell-derived airway basal stem cells (iBCs) and methods for their intra-airway transplantation into polidocanol-conditioned murine recipients to achieve durable in vivo airway regeneration. Reconstitution of the airway tissue resident epithelial stem cell compartment of immunocompetent mice with syngeneic donor cells leverages the extensive self-renewal and multipotent differentiation properties of basal stem cells (BCs) to durably generate a broad diversity of mature airway epithelial lineages in vivo. Engrafted donor-derived cells re-establish planar cell polarity as well as functional ciliary transport. By using this same approach, human primary BCs or iBCs transplanted into NOD-SCID gamma recipient mice similarly display engraftment and multilineage airway epithelial differentiation in vivo. The time to generate mouse or human iBCs takes ~60 d, which can be reduced to ~20 d if previously differentiated cells are thawed from cryopreserved iBC archives. The tracheal conditioning regimen and cell transplantation procedure is completed in 1 d. A competent graduate student or postdoctoral trainee should be able to perform the procedures listed in this protocol.
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Grants
- R21 HD094012 NICHD NIH HHS
- R21HD094012 U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
- P01HL170952 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- HAWKIN20XX2 Cystic Fibrosis Foundation (CF Foundation)
- U01HL134766 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- R01HL139799 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- R01HL095993 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- R01 HL139799 NHLBI NIH HHS
- R01HL124392 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- R01 HL124392 NHLBI NIH HHS
- NO1: 75N92020C00005 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- PCTC Jumpstart Award U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
- U01HL148692 U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
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Affiliation(s)
- Liang Ma
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
| | - Bibek R Thapa
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
- Department of Biology, Boston University, Boston, MA, USA
| | - Jake A Le Suer
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
| | - Andrew Tilston-Lünel
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Michael J Herriges
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
| | - Feiya Wang
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Pushpinder S Bawa
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Xaralabos Varelas
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- Department of Biochemistry and Cell Biology, Boston University School of Medicine, Boston, MA, USA
| | - Finn J Hawkins
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
| | - Darrell N Kotton
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA.
- The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA, USA.
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2
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Schüle KM, Probst S. Epigenetic control of cell identities from epiblast to gastrulation. FEBS J 2025. [PMID: 39985220 DOI: 10.1111/febs.70024] [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: 08/22/2024] [Revised: 01/20/2025] [Accepted: 02/04/2025] [Indexed: 02/24/2025]
Abstract
Epigenetic modifications of chromatin are essential for the establishment of cell identities during embryogenesis. Between embryonic days 3.5-7.5 of murine development, major cell lineage decisions are made that discriminate extraembryonic and embryonic tissues, and the embryonic primary germ layers are formed, thereby laying down the basic body plan. In this review, we cover the contribution of dynamic chromatin modifications by DNA methylation, changes of chromatin accessibility, and histone modifications, that in combination with transcription factors control gene expression programs of different cell types. We highlight the differences in regulation of enhancer and promoter marks and discuss their requirement in cell lineage specification. Importantly, in many cases, lineage-specific targeting of epigenetic modifiers is carried out by pioneer or master transcription factors, that in sum mediate the chromatin landscape and thereby control the transcription of cell-type-specific gene programs and thus, cell identities.
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Affiliation(s)
- Katrin M Schüle
- Faculty of Medicine, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Germany
- Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Germany
| | - Simone Probst
- Faculty of Medicine, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Germany
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3
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Schaaf C, Sussel L. A Cure for Type 1 Diabetes: Are We There Yet? Diabetes Technol Ther 2025. [PMID: 39911033 DOI: 10.1089/dia.2024.0498] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2025]
Abstract
Type 1 diabetes (T1D) affects over 2 million people in the United States and has no known cure. The discovery and first use of insulin in humans 102 years ago marked a revolutionary course of treatment for the disease, and although the formulations and delivery systems have advanced, insulin administration remains the standard of care today. While improved treatment options represent notable progress in T1D management, finding a functional cure for the disease remains the ultimate goal. Approaches to curing T1D have historically focused on blunting the autoimmune response, although sustained effects of immune modulation have proven elusive. Islet transplant therapies have also proven effective, although a lack of available donor tissue and the need for immunosuppression to prevent both host-graft rejection and the autoimmune response have reserved such treatments for those who already require immunosuppressive regimens for other reasons or undergo severe hypoglycemic events in conjunction with hypoglycemic unawareness. With the advent of human stem cell research, the focus has shifted toward generating an abundance of allogeneic, functional beta-like cells that can be transplanted into the patients. Immunoisolation devices have also shown some promise as a method of preventing immune rejection and the autoimmune destruction of transplanted cells. Finally, advances in new immune therapies, if used in the early stages of T1D progression, have proven to delay the onset of diabetes. Stem cell-based therapies are a promising approach to curing T1D. The ongoing clinical trials show some success, although they currently require immunosuppressant agents. Encapsulation devices provide a method of immunoisolation that does not require immunosuppression; however, the devices tested thus far eventually lead to cell death and fibrotic tissue growth. Substantial research efforts are underway to develop new approaches to protect the stem cell-derived beta cells upon transplantation.
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Affiliation(s)
- Christopher Schaaf
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Center, Denver, Colorado, USA
| | - Lori Sussel
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Center, Denver, Colorado, USA
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4
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Yamada T, Trentesaux C, Brunger JM, Xiao Y, Stevens AJ, Martyn I, Kasparek P, Shroff NP, Aguilar A, Bruneau BG, Boffelli D, Klein OD, Lim WA. Synthetic organizer cells guide development via spatial and biochemical instructions. Cell 2025; 188:778-795.e18. [PMID: 39706189 PMCID: PMC12027307 DOI: 10.1016/j.cell.2024.11.017] [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: 09/26/2023] [Revised: 07/10/2024] [Accepted: 11/08/2024] [Indexed: 12/23/2024]
Abstract
In vitro development relies primarily on treating progenitor cells with media-borne morphogens and thus lacks native-like spatial information. Here, we engineer morphogen-secreting organizer cells programmed to self-assemble, via cell adhesion, around mouse embryonic stem (ES) cells in defined architectures. By inducing the morphogen WNT3A and its antagonist DKK1 from organizer cells, we generated diverse morphogen gradients, varying in range and steepness. These gradients were strongly correlated with morphogenetic outcomes: the range of minimum-maximum WNT activity determined the resulting range of anterior-to-posterior (A-P) axis cell lineages. Strikingly, shallow WNT activity gradients, despite showing truncated A-P lineages, yielded higher-resolution tissue morphologies, such as a beating, chambered cardiac-like structure associated with an endothelial network. Thus, synthetic organizer cells, which integrate spatial, temporal, and biochemical information, provide a powerful way to systematically and flexibly direct the development of ES or other progenitor cells in different directions within the morphogenetic landscape.
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Affiliation(s)
- Toshimichi Yamada
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Coralie Trentesaux
- Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Jonathan M Brunger
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yini Xiao
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Adam J Stevens
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Iain Martyn
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Petr Kasparek
- Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Neha P Shroff
- Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Angelica Aguilar
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Benoit G Bruneau
- Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA; Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Dario Boffelli
- Department of Pediatrics, Cedars-Sinai Guerin Children's, Los Angeles, CA 90048, USA
| | - Ophir D Klein
- Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pediatrics, Cedars-Sinai Guerin Children's, Los Angeles, CA 90048, USA.
| | - Wendell A Lim
- Cell Design Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
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Undeutsch HJ, Posabella A, Alber AB, Bawa PS, Villacorta-Martin C, Wang F, Ikonomou L, Kotton DN, Hollenberg AN. Derivation of transplantable human thyroid follicular epithelial cells from induced pluripotent stem cells. Stem Cell Reports 2024; 19:1690-1705. [PMID: 39515316 PMCID: PMC11751801 DOI: 10.1016/j.stemcr.2024.10.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 10/08/2024] [Accepted: 10/09/2024] [Indexed: 11/16/2024] Open
Abstract
The production of mature functioning thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) is critical for potential novel therapeutic approaches to post-surgical and congenital hypothyroidism. To accomplish this, we developed a novel human iPSC line that expresses fluorophores targeted to the NKX2-1 and PAX8 loci, allowing for the identification and purification of cells destined to become TFCs. Optimizing a sequence of defined, serum-free media to promote stepwise developmental directed differentiation, we found that bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) stimulated lineage specification into TFCs from multiple iPSC lines. Single-cell RNA sequencing demonstrated that BMP4 withdrawal after lineage specification promoted TFC maturation, with mature TFCs representing the majority of cells present within 1 month. After xenotransplantation into athyreotic immunodeficient mice, engrafted cells exhibited thyroid follicular organization with thyroglobulin protein detected in the lumens of NKX2-1-positive follicles. While our iPSC-derived TFCs presented durable expression of thyroid-specific proteins, they were unable to rescue hypothyroidism in vivo.
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Affiliation(s)
- Hendrik J Undeutsch
- Division of Endocrinology, Diabetes and Metabolism, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA; Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Alberto Posabella
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA; University Center of Gastrointestinal and Liver Diseases - Clarunis, University of Basel Faculty of Medicine, Basel, Switzerland
| | - Andrea B Alber
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Pushpinder S Bawa
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Carlos Villacorta-Martin
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Feiya Wang
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA
| | - Laertis Ikonomou
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA; Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY, USA
| | - Darrell N Kotton
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA; The Pulmonary Center, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, USA
| | - Anthony N Hollenberg
- Division of Endocrinology, Diabetes and Metabolism, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA; Department of Medicine, Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center, Boston, MA, USA; Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA, USA.
