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Habecker BA, Bers DM, Birren SJ, Chang R, Herring N, Kay MW, Li D, Mendelowitz D, Mongillo M, Montgomery JM, Ripplinger CM, Tampakakis E, Winbo A, Zaglia T, Zeltner N, Paterson DJ. Molecular and cellular neurocardiology in heart disease. J Physiol 2025; 603:1689-1728. [PMID: 38778747 PMCID: PMC11582088 DOI: 10.1113/jp284739] [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: 02/23/2024] [Accepted: 04/16/2024] [Indexed: 05/25/2024] Open
Abstract
This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.
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Affiliation(s)
- Beth A Habecker
- Department of Chemical Physiology & Biochemistry, Department of Medicine Knight Cardiovascular Institute, Oregon Health and Science University, Portland, OR, USA
| | - Donald M Bers
- Department of Pharmacology, University of California, Davis School of Medicine, Davis, CA, USA
| | - Susan J Birren
- Department of Biology, Volen Center for Complex Systems, Brandeis University, Waltham, MA, USA
| | - Rui Chang
- Department of Neuroscience, Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
| | - Neil Herring
- Burdon Sanderson Cardiac Science Centre and BHF Centre of Research Excellence, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
| | - Matthew W Kay
- Department of Biomedical Engineering, George Washington University, Washington, DC, USA
| | - Dan Li
- Burdon Sanderson Cardiac Science Centre and BHF Centre of Research Excellence, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
| | - David Mendelowitz
- Department of Pharmacology and Physiology, George Washington University, Washington, DC, USA
| | - Marco Mongillo
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Johanna M Montgomery
- Department of Physiology and Manaaki Manawa Centre for Heart Research, University of Auckland, Auckland, New Zealand
| | - Crystal M Ripplinger
- Department of Pharmacology, University of California, Davis School of Medicine, Davis, CA, USA
| | | | - Annika Winbo
- Department of Physiology and Manaaki Manawa Centre for Heart Research, University of Auckland, Auckland, New Zealand
| | - Tania Zaglia
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Nadja Zeltner
- Departments of Biochemistry and Molecular Biology, Cell Biology, and Center for Molecular Medicine, University of Georgia, Athens, GA, USA
| | - David J Paterson
- Burdon Sanderson Cardiac Science Centre and BHF Centre of Research Excellence, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
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Rothstein M, Azambuja AP, Kanno TY, Breen C, Simoes-Costa M. TGF-β signaling controls neural crest developmental plasticity via SMAD2/3. Dev Cell 2025:S1534-5807(25)00059-0. [PMID: 39983721 DOI: 10.1016/j.devcel.2025.01.018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 09/25/2024] [Accepted: 01/28/2025] [Indexed: 02/23/2025]
Abstract
The neural crest is a highly plastic stem cell population that represents an exception to the germ layer theory. Despite being of ectodermal origin, cranial neural crest cells can differentiate into skeletal derivatives typically formed by mesoderm. Here, we report that SMAD2/3-mediated transforming growth factor β (TGF-β) signaling enhances neural crest developmental potential in the chicken embryo. Our results show that TGF-β signaling modulates neural crest axial identity and directly controls the gene circuits that support skeletal differentiation. Cooperation between TGF-β and low levels of WNT signaling in the embryonic head activates cranial-specific cis-regulatory elements. Activation of TGF-β signaling reprogrammed trunk neural crest cells into adopting an anterior identity and led to the development of an improved protocol for the generation of human cranial neural crest cells. Our findings indicate TGF-β signaling is required for the specification of cranial neural crest cells, endowing them with the potential to give rise to the craniofacial skeleton.
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Affiliation(s)
- Megan Rothstein
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Ana Paula Azambuja
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA; Department of Pathology, Boston Children's Hospital, Boston, MA, USA; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Tatiane Y Kanno
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA; Department of Pathology, Boston Children's Hospital, Boston, MA, USA; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Catriona Breen
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Marcos Simoes-Costa
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA; Department of Pathology, Boston Children's Hospital, Boston, MA, USA; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
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Hojo H, Tani S, Ohba S. Modeling of skeletal development and diseases using human pluripotent stem cells. J Bone Miner Res 2024; 40:5-19. [PMID: 39498496 DOI: 10.1093/jbmr/zjae178] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 09/28/2024] [Accepted: 11/02/2024] [Indexed: 01/07/2025]
Abstract
Human skeletal elements are formed from distinct origins at distinct positions of the embryo. For example, the neural crest produces the facial bones, the paraxial mesoderm produces the axial skeleton, and the lateral plate mesoderm produces the appendicular skeleton. During skeletal development, different combinations of signaling pathways are coordinated from distinct origins during the sequential developmental stages. Models for human skeletal development have been established using human pluripotent stem cells (hPSCs) and by exploiting our understanding of skeletal development. Stepwise protocols for generating skeletal cells from different origins have been designed to mimic developmental trails. Recently, organoid methods have allowed the multicellular organization of skeletal cell types to recapitulate complicated skeletal development and metabolism. Similarly, several genetic diseases of the skeleton have been modeled using patient-derived induced pluripotent stem cells and genome-editing technologies. Model-based drug screening is a powerful tool for identifying drug candidates. This review briefly summarizes our current understanding of the embryonic development of skeletal tissues and introduces the current state-of-the-art hPSC methods for recapitulating skeletal development, metabolism, and diseases. We also discuss the current limitations and future perspectives for applications of the hPSC-based modeling system in precision medicine in this research field.
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Affiliation(s)
- Hironori Hojo
- Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8655, Japan
| | - Shoichiro Tani
- Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States
| | - Shinsuke Ohba
- Department of Tissue and Developmental Biology, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan
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4
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Visintin PV, Zampieri BL, Griesi-Oliveira K. Chemical transdifferentiation of somatic cells to neural cells: a systematic review. EINSTEIN-SAO PAULO 2024; 22:eRW0423. [PMID: 39661857 PMCID: PMC11634374 DOI: 10.31744/einstein_journal/2024rw0423] [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: 01/19/2023] [Accepted: 02/21/2024] [Indexed: 12/13/2024] Open
Abstract
INTRODUCTION Transdifferentiation is the conversion of a specific somatic cell into another cell type, bypassing a transient pluripotent state. This implies a faster method to generate cells of interest with the additional benefit of reduced tumorigenic risk for clinical use. OBJECTIVE We describe protocols that use small molecules as direct conversion inducers, without the need for exogenous factors, to evaluate the potential of cell transdifferentiation for pharmacological and clinical applications. METHODS In this systematic review, using PRISMA guidelines, we conducted a personalized search strategy in four databases (PubMed, Scopus, Embase, and Web Of Science), looking for experimental works that used exclusively small molecules for transdifferentiation of non-neural cell types into neural lineage cells. RESULTS We explored the main biological mechanisms involved in direct cell conversion induced by different small molecules used in 33 experimental in vitro and in vitro transdifferentiation protocols. We also summarize the main characteristics of these protocols, such as the chemical cocktails used, time for transdifferentiation, and conversion efficiency. CONCLUSION Small molecules-based protocols for neuronal transdifferentiation are reasonably safe, economical, accessible, and are a promising alternative for future use in regenerative medicine and pharmacology.
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Affiliation(s)
- Paulo Victor Visintin
- Hospital Israelita Albert EinsteinSão PauloSPBrazilHospital Israelita Albert Einstein, São Paulo, SP, Brazil.
| | - Bruna Lancia Zampieri
- Hospital Israelita Albert EinsteinSão PauloSPBrazilHospital Israelita Albert Einstein, São Paulo, SP, Brazil.
| | - Karina Griesi-Oliveira
- Hospital Israelita Albert EinsteinSão PauloSPBrazilHospital Israelita Albert Einstein, São Paulo, SP, Brazil.
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Lisboa MDO, Selenko AH, Hochuli AHD, Senegaglia AC, Fracaro L, Brofman PRS. The influence of fetal bovine serum concentration on stemness and neuronal differentiation markers in stem cells from human exfoliated deciduous teeth. Tissue Cell 2024; 91:102571. [PMID: 39353229 DOI: 10.1016/j.tice.2024.102571] [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: 05/14/2024] [Revised: 07/26/2024] [Accepted: 09/18/2024] [Indexed: 10/04/2024]
Abstract
Dental Stem Cells (DSCs) from discarded teeth are a non-invasive and ethically favorable source with the potential for neurogenesis due to their ectodermal origin. Stem cells from human exfoliated deciduous teeth (SHED) are particularly promising due to their high differentiation potential and relative immaturity compared to other Mesenchymal Stromal Cells (MSCs). Markers like CD56 and CD271 are critical in identifying MSC subpopulations for therapeutic applications because of their roles in neurodevelopment and maintaining stemness. This study investigates how fetal bovine serum (FBS) concentrations affect the expression of CD56 and CD271 in SHED, influencing their stemness and neuronal differentiation potential. SHEDs were isolated from various donors, cultured, and characterized for MSC traits using markers such as CD14, CD19, CD29, CD34, CD45, CD73, CD90, CD105, CD56, and CD271. Culturing SHED in different FBS conditions (standard 15 %, reduced 1 % and 5 %, and FBS-free) showed that lower FBS concentrations increase CD271 and CD56 expression while maintaining the standard MSC immunophenotype. Importantly, the enhanced expression of these markers can be induced even after SHEDs have been expanded in standard FBS concentrations. These findings suggest that FBS concentration can optimize SHED culture conditions, enhancing their suitability for regenerative medicine and tissue engineering applications.
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Affiliation(s)
- Mateus de Oliveira Lisboa
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil.
| | - Ana Helena Selenko
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil
| | - Agner Henrique Dorigo Hochuli
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil
| | - Alexandra Cristina Senegaglia
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil
| | - Letícia Fracaro
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil.
| | - Paulo Roberto Slud Brofman
- Core for Cell Technology, School of Medicine and Health Sciences - Pontifícia Universidade Católica do Paraná, Curitiba, Paraná 80215-901, Brazil; National Institute of Science and Technology for Regenerative Medicine, INCT-REGENERA, 21941-599, Brazil
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Yoshimaru K, Matsuura T, Uchida Y, Sonoda S, Maeda S, Kajihara K, Kawano Y, Shirai T, Toriigahara Y, Kalim AS, Zhang XY, Takahashi Y, Kawakubo N, Nagata K, Yamaza H, Yamaza T, Taguchi T, Tajiri T. Cutting-edge regenerative therapy for Hirschsprung disease and its allied disorders. Surg Today 2024; 54:977-994. [PMID: 37668735 DOI: 10.1007/s00595-023-02741-6] [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: 05/12/2023] [Accepted: 08/06/2023] [Indexed: 09/06/2023]
Abstract
Hirschsprung disease (HSCR) and its associated disorders (AD-HSCR) often result in severe hypoperistalsis caused by enteric neuropathy, mesenchymopathy, and myopathy. Notably, HSCR involving the small intestine, isolated hypoganglionosis, chronic idiopathic intestinal pseudo-obstruction, and megacystis-microcolon-intestinal hypoperistalsis syndrome carry a poor prognosis. Ultimately, small-bowel transplantation (SBTx) is necessary for refractory cases, but it is highly invasive and outcomes are less than optimal, despite advances in surgical techniques and management. Thus, regenerative therapy has come to light as a potential form of treatment involving regeneration of the enteric nervous system, mesenchyme, and smooth muscle in affected areas. We review the cutting-edge regenerative therapeutic approaches for managing HSCR and AD-HSCR, including the use of enteric nervous system progenitor cells, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells as cell sources, the recipient intestine's microenvironment, and transplantation methods. Perspectives on the future of these treatments are also discussed.
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Affiliation(s)
- Koichiro Yoshimaru
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Toshiharu Matsuura
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
| | - Yasuyuki Uchida
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Soichiro Sonoda
- Department of Molecular Cell Biology and Oral Anatomy, Kyushu University Graduate School of Dental Science, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Shohei Maeda
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Keisuke Kajihara
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Yuki Kawano
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Takeshi Shirai
- Department of Pediatric Surgery, Miyazaki Prefectural Miyazaki Hospital, 5-30 Kitatakamatsu-cho, Miyazaki, Miyazaki, 880-8510, Japan
| | - Yukihiro Toriigahara
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Alvin Santoso Kalim
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Xiu-Ying Zhang
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Yoshiaki Takahashi
- Department of Pediatric Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Chuo-ku, Niigata, Japan
| | - Naonori Kawakubo
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Kouji Nagata
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Haruyoshi Yamaza
- Department of Pediatric Dentistry, Kyushu University Graduate School of Dental Science, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Takayoshi Yamaza
- Department of Molecular Cell Biology and Oral Anatomy, Kyushu University Graduate School of Dental Science, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Tomoaki Taguchi
- Fukuoka College of Health Sciences, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan
| | - Tatsuro Tajiri
- Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
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7
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de Jaime-Soguero A, Hattemer J, Bufe A, Haas A, van den Berg J, van Batenburg V, Das B, di Marco B, Androulaki S, Böhly N, Landry JJM, Schoell B, Rosa VS, Villacorta L, Baskan Y, Trapp M, Benes V, Chabes A, Shahbazi M, Jauch A, Engel U, Patrizi A, Sotillo R, van Oudenaarden A, Bageritz J, Alfonso J, Bastians H, Acebrón SP. Developmental signals control chromosome segregation fidelity during pluripotency and neurogenesis by modulating replicative stress. Nat Commun 2024; 15:7404. [PMID: 39191776 DOI: 10.1038/s41467-024-51821-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Accepted: 08/09/2024] [Indexed: 08/29/2024] Open
Abstract
Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals-including WNT, BMP, and FGF-converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development.
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Affiliation(s)
| | - Janina Hattemer
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
| | - Anja Bufe
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
| | - Alexander Haas
- Department of Molecular Oncology, Section for Cellular Oncology, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Jeroen van den Berg
- Oncode Institute, Utrecht, The Netherlands
- Hubrecht Institute, Utrecht, The Netherlands
- KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, The Netherlands
- University Medical Center Utrecht, Utrecht, The Netherlands
| | - Vincent van Batenburg
- Oncode Institute, Utrecht, The Netherlands
- Hubrecht Institute, Utrecht, The Netherlands
- KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, The Netherlands
- University Medical Center Utrecht, Utrecht, The Netherlands
| | - Biswajit Das
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
| | - Barbara di Marco
- Department of Clinical Neurobiology, University Hospital Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Stefania Androulaki
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
| | - Nicolas Böhly
- Department of Molecular Oncology, Section for Cellular Oncology, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Jonathan J M Landry
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Brigitte Schoell
- Institute of Human Genetics, Heidelberg University, Heidelberg, Germany
| | | | - Laura Villacorta
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Yagmur Baskan
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
| | - Marleen Trapp
- Schaller Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Vladimir Benes
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Andrei Chabes
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
| | | | - Anna Jauch
- Institute of Human Genetics, Heidelberg University, Heidelberg, Germany
| | - Ulrike Engel
- Nikon Imaging Center at the University of Heidelberg, Bioquant, Heidelberg, Germany
| | - Annarita Patrizi
- Schaller Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Rocio Sotillo
- Division of Molecular Thoracic Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Alexander van Oudenaarden
- Oncode Institute, Utrecht, The Netherlands
- Hubrecht Institute, Utrecht, The Netherlands
- KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, The Netherlands
- University Medical Center Utrecht, Utrecht, The Netherlands
| | - Josephine Bageritz
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
| | - Julieta Alfonso
- Department of Clinical Neurobiology, University Hospital Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Holger Bastians
- Department of Molecular Oncology, Section for Cellular Oncology, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Sergio P Acebrón
- Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany.
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8
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Vignard V, Baruteau AE, Toutain B, Mercier S, Isidor B, Redon R, Schott JJ, Küry S, Bézieau S, Monsoro-Burq AH, Ebstein F. Exploring the origins of neurodevelopmental proteasomopathies associated with cardiac malformations: are neural crest cells central to certain pathological mechanisms? Front Cell Dev Biol 2024; 12:1370905. [PMID: 39071803 PMCID: PMC11272537 DOI: 10.3389/fcell.2024.1370905] [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: 01/15/2024] [Accepted: 06/05/2024] [Indexed: 07/30/2024] Open
Abstract
Neurodevelopmental proteasomopathies constitute a recently defined class of rare Mendelian disorders, arising from genomic alterations in proteasome-related genes. These alterations result in the dysfunction of proteasomes, which are multi-subunit protein complexes essential for maintaining cellular protein homeostasis. The clinical phenotype of these diseases manifests as a syndromic association involving impaired neural development and multisystem abnormalities, notably craniofacial anomalies and malformations of the cardiac outflow tract (OFT). These observations suggest that proteasome loss-of-function variants primarily affect specific embryonic cell types which serve as origins for both craniofacial structures and the conotruncal portion of the heart. In this hypothesis article, we propose that neural crest cells (NCCs), a highly multipotent cell population, which generates craniofacial skeleton, mesenchyme as well as the OFT of the heart, in addition to many other derivatives, would exhibit a distinctive vulnerability to protein homeostasis perturbations. Herein, we introduce the diverse cellular compensatory pathways activated in response to protein homeostasis disruption and explore their potential implications for NCC physiology. Altogether, the paper advocates for investigating proteasome biology within NCCs and their early cranial and cardiac derivatives, offering a rationale for future exploration and laying the initial groundwork for therapeutic considerations.
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Affiliation(s)
- Virginie Vignard
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
| | - Alban-Elouen Baruteau
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
- CHU Nantes, Department of Pediatric Cardiology and Pediatric Cardiac Surgery, FHU PRECICARE, Nantes Université, Nantes, France
- Nantes Université, CHU Nantes, INSERM, CIC FEA 1413, Nantes, France
| | - Bérénice Toutain
- Nantes Université, CNRS, INSERM, l’institut du thorax, Nantes, France
| | - Sandra Mercier
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
- CHU Nantes, Service de Génétique Médicale, Nantes Université, Nantes, France
| | - Bertrand Isidor
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
- CHU Nantes, Service de Génétique Médicale, Nantes Université, Nantes, France
| | - Richard Redon
- Nantes Université, CNRS, INSERM, l’institut du thorax, Nantes, France
| | | | - Sébastien Küry
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
- CHU Nantes, Service de Génétique Médicale, Nantes Université, Nantes, France
| | - Stéphane Bézieau
- Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France
- CHU Nantes, Service de Génétique Médicale, Nantes Université, Nantes, France
| | - Anne H. Monsoro-Burq
- Faculté des Sciences d'Orsay, CNRS, UMR 3347, INSERM, Université Paris-Saclay, Orsay, France
- Institut Curie, PSL Research University, CNRS, UMR 3347, INSERM, Orsay, France
- Institut Universitaire de France, Paris, France
| | - Frédéric Ebstein
- Nantes Université, CNRS, INSERM, l’institut du thorax, Nantes, France
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9
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Takahashi K, Aritomi S, Honkawa F, Asari S, Hirose K, Konishi A. Efficient and cost-effective differentiation of induced neural crest cells from induced pluripotent stem cells using laminin 211. Regen Ther 2024; 26:749-759. [PMID: 39290629 PMCID: PMC11406167 DOI: 10.1016/j.reth.2024.08.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2024] [Revised: 08/27/2024] [Accepted: 08/29/2024] [Indexed: 09/19/2024] Open
Abstract
Introduction Neural crest cells (NCCs) are cell populations that originate during the formation of neural crest in developmental stages. They are characterized by their multipotency, self-renewal and migration potential. Given their ability to differentiate into various types of cells such as neurons and Schwann cells, NCCs hold promise for cell therapy applications. The conventional method for obtaining NCCs involves inducing them from stem cells like induced pluripotent stem cells (iPSCs), followed by a long-term passage or purification using fluorescence-activated cell sorting (FACS). Although FACS allows high purity induced neural crest cells (iNCCs) to be obtained quickly, it is complex and costly. Therefore, there is a need for a simpler, cost-effective and less time-consuming method for cell therapy application. Methods To select differentiated iNCCs from heterogeneous cell populations quickly without using FACS, we adopted the use of scaffold material full-length laminin 211 (LN211), a recombinant, xeno-free protein suitable for cell therapy. After fist passage on LN211, iNCCs characterization was performed using polymerase chain reaction and flow cytometry. Additionally, proliferation and multipotency to various cells were evaluated. Result The iNCCs obtained using our new method expressed cranial NCC- related genes and exhibited stable proliferation ability for at least 57 days, while maintaining high expression level of the NCCs marker CD271. They demonstrated differentiation ability into several cell types: neurons, astrocytes, melanocytes, smooth muscle cells, osteoblasts, adipocytes and chondrocytes. Furthermore, they could be induced to differentiate into induced mesenchymal stem cells (iMSCs) which retain the essential functions of somatic MSCs. Conclusion In this study, we have developed novel method for obtaining high purity iNCCs differentiated from iPSCs in a short time using LN211 under xeno-free condition. Compared with traditional methods, like FACS or long-term passage, this approach enables the acquisition of a large amount of cells at a lower cost and labor, and it is expected to contribute to stable supply of large scale iNCCs for future cell therapy applications.
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Affiliation(s)
- Kazuma Takahashi
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
| | - Shizuka Aritomi
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
| | - Fumie Honkawa
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
| | - Sayaka Asari
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
| | - Ken Hirose
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
| | - Atsushi Konishi
- Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kanagawa, Kawasaki, 210-8681, Japan
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10
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Seto Y, Ogihara R, Takizawa K, Eiraku M. In vitro induction of patterned branchial arch-like aggregate from human pluripotent stem cells. Nat Commun 2024; 15:1351. [PMID: 38355589 PMCID: PMC10867012 DOI: 10.1038/s41467-024-45285-0] [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: 05/16/2023] [Accepted: 01/19/2024] [Indexed: 02/16/2024] Open
Abstract
Early patterning of neural crest cells (NCCs) in the craniofacial primordium is important for subsequent development of proper craniofacial structures. However, because of the complexity of the environment of developing tissues, surveying the early specification and patterning of NCCs is difficult. In this study, we develop a simplified in vitro 3D model using human pluripotent stem cells to analyze the early stages of facial development. In this model, cranial NCC-like cells spontaneously differentiate from neural plate border-like cells into maxillary arch-like mesenchyme after a long-term culture. Upon the addition of EDN1 and BMP4, these aggregates are converted into a mandibular arch-like state. Furthermore, temporary treatment with EDN1 and BMP4 induces the formation of spatially separated domains expressing mandibular and maxillary arch markers within a single aggregate. These results suggest that this in vitro model is useful for determining the mechanisms underlying cell fate specification and patterning during early facial development.