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6
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Liu S, Ren J, Hu Y, Zhou F, Zhang L. TGFβ family signaling in human stem cell self-renewal and differentiation. CELL REGENERATION (LONDON, ENGLAND) 2024; 13:26. [PMID: 39604763 PMCID: PMC11602941 DOI: 10.1186/s13619-024-00207-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Revised: 10/16/2024] [Accepted: 11/01/2024] [Indexed: 11/29/2024]
Abstract
Human stem cells are undifferentiated cells with the capacity for self-renewal and differentiation into distinct cell lineages, playing important role in the development and maintenance of diverse tissues and organs. The microenvironment of stem cell provides crucial factors and components that exert significant influence over the determination of cell fate. Among these factors, cytokines from the transforming growth factor β (TGFβ) superfamily, including TGFβ, bone morphogenic protein (BMP), Activin and Nodal, have been identified as important regulators governing stem cell maintenance and differentiation. In this review, we present a comprehensive overview of the pivotal roles played by TGFβ superfamily signaling in governing human embryonic stem cells, somatic stem cells, induced pluripotent stem cells, and cancer stem cells. Furthermore, we summarize the latest research and advancements of TGFβ family in various cancer stem cells and stem cell-based therapy, discussing their potential clinical applications in cancer therapy and regeneration medicine.
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Affiliation(s)
- Sijia Liu
- International Biomed-X Research Center, Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China
| | - Jiang Ren
- The First Affiliated Hospital, MOE Basic Research and Innovation Center for the Targeted Therapeutics of Solid Tumors, Institute of Biomedical Innovation, School of Basic Medical Sciences, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Yanmei Hu
- Department of Endocrinology and Metabolism, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
| | - Fangfang Zhou
- The First Affiliated Hospital, the Institutes of Biology and Medical Sciences, Suzhou Medical College, Soochow University, Suzhou, China.
| | - Long Zhang
- International Biomed-X Research Center, Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China.
- The First Affiliated Hospital, MOE Basic Research and Innovation Center for the Targeted Therapeutics of Solid Tumors, Institute of Biomedical Innovation, School of Basic Medical Sciences, Jiangxi Medical College, Nanchang University, Nanchang, China.
- MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China.
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7
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Farag N, Sacharen C, Avni L, Nachman I. Coordination between endoderm progression and mouse gastruloid elongation controls endodermal morphotype choice. Dev Cell 2024; 59:2364-2374.e4. [PMID: 38838673 DOI: 10.1016/j.devcel.2024.05.017] [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] [Received: 03/24/2023] [Revised: 09/11/2023] [Accepted: 05/15/2024] [Indexed: 06/07/2024]
Abstract
Embryonic development is highly robust. Morphogenetic variability between embryos (under ideal conditions) is largely quantitative. This robustness stands in contrast to in vitro embryo-like models, which, like most organoids, can display a high degree of tissue morphogenetic variability. The source of this difference is not fully understood. We use the mouse gastruloid model to study the morphogenetic progression of definitive endoderm (DE) and its divergence. We first catalog the different morphologies and characterize their statistics. We then learn predictive models for DE morphotype based on earlier expression and morphology measurements. Finally, we analyze these models to identify key drivers of morphotype variability and devise gastruloid-specific and global interventions that can lower this variability and steer morphotype choice. In the process, we identify two types of coordination lacking in the in vitro model but required for robust gut-tube formation. This approach can help improve the quality and usability of 3D embryo-like models.
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Affiliation(s)
- Naama Farag
- School of Neurobiology, Biochemistry and Biophysics, Tel Aviv University, Tel Aviv, Israel
| | - Chen Sacharen
- School of Neurobiology, Biochemistry and Biophysics, Tel Aviv University, Tel Aviv, Israel
| | - Lara Avni
- School of Neurobiology, Biochemistry and Biophysics, Tel Aviv University, Tel Aviv, Israel
| | - Iftach Nachman
- School of Neurobiology, Biochemistry and Biophysics, Tel Aviv University, Tel Aviv, Israel.
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8
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Turner DL, Amoozadeh S, Baric H, Stanley E, Werder RB. Building a human lung from pluripotent stem cells to model respiratory viral infections. Respir Res 2024; 25:277. [PMID: 39010108 PMCID: PMC11251358 DOI: 10.1186/s12931-024-02912-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Accepted: 07/08/2024] [Indexed: 07/17/2024] Open
Abstract
To protect against the constant threat of inhaled pathogens, the lung is equipped with cellular defenders. In coordination with resident and recruited immune cells, this defence is initiated by the airway and alveolar epithelium following their infection with respiratory viruses. Further support for viral clearance and infection resolution is provided by adjacent endothelial and stromal cells. However, even with these defence mechanisms, respiratory viral infections are a significant global health concern, causing substantial morbidity, socioeconomic losses, and mortality, underlining the need to develop effective vaccines and antiviral medications. In turn, the identification of new treatment options for respiratory infections is critically dependent on the availability of tractable in vitro experimental models that faithfully recapitulate key aspects of lung physiology. For such models to be informative, it is important these models incorporate human-derived, physiologically relevant versions of all cell types that normally form part of the lungs anti-viral response. This review proposes a guideline using human induced pluripotent stem cells (iPSCs) to create all the disease-relevant cell types. iPSCs can be differentiated into lung epithelium, innate immune cells, endothelial cells, and fibroblasts at a large scale, recapitulating in vivo functions and providing genetic tractability. We advocate for building comprehensive iPSC-derived in vitro models of both proximal and distal lung regions to better understand and model respiratory infections, including interactions with chronic lung diseases.
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Affiliation(s)
- Declan L Turner
- Murdoch Children's Research Institute, Melbourne, 3056, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, 3056, Australia
- Novo Nordisk Foundation Centre for Stem Cell Medicine, reNEW Melbourne, Melbourne, 3056, Australia
| | - Sahel Amoozadeh
- Murdoch Children's Research Institute, Melbourne, 3056, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, 3056, Australia
- Novo Nordisk Foundation Centre for Stem Cell Medicine, reNEW Melbourne, Melbourne, 3056, Australia
| | - Hannah Baric
- Murdoch Children's Research Institute, Melbourne, 3056, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, 3056, Australia
- Novo Nordisk Foundation Centre for Stem Cell Medicine, reNEW Melbourne, Melbourne, 3056, Australia
| | - Ed Stanley
- Murdoch Children's Research Institute, Melbourne, 3056, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, 3056, Australia
- Novo Nordisk Foundation Centre for Stem Cell Medicine, reNEW Melbourne, Melbourne, 3056, Australia
| | - Rhiannon B Werder
- Murdoch Children's Research Institute, Melbourne, 3056, Australia.
- Department of Paediatrics, University of Melbourne, Melbourne, 3056, Australia.
- Novo Nordisk Foundation Centre for Stem Cell Medicine, reNEW Melbourne, Melbourne, 3056, Australia.
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9
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Simpson L, Strange A, Klisch D, Kraunsoe S, Azami T, Goszczynski D, Le Minh T, Planells B, Holmes N, Sang F, Henson S, Loose M, Nichols J, Alberio R. A single-cell atlas of pig gastrulation as a resource for comparative embryology. Nat Commun 2024; 15:5210. [PMID: 38890321 PMCID: PMC11189408 DOI: 10.1038/s41467-024-49407-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2023] [Accepted: 06/04/2024] [Indexed: 06/20/2024] Open
Abstract
Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.
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Affiliation(s)
- Luke Simpson
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Andrew Strange
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Doris Klisch
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Sophie Kraunsoe
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
- The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
| | - Takuya Azami
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK
| | - Daniel Goszczynski
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Triet Le Minh
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Benjamin Planells
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK
| | - Nadine Holmes
- School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
| | - Fei Sang
- School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
| | - Sonal Henson
- School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
| | - Matthew Loose
- School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
| | - Jennifer Nichols
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham, LE12 5RD, UK.
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10
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Magro-Lopez E, Vazquez-Alejo E, Espinar-Buitrago MDLS, Muñoz-Fernández MÁ. Optimizing Nodal, Wnt and BMP signaling pathways for robust and efficient differentiation of human induced pluripotent stem cells to intermediate mesoderm cells. Front Cell Dev Biol 2024; 12:1395723. [PMID: 38887514 PMCID: PMC11182123 DOI: 10.3389/fcell.2024.1395723] [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: 03/04/2024] [Accepted: 05/06/2024] [Indexed: 06/20/2024] Open
Abstract
Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 μM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 μM CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.