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Affiliation(s)
- Yusuke Seto
- Laboratory of Developmental Systems, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan.
- Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
| | - Ryoma Ogihara
- Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
| | - Kaori Takizawa
- Laboratory of Developmental Systems, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Mototsugu Eiraku
- Laboratory of Developmental Systems, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan.
- Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
- Institute for Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan.
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11
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Van Haver S, Fan Y, Bekaert SL, Everaert C, Van Loocke W, Zanzani V, Deschildre J, Maestre IF, Amaro A, Vermeirssen V, De Preter K, Zhou T, Kentsis A, Studer L, Speleman F, Roberts SS. Human iPSC modeling recapitulates in vivo sympathoadrenal development and reveals an aberrant developmental subpopulation in familial neuroblastoma. iScience 2024; 27:108096. [PMID: 38222111 PMCID: PMC10784699 DOI: 10.1016/j.isci.2023.108096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Revised: 06/12/2023] [Accepted: 09/26/2023] [Indexed: 01/16/2024] Open
Abstract
Studies defining normal and disrupted human neural crest cell development have been challenging given its early timing and intricacy of development. Consequently, insight into the early disruptive events causing a neural crest related disease such as pediatric cancer neuroblastoma is limited. To overcome this problem, we developed an in vitro differentiation model to recapitulate the normal in vivo developmental process of the sympathoadrenal lineage which gives rise to neuroblastoma. We used human in vitro pluripotent stem cells and single-cell RNA sequencing to recapitulate the molecular events during sympathoadrenal development. We provide a detailed map of dynamically regulated transcriptomes during sympathoblast formation and illustrate the power of this model to study early events of the development of human neuroblastoma, identifying a distinct subpopulation of cell marked by SOX2 expression in developing sympathoblast obtained from patient derived iPSC cells harboring a germline activating mutation in the anaplastic lymphoma kinase (ALK) gene.
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Affiliation(s)
- Stéphane Van Haver
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
| | - Yujie Fan
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY, USA
- Developmental Biology Program, MSKCC, New York, NY 10065, USA
- Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10065, USA
| | - Sarah-Lee Bekaert
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
| | - Celine Everaert
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
| | - Wouter Van Loocke
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
| | - Vittorio Zanzani
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Lab for Computational Biology, Integromics and Gene Regulation (CBIGR), Cancer Research Institute Ghent (CRIG), Ghent, Belgium
- Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium
| | - Joke Deschildre
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Lab for Computational Biology, Integromics and Gene Regulation (CBIGR), Cancer Research Institute Ghent (CRIG), Ghent, Belgium
- Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium
| | - Inés Fernandez Maestre
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Louis V. Gerstner Jr Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Adrianna Amaro
- Department of Pediatrics, MSKCC, New York, NY 10065, USA
| | - Vanessa Vermeirssen
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Lab for Computational Biology, Integromics and Gene Regulation (CBIGR), Cancer Research Institute Ghent (CRIG), Ghent, Belgium
- Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium
| | - Katleen De Preter
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
| | - Ting Zhou
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA
| | - Alex Kentsis
- Department of Pediatrics, MSKCC, New York, NY 10065, USA
- Molecular Pharmacology Program, MSKCC, New York, NY, USA
- Tow Center for Developmental Oncology, MSKCC, New York, NY 10065, USA
- Departments of Pediatrics, Pharmacology and Physiology & Biophysics, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY, USA
- Developmental Biology Program, MSKCC, New York, NY 10065, USA
| | - Frank Speleman
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium
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12
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Huang B, Fu S, Hao Y, Yeung CK, Zhang X, Li E, Xu X, Shao N, Xu RH. Developmental potency of human ES cell-derived mesenchymal stem cells revealed in mouse embryos following blastocyst injection. Cell Rep 2023; 42:113459. [PMID: 37988266 DOI: 10.1016/j.celrep.2023.113459] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Revised: 08/26/2023] [Accepted: 11/02/2023] [Indexed: 11/23/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are present in almost all the tissues in the body, critical for their homeostasis and regeneration. However, the stemness of MSCs is mainly an in vitro observation, and lacking exclusive markers for endogenous MSCs makes it difficult to study the multipotency of MSCs in vivo, especially for human MSCs. To address this hurdle, we injected GFP-tagged human embryonic stem cell (hESC)-derived MSCs (EMSCs) into mouse blastocysts. EMSCs survived well and penetrated both the inner cell mass and trophectoderm, correlating to the higher anti-apoptotic capability of EMSCs than hESCs. Injected EMSCs contributed to skeletal, dermal, and extraembryonic tissues in the resultant chimera and partially rescued skeletal defects in Sox9+/- mouse fetuses. Thus, this study provides evidence for the stemness and developmental capability of human MSCs through chimerization with the mouse blastocyst, serving as a model for studying human mesenchymal and skeletal development.
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Affiliation(s)
- Borong Huang
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Siyi Fu
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Yanan Hao
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Cheung Kwan Yeung
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Xin Zhang
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Enqin Li
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Xiaoling Xu
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Ningyi Shao
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Ren-He Xu
- Center of Reproduction, Development & Aging, and Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China.
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13
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Charney RM, Prasad MS, Juan-Sing C, Patel LJ, Hernandez JC, Wu J, García-Castro MI. Mowat-Wilson syndrome factor ZEB2 controls early formation of human neural crest through BMP signaling modulation. Stem Cell Reports 2023; 18:2254-2267. [PMID: 37890485 PMCID: PMC10679662 DOI: 10.1016/j.stemcr.2023.10.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Revised: 09/30/2023] [Accepted: 10/02/2023] [Indexed: 10/29/2023] Open
Abstract
Mowat-Wilson syndrome is caused by mutations in ZEB2, with patients exhibiting characteristics indicative of neural crest (NC) defects. We examined the contribution of ZEB2 to human NC formation using a model based on human embryonic stem cells. We found ZEB2 to be one of the earliest factors expressed in prospective human NC, and knockdown revealed a role for ZEB2 in establishing the NC state while repressing pre-placodal and non-neural ectoderm genes. Examination of ZEB2 N-terminal mutant NC cells demonstrates its requirement for the repression of enhancers in the NC gene network and proper NC cell terminal differentiation into osteoblasts and peripheral neurons and neuroglia. This ZEB2 mutation causes early misexpression of BMP signaling ligands, which can be rescued by the attenuation of BMP. Our findings suggest that ZEB2 regulates early human NC specification by modulating proper BMP signaling and further elaborate the molecular defects underlying Mowat-Wilson syndrome.
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Affiliation(s)
- Rebekah M Charney
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA.
| | - Maneeshi S Prasad
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA
| | - Czarina Juan-Sing
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA
| | - Lipsa J Patel
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA
| | - Jacqueline C Hernandez
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA
| | - Jie Wu
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA
| | - Martín I García-Castro
- Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA.
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14
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Militi S, Nibhani R, Jalali M, Pauklin S. RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling. Cell Rep 2023; 42:113146. [PMID: 37725511 DOI: 10.1016/j.celrep.2023.113146] [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: 01/15/2023] [Revised: 05/30/2023] [Accepted: 08/31/2023] [Indexed: 09/21/2023] Open
Abstract
The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/β-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.
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Affiliation(s)
- Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Morteza Jalali
- Anne McLaren Laboratory for Regenerative Medicine, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK.
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15
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Arakawa M, Sakamoto Y, Miyagawa Y, Nito C, Takahashi S, Nitahara-Kasahara Y, Suda S, Yamazaki Y, Sakai M, Kimura K, Okada T. iPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress. Mol Ther Methods Clin Dev 2023; 30:333-349. [PMID: 37637385 PMCID: PMC10448333 DOI: 10.1016/j.omtm.2023.07.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 07/11/2023] [Indexed: 08/29/2023]
Abstract
Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.
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Affiliation(s)
- Masafumi Arakawa
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yuki Sakamoto
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yoshitaka Miyagawa
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Chikako Nito
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Laboratory for Clinical Research, Collaborative Research Center, Nippon Medical School, Tokyo, Japan
| | - Shiro Takahashi
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yuko Nitahara-Kasahara
- Division of Molecular and Medical Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Satoshi Suda
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Yoshiyuki Yamazaki
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Mashito Sakai
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Kazumi Kimura
- Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Takashi Okada
- Division of Molecular and Medical Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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16
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Yan M, Tsukasaki M, Muro R, Ando Y, Nakamura K, Komatsu N, Nitta T, Okamura T, Okamoto K, Takayanagi H. Identification of an intronic enhancer regulating RANKL expression in osteocytic cells. Bone Res 2023; 11:43. [PMID: 37563119 PMCID: PMC10415388 DOI: 10.1038/s41413-023-00277-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Revised: 06/22/2023] [Accepted: 07/05/2023] [Indexed: 08/12/2023] Open
Abstract
The bony skeleton is continuously renewed throughout adult life by the bone remodeling process, in which old or damaged bone is removed by osteoclasts via largely unknown mechanisms. Osteocytes regulate bone remodeling by producing the osteoclast differentiation factor RANKL (encoded by the TNFSF11 gene). However, the precise mechanisms underlying RANKL expression in osteocytes are still elusive. Here, we explored the epigenomic landscape of osteocytic cells and identified a hitherto-undescribed osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus. Bioinformatics analyses showed that transcription factors involved in cell death and senescence act on this intronic enhancer region. Single-cell transcriptomic data analysis demonstrated that cell death signaling increased RANKL expression in osteocytic cells. Genetic deletion of the intronic enhancer led to a high-bone-mass phenotype with decreased levels of RANKL in osteocytic cells and osteoclastogenesis in the adult stage, while RANKL expression was not affected in osteoblasts or lymphocytes. These data suggest that osteocytes may utilize a specialized regulatory element to facilitate osteoclast formation at the bone surface to be resorbed by linking signals from cellular senescence/death and RANKL expression.
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Affiliation(s)
- Minglu Yan
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Masayuki Tsukasaki
- Department of Osteoimmunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
| | - Ryunosuke Muro
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yutaro Ando
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
- Department of Microbiology, Tokyo Dental College, Tokyo, Japan
| | - Kazutaka Nakamura
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
- Department of Oral and Maxillofacial Surgery, Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Noriko Komatsu
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Takeshi Nitta
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Tadashi Okamura
- Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Kazuo Okamoto
- Department of Osteoimmunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hiroshi Takayanagi
- Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
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17
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Hong H, Yoon SB, Park JE, Lee JI, Kim HY, Nam HJ, Cho H. MeCP2 dysfunction prevents proper BMP signaling and neural progenitor expansion in brain organoid. Ann Clin Transl Neurol 2023. [PMID: 37302988 DOI: 10.1002/acn3.51799] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 04/26/2023] [Accepted: 05/10/2023] [Indexed: 06/13/2023] Open
Abstract
OBJECTIVES Sporadic mutations in MeCP2 are a hallmark of Rett syndrome (RTT). Many RTT brain organoid models have exhibited pathogenic phenotypes such as decreased spine density and small size of soma with altered electrophysiological signals. However, previous models are mainly focused on the phenotypes observed in the late phase and rarely provide clues for the defect of neural progenitors which generate different types of neurons and glial cells. METHODS We newly established the RTT brain organoid model derived from MeCP2-truncated iPS cells which were genetically engineered by CRISPR/Cas9 technology. By immunofluorescence imaging, we studied the development of NPC pool and its fate specification into glutamatergic neurons or astrocytes in RTT organoids. By total RNA sequencing, we investigated which signaling pathways were altered during the early brain development in RTT organoids. RESULTS Dysfunction of MeCP2 caused the defect of neural rosette formation in the early phase of cortical development. In total transcriptome analysis, BMP pathway-related genes are highly associated with MeCP2 depletion. Moreover, levels of pSMAD1/5 and BMP target genes are excessively increased, and treatment of BMP inhibitors partially rescues the cell cycle progression of neural progenitors. Subsequently, MeCP2 dysfunction reduced the glutamatergic neurogenesis and induced overproduction of astrocytes. Nevertheless, early inhibition of BMP pathway rescued VGLUT1 expression and suppressed astrocyte maturation. INTERPRETATION Our results demonstrate that MeCP2 is required for the expansion of neural progenitor cells by modulating BMP pathway at early stages of development, and this influence persists during neurogenesis and gliogenesis at later stages of brain organoid development.
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Affiliation(s)
- Hyowon Hong
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Sae-Bom Yoon
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Jung Eun Park
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Jung In Lee
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Hyun Young Kim
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Hye Jin Nam
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Heeyeong Cho
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
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18
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Sampayo RG, Sakamoto M, Wang M, Kumar S, Schaffer DV. Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases. Proc Natl Acad Sci U S A 2023; 120:e2219854120. [PMID: 37216516 PMCID: PMC10235963 DOI: 10.1073/pnas.2219854120] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Accepted: 04/24/2023] [Indexed: 05/24/2023] Open
Abstract
During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.
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Affiliation(s)
- Rocío G. Sampayo
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Mason Sakamoto
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Madeline Wang
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - Sanjay Kumar
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
| | - David V. Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720
- Department of Bioengineering, University of California, Berkeley, CA94720
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19
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Takayama Y, Akagi Y, Kida YS. Deciphering the Molecular Mechanisms of Autonomic Nervous System Neuron Induction through Integrative Bioinformatics Analysis. Int J Mol Sci 2023; 24:ijms24109053. [PMID: 37240399 DOI: 10.3390/ijms24109053] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2023] [Revised: 05/18/2023] [Accepted: 05/19/2023] [Indexed: 05/28/2023] Open
Abstract
In vitro derivation of human neurons in the autonomic nervous system (ANS) is an important technology, given its regulatory roles in maintaining homeostasis in the human body. Although several induction protocols for autonomic lineages have been reported, the regulatory machinery remains largely undefined, primarily due to the absence of a comprehensive understanding of the molecular mechanism regulating human autonomic induction in vitro. In this study, our objective was to pinpoint key regulatory components using integrated bioinformatics analysis. A protein-protein interaction network construction for the proteins encoded by the differentially expressed genes from our RNA sequencing data, and conducting subsequent module analysis, we identified distinct gene clusters and hub genes involved in the induction of autonomic lineages. Moreover, we analyzed the impact of transcription factor (TF) activity on target gene expression, revealing enhanced autonomic TF activity that could lead to the induction of autonomic lineages. The accuracy of this bioinformatics analysis was corroborated by employing calcium imaging to observe specific responses to certain ANS agonists. This investigation offers novel insights into the regulatory machinery in the generation of neurons in the ANS, which would be valuable for further understanding and precise regulation of autonomic induction and differentiation.
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Affiliation(s)
- Yuzo Takayama
- Cellular and Molecular Biotechnology Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-41, 1-1-1 Higashi, Tsukuba 305-8565, Japan
| | - Yuka Akagi
- Cellular and Molecular Biotechnology Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-41, 1-1-1 Higashi, Tsukuba 305-8565, Japan
- Tsukuba Life Science Innovation Program (T-LSI), School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8572, Japan
| | - Yasuyuki S Kida
- Cellular and Molecular Biotechnology Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-41, 1-1-1 Higashi, Tsukuba 305-8565, Japan
- School of Integrative & Global Majors, University of Tsukuba, Tsukuba 305-8572, Japan
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20
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Zujur D, Al-Akashi Z, Nakamura A, Zhao C, Takahashi K, Aritomi S, Theoputra W, Kamiya D, Nakayama K, Ikeya M. Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair. Front Cell Dev Biol 2023; 11:1140717. [PMID: 37234772 PMCID: PMC10206169 DOI: 10.3389/fcell.2023.1140717] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Accepted: 04/24/2023] [Indexed: 05/28/2023] Open
Abstract
Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.
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Affiliation(s)
- Denise Zujur
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Ziadoon Al-Akashi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Anna Nakamura
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Chengzhu Zhao
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Laboratory of Skeletal Development and Regeneration, Institute of Life Sciences, Chongqing Medical University, Chongqing, China
| | - Kazuma Takahashi
- Research Institute for Bioscience Product and Fine Chemicals, Ajinomoto Co., Inc, Kawasaki, Japan
| | - Shizuka Aritomi
- Research Institute for Bioscience Product and Fine Chemicals, Ajinomoto Co., Inc, Kawasaki, Japan
| | - William Theoputra
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Daisuke Kamiya
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Takeda-CiRA Joint Program (T-CiRA), Kanagawa, Japan
| | - Koichi Nakayama
- Center for Regenerative Medicine Research, Faculty of Medicine, Saga University, Saga, Japan
| | - Makoto Ikeya
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Takeda-CiRA Joint Program (T-CiRA), Kanagawa, Japan
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21
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Majd H, Amin S, Ghazizadeh Z, Cesiulis A, Arroyo E, Lankford K, Majd A, Farahvashi S, Chemel AK, Okoye M, Scantlen MD, Tchieu J, Calder EL, Le Rouzic V, Shibata B, Arab A, Goodarzi H, Pasternak G, Kocsis JD, Chen S, Studer L, Fattahi F. Deriving Schwann cells from hPSCs enables disease modeling and drug discovery for diabetic peripheral neuropathy. Cell Stem Cell 2023; 30:632-647.e10. [PMID: 37146583 PMCID: PMC10249419 DOI: 10.1016/j.stem.2023.04.006] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 01/11/2023] [Accepted: 04/10/2023] [Indexed: 05/07/2023]
Abstract
Schwann cells (SCs) are the primary glia of the peripheral nervous system. SCs are involved in many debilitating disorders, including diabetic peripheral neuropathy (DPN). Here, we present a strategy for deriving SCs from human pluripotent stem cells (hPSCs) that enables comprehensive studies of SC development, physiology, and disease. hPSC-derived SCs recapitulate the molecular features of primary SCs and are capable of in vitro and in vivo myelination. We established a model of DPN that revealed the selective vulnerability of SCs to high glucose. We performed a high-throughput screen and found that an antidepressant drug, bupropion, counteracts glucotoxicity in SCs. Treatment of hyperglycemic mice with bupropion prevents their sensory dysfunction, SC death, and myelin damage. Further, our retrospective analysis of health records revealed that bupropion treatment is associated with a lower incidence of neuropathy among diabetic patients. These results highlight the power of this approach for identifying therapeutic candidates for DPN.
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Affiliation(s)
- Homa Majd
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Sadaf Amin
- Department of Surgery, Weill Cornell Medicine, New York, NY 10065, USA
| | - Zaniar Ghazizadeh
- Department of Surgery, Weill Cornell Medicine, New York, NY 10065, USA
| | - Andrius Cesiulis
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Edgardo Arroyo
- Neuroscience Research Center, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA; Department of Neurology, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA
| | - Karen Lankford
- Neuroscience Research Center, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA; Department of Neurology, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA
| | - Alireza Majd
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Sina Farahvashi
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Angeline K Chemel
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Mesomachukwu Okoye
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Megan D Scantlen
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA
| | - Jason Tchieu
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Elizabeth L Calder
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Valerie Le Rouzic
- Molecular Pharmacology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA; Department of Neurology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Bradley Shibata
- Biological Electron Microscopy Facility, UCD, Davis, CA 95616, USA
| | - Abolfazl Arab
- Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94158, USA
| | - Hani Goodarzi
- Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94158, USA; Department of Urology, UCSF, San Francisco, CA 94158, USA
| | - Gavril Pasternak
- Molecular Pharmacology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA; Department of Neurology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Jeffery D Kocsis
- Neuroscience Research Center, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA; Department of Neurology, Yale University School of Medicine and VA Healthcare System, West Haven, CT 06516, USA
| | - Shuibing Chen
- Department of Surgery, Weill Cornell Medicine, New York, NY 10065, USA; Center of Genomic Health, Weill Cornell Medicine, New York, NY 10065, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA.
| | - Faranak Fattahi
- Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94158, USA; Program in Craniofacial Biology, UCSF, San Francisco, CA 94110, USA.
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22
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Zhang Y, Yi Y, Xiao X, Hu L, Xu J, Zheng D, Koc HC, Chan UI, Meng Y, Lu L, Liu W, Xu X, Shao N, Cheung ECW, Xu RH, Chen G. Definitive Endodermal Cells Supply an in vitro Source of Mesenchymal Stem/Stromal Cells. Commun Biol 2023; 6:476. [PMID: 37127734 PMCID: PMC10151361 DOI: 10.1038/s42003-023-04810-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Accepted: 04/05/2023] [Indexed: 05/03/2023] Open
Abstract
Mesenchymal stem/Stromal cells (MSCs) have great therapeutic potentials, and they have been isolated from various tissues and organs including definitive endoderm (DE) organs, such as the lung, liver and intestine. MSCs have been induced from human pluripotent stem cells (hPSCs) through multiple embryonic lineages, including the mesoderm, neural crest, and extraembryonic cells. However, it remains unclear whether hPSCs could give rise to MSCs in vitro through the endodermal lineage. Here, we report that hPSC-derived, SOX17+ definitive endoderm progenitors can further differentiate to cells expressing classic MSC markers, which we name definitive endoderm-derived MSCs (DE-MSCs). Single cell RNA sequencing demonstrates the stepwise emergence of DE-MSCs, while endoderm-specific gene expression can be elevated by signaling modulation. DE-MSCs display multipotency and immunomodulatory activity in vitro and possess therapeutic effects in a mouse ulcerative colitis model. This study reveals that, in addition to the other germ layers, the definitive endoderm can also contribute to MSCs and DE-MSCs could be a cell source for regenerative medicine.