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Affiliation(s)
- Esmeralda Magro-Lopez
- Molecular Immuno-Biology Laboratory, Immunology Section, Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Spain
- Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
| | - Elena Vazquez-Alejo
- Molecular Immuno-Biology Laboratory, Immunology Section, Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Spain
- Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
| | - María de la Sierra Espinar-Buitrago
- Molecular Immuno-Biology Laboratory, Immunology Section, Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Spain
- Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
| | - María Ángeles Muñoz-Fernández
- Molecular Immuno-Biology Laboratory, Immunology Section, Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Spain
- Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
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11
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Asashima M, Satou-Kobayashi Y. Spemann-Mangold organizer and mesoderm induction. Cells Dev 2024; 178:203903. [PMID: 38295873 DOI: 10.1016/j.cdev.2024.203903] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 01/23/2024] [Accepted: 01/24/2024] [Indexed: 02/09/2024]
Abstract
The discovery of the Spemann-Mangold organizer strongly influenced subsequent research on embryonic induction, with research aiming to elucidate the molecular characteristics of organizer activity being currently underway. Herein, we review the history of research on embryonic induction, and describe how the mechanisms of induction phenomena and developmental processes have been investigated. Classical experiments investigating the differentiation capacity and inductive activity of various embryonic regions were conducted by many researchers, and important theories of region-specific induction and the concept for chain of induction were proposed. The transition from experimental embryology to developmental biology has enabled us to understand the mechanisms of embryonic induction at the molecular level. Consequently, many inducing substances and molecules such as transcriptional factors and peptide growth factors involved in the organizer formation were identified. One of peptide growth factors, activin, acts as a mesoderm- and endoderm-inducing substance. Activin induces several tissues and organs from the undifferentiated cell mass of amphibian embryos in a concentration-dependent manner. We review the extent to which we can control in vitro organogenesis from undifferentiated cells, and discuss the application to stem cell-based regenerative medicine based on insights gained from animal experiments, such as in amphibians.
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Affiliation(s)
- Makoto Asashima
- Advanced Comprehensive Research Organization, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan.
| | - Yumeko Satou-Kobayashi
- Advanced Comprehensive Research Organization, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan
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12
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Basil MC, Alysandratos KD, Kotton DN, Morrisey EE. Lung repair and regeneration: Advanced models and insights into human disease. Cell Stem Cell 2024; 31:439-454. [PMID: 38492572 PMCID: PMC11070171 DOI: 10.1016/j.stem.2024.02.009] [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: 12/05/2023] [Revised: 02/07/2024] [Accepted: 02/22/2024] [Indexed: 03/18/2024]
Abstract
The respiratory system acts as both the primary site of gas exchange and an important sensor and barrier to the external environment. The increase in incidences of respiratory disease over the past decades has highlighted the importance of developing improved therapeutic approaches. This review will summarize recent research on the cellular complexity of the mammalian respiratory system with a focus on gas exchange and immunological defense functions of the lung. Different models of repair and regeneration will be discussed to help interpret human and animal data and spur the investigation of models and assays for future drug development.
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Affiliation(s)
- Maria C Basil
- Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn, Children's Hospital of Philadelphia (CHOP) Lung Biology Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
| | - Konstantinos-Dionysios Alysandratos
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA.
| | - Darrell N Kotton
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA.
| | - Edward E Morrisey
- Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn, Children's Hospital of Philadelphia (CHOP) Lung Biology Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
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13
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Ghoneim MA, Gabr MM, El-Halawani SM, Refaie AF. Current status of stem cell therapy for type 1 diabetes: a critique and a prospective consideration. Stem Cell Res Ther 2024; 15:23. [PMID: 38281991 PMCID: PMC10823744 DOI: 10.1186/s13287-024-03636-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 01/10/2024] [Indexed: 01/30/2024] Open
Abstract
Over the past decade, there had been progress in the development of cell therapy for insulin-dependent diabetes. Nevertheless, important hurdles that need to be overcome still remain. Protocols for the differentiation of pluripotent stem cells into pancreatic progenitors or fully differentiated β-cells have been developed. The resulting insulin-producing cells can control chemically induced diabetes in rodents and were the subject of several clinical trials. However, these cells are immunogenic and possibly teratogenic for their transplantation, and an immunoisolation device and/or immunosuppression is needed. A growing number of studies have utilized genetic manipulations to produce immune evasive cells. Evidence must be provided that in addition to the expected benefit, gene manipulations should not lead to any unforeseen complications. Mesenchymal stem/stromal cells (MSCs) can provide a viable alternative. MSCs are widely available from many tissues. They can form insulin-producing cells by directed differentiation. Experimentally, evidence has shown that the transplantation of allogenic insulin-producing cells derived from MSCs is associated with a muted allogeneic response that does not interfere with their functionality. This can be explained by the immunomodulatory functions of the MSC subpopulation that did not differentiate into insulin-producing cells. Recently, exosomes derived from naive MSCs have been used in the experimental domain to treat diabetes in rodents with varying degrees of success. Several mechanisms for their beneficial functions were proposed including a reduction in insulin resistance, the promotion of autophagy, and an increase in the T regulatory population. However, euglycemia was not achieved in any of these experiments. We suggest that exosomes derived from β-cells or insulin-producing cells (educated) can provide a better therapeutic effect than those derived from undifferentiated cells.
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14
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Wildey A, Harrington S, Stehno-Bittel L, Karanu F. Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers. Regen Med 2024; 19:47-63. [PMID: 38240144 DOI: 10.2217/rme-2023-0187] [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] [Indexed: 02/10/2024] Open
Abstract
Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.
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Affiliation(s)
| | | | - Lisa Stehno-Bittel
- Likarda LLC, Kansas City, MO 64137, USA
- University of Kansas Medical Center, Kansas City, KS, USA
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15
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Simpson L, Alberio R. Interspecies control of development during mammalian gastrulation. Emerg Top Life Sci 2023; 7:397-408. [PMID: 37933589 PMCID: PMC10754326 DOI: 10.1042/etls20230083] [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] [Received: 08/28/2023] [Revised: 10/19/2023] [Accepted: 10/23/2023] [Indexed: 11/08/2023]
Abstract
Gastrulation represents a pivotal phase of development and aberrations during this period can have major consequences, from minor anatomical deviations to severe congenital defects. Animal models are used to study gastrulation, however, there is considerable morphological and molecular diversity of gastrula across mammalian species. Here, we provide an overview of the latest research on interspecies developmental control across mammals. This includes single-cell atlases of several mammalian gastrula which have enabled comparisons of the temporal and molecular dynamics of differentiation. These studies highlight conserved cell differentiation regulators and both absolute and relative differences in differentiation dynamics between species. Recent advances in in vitro culture techniques have facilitated the derivation, maintenance and differentiation of cell lines from a range of species and the creation of multi-species models of gastrulation. Gastruloids are three-dimensional aggregates capable of self-organising and recapitulating aspects of gastrulation. Such models enable species comparisons outside the confines of the embryo. We highlight recent in vitro evidence that differentiation processes such as somitogenesis and neuronal maturation scale with known in vivo differences in developmental tempo across species. This scaling is likely due to intrinsic differences in cell biochemistry. We also highlight several studies which provide examples of cell differentiation dynamics being influenced by extrinsic factors, including culture conditions, chimeric co-culture, and xenotransplantation. These collective studies underscore the complexity of gastrulation across species, highlighting the necessity of additional datasets and studies to decipher the intricate balance between intrinsic cellular programs and extrinsic signals in shaping embryogenesis.
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Affiliation(s)
- Luke Simpson
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, U.K
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, U.K
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16
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Li Q, Li J, Wang P, He X, Hong M, Liu F. A Comparative Study of Endoderm Differentiation Between Activin A and Small Molecules. Exp Clin Endocrinol Diabetes 2023; 131:667-675. [PMID: 38056491 DOI: 10.1055/a-2182-8936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/08/2023]
Abstract
Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm.
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Affiliation(s)
- Qiang Li
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Jin Li
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha 410078, Hunan, PR China
| | - Ping Wang
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Xiaoqun He
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Mingzhao Hong
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Feng Liu
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
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17
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Kuo YC, Lin SY, De S, Rajesh R. Regeneration of Pancreatic Cells Using Optimized Nanoparticles and l-Glutamic Acid-Gelatin Scaffolds with Controlled Topography and Grafted Activin A/BMP4. ACS Biomater Sci Eng 2023; 9:6208-6224. [PMID: 37882705 DOI: 10.1021/acsbiomaterials.3c00791] [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] [Indexed: 10/27/2023]
Abstract
Regeneration of insulin-producing cells (IPCs) from induced pluripotent stem cells (iPSCs) under controlled conditions has a lot of promise to emulate the pancreatic mechanism in vivo as a foundation of cell-based diabetic therapy. l-Glutamic acid-gelatin scaffolds with orderly pore sizes of 160 and 200 μm were grafted with activin A and bone morphogenic proteins 4 (BMP4) to differentiate iPSCs into definitive endoderm (DE) cells, which were then guided with fibroblast growth factor 7 (FGF7)-grafted retinoic acid (RA)-loaded solid lipid nanoparticles (FR-SLNs) to harvest IPCs. Response surface methodology was adopted to optimize the l-glutamic acid-to-gelatin ratio of scaffolds and to optimize surfactant concentration and lipid proportion in FR-SLNs. Experimental results of immunofluorescence, flow cytometry, and western blots revealed that activin A (100 ng/mL)-BMP4 (50 ng/mL)-l-glutamic acid (5%)-gelatin (95%) scaffolds provoked the largest number of SOX17-positive DE cells from iPSCs. Treatment with FGF7 (50 ng/mL)-RA (600 ng/mL)-SLNs elicited the highest number of PDX1-positive β-cells from differentiated DE cells. To imitate the natural pancreas, the scaffolds with controlled topography were appropriate for IPC production with sufficient insulin secretion. Hence, the current scheme using FR-SLNs and activin A-BMP4-l-glutamic acid-gelatin scaffolds in the two-stage differentiation of iPSCs can be promising for replacing impaired β-cells in diabetic management.
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Affiliation(s)
- Yung-Chih Kuo
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC
- Advanced Institute of Manufacturing with High-tech Innovations, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC
| | - Sheng-Yuan Lin
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC
| | - Sourav De
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC
| | - Rajendiran Rajesh
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC
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18
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Lo EKW, Velazquez JJ, Peng D, Kwon C, Ebrahimkhani MR, Cahan P. Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols. Stem Cell Reports 2023; 18:1721-1742. [PMID: 37478860 PMCID: PMC10444577 DOI: 10.1016/j.stemcr.2023.06.008] [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: 09/07/2022] [Revised: 06/16/2023] [Accepted: 06/17/2023] [Indexed: 07/23/2023] Open
Abstract
Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.
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Affiliation(s)
- Emily K W Lo
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Jeremy J Velazquez
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Da Peng
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Chulan Kwon
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Mo R Ebrahimkhani
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
| | - Patrick Cahan
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
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19
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Alsobaie S, Alsobaie T, Alshammary A, Mantalaris S. Differentiation of human induced pluripotent stem cells into functional lung alveolar epithelial cells in 3D dynamic culture. Front Bioeng Biotechnol 2023; 11:1173149. [PMID: 37388774 PMCID: PMC10303808 DOI: 10.3389/fbioe.2023.1173149] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Accepted: 05/15/2023] [Indexed: 07/01/2023] Open
Abstract
Introduction: Understanding lung epithelium cell development from human induced pluripotent stem cells (IPSCs) in vitro can lead to an individualized model for lung engineering, therapy, and drug testing. Method: We developed a protocol to produce lung mature type I pneumocytes using encapsulation of human IPSCs in 1.1% (w/v) alginate solution within a rotating wall bioreactor system in only 20 days without using feeder cells. The aim was to reduce exposure to animal products and laborious interventions in the future. Results: The three-dimensional (3D) bioprocess allowed cell derivation into endoderm, and subsequently into type II alveolar epithelial cells within a very short period. Cells successfully expressed surfactant proteins C and B associated with type II alveolar epithelial cells, and the key structure of lamellar bodies and microvilli was shown by transmission electron microscopy. The survival rate was the highest under dynamic conditions, which reveal the possibility of adapting this integration for large-scale cell production of alveolar epithelial cells from human IPSCs. Discussion: We were able to develop a strategy for the culture and differentiation of human IPSCs into alveolar type II cells using an in vitro system that mimics the in vivo environment. Hydrogel beads would offer a suitable matrix for 3D cultures and that the high-aspect-ratio vessel bioreactor can be used to increase the differentiation of human IPSCs relative to the results obtained with traditional monolayer cultures.
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Affiliation(s)
- Sarah Alsobaie
- Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia
| | - Tamador Alsobaie
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, United Kingdom
| | - Amal Alshammary
- Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia
| | - Sakis Mantalaris
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, United States
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20
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Deguchi K, Zambaiti E, De Coppi P. Regenerative medicine: current research and perspective in pediatric surgery. Pediatr Surg Int 2023; 39:167. [PMID: 37014468 PMCID: PMC10073065 DOI: 10.1007/s00383-023-05438-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/01/2023] [Indexed: 04/05/2023]
Abstract
The field of regenerative medicine, encompassing several disciplines including stem cell biology and tissue engineering, continues to advance with the accumulating research on cell manipulation technologies, gene therapy and new materials. Recent progress in preclinical and clinical studies may transcend the boundaries of regenerative medicine from laboratory research towards clinical reality. However, for the ultimate goal to construct bioengineered transplantable organs, a number of issues still need to be addressed. In particular, engineering of elaborate tissues and organs requires a fine combination of different relevant aspects; not only the repopulation of multiple cell phenotypes in an appropriate distribution but also the adjustment of the host environmental factors such as vascularisation, innervation and immunomodulation. The aim of this review article is to provide an overview of the recent discoveries and development in stem cells and tissue engineering, which are inseparably interconnected. The current status of research on tissue stem cells and bioengineering, and the possibilities for application in specific organs relevant to paediatric surgery have been specifically focused and outlined.
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Affiliation(s)
- Koichi Deguchi
- Stem Cells and Regenerative Medicine Section, University College London Great Ormond Street Institute of Child Health, London, UK
- Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Elisa Zambaiti
- Stem Cells and Regenerative Medicine Section, University College London Great Ormond Street Institute of Child Health, London, UK
- UOC Chirurgia Pediatrica, Ospedale Infantile Regina Margherita, Turin, Italy
| | - Paolo De Coppi
- Stem Cells and Regenerative Medicine Section, University College London Great Ormond Street Institute of Child Health, London, UK.
- NIHR BRC SNAPS Great Ormond Street Hospitals, London, UK.
- Stem Cells and Regenerative Medicine Section, Faculty of Population Health Sciences, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK.
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21
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Chamorro-Herrero I, Zambrano A. Modeling of Respiratory Diseases Evolving with Fibrosis from Organoids Derived from Human Pluripotent Stem Cells. Int J Mol Sci 2023; 24:ijms24054413. [PMID: 36901843 PMCID: PMC10002124 DOI: 10.3390/ijms24054413] [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] [Received: 12/15/2022] [Revised: 02/03/2023] [Accepted: 02/21/2023] [Indexed: 02/25/2023] Open
Abstract
Respiratory disease is one of the leading causes of morbidity and mortality worldwide. There is no cure for most diseases, which are treated symptomatically. Hence, new strategies are required to deepen the understanding of the disease and development of therapeutic strategies. The advent of stem cell and organoid technology has enabled the development of human pluripotent stem cell lines and adequate differentiation protocols for developing both airways and lung organoids in different formats. These novel human-pluripotent-stem-cell-derived organoids have enabled relatively accurate disease modeling. Idiopathic pulmonary fibrosis is a fatal and debilitating disease that exhibits prototypical fibrotic features that may be, to some extent, extrapolated to other conditions. Thus, respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or the one caused by SARS-CoV-2 may reflect some fibrotic aspects reminiscent of those present in idiopathic pulmonary fibrosis. Modeling of fibrosis of the airways and the lung is a real challenge due to the large number of epithelial cells involved and interaction with other cell types of mesenchymal origin. This review will focus on the status of respiratory disease modeling from human-pluripotent-stem-cell-derived organoids, which are being used to model several representative respiratory diseases, such as idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease, and COVID-19.
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22
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Leibel SL, McVicar RN, Murad R, Kwong EM, Clark AE, Alvarado A, Grimmig BA, Nuryyev R, Young RE, Lee JC, Peng W, Zhu YP, Griffis E, Nowell CJ, Liu K, James B, Alarcon S, Malhotra A, Gearing LJ, Hertzog PJ, Galapate CM, Galenkamp KM, Commisso C, Smith DM, Sun X, Carlin AF, Croker BA, Snyder EY. The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.26.525578. [PMID: 36747824 PMCID: PMC9900938 DOI: 10.1101/2023.01.26.525578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.
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23
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Legier T, Rattier D, Llewellyn J, Vannier T, Sorre B, Maina F, Dono R. Epithelial disruption drives mesendoderm differentiation in human pluripotent stem cells by enabling TGF-β protein sensing. Nat Commun 2023; 14:349. [PMID: 36681697 PMCID: PMC9867713 DOI: 10.1038/s41467-023-35965-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Accepted: 01/10/2023] [Indexed: 01/22/2023] Open
Abstract
The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.
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Affiliation(s)
- Thomas Legier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Diane Rattier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Jack Llewellyn
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Thomas Vannier
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Benoit Sorre
- Institut Curie, Universite ́PSL, Sorbonne Universite ́, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
| | - Flavio Maina
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France
| | - Rosanna Dono
- Aix Marseille Univ, CNRS, IBDM, Turing Center for Living Systems, NeuroMarseille, Marseille, France.
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24
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The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation. Curr Issues Mol Biol 2022; 44:6104-6116. [PMID: 36547077 PMCID: PMC9776753 DOI: 10.3390/cimb44120416] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2022] [Revised: 12/02/2022] [Accepted: 12/02/2022] [Indexed: 12/12/2022] Open
Abstract
Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.
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25
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Verhoeff K, Cuesta-Gomez N, Jasra I, Marfil-Garza B, Dadheech N, Shapiro AMJ. Optimizing Generation of Stem Cell-Derived Islet Cells. Stem Cell Rev Rep 2022; 18:2683-2698. [PMID: 35639237 DOI: 10.1007/s12015-022-10391-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/13/2022] [Indexed: 02/06/2023]
Abstract
Islet transplantation is a highly effective treatment for select patients with type 1 diabetes. Unfortunately, current use is limited to those with brittle disease due to donor limitations and immunosuppression requirements. Discovery of factors for induction of pluripotent stem cells from adult somatic cells into a malleable state has reinvigorated the possibility of autologous-based regenerative cell therapies. Similarly, recent progress in allogeneic human embryonic stem cell islet products is showing early success in clinical trials. Describing safe and standardized differentiation protocols with clear pathways to optimize yield and minimize off-target growth is needed to efficiently move the field forward. This review discusses current islet differentiation protocols with a detailed break-down of differentiation stages to guide step-wise controlled generation of functional islet products.