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Affiliation(s)
- Yumeng Zhang
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Ye Yi
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Xia Xiao
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Lingling Hu
- Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Jiaqi Xu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Dejin Zheng
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Ho Cheng Koc
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Un In Chan
- Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Ya Meng
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Zhuhai Precision Medical Center, Zhuhai People's Hospital, Jinan University, Zhuhai, Guangdong, China
| | - Ligong Lu
- Zhuhai Precision Medical Center, Zhuhai People's Hospital, Jinan University, Zhuhai, Guangdong, China
| | - Weiwei Liu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- Biological Imaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Macau SAR, China
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China
| | - Xiaoling Xu
- Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China
| | - Ningyi Shao
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China
| | - Edwin Chong Wing Cheung
- Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China
| | - Ren-He Xu
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China.
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China.
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China.
| | - Guokai Chen
- Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China.
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China.
- MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China.
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23
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Aldoghachi AF, Loh JK, Wang ML, Yang YP, Chien CS, Teh HX, Omar AH, Cheong SK, Yeap SK, Ho WY, Ong AHK. Current developments and therapeutic potentials of exosomes from induced pluripotent stem cells-derived mesenchymal stem cells. J Chin Med Assoc 2023; 86:356-365. [PMID: 36762931 DOI: 10.1097/jcma.0000000000000899] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/11/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are multipotent cells derived from adult human tissues that have the ability to proliferate in vitro and maintain their multipotency, making them attractive cell sources for regenerative medicine. However, MSCs reportedly show limited proliferative capacity with inconsistent therapeutic outcomes due to their heterogeneous nature. On the other hand, induced pluripotent stem cells (iPSC) have emerged as an alternative source for the production of various specialized cell types via their ability to differentiate from all three primary germ layers, leading to applications in regenerative medicine, disease modeling, and drug therapy. Notably, iPSCs can differentiate into MSCs in monolayer, commonly referred to as induced mesenchymal stem cells (iMSCs). These cells show superior therapeutic qualities compared with adult MSCs as the applications of the latter are restricted by passage number and autoimmune rejection when applied in tissue regeneration trials. Furthermore, increasing evidence shows that the therapeutic properties of stem cells are a consequence of the paracrine effects mediated by their secretome such as from exosomes, a type of extracellular vesicle secreted by most cell types. Several studies that investigated the potential of exosomes in regenerative medicine and therapy have revealed promising results. Therefore, this review focuses on the recent findings of exosomes secreted from iMSCs as a potential noncell-based therapy.
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Affiliation(s)
- Ahmed Faris Aldoghachi
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Jit-Kai Loh
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, ROC
| | - Mong-Lien Wang
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, ROC
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
| | - Yi-Ping Yang
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, ROC
| | - Chian-Shiu Chien
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, ROC
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
| | - Hui Xin Teh
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Alfaqih Hussain Omar
- Biomedicine Programme, School of Health Sciences, Universiti Sains Malaysia, Malaysia
| | - Soon-Keng Cheong
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
- National Cancer Council (MAKNA), Kuala Lumpur, Malaysia
| | - Swee Keong Yeap
- Marine Biotechnology, China-ASEAN College of Marine Sciences, Xiamen University Malaysia Campus, Jalan Sunsuria, Bandar Sunsuria, Sepang, Selangor, Malaysia
| | - Wan Yong Ho
- Faculty of Sciences and Engineering, University of Nottingham Malaysia, Semenyih, Malaysia
| | - Alan Han-Kiat Ong
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
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24
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Fan Y, Hackland J, Baggiolini A, Hung LY, Zhao H, Zumbo P, Oberst P, Minotti AP, Hergenreder E, Najjar S, Huang Z, Cruz NM, Zhong A, Sidharta M, Zhou T, de Stanchina E, Betel D, White RM, Gershon M, Margolis KG, Studer L. hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease. Cell Stem Cell 2023; 30:264-282.e9. [PMID: 36868194 PMCID: PMC10034921 DOI: 10.1016/j.stem.2023.02.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 11/22/2022] [Accepted: 02/02/2023] [Indexed: 03/05/2023]
Abstract
The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.
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Affiliation(s)
- Yujie Fan
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10065, USA
| | - James Hackland
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Arianna Baggiolini
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Lin Y Hung
- Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA
| | - Huiyong Zhao
- Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Paul Zumbo
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA; Applied Bioinformatics Core, Weill Cornell Medicine, New York, NY 10065, USA
| | - Polina Oberst
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Andrew P Minotti
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10065, USA
| | - Emiliano Hergenreder
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10065, USA
| | - Sarah Najjar
- Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA
| | - Zixing Huang
- Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA
| | - Nelly M Cruz
- Cancer Biology and Genetics and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Aaron Zhong
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; The SKI Stem Cell Research Facility, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Mega Sidharta
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; The SKI Stem Cell Research Facility, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Ting Zhou
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; The SKI Stem Cell Research Facility, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Elisa de Stanchina
- Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Doron Betel
- Applied Bioinformatics Core, Weill Cornell Medicine, New York, NY 10065, USA; Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Richard M White
- Cancer Biology and Genetics and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Michael Gershon
- Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA
| | - Kara Gross Margolis
- Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA; Department of Pediatrics, NYU Grossman School of Medicine, New York, NY 10010, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
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25
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Ahmad MH, Ghosh B, Rizvi MA, Ali M, Kaur L, Mondal AC. Neural crest cells development and neuroblastoma progression: Role of Wnt signaling. J Cell Physiol 2023; 238:306-328. [PMID: 36502519 DOI: 10.1002/jcp.30931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Revised: 09/19/2022] [Accepted: 11/29/2022] [Indexed: 12/14/2022]
Abstract
Neuroblastoma (NB) is one of the most common heterogeneous extracranial cancers in infancy that arises from neural crest (NC) cells of the sympathetic nervous system. The Wnt signaling pathway, both canonical and noncanonical pathway, is a highly conserved signaling pathway that regulates the development and differentiation of the NC cells during embryogenesis. Reports suggest that aberrant activation of Wnt ligands/receptors in Wnt signaling pathways promote progression and relapse of NB. Wnt signaling pathways regulate NC induction and migration in a similar manner; it regulates proliferation and metastasis of NB. Inhibiting the Wnt signaling pathway or its ligands/receptors induces apoptosis and abrogates proliferation and tumorigenicity in all major types of NB cells. Here, we comprehensively discuss the Wnt signaling pathway and its mechanisms in regulating the development of NC and NB pathogenesis. This review highlights the implications of aberrant Wnt signaling in the context of etiology, progression, and relapse of NB. We have also described emerging strategies for Wnt-based therapies against the progression of NB that will provide new insights into the development of Wnt-based therapeutic strategies for NB.
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Affiliation(s)
- Mir Hilal Ahmad
- School of Life Sciences, Laboratory of Cellular and Molecular Neurobiology, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.,Genome Biology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi, India
| | - Balaram Ghosh
- Department of Clinical Pharmacology, Midnapore Medical College & Hospital, West Bengal, Medinipur, India
| | - Moshahid Alam Rizvi
- Genome Biology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi, India
| | - Mansoor Ali
- School of Life Sciences, Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
| | - Loveleena Kaur
- Division of Cancer Pharmacology, Indian Institute of Integrative Medicine (IIIM), Srinagar, India
| | - Amal Chandra Mondal
- School of Life Sciences, Laboratory of Cellular and Molecular Neurobiology, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
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26
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Neaverson A, Andersson MHL, Arshad OA, Foulser L, Goodwin-Trotman M, Hunter A, Newman B, Patel M, Roth C, Thwaites T, Kilpinen H, Hurles ME, Day A, Gerety SS. Differentiation of human induced pluripotent stem cells into cortical neural stem cells. Front Cell Dev Biol 2023; 10:1023340. [PMID: 36684426 PMCID: PMC9849742 DOI: 10.3389/fcell.2022.1023340] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Accepted: 12/15/2022] [Indexed: 01/07/2023] Open
Abstract
Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.
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Affiliation(s)
- Alexandra Neaverson
- Wellcome Sanger Institute, Cambridge, United Kingdom
- Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom
| | | | | | - Luke Foulser
- Wellcome Sanger Institute, Cambridge, United Kingdom
- Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom
| | | | - Adam Hunter
- Wellcome Sanger Institute, Cambridge, United Kingdom
| | - Ben Newman
- Wellcome Sanger Institute, Cambridge, United Kingdom
| | - Minal Patel
- Wellcome Sanger Institute, Cambridge, United Kingdom
| | - Charlotte Roth
- UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom
| | | | - Helena Kilpinen
- Wellcome Sanger Institute, Cambridge, United Kingdom
- Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom
- UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom
- Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
- Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Matthew E Hurles
- Wellcome Sanger Institute, Cambridge, United Kingdom
- Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom
| | - Andrew Day
- Wellcome Sanger Institute, Cambridge, United Kingdom
| | - Sebastian S Gerety
- Wellcome Sanger Institute, Cambridge, United Kingdom
- Open Targets, Wellcome Genome Campus, Hinxton, United Kingdom
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27
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Current Application of iPS Cells in the Dental Tissue Regeneration. Biomedicines 2022; 10:biomedicines10123269. [PMID: 36552025 PMCID: PMC9775967 DOI: 10.3390/biomedicines10123269] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2022] [Revised: 12/12/2022] [Accepted: 12/14/2022] [Indexed: 12/24/2022] Open
Abstract
When teeth and periodontal tissues are severely damaged by severe caries, trauma, and periodontal disease, such cases may be subject to tooth extraction. As tooth loss leads to the deterioration of quality of life, the development of regenerative medicine for tooth and periodontal tissue is desired. Induced pluripotent stem cells (iPS cells) are promising cell resources for dental tissue regeneration because they offer high self-renewal and pluripotency, along with fewer ethical issues than embryonic stem cells. As iPS cells retain the epigenetic memory of donor cells, they have been established from various dental tissues for dental tissue regeneration. This review describes the regeneration of dental tissue using iPS cells. It is important to mimic the process of tooth development in dental tissue regeneration using iPS cells. Although iPS cells had safety issues in clinical applications, they have been overcome in recent years. Dental tissue regeneration using iPS cells has not yet been established, but it is expected in the future.
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28
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Badiola-Mateos M, Osaki T, Kamm RD, Samitier J. In vitro modelling of human proprioceptive sensory neurons in the neuromuscular system. Sci Rep 2022; 12:21318. [PMID: 36494423 PMCID: PMC9734133 DOI: 10.1038/s41598-022-23565-3] [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: 04/21/2022] [Accepted: 11/02/2022] [Indexed: 12/13/2022] Open
Abstract
Proprioceptive sensory neurons (pSN) are an essential and undervalued part of the neuromuscular circuit. A protocol to differentiate healthy and amyotrophic lateral sclerosis (ALS) human neural stem cells (hNSC) into pSN, and their comparison with the motor neuron (MN) differentiation process from the same hNSC sources, facilitated the development of in vitro co-culture platforms. The obtained pSN spheroids cultured interact with human skeletal myocytes showing the formation of annulospiral wrapping-like structures between TrkC + neurons and a multinucleated muscle fibre, presenting synaptic bouton-like structures in the contact point. The comparative analysis of the genetic profile performed in healthy and sporadic ALS hNSC differentiated to pSN suggested that basal levels of ETV1, critical for motor feedback from pSN, were much lower for ALS samples and that the differences between healthy and ALS samples, suggest the involvement of pSN in ALS pathology development and progression.