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Affiliation(s)
- Kevin Verhoeff
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Nerea Cuesta-Gomez
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Ila Jasra
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Braulio Marfil-Garza
- National Institute of Medical Sciences and Nutrition Salvador Zubiran, Mexico City, and CHRISTUS-LatAm Hub - Excellence and Innovation Center, Monterrey, Mexico
| | - Nidheesh Dadheech
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - A M James Shapiro
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.
- 1-002 Li Ka Shing Centre for Health Research Innovation, 112 St. NW & 87 Ave NW, Edmonton, Alberta, T6G 2E1, Canada.
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26
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Wehmeyer AE, Schüle KM, Conrad A, Schröder CM, Probst S, Arnold SJ. Chimeric 3D gastruloids - a versatile tool for studies of mammalian peri-gastrulation development. Development 2022; 149:280536. [PMID: 36326003 DOI: 10.1242/dev.200812] [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: 03/29/2022] [Accepted: 10/18/2022] [Indexed: 11/06/2022]
Abstract
Stem cell-derived three-dimensional (3D) gastruloids show a remarkable capacity of self-organisation and recapitulate many aspects of gastrulation stage mammalian development. Gastruloids can be rapidly generated and offer several experimental advantages, such as scalability, observability and accessibility for manipulation. Here, we present approaches to further expand the experimental potency of murine 3D gastruloids by using functional genetics in mouse embryonic stem cells (mESCs) to generate chimeric gastruloids. In chimeric gastruloids, fluorescently labelled cells of different genotypes harbouring inducible gene expression or loss-of-function alleles are combined with wild-type cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factor brachyury or inducible expression of Eomes. Resulting chimeric gastruloids recapitulate reported Eomes and brachyury functions, such as instructing cardiac fate and promoting posterior axial extension, respectively. Additionally, chimeric gastruloids revealed previously unrecognised phenotypes, such as the tissue sorting preference of brachyury deficient cells to endoderm and the cell non-autonomous effects of brachyury deficiency on Wnt3a patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as an efficient tool for studies of mammalian gastrulation.
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Affiliation(s)
- Alexandra E Wehmeyer
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany
| | - Katrin M Schüle
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany
| | - Alexandra Conrad
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany
| | - Chiara M Schröder
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.,Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstrasse 19a, D-79104 Freiburg, Germany.,Faculty of Biology, University of Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany.,Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Schänzlestrasse 18, D-79104 Freiburg, Germany
| | - Simone Probst
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany
| | - Sebastian J Arnold
- Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.,Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Schänzlestrasse 18, D-79104 Freiburg, Germany
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27
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Hein RFC, Conchola AS, Fine AS, Xiao Z, Frum T, Brastrom LK, Akinwale MA, Childs CJ, Tsai YH, Holloway EM, Huang S, Mahoney J, Heemskerk I, Spence JR. Stable iPSC-derived NKX2-1+ lung bud tip progenitor organoids give rise to airway and alveolar cell types. Development 2022; 149:dev200693. [PMID: 36039869 PMCID: PMC9534489 DOI: 10.1242/dev.200693] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 07/28/2022] [Indexed: 12/13/2022]
Abstract
Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.
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Affiliation(s)
- Renee F. C. Hein
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Ansley S. Conchola
- Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Alexis S. Fine
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Zhiwei Xiao
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Tristan Frum
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Lindy K. Brastrom
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Mayowa A. Akinwale
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Charlie J. Childs
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Yu-Hwai Tsai
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Emily M. Holloway
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Sha Huang
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - John Mahoney
- Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, MA 02421, USA
| | - Idse Heemskerk
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Jason R. Spence
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Biomedical Engineering Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
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28
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Kotasová H, Capandová M, Pelková V, Dumková J, Koledová Z, Remšík J, Souček K, Garlíková Z, Sedláková V, Rabata A, Vaňhara P, Moráň L, Pečinka L, Porokh V, Kučírek M, Streit L, Havel J, Hampl A. Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells. Tissue Eng Regen Med 2022; 19:1033-1050. [PMID: 35670910 PMCID: PMC9478014 DOI: 10.1007/s13770-022-00458-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 03/09/2022] [Accepted: 04/08/2022] [Indexed: 11/30/2022] Open
Abstract
BACKGROUND The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
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Affiliation(s)
- Hana Kotasová
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
| | - Michaela Capandová
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Vendula Pelková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Jana Dumková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Zuzana Koledová
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Ján Remšík
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
- Institute of Biophysics, The Czech Academy of Sciences, Brno, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
- Current Address: Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
| | - Karel Souček
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
- Institute of Biophysics, The Czech Academy of Sciences, Brno, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
| | - Zuzana Garlíková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Veronika Sedláková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Anas Rabata
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Petr Vaňhara
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
| | - Lukáš Moráň
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
- Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Brno, Czech Republic
| | - Lukáš Pečinka
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
- Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
| | - Volodymyr Porokh
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Martin Kučírek
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic
| | - Libor Streit
- Department of Plastic and Cosmetic Surgery, Faculty of Medicine, Masaryk University, Brno, Czech Republic
- Department of Plastic and Cosmetic Surgery, St. Anne's Faculty Hospital, Brno, Czech Republic
| | - Josef Havel
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
- Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
| | - Aleš Hampl
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic.
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic.
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29
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Bricker RL, Bhaskar U, Titone R, Carless MA, Barberi T. A Molecular Analysis of Neural Olfactory Placode Differentiation in Human Pluripotent Stem Cells. Stem Cells Dev 2022; 31:507-520. [PMID: 35592997 PMCID: PMC9641992 DOI: 10.1089/scd.2021.0257] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Accepted: 05/19/2022] [Indexed: 11/12/2022] Open
Abstract
During embryonic development, the olfactory sensory neurons (OSNs) and the gonadotropic-releasing hormone neurons (GNRHNs) migrate from the early nasal cavity, known as the olfactory placode, to the brain. Defects in the development of OSNs and GNRHNs result in neurodevelopmental disorders such as anosmia and congenital hypogonadotropic hypogonadism, respectively. Treatments do not restore the defective neurons in these disorders, and as a result, patients have a diminished sense of smell or a gonadotropin hormone deficiency. Human pluripotent stem cells (hPSCs) can produce any cell type in the body; therefore, they are an invaluable tool for cell replacement therapies. Transplantation of olfactory placode progenitors, derived from hPSCs, is a promising therapeutic to replace OSNs and GNRHNs and restore tissue function. Protocols to generate olfactory placode progenitors are limited, and thus, we describe, in this study, a novel in vitro model for olfactory placode differentiation in hPSCs, which is capable of producing both OSNs and GNRHNs. Our study investigates the major developmental signaling factors that recapitulate the embryonic development of the olfactory tissue. We demonstrate that induction of olfactory placode in hPSCs requires bone morphogenetic protein inhibition, wingless/integrated protein inhibition, retinoic acid inhibition, transforming growth factor alpha activation, and fibroblast growth factor 8 activation. We further show that the protocol transitions hPSCs through the anterior pan-placode ectoderm and neural ectoderm regions in early development while preventing neural crest and non-neural ectoderm regions. Finally, we demonstrate production of OSNs and GNRHNs by day 30 of differentiation. Our study is the first to report on OSN differentiation in hPSCs.
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Affiliation(s)
- Rebecca L. Bricker
- Population Health Program, Texas Biomedical Research Institute, San Antonio, Texas, USA
- Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
| | - Uchit Bhaskar
- Department of Neuroscience, Developmental and Regenerative Biology, University of Texas at San Antonio, San Antonio, Texas, USA
| | - Rossella Titone
- Population Health Program, Texas Biomedical Research Institute, San Antonio, Texas, USA
| | - Melanie A. Carless
- Population Health Program, Texas Biomedical Research Institute, San Antonio, Texas, USA
- Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
- Department of Neuroscience, Developmental and Regenerative Biology, University of Texas at San Antonio, San Antonio, Texas, USA
| | - Tiziano Barberi
- Population Health Program, Texas Biomedical Research Institute, San Antonio, Texas, USA
- Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
- Lab Farm Foods, Inc., New York City, New York, USA
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30
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Arias AM, Marikawa Y, Moris N. Gastruloids: Pluripotent stem cell models of mammalian gastrulation and embryo engineering. Dev Biol 2022; 488:35-46. [PMID: 35537519 PMCID: PMC9477185 DOI: 10.1016/j.ydbio.2022.05.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 04/29/2022] [Accepted: 05/02/2022] [Indexed: 12/12/2022]
Abstract
Gastrulation is a fundamental and critical process of animal development whereby the mass of cells that results from the proliferation of the zygote transforms itself into a recognizable outline of an organism. The last few years have seen the emergence of a number of experimental models of early mammalian embryogenesis based on Embryonic Stem (ES) cells. One of this is the Gastruloid model. Gastruloids are aggregates of defined numbers of ES cells that, under defined culture conditions, undergo controlled proliferation, symmetry breaking, and the specification of all three germ layers characteristic of vertebrate embryos, and their derivatives. However, they lack brain structures and, surprisingly, reveal a disconnect between cell type specific gene expression and tissue morphogenesis, for example during somitogenesis. Gastruloids have been derived from mouse and human ES cells and several variations of the original model have emerged that reveal a hereto unknown modularity of mammalian embryos. We discuss the organization and development of gastruloids in the context of the embryonic stages that they represent, pointing out similarities and differences between the two. We also point out their potential as a reproducible, scalable and searchable experimental system and highlight some questions posed by the current menagerie of gastruloids.
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Affiliation(s)
- Alfonso Martinez Arias
- Systems Bioengineering, MELIS, Universidad Pompeu Fabra, Doctor Aiguader, 88, ICREA, Pag Lluis Companys 23, Barcelona, Spain.
| | - Yusuke Marikawa
- Institute for Biogenesis Research, University of Hawaii John A. Burns School of Medicine, Honolulu, HI, 96813, USA
| | - Naomi Moris
- The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
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31
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Asadi Jozani K, Kouthouridis S, Hirota JA, Zhang B. Next generation preclinical models of lung development, physiology and disease. CAN J CHEM ENG 2022. [DOI: 10.1002/cjce.24581] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Kimia Asadi Jozani
- School of Biomedical Engineering, McMaster University 1280 Main Street West, Hamilton Ontario Canada
| | - Sonya Kouthouridis
- Department of Chemical Engineering McMaster University Hamilton Ontario Canada
| | - Jeremy Alexander Hirota
- School of Biomedical Engineering, McMaster University 1280 Main Street West, Hamilton Ontario Canada
- Department of Medicine, Division of Respirology McMaster University Hamilton Ontario Canada
- Firestone Institute for Respiratory Health St. Joseph’s Hospital, Hamilton Ontario Canada
| | - Boyang Zhang
- School of Biomedical Engineering, McMaster University 1280 Main Street West, Hamilton Ontario Canada
- Department of Chemical Engineering McMaster University Hamilton Ontario Canada
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32
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Berical A, Lee RE, Lu J, Beermann ML, Le Suer JA, Mithal A, Thomas D, Ranallo N, Peasley M, Stuffer A, Bukis K, Seymour R, Harrington J, Coote K, Valley H, Hurley K, McNally P, Mostoslavsky G, Mahoney J, Randell SH, Hawkins FJ. A multimodal iPSC platform for cystic fibrosis drug testing. Nat Commun 2022; 13:4270. [PMID: 35906215 PMCID: PMC9338271 DOI: 10.1038/s41467-022-31854-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2021] [Accepted: 07/06/2022] [Indexed: 01/27/2023] Open
Abstract
Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants.
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Affiliation(s)
- Andrew Berical
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
- The Pulmonary Center and Department of Medicine, Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Rhianna E Lee
- Marsico Lung Institute and Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Junjie Lu
- Cystic Fibrosis Foundation, Lexington, MA, 02421, USA
| | - Mary Lou Beermann
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Jake A Le Suer
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Aditya Mithal
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Dylan Thomas
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Nicole Ranallo
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - Megan Peasley
- Cystic Fibrosis Foundation, Lexington, MA, 02421, USA
| | - Alex Stuffer
- Cystic Fibrosis Foundation, Lexington, MA, 02421, USA
| | | | | | | | - Kevin Coote
- Cystic Fibrosis Foundation, Lexington, MA, 02421, USA
| | | | - Killian Hurley
- Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin, Ireland
- Tissue Engineering Research Group, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Paul McNally
- RCSI University of Medicine and Health Sciences, Dublin, Ireland
- Children's Health Ireland, Dublin, Ireland
| | - Gustavo Mostoslavsky
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA
| | - John Mahoney
- Cystic Fibrosis Foundation, Lexington, MA, 02421, USA
| | - Scott H Randell
- Marsico Lung Institute and Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Finn J Hawkins
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA, 02118, USA.
- The Pulmonary Center and Department of Medicine, Boston University and Boston Medical Center, Boston, MA, 02118, USA.
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Lin Y, Wang D, Zeng Y. A Maverick Review of Common Stem/Progenitor Markers in Lung Development. Stem Cell Rev Rep 2022; 18:2629-2645. [DOI: 10.1007/s12015-022-10422-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/26/2022] [Indexed: 10/16/2022]
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Dang Le Q, Rodprasert W, Kuncorojakti S, Pavasant P, Osathanon T, Sawangmake C. In vitro generation of transplantable insulin-producing cells from canine adipose-derived mesenchymal stem cells. Sci Rep 2022; 12:9127. [PMID: 35650303 PMCID: PMC9160001 DOI: 10.1038/s41598-022-13114-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2022] [Accepted: 05/20/2022] [Indexed: 11/27/2022] Open
Abstract
Canine mesenchymal stem cells (cMSCs) have potential applications for regenerative therapy, including the generation of insulin-producing cells (IPCs) for studying and treating diabetes. In this study, we established a useful protocol for generating IPCs from canine adipose mesenchymal stem cells (cAD-MSCs). Subsequently, in vitro preservation of pluronic F127-coated alginate (ALGPA)-encapsulated cAD-MSC-derived IPCs was performed to verify ready-to-use IPCs. IPCs were induced from cAD-MSCs with the modulated three-stepwise protocol. The first step of definitive endoderm (DE) induction showed that the cooperation of Chir99021 and Activin A created the effective production of Sox17-expressed DE cells. The second step for pancreatic endocrine (PE) progenitor induction from DE indicated that the treatment with taurine, retinoic acid, FGF2, EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT showed the significant upregulation of the pancreatic endocrine precursor markers Pdx1 and Ngn3. The last step of IPC production, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor, yielded efficiently functional IPCs from PE precursors. Afterward, the maintenance of ALGPA-encapsulated cAD-MSC-derived IPCs with VSCBIC-1, a specialized medium, enhanced IPC properties. Conclusion, the modulated three-stepwise protocol generates the functional IPCs. Together, the encapsulation of cAD-MSC-derived IPCs and the cultivation with VSCBIC-1 enrich the maturation of generated IPCs.
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Affiliation(s)
- Quynh Dang Le
- International Program of Veterinary Science and Technology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
- Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
- Veterinary Stem Cell and Bioengineering Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Watchareewan Rodprasert
- Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Suryo Kuncorojakti
- Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
- Department of Veterinary Science, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia
| | - Prasit Pavasant
- Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
- Center of Excellence in Regenerative Dentistry (CERD), Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
| | - Thanaphum Osathanon
- Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
- Dental Stem Cell Biology Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
| | - Chenphop Sawangmake
- Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
- Veterinary Stem Cell and Bioengineering Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
- Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
- Center of Excellence in Regenerative Dentistry (CERD), Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
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35
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Regeneration of insulin-producing cells from iPS cells using functionalized scaffolds and solid lipid nanoparticles. J Taiwan Inst Chem Eng 2022. [DOI: 10.1016/j.jtice.2022.104387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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36
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Graffmann N, Scherer B, Adjaye J. In vitro differentiation of pluripotent stem cells into hepatocyte like cells - basic principles and current progress. Stem Cell Res 2022; 61:102763. [DOI: 10.1016/j.scr.2022.102763] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 03/08/2022] [Accepted: 03/22/2022] [Indexed: 12/11/2022] Open
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Pour M, Kumar AS, Farag N, Bolondi A, Kretzmer H, Walther M, Wittler L, Meissner A, Nachman I. Emergence and patterning dynamics of mouse-definitive endoderm. iScience 2022; 25:103556. [PMID: 34988400 PMCID: PMC8693470 DOI: 10.1016/j.isci.2021.103556] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 10/21/2021] [Accepted: 12/01/2021] [Indexed: 11/30/2022] Open
Abstract
The segregation of definitive endoderm (DE) from bipotent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatiotemporal window in the embryo. Here, we employ a dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in vivo in isolated cells within a temporally restricted window. In 2D and 3D in vitro models, DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17 + cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with upregulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.
Sox17 onsets in a few isolated cells within Bra-expressing population Sox17 onset followed by expansion and self-sorting Final number of Sox17+ cells does not depend on self-sorting or cell movement The DE segregation pattern is similar in in vivo and in 2D, 3D in vitro systems
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Affiliation(s)
- Maayan Pour
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Abhishek Sampath Kumar
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Naama Farag
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Adriano Bolondi
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Helene Kretzmer
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Maria Walther
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Lars Wittler
- Department of Developmental Genetics, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
| | - Alexander Meissner
- Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.,Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.,Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Iftach Nachman
- School of Neurobiology, Biochemistry and Biophysics, Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
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Moreira A, Müller M, Costa PF, Kohl Y. Advanced In Vitro Lung Models for Drug and Toxicity Screening: The Promising Role of Induced Pluripotent Stem Cells. Adv Biol (Weinh) 2021; 6:e2101139. [PMID: 34962104 DOI: 10.1002/adbi.202101139] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 11/25/2021] [Indexed: 12/24/2022]
Abstract
The substantial socioeconomic burden of lung diseases, recently highlighted by the disastrous impact of the coronavirus disease 2019 (COVID-19) pandemic, accentuates the need for interventive treatments capable of decelerating disease progression, limiting organ damage, and contributing to a functional tissue recovery. However, this is hampered by the lack of accurate human lung research models, which currently fail to reproduce the human pulmonary architecture and biochemical environment. Induced pluripotent stem cells (iPSCs) and organ-on-chip (OOC) technologies possess suitable characteristics for the generation of physiologically relevant in vitro lung models, allowing for developmental studies, disease modeling, and toxicological screening. Importantly, these platforms represent potential alternatives for animal testing, according to the 3Rs (replace, reduce, refine) principle, and hold promise for the identification and approval of new chemicals under the European REACH (registration, evaluation, authorization and restriction of chemicals) framework. As such, this review aims to summarize recent progress made in human iPSC- and OOC-based in vitro lung models. A general overview of the present applications of in vitro lung models is presented, followed by a summary of currently used protocols to generate different lung cell types from iPSCs. Lastly, recently developed iPSC-based lung models are discussed.