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Affiliation(s)
- Maider Badiola-Mateos
- grid.424736.00000 0004 0536 2369Institute for Bioengineering of Catalonia (IBEC)—Barcelona Institute of Science and Technology, 08028 Barcelona, Spain ,grid.5841.80000 0004 1937 0247Department of Electronic and Biomedical Engineering, Universitat de Barcelona, 08028 Barcelona, Spain ,grid.116068.80000 0001 2341 2786Department of Biological Engineering, Massachusetts Institute of Technology (MIT), 500 Technology Square, MIT Building, Cambridge, MA 02139 USA ,grid.263145.70000 0004 1762 600XPresent Address: The BioRobotics Institute, Department of Excellence in Robotics and AI, Scuola Superiore Sant’Anna, 56127 Pisa, Italy
| | - Tatsuya Osaki
- grid.116068.80000 0001 2341 2786Department of Biological Engineering, Massachusetts Institute of Technology (MIT), 500 Technology Square, MIT Building, Cambridge, MA 02139 USA ,grid.26999.3d0000 0001 2151 536XPresent Address: Institute of Industrial Science, The University of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo, 153-8505 Japan
| | - Roger Dale Kamm
- grid.116068.80000 0001 2341 2786Department of Biological Engineering, Massachusetts Institute of Technology (MIT), 500 Technology Square, MIT Building, Cambridge, MA 02139 USA ,grid.116068.80000 0001 2341 2786Department of Mechanical Engineering, Massachusetts Institute of Technology, 500 Technology Square, MIT Building, Cambridge, MA 02139 USA
| | - Josep Samitier
- grid.424736.00000 0004 0536 2369Institute for Bioengineering of Catalonia (IBEC)—Barcelona Institute of Science and Technology, 08028 Barcelona, Spain ,grid.5841.80000 0004 1937 0247Department of Electronic and Biomedical Engineering, Universitat de Barcelona, 08028 Barcelona, Spain ,grid.512890.7Centro de Investigación Biomédica en Red (CIBER-BBN), 28029 Madrid, Spain
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29
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Migration deficits of the neural crest caused by CXADR triplication in a human Down syndrome stem cell model. Cell Death Dis 2022; 13:1018. [PMID: 36470861 PMCID: PMC9722909 DOI: 10.1038/s41419-022-05481-6] [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: 02/06/2022] [Revised: 11/24/2022] [Accepted: 11/28/2022] [Indexed: 12/07/2022]
Abstract
Down syndrome (DS) is the most common chromosomal abnormality in live-born infants and is caused by trisomy of chromosome 21. Most individuals with DS display craniofacial dysmorphology, including reduced sizes of the skull, maxilla, and mandible. However, the underlying pathogenesis remains largely unknown. Since the craniofacial skeleton is mainly formed by the neural crest, whether neural crest developmental defects are involved in the craniofacial anomalies of individuals with DS needs to be investigated. Here, we successfully derived DS-specific human induced pluripotent stem cells (hiPSCs) using a Sendai virus vector. When DS-hiPSCs were induced to differentiate into the neural crest, we found that trisomy 21 (T21) did not influence cell proliferation or apoptosis. However, the migratory ability of differentiated cells was significantly compromised, thus resulting in a substantially lower number of postmigratory cranial neural crest stem cells (NCSCs) in the DS group than in the control group. We further discovered that the migration defects could be partially attributed to the triplication of the coxsackievirus and adenovirus receptor gene (CXADR; an adhesion protein) in the DS group cells, since knockdown of CXADR substantially recovered the cell migratory ability and generation of postmigratory NCSCs in the DS group. Thus, the migratory deficits of neural crest cells may be an underlying cause of craniofacial dysmorphology in individuals with DS, which may suggest potential targets for therapeutic intervention to ameliorate craniofacial or other neural crest-related anomalies in DS.
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30
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Hörner SJ, Couturier N, Gueiber DC, Hafner M, Rudolf R. Development and In Vitro Differentiation of Schwann Cells. Cells 2022; 11:3753. [PMID: 36497014 PMCID: PMC9739763 DOI: 10.3390/cells11233753] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 11/16/2022] [Accepted: 11/22/2022] [Indexed: 11/25/2022] Open
Abstract
Schwann cells are glial cells of the peripheral nervous system. They exist in several subtypes and perform a variety of functions in nerves. Their derivation and culture in vitro are interesting for applications ranging from disease modeling to tissue engineering. Since primary human Schwann cells are challenging to obtain in large quantities, in vitro differentiation from other cell types presents an alternative. Here, we first review the current knowledge on the developmental signaling mechanisms that determine neural crest and Schwann cell differentiation in vivo. Next, an overview of studies on the in vitro differentiation of Schwann cells from multipotent stem cell sources is provided. The molecules frequently used in those protocols and their involvement in the relevant signaling pathways are put into context and discussed. Focusing on hiPSC- and hESC-based studies, different protocols are described and compared, regarding cell sources, differentiation methods, characterization of cells, and protocol efficiency. A brief insight into developments regarding the culture and differentiation of Schwann cells in 3D is given. In summary, this contribution provides an overview of the current resources and methods for the differentiation of Schwann cells, it supports the comparison and refinement of protocols and aids the choice of suitable methods for specific applications.
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Affiliation(s)
- Sarah Janice Hörner
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
- Center for Mass Spectrometry and Optical Spectroscopy, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
| | - Nathalie Couturier
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
- Center for Mass Spectrometry and Optical Spectroscopy, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
| | - Daniele Caroline Gueiber
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
- Center for Mass Spectrometry and Optical Spectroscopy, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Department of Electronics Engineering, Federal University of Technology Paraná, Ponta Grossa 84017-220, Brazil
| | - Mathias Hafner
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Institute of Medical Technology, Heidelberg University and Mannheim University of Applied Sciences, 69117 Heidelberg, Germany
| | - Rüdiger Rudolf
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
- Center for Mass Spectrometry and Optical Spectroscopy, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
- Institute of Medical Technology, Heidelberg University and Mannheim University of Applied Sciences, 69117 Heidelberg, Germany
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31
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iPSC-neural crest derived cells embedded in 3D printable bio-ink promote cranial bone defect repair. Sci Rep 2022; 12:18701. [PMID: 36333414 PMCID: PMC9636385 DOI: 10.1038/s41598-022-22502-8] [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: 06/27/2022] [Accepted: 10/17/2022] [Indexed: 11/06/2022] Open
Abstract
Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stromal cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-derived mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The study aimed to evaluate the potential of iPSC-derived cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Cell viability, osteogenic potential of cells, and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. The osteogenic potential of bio-ink-cell constructs were evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro μCT. For in vivo testing, two 2 mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6, Ink-Bone + iNCC-MPC-BMP6, or left untreated. For follow-up, µCT was performed at weeks 0, 4, and 8 weeks. At the time of sacrifice (week 8), histological and immunofluorescent analyses were performed. Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. The combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between the implant and host bone compared to the other groups. The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.
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32
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Gao P, Liu S, Wang X, Ikeya M. Dental applications of induced pluripotent stem cells and their derivatives. JAPANESE DENTAL SCIENCE REVIEW 2022; 58:162-171. [PMID: 35516907 PMCID: PMC9065891 DOI: 10.1016/j.jdsr.2022.03.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2021] [Revised: 02/24/2022] [Accepted: 03/17/2022] [Indexed: 11/26/2022] Open
Abstract
Periodontal tissue regeneration is the ideal tactic for treating periodontitis. Tooth regeneration is the potential strategy to restore the lost teeth. With infinite self-renewal, broad differentiation potential, and less ethical issues than embryonic stem cells, induced pluripotent stem cells (iPSCs) are promising cell resource for periodontal and tooth regeneration. This review summarized the optimized technologies of generating iPSC lines and application of iPSC derivatives, which reduce the risk of tumorigenicity. Given that iPSCs may have epigenetic memory from the donor tissue and tend to differentiate into lineages along with the donor cells, iPSCs derived from dental tissues may benefit for personalized dental application. Neural crest cells (NCCs) and mesenchymal stem or stomal cells (MSCs) are lineage-specific progenitor cells derived from iPSCs and can differentiate into multilineage cell types. This review introduced the updated technologies of inducing iPSC-derived NCCs and iPSC-derived MSCs and their application in periodontal and tooth regeneration. Given the complexity of periodontal tissues and teeth, it is crucial to elucidate the integrated mechanisms of all constitutive cells and the spatio-temporal interactions among them to generate structural periodontal tissues and functional teeth. Thus, more sophisticated studies in vitro and in vivo and even preclinical investigations need to be conducted.
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Affiliation(s)
- Pan Gao
- State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of General and Emergency Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Shan Liu
- State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
- Division of Oral Ecology and Biochemistry, Oral Biology, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Xiaoyi Wang
- State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
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33
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Ducos B, Bensimon D, Scerbo P. Vertebrate Cell Differentiation, Evolution, and Diseases: The Vertebrate-Specific Developmental Potential Guardians VENTX/ NANOG and POU5/ OCT4 Enter the Stage. Cells 2022; 11:cells11152299. [PMID: 35892595 PMCID: PMC9331430 DOI: 10.3390/cells11152299] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Revised: 07/09/2022] [Accepted: 07/13/2022] [Indexed: 01/02/2023] Open
Abstract
During vertebrate development, embryonic cells pass through a continuum of transitory pluripotent states that precede multi-lineage commitment and morphogenesis. Such states are referred to as “refractory/naïve” and “competent/formative” pluripotency. The molecular mechanisms maintaining refractory pluripotency or driving the transition to competent pluripotency, as well as the cues regulating multi-lineage commitment, are evolutionarily conserved. Vertebrate-specific “Developmental Potential Guardians” (vsDPGs; i.e., VENTX/NANOG, POU5/OCT4), together with MEK1 (MAP2K1), coordinate the pluripotency continuum, competence for multi-lineage commitment and morphogenesis in vivo. During neurulation, vsDPGs empower ectodermal cells of the neuro-epithelial border (NEB) with multipotency and ectomesenchyme potential through an “endogenous reprogramming” process, giving rise to the neural crest cells (NCCs). Furthermore, vsDPGs are expressed in undifferentiated-bipotent neuro-mesodermal progenitor cells (NMPs), which participate in posterior axis elongation and growth. Finally, vsDPGs are involved in carcinogenesis, whereby they confer selective advantage to cancer stem cells (CSCs) and therapeutic resistance. Intriguingly, the heterogenous distribution of vsDPGs in these cell types impact on cellular potential and features. Here, we summarize the findings about the role of vsDPGs during vertebrate development and their selective advantage in evolution. Our aim to present a holistic view regarding vsDPGs as facilitators of both cell plasticity/adaptability and morphological innovation/variation. Moreover, vsDPGs may also be at the heart of carcinogenesis by allowing malignant cells to escape from physiological constraints and surveillance mechanisms.