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Affiliation(s)
| | - Michelle Müller
- Department of Bioprocessing and Bioanalytics, Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280, Sulzbach, Germany
| | - Pedro F Costa
- BIOFABICS, Rua Alfredo Allen 455, Porto, 4200-135, Portugal
| | - Yvonne Kohl
- Department of Bioprocessing and Bioanalytics, Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280, Sulzbach, Germany.,Postgraduate Course for Toxicology and Environmental Toxicology, Medical Faculty, University of Leipzig, Johannisallee 28, 04103, Leipzig, Germany
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39
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From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish. Int J Mol Sci 2021; 22:ijms222413180. [PMID: 34947977 PMCID: PMC8708686 DOI: 10.3390/ijms222413180] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 12/02/2021] [Accepted: 12/05/2021] [Indexed: 12/12/2022] Open
Abstract
Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.
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Gwon K, Hong HJ, Gonzalez-Suarez AM, Slama MQ, Choi D, Hong J, Baskaran H, Stybayeva G, Peterson QP, Revzin A. Bioactive hydrogel microcapsules for guiding stem cell fate decisions by release and reloading of growth factors. Bioact Mater 2021; 15:1-14. [PMID: 35386345 PMCID: PMC8941170 DOI: 10.1016/j.bioactmat.2021.12.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2021] [Revised: 11/22/2021] [Accepted: 12/12/2021] [Indexed: 12/21/2022] Open
Abstract
Human pluripotent stem cells (hPSC) hold considerable promise as a source of adult cells for treatment of diseases ranging from diabetes to liver failure. Some of the challenges that limit the clinical/translational impact of hPSCs are high cost and difficulty in scaling-up of existing differentiation protocols. In this paper, we sought to address these challenges through the development of bioactive microcapsules. A co-axial flow focusing microfluidic device was used to encapsulate hPSCs in microcapsules comprised of an aqueous core and a hydrogel shell. Importantly, the shell contained heparin moieties for growth factor (GF) binding and release. The aqueous core enabled rapid aggregation of hPSCs into 3D spheroids while the bioactive hydrogel shell was used to load inductive cues driving pluripotency maintenance and endodermal differentiation. Specifically, we demonstrated that one-time, 1 h long loading of pluripotency signals, fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, into bioactive microcapsules was sufficient to induce and maintain pluripotency of hPSCs over the course of 5 days at levels similar to or better than a standard protocol with soluble GFs. Furthermore, stem cell-carrying microcapsules that previously contained pluripotency signals could be reloaded with an endodermal cue, Nodal, resulting in higher levels of endodermal markers compared to stem cells differentiated in a standard protocol. Overall, bioactive heparin-containing core-shell microcapsules decreased GF usage five-fold while improving stem cell phenotype and are well suited for 3D cultivation of hPSCs.
Heparin-containing microcapsules enable sustained release of inductive cues (growth factors) over the course of seven to nine days. Heparin-growth factor binding is reversible which means that different growth factors may be loaded in a sequential manner. Loading inductive cues into microcapsules results in better differentiation of pluripotent stem cells. Loading inductive cues into microcapsules allows to decrease the usage of growth factors by several fold.
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Affiliation(s)
- Kihak Gwon
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
| | - Hye Jin Hong
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
| | | | - Michael Q. Slama
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
| | - Daheui Choi
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, 03722, Republic of Korea
| | - Jinkee Hong
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, 03722, Republic of Korea
| | - Harihara Baskaran
- Department of Chemical and Biomolecular Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
| | - Quinn P. Peterson
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA
- Corresponding author.
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Alvarez Fallas ME, Pedraza-Arevalo S, Cujba AM, Manea T, Lambert C, Morrugares R, Sancho R. Stem/progenitor cells in normal physiology and disease of the pancreas. Mol Cell Endocrinol 2021; 538:111459. [PMID: 34543699 PMCID: PMC8573583 DOI: 10.1016/j.mce.2021.111459] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Revised: 03/19/2021] [Accepted: 09/13/2021] [Indexed: 02/08/2023]
Abstract
Though embryonic pancreas progenitors are well characterised, the existence of stem/progenitor cells in the postnatal mammalian pancreas has been long debated, mainly due to contradicting results on regeneration after injury or disease in mice. Despite these controversies, sequencing advancements combined with lineage tracing and organoid technologies indicate that homeostatic and trigger-induced regenerative responses in mice could occur. The presence of putative progenitor cells in the adult pancreas has been proposed during homeostasis and upon different stress challenges such as inflammation, tissue damage and oncogenic stress. More recently, single cell transcriptomics has revealed a remarkable heterogeneity in all pancreas cell types, with some cells showing the signature of potential progenitors. In this review we provide an overview on embryonic and putative adult pancreas progenitors in homeostasis and disease, with special emphasis on in vitro culture systems and scRNA-seq technology as tools to address the progenitor nature of different pancreatic cells.
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Affiliation(s)
- Mario Enrique Alvarez Fallas
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Sergio Pedraza-Arevalo
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Ana-Maria Cujba
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Teodora Manea
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Christopher Lambert
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Rosario Morrugares
- Instituto Maimonides de Investigacion Biomedica de Cordoba (IMIBIC), Cordoba, Spain; Departamento de Biologia Celular, Fisiologia e Inmunologia, Universidad de Cordoba, Cordoba, Spain; Hospital Universitario Reina Sofia, Cordoba, Spain
| | - Rocio Sancho
- Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK; Department of Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany.
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Xiao Y, Sosa F, Ross PJ, Diffenderfer KE, Hansen PJ. Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts. Biol Open 2021; 10:bio058669. [PMID: 34643229 PMCID: PMC8649639 DOI: 10.1242/bio.058669] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Accepted: 10/08/2021] [Indexed: 12/12/2022] Open
Abstract
Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Yao Xiao
- Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, Shandong 250100, China
- Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, FL 32611-0910, USA
| | - Froylan Sosa
- Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, FL 32611-0910, USA
| | - Pablo J. Ross
- Department of Animal Science, University of California, Davis, CA 95616, USA
| | | | - Peter J. Hansen
- Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, FL 32611-0910, USA
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Hosseini V, Kalantary-Charvadeh A, Hajikarami M, Fayyazpour P, Rahbarghazi R, Totonchi M, Darabi M. A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation. Stem Cell Res Ther 2021; 12:550. [PMID: 34674740 PMCID: PMC8532309 DOI: 10.1186/s13287-021-02617-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2021] [Accepted: 10/07/2021] [Indexed: 12/11/2022] Open
Abstract
Background Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). Methods The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. Results When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. Conclusion The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.![]() Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02617-x.
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Affiliation(s)
- Vahid Hosseini
- Student Research Committee, Tabriz University of Medical Sciences, 5166615573, Tabriz, Iran.,Stem Cell Research Center, Tabriz University of Medical Sciences, 516615731, Tabriz, Iran.,Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ashkan Kalantary-Charvadeh
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
| | - Maryam Hajikarami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Parisa Fayyazpour
- Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Reza Rahbarghazi
- Stem Cell Research Center, Tabriz University of Medical Sciences, 516615731, Tabriz, Iran.,Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mehdi Totonchi
- Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Masoud Darabi
- Stem Cell Research Center, Tabriz University of Medical Sciences, 516615731, Tabriz, Iran. .,Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. .,Department of Internal Medicine IV, Heidelberg University Hospital, Heidelberg, Germany.
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Directed Differentiation of Mouse Embryonic Stem Cells to Mesoderm, Endoderm, and Neuroectoderm Lineages. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2021; 2520:295-307. [PMID: 34611822 DOI: 10.1007/7651_2021_439] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
The self-renewal and pluripotency features of mouse embryonic stem cells (mESCs) make them a great tool to study early mammalian development. Various signaling pathways that shape early mammalian development can be mimicked for in vitro mESC differentiation toward primitive lineages first and more specialized cell types later. Since the precise nature of the molecular mechanisms and the crosstalk between these signaling pathways is yet to be fully understood, there is a high level of variability in the efficiency and synchronicity among available differentiation protocols. Commitment of mESCs toward mesoderm, endoderm, or neuroectoderm lineages happens over only a few days and is highly efficient. Here, we provide protocols for the directed differentiation of mESCs toward these lineages in vitro.