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Affiliation(s)
- Bertrand Ducos
- LPENS, PSL, CNRS, 24 rue Lhomond, 75005 Paris, France
- IBENS, PSL, CNRS, 46 rue d’Ulm, 75005 Paris, France
- High Throughput qPCR Core Facility, ENS, PSL, 46 rue d’Ulm, 75005 Paris, France
- Correspondence: (B.D.); (D.B.); (P.S.)
| | - David Bensimon
- LPENS, PSL, CNRS, 24 rue Lhomond, 75005 Paris, France
- IBENS, PSL, CNRS, 46 rue d’Ulm, 75005 Paris, France
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90094, USA
- Correspondence: (B.D.); (D.B.); (P.S.)
| | - Pierluigi Scerbo
- LPENS, PSL, CNRS, 24 rue Lhomond, 75005 Paris, France
- IBENS, PSL, CNRS, 46 rue d’Ulm, 75005 Paris, France
- Correspondence: (B.D.); (D.B.); (P.S.)
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34
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On the evolutionary origins and regionalization of the neural crest. Semin Cell Dev Biol 2022; 138:28-35. [PMID: 35787974 DOI: 10.1016/j.semcdb.2022.06.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Revised: 04/19/2022] [Accepted: 06/19/2022] [Indexed: 11/22/2022]
Abstract
The neural crest is a vertebrate-specific embryonic stem cell population that gives rise to a vast array of cell types throughout the animal body plan. These cells are first born at the edges of the central nervous system, from which they migrate extensively and differentiate into multiple cellular derivatives. Given the unique set of structures these cells comprise, the origin of the neural crest is thought to have important implications for the evolution and diversification of the vertebrate clade. In jawed vertebrates, neural crest cells exist as distinct subpopulations along the anterior-posterior axis. These subpopulations differ in terms of their respective differentiation potential and cellular derivatives. Thus, the modern neural crest is characterized as multipotent, migratory, and regionally segregated throughout the embryo. Here, we retrace the evolutionary origins of the neural crest, from the appearance of conserved regulatory circuitry in basal chordates to the emergence of neural crest subpopulations in higher vertebrates. Finally, we discuss a stepwise trajectory by which these cells may have arisen and diversified throughout vertebrate evolution.
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35
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Zhang W, Ross PJ, Ellis J, Salter MW. Targeting NMDA receptors in neuropsychiatric disorders by drug screening on human neurons derived from pluripotent stem cells. Transl Psychiatry 2022; 12:243. [PMID: 35680847 PMCID: PMC9184461 DOI: 10.1038/s41398-022-02010-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 05/25/2022] [Accepted: 05/27/2022] [Indexed: 01/04/2023] Open
Abstract
NMDA receptors (NMDARs), a prominent subtype of glutamatergic receptors, are implicated in the pathogenesis and development of neuropsychiatric disorders such as epilepsy, intellectual disability, autism spectrum disorder, and schizophrenia, and are therefore a potential therapeutic target in treating these disorders. Neurons derived from induced pluripotent stem cells (iPSCs) have provided the opportunity to investigate human NMDARs in their native environment. In this review, we describe the expression, function, and regulation of NMDARs in human iPSC-derived neurons and discuss approaches for utilizing human neurons for identifying potential drugs that target NMDARs in the treatment of neuropsychiatric disorders. A challenge in studying NMDARs in human iPSC-derived neurons is a predominance of those receptors containing the GluN2B subunit and low synaptic expression, suggesting a relatively immature phenotype of these neurons and delayed development of functional NMDARs. We outline potential approaches for improving neuronal maturation of human iPSC-derived neurons and accelerating the functional expression of NMDARs. Acceleration of functional expression of NMDARs in human iPSC-derived neurons will improve the modeling of neuropsychiatric disorders and facilitate the discovery and development of novel therapeutics targeting NMDARs for the treatment of these disorders.
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Affiliation(s)
- Wenbo Zhang
- Program in Neurosciences & Mental Health, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada
| | - P Joel Ross
- Biology Department, University of Prince Edward Island, Charlottetown, PE, C1A 4P3, Canada
| | - James Ellis
- Program in Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, M5S 1A8, Canada
| | - Michael W Salter
- Program in Neurosciences & Mental Health, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada.
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.
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36
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Jin W, He Y, Li T, Long F, Qin X, Yuan Y, Gao G, Shakhawat HM, Liu X, Jin G, Zhou Z. Rapid and robust derivation of mesenchymal stem cells from human pluripotent stem cells via temporal induction of neuralized ectoderm. Cell Biosci 2022; 12:31. [PMID: 35292115 PMCID: PMC8922747 DOI: 10.1186/s13578-022-00753-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Accepted: 01/31/2022] [Indexed: 11/10/2022] Open
Abstract
Background Mesenchymal stem cells (MSCs) are emerging as the mainstay of regenerative medicine because of their ability to differentiate into multiple cell lineages. The infinite proliferative potential of human pluripotent stem cells (PSCs) grants an unlimited supply of MSCs. Despite their great potential in therapeutic applications, several drawbacks have hindered its clinical translation, including limited number of replication, compromised potential and altered function in late passages. The aim of this study is to establish an efficient method for the production of MSCs from pluripotent stem cells for potential clinical application in rare human disease Hutchinson-Gilford progeria syndrome. Results We established a robust method allowing rapid derivation of MSCs from both human iPSCs and ESCs via a temporal induction of neural ectoderm in chemically defined media. The iPSC- and ESC-derived MSCs satisfy the standard criteria of surface markers. They exhibited a high tri-lineage differentiation potential with over 90% transcriptional similarity to the primary MSCs derived from bone marrow. To evaluate the potential application of this method in disease modeling, MSCs were generated from iPSCs derived from a patient with Hutchinson-Gilford progeria syndrome (HGPS-MSCs) and from mutation-rectified HGPS-iPSCs (cHGPS-MSCs). HGPS-MSCs manifested accelerated senescence whereas mutation rectification rescued cellular senescence in HGPS-MSCs. Conclusions The robust method of MSC derivation from ESCs and iPSCs provides an efficient approach to rapidly generate sufficient MSCs for in vitro disease modeling and clinical applications. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-022-00753-2.
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Affiliation(s)
- Wei Jin
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.,Chinese Academy of Sciences Regenerative Medicine of Hong Kong, Hong Kong, China
| | - Yi He
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Tuo Li
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.,Department of Endocrinology, Chang Zheng Hospital, Shanghai, 200003, China
| | - Fei Long
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Xin Qin
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Yuan Yuan
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.,Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Institute for Aging Research, Guangdong Medical University, Dongguan, China
| | - Ge Gao
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Hosen Md Shakhawat
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Xinguang Liu
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Institute for Aging Research, Guangdong Medical University, Dongguan, China
| | - Guoxiang Jin
- Medical Research Center, Guangdong Provincial People's Hospital, Guangzhou, China.
| | - Zhongjun Zhou
- School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China. .,Shenzhen Hospital, The University of Hong Kong, Shenzhen, China.
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37
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Cooper F, Tsakiridis A. Shaping axial identity during human pluripotent stem cell differentiation to neural crest cells. Biochem Soc Trans 2022; 50:499-511. [PMID: 35015077 PMCID: PMC9022984 DOI: 10.1042/bst20211152] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2021] [Revised: 12/07/2021] [Accepted: 12/21/2021] [Indexed: 12/18/2022]
Abstract
The neural crest (NC) is a multipotent cell population which can give rise to a vast array of derivatives including neurons and glia of the peripheral nervous system, cartilage, cardiac smooth muscle, melanocytes and sympathoadrenal cells. An attractive strategy to model human NC development and associated birth defects as well as produce clinically relevant cell populations for regenerative medicine applications involves the in vitro generation of NC from human pluripotent stem cells (hPSCs). However, in vivo, the potential of NC cells to generate distinct cell types is determined by their position along the anteroposterior (A-P) axis and, therefore the axial identity of hPSC-derived NC cells is an important aspect to consider. Recent advances in understanding the developmental origins of NC and the signalling pathways involved in its specification have aided the in vitro generation of human NC cells which are representative of various A-P positions. Here, we explore recent advances in methodologies of in vitro NC specification and axis patterning using hPSCs.
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Affiliation(s)
- Fay Cooper
- Centre for Stem Cell Biology, School of Biosciences, The University of Sheffield, Western Bank, Sheffield S10 2TN, U.K
- Neuroscience Institute, The University of Sheffield, Western Bank, Sheffield S10 2TN, U.K
| | - Anestis Tsakiridis
- Centre for Stem Cell Biology, School of Biosciences, The University of Sheffield, Western Bank, Sheffield S10 2TN, U.K
- Neuroscience Institute, The University of Sheffield, Western Bank, Sheffield S10 2TN, U.K
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38
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Solis-Castro OO, Rivolta MN, Boissonade FM. Neural Crest-Derived Stem Cells (NCSCs) Obtained from Dental-Related Stem Cells (DRSCs): A Literature Review on Current Knowledge and Directions toward Translational Applications. Int J Mol Sci 2022; 23:ijms23052714. [PMID: 35269856 PMCID: PMC8911272 DOI: 10.3390/ijms23052714] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 02/21/2022] [Accepted: 02/27/2022] [Indexed: 11/21/2022] Open
Abstract
Evidence from dental-related stem cells (DRSCs) suggests an enhanced potential for ectodermal lineage differentiation due to their neural crest origin. Growing evidence that DRSC cultures can produce cells with a neural crest-derived stem cell (NCSC)-like phenotype supports their potential for future therapeutic approaches for neurodegenerative diseases and nerve injuries. However, most of the evidence is limited to the characterization of DRSCs as NCSCs by detecting the expression of neural crest markers. Only a few studies have provided proof of concept of an improved neuro-glial differentiation or direct applicability in relevant models. In addition, a current problem is that several of the existing protocols do not meet manufacturing standards for transferability to a clinical scenario. This review describes the current protocols to obtain NCSCs from DRSCs and their characterization. Also, it provides important considerations from previous work where DRSCs were established and characterized as mesenchymal stromal cells but studied for their neuro-glial differentiation potential. The therapeutic advancement of DRSCs would depend on establishing protocols that can yield a neural crest-like phenotype efficiently, using appropriate manufacturing standards and testing them in relevant models of disease or injury. Achieving these conditions could then facilitate and validate the therapeutic potential of DRSC-NCSCs in regenerative therapies.
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Affiliation(s)
- Oscar O. Solis-Castro
- School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, UK;
- The Neuroscience Institute, University of Sheffield, Sheffield S10 2TN, UK;
| | - Marcelo N. Rivolta
- The Neuroscience Institute, University of Sheffield, Sheffield S10 2TN, UK;
- Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK
| | - Fiona M. Boissonade
- School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, UK;
- The Neuroscience Institute, University of Sheffield, Sheffield S10 2TN, UK;
- Correspondence:
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39
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Schonkeren SL, Küthe TT, Idris M, Bon-Frauches AC, Boesmans W, Melotte V. The gut brain in a dish: Murine primary enteric nervous system cell cultures. Neurogastroenterol Motil 2022; 34:e14215. [PMID: 34236124 PMCID: PMC9285479 DOI: 10.1111/nmo.14215] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 05/22/2021] [Accepted: 06/01/2021] [Indexed: 01/09/2023]
Abstract
BACKGROUND The enteric nervous system (ENS) is an extensive neural network embedded in the wall of the gastrointestinal tract that regulates digestive function and gastrointestinal homeostasis. The ENS consists of two main cell types; enteric neurons and enteric glial cells. In vitro techniques allow simplified investigation of ENS function, and different culture methods have been developed over the years helping to understand the role of ENS cells in health and disease. PURPOSE This review focuses on summarizing and comparing available culture protocols for the generation of primary ENS cells from adult mice, including dissection of intestinal segments, enzymatic digestions, surface coatings, and culture media. In addition, the potential of human ENS cultures is also discussed.
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Affiliation(s)
- Simone L Schonkeren
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands
| | - Tara T Küthe
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands
| | - Musa Idris
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands.,Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, Netherlands
| | - Ana C Bon-Frauches
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands
| | - Werend Boesmans
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands.,Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium
| | - Veerle Melotte
- Department of Pathology, Maastricht University Medical Center, Maastricht, Netherlands.,Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, Netherlands
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40
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Cerrizuela S, Vega-Lopez GA, Méndez-Maldonado K, Velasco I, Aybar MJ. The crucial role of model systems in understanding the complexity of cell signaling in human neurocristopathies. WIREs Mech Dis 2022; 14:e1537. [PMID: 35023327 DOI: 10.1002/wsbm.1537] [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: 03/30/2021] [Revised: 08/26/2021] [Accepted: 08/30/2021] [Indexed: 11/07/2022]
Abstract
Animal models are useful to study the molecular, cellular, and morphogenetic mechanisms underlying normal and pathological development. Cell-based study models have emerged as an alternative approach to study many aspects of human embryonic development and disease. The neural crest (NC) is a transient, multipotent, and migratory embryonic cell population that generates a diverse group of cell types that arises during vertebrate development. The abnormal formation or development of the NC results in neurocristopathies (NCPs), which are characterized by a broad spectrum of functional and morphological alterations. The impaired molecular mechanisms that give rise to these multiphenotypic diseases are not entirely clear yet. This fact, added to the high incidence of these disorders in the newborn population, has led to the development of systematic approaches for their understanding. In this article, we have systematically reviewed the ways in which experimentation with different animal and cell model systems has improved our knowledge of NCPs, and how these advances might contribute to the development of better diagnostic and therapeutic tools for the treatment of these pathologies. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Stem Cells and Development Congenital Diseases > Molecular and Cellular Physiology Neurological Diseases > Genetics/Genomics/Epigenetics.
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Affiliation(s)
- Santiago Cerrizuela
- Division of Molecular Neurobiology, German Cancer Research Center (DKFZ), Heidelberg, Germany.,Instituto Superior de Investigaciones Biológicas (INSIBIO, CONICET-UNT), Tucumán, Argentina
| | - Guillermo A Vega-Lopez
- Instituto Superior de Investigaciones Biológicas (INSIBIO, CONICET-UNT), Tucumán, Argentina.,Instituto de Biología "Dr. Francisco D. Barbieri", Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina
| | - Karla Méndez-Maldonado
- Instituto de Fisiología Celular - Neurociencias, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.,Departamento de Fisiología y Farmacología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| | - Iván Velasco
- Instituto de Fisiología Celular - Neurociencias, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.,Laboratorio de Reprogramación Celular del Instituto de Fisiología Celular, UNAM en el Instituto Nacional de Neurología y Neurocirugía "Manuel Velasco Suárez", Ciudad de México, Mexico
| | - Manuel J Aybar
- Instituto Superior de Investigaciones Biológicas (INSIBIO, CONICET-UNT), Tucumán, Argentina.,Instituto de Biología "Dr. Francisco D. Barbieri", Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina
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41
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Liu TM. Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine. World J Stem Cells 2021; 13:1826-1844. [PMID: 35069985 PMCID: PMC8727229 DOI: 10.4252/wjsc.v13.i12.1826] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 06/29/2021] [Accepted: 11/30/2021] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) represent the most clinically used stem cells in regenerative medicine. However, due to the disadvantages with primary MSCs, such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs, gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application, and variation among donors increasing the uncertainty of MSC efficacy, the clinical application of MSCs has been greatly hampered. MSCs derived from human pluripotent stem cells (hPSC-MSCs) can circumvent these problems associated with primary MSCs. Due to the infinite self-renewal of hPSCs and their differentiation potential towards MSCs, hPSC-MSCs are emerging as an attractive alternative for regenerative medicine. This review summarizes the progress on derivation of MSCs from human pluripotent stem cells, disease modelling and drug screening using hPSC-MSCs, and various applications of hPSC-MSCs in regenerative medicine. In the end, the challenges and concerns with hPSC-MSC applications are also discussed.
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Affiliation(s)
- Tong-Ming Liu
- Agency for Science, Technology and Research, Institute of Molecular and Cell Biology, Singapore 138648, Singapore.
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42
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Hörner SJ, Couturier N, Bruch R, Koch P, Hafner M, Rudolf R. hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures. Cells 2021; 10:cells10123292. [PMID: 34943800 PMCID: PMC8699767 DOI: 10.3390/cells10123292] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Revised: 11/20/2021] [Accepted: 11/21/2021] [Indexed: 12/13/2022] Open
Abstract
Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.
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Affiliation(s)
- Sarah Janice Hörner
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany; (S.J.H.); (N.C.); (R.B.); (M.H.)
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
| | - Nathalie Couturier
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany; (S.J.H.); (N.C.); (R.B.); (M.H.)
| | - Roman Bruch
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany; (S.J.H.); (N.C.); (R.B.); (M.H.)
| | - Philipp Koch
- Central Institute of Mental Health, Medical Faculty Mannheim of Heidelberg University, 68159 Mannheim, Germany;
- Hector Institute for Translational Brain Research (HITBR gGmbH), 68159 Mannheim, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Mathias Hafner
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany; (S.J.H.); (N.C.); (R.B.); (M.H.)
- Institute of Medical Technology, Mannheim University of Applied Sciences and Heidelberg University, 68163 Mannheim, Germany
| | - Rüdiger Rudolf
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany; (S.J.H.); (N.C.); (R.B.); (M.H.)
- Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
- Institute of Medical Technology, Mannheim University of Applied Sciences and Heidelberg University, 68163 Mannheim, Germany
- Correspondence:
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43
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Pluripotent-derived Mesenchymal Stem/stromal Cells: an Overview of the Derivation Protocol Efficacies and the Differences Among the Derived Cells. Stem Cell Rev Rep 2021; 18:94-125. [PMID: 34545529 DOI: 10.1007/s12015-021-10258-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/02/2021] [Indexed: 10/20/2022]
Abstract
Mesenchymal stem/stromal cells (MSCs) are remarkable tools for regenerative medicine. Therapeutic approaches using these cells can promote increased activity and viability in several cell types through diverse mechanisms such as paracrine and immunomodulatory activities, contributing substantially to tissue regeneration and functional recovery. However, biological samples of human MSCs, usually obtained from adult tissues, often exhibit variable behavior during in vitro culture, especially with respect to cell population heterogeneity, replicative senescence, and consequent loss of functionality. Accordingly, it is necessary to establish standard protocols to generate high-quality, stable cell cultures, for example, by using pluripotent stem cells (PSCs) in derivation protocols of MSC-like cells since PSCs maintain their characteristics consistently during culture. However, the available protocols seem to generate distinct populations of PSC-derivedMSCs (PSC-MSCs) with peculiar attributes, which do not always resemble bona fide primary MSCs. The present review addresses the developmental basis behind some of these derivation protocols, exposing the differences among them and discussing the functional properties of PSC-MSCs, shedding light on elements that may help determine standard characterizations and criteria to evaluate and define these cells.
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44
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Mo J, Anastasaki C, Chen Z, Shipman T, Papke J, Yin K, Gutmann DH, Le LQ. Humanized neurofibroma model from induced pluripotent stem cells delineates tumor pathogenesis and developmental origins. J Clin Invest 2021; 131:139807. [PMID: 33108355 DOI: 10.1172/jci139807] [Citation(s) in RCA: 52] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Accepted: 10/21/2020] [Indexed: 02/06/2023] Open
Abstract
Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by NF1 gene mutation, in which affected patients develop Schwann cell lineage peripheral nerve sheath tumors (neurofibromas). To investigate human neurofibroma pathogenesis, we differentiated a series of isogenic, patient-specific NF1-mutant human induced pluripotent stem cells (hiPSCs) into Schwannian lineage cells (SLCs). We found that, although WT and heterozygous NF1-mutant hiPSCs-SLCs did not form tumors following mouse sciatic nerve implantation, NF1-null SLCs formed bona fide neurofibromas with high levels of SOX10 expression. To confirm that SOX10+ SLCs contained the cells of origin for neurofibromas, both Nf1 alleles were inactivated in mouse Sox10+ cells, leading to classic nodular cutaneous and plexiform neurofibroma formation that completely recapitulated their human counterparts. Moreover, we discovered that NF1 loss impaired Schwann cell differentiation by inducing a persistent stem-like state to expand the pool of progenitors required to initiate tumor formation, indicating that, in addition to regulating MAPK-mediated cell growth, NF1 loss also altered Schwann cell differentiation to promote neurofibroma development. Taken together, we established a complementary humanized neurofibroma explant and, to our knowledge, first-in-kind genetically engineered nodular cutaneous neurofibroma mouse models that delineate neurofibroma pathogenesis amenable to future therapeutic target discovery and evaluation.
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Affiliation(s)
- Juan Mo
- Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, USA
| | - Corina Anastasaki
- Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA
| | - Zhiguo Chen
- Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, USA
| | - Tracey Shipman
- Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, USA
| | - Jason Papke
- Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA
| | - Kevin Yin
- Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, USA
| | - David H Gutmann
- Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA
| | - Lu Q Le
- Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, USA.,Simmons Comprehensive Cancer Center and.,Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, Texas, USA
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45
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Kim H, Noh HB, Lee S, Lee K, Chang B, Cheong E, Lee CJ, Hwang D. Fine-tuning of dual-SMAD inhibition to differentiate human pluripotent stem cells into neural crest stem cells. Cell Prolif 2021; 54:e13103. [PMID: 34323338 PMCID: PMC8450125 DOI: 10.1111/cpr.13103] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Revised: 05/27/2021] [Accepted: 07/09/2021] [Indexed: 01/15/2023] Open
Abstract
OBJECTIVES The derivation of neural crest stem cells (NCSCs) from human pluripotent stem cells (hPSCs) has been commonly induced by WNT activation in combination with dual-SMAD inhibition. In this study, by fine-tuning BMP signalling in the conventional dual-SMAD inhibition, we sought to generate large numbers of NCSCs without WNT activation. MATERIALS AND METHODS In the absence of WNT activation, we modulated the level of BMP signalling in the dual-SMAD inhibition system to identify conditions that efficiently drove the differentiation of hPSCs into NCSCs. We isolated two NCSC populations separately and characterized them in terms of global gene expression profiles and differentiation ability. RESULTS Our modified dual-SMAD inhibition containing a lower dose of BMP inhibitor than that of the conventional dual-SMAD inhibition drove hPSCs into mainly NCSCs, which consisted of HNK+ p75high and HNK+ p75low cell populations. We showed that the p75high population formed