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Hirota A, AlMusawi S, Nateri AS, Ordóñez-Morán P, Imajo M. Biomaterials for intestinal organoid technology and personalized disease modeling. Acta Biomater 2021; 132:272-287. [PMID: 34023456 DOI: 10.1016/j.actbio.2021.05.010] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 04/08/2021] [Accepted: 05/07/2021] [Indexed: 12/20/2022]
Abstract
Recent advances in intestinal organoid technologies have paved the way for in vitro recapitulation of the homeostatic renewal of adult tissues, tissue or organ morphogenesis during development, and pathogenesis of many disorders. In vitro modelling of individual patient diseases using organoid systems have been considered key in establishing rational design of personalized treatment strategies and in improving therapeutic outcomes. In addition, the transplantation of organoids into diseased tissues represents a novel approach to treat currently incurable diseases. Emerging evidence from intensive studies suggests that organoid systems' development and functional maturation depends on the presence of an extracellular matrix with suitable biophysical properties, where advanced synthetic hydrogels open new avenues for theoretical control of organoid phenotypes and potential applications of organoids in therapeutic purposes. In this review, we discuss the status, applications, challenges and perspectives of intestinal organoid systems emphasising on hydrogels and their properties suitable for intestinal organoid culture. We provide an overview of hydrogels used for intestinal organoid culture and key factors regulating their biological activity. The comparison of different hydrogels would be a theoretical basis for establishing design principles of synthetic niches directing intestinal cell fates and functions. STATEMENT OF SIGNIFICANCE: Intestinal organoid is an in vitro recapitulation of the gut, which self-organizes from intestinal stem cells and maintains many features of the native tissue. Since the development of this technology, intestinal organoid systems have made significant contribution to rapid progress in intestinal biology. Prevailing methodology for organoid culture, however, depends on animal-derived matrices and suffers from variability and potential risk for contamination of pathogens, limiting their therapeutic application. Synthetic scaffold matrices, hydrogels, might provide solutions to these issues and deepen our understanding on how intestinal cells sense and respond to key biophysical properties of the surrounding matrices. This review provides an overview of developing intestinal models and biomaterials, thereby leading to better understanding of current intestinal organoid systems for both biologists and materials scientists.
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Affiliation(s)
- Akira Hirota
- Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University, N15, W7, Kita-ku, Sapporo 060-8638, Japan
| | - Shaikha AlMusawi
- Cancer Genetic and Stem Cell group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom; Stem Cell biology and Cancer group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom
| | - Abdolrahman S Nateri
- Cancer Genetic and Stem Cell group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom
| | - Paloma Ordóñez-Morán
- Stem Cell biology and Cancer group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom.
| | - Masamichi Imajo
- Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University, N15, W7, Kita-ku, Sapporo 060-8638, Japan.
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Wang YF, Liu C, Xu PF. Deciphering and reconstitution of positional information in the human brain development. ACTA ACUST UNITED AC 2021; 10:29. [PMID: 34467458 PMCID: PMC8408296 DOI: 10.1186/s13619-021-00091-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Accepted: 08/02/2021] [Indexed: 12/29/2022]
Abstract
Organoid has become a novel in vitro model to research human development and relevant disorders in recent years. With many improvements on the culture protocols, current brain organoids could self-organize into a complicated three-dimensional organization that mimics most of the features of the real human brain at the molecular, cellular, and further physiological level. However, lacking positional information, an important characteristic conveyed by gradients of signaling molecules called morphogens, leads to the deficiency of spatiotemporally regulated cell arrangements and cell–cell interactions in the brain organoid development. In this review, we will overview the role of morphogen both in the vertebrate neural development in vivo as well as the brain organoid culture in vitro, the strategies to apply morphogen concentration gradients in the organoid system and future perspectives of the brain organoid technology.
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Affiliation(s)
- Yi-Fan Wang
- Women's Hospital, and Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.,Institute of Zhejiang University and University of Edinburgh, Jiaxing, Zhejiang, China.,Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Dr, Singapore, 117599, Singapore
| | - Cong Liu
- Women's Hospital, and Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Peng-Fei Xu
- Women's Hospital, and Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
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Maurya S, Yang W, Tamai M, Zhang Q, Erdmann-Gilmore P, Bystry A, Martins Rodrigues F, Valentine MC, Wong WH, Townsend R, Druley TE. Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification. Epigenetics 2021; 17:220-238. [PMID: 34304711 PMCID: PMC8865227 DOI: 10.1080/15592294.2021.1954780] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.
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Affiliation(s)
- Shailendra Maurya
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | - Wei Yang
- McDonnell Genome Institute, Genome Technology Access Center, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | - Minori Tamai
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | - Qiang Zhang
- Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Petra Erdmann-Gilmore
- Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Amelia Bystry
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | | | - Mark C Valentine
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | - Wing H Wong
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
| | - Reid Townsend
- Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Todd E Druley
- Department of Pediatrics, Division of Pediatric Hematology and Oncology, Washington University in St Louis School of Medicine, St. Louis, Missouri, United States
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Satou-Kobayashi Y, Kim JD, Fukamizu A, Asashima M. Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment. Sci Rep 2021; 11:14537. [PMID: 34267234 PMCID: PMC8282838 DOI: 10.1038/s41598-021-93524-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 06/23/2021] [Indexed: 02/06/2023] Open
Abstract
Activin, a member of the transforming growth factor-β (TGF-β) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.
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Affiliation(s)
- Yumeko Satou-Kobayashi
- grid.264706.10000 0000 9239 9995Strategic Innovation and Research Center, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605 Japan ,grid.264706.10000 0000 9239 9995Advanced Comprehensive Research Organization, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605 Japan ,grid.20515.330000 0001 2369 4728Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1, Tsukuba, Tennoudai Ibaraki 305-8577 Japan
| | - Jun-Dal Kim
- grid.20515.330000 0001 2369 4728Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1, Tsukuba, Tennoudai Ibaraki 305-8577 Japan ,grid.267346.20000 0001 2171 836XDivision of Complex Bioscience Research, Department of Research and Development, Institute of National Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194 Japan
| | - Akiyoshi Fukamizu
- grid.20515.330000 0001 2369 4728Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1, Tsukuba, Tennoudai Ibaraki 305-8577 Japan
| | - Makoto Asashima
- grid.264706.10000 0000 9239 9995Strategic Innovation and Research Center, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605 Japan ,grid.264706.10000 0000 9239 9995Advanced Comprehensive Research Organization, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605 Japan ,grid.20515.330000 0001 2369 4728Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1, Tsukuba, Tennoudai Ibaraki 305-8577 Japan
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Kishimoto K, Morimoto M. Mammalian tracheal development and reconstruction: insights from in vivo and in vitro studies. Development 2021; 148:dev198192. [PMID: 34228796 PMCID: PMC8276987 DOI: 10.1242/dev.198192] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The trachea delivers inhaled air into the lungs for gas exchange. Anomalies in tracheal development can result in life-threatening malformations, such as tracheoesophageal fistula and tracheomalacia. Given the limitations of current therapeutic approaches, development of technologies for the reconstitution of a three-dimensional trachea from stem cells is urgently required. Recently, single-cell sequencing technologies and quantitative analyses from cell to tissue scale have been employed to decipher the cellular basis of tracheal morphogenesis. In this Review, recent advances in mammalian tracheal development and the generation of tracheal tissues from pluripotent stem cells are summarized.
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Affiliation(s)
- Keishi Kishimoto
- Laboratory for Lung Development and Regeneration, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe 650-0047, Japan
- RIKEN BDR–CuSTOM Joint Laboratory, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Stem Cell & Organoid Medicine (CuSTOM), Perinatal Institute, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Mitsuru Morimoto
- Laboratory for Lung Development and Regeneration, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe 650-0047, Japan
- RIKEN BDR–CuSTOM Joint Laboratory, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
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Lyadova I, Gerasimova T, Nenasheva T. Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions. Front Cell Dev Biol 2021; 9:640703. [PMID: 34150747 PMCID: PMC8207294 DOI: 10.3389/fcell.2021.640703] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Accepted: 03/25/2021] [Indexed: 12/23/2022] Open
Abstract
Macrophages (Mφ) derived from induced pluripotent stem cells (iMphs) represent a novel and promising model for studying human Mφ function and differentiation and developing new therapeutic strategies based on or oriented at Mφs. iMphs have several advantages over the traditionally used human Mφ models, such as immortalized cell lines and monocyte-derived Mφs. The advantages include the possibility of obtaining genetically identical and editable cells in a potentially scalable way. Various applications of iMphs are being developed, and their number is rapidly growing. However, the protocols of iMph differentiation that are currently used vary substantially, which may lead to differences in iMph differentiation trajectories and properties. Standardization of the protocols and identification of minimum required conditions that would allow obtaining iMphs in a large-scale, inexpensive, and clinically suitable mode are needed for future iMph applications. As a first step in this direction, the current review discusses the fundamental basis for the generation of human iMphs, performs a detailed analysis of the generalities and the differences between iMph differentiation protocols currently employed, and discusses the prospects of iMph applications.
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Affiliation(s)
- Irina Lyadova
- Laboratory of Cellular and Molecular Basis of Histogenesis, Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
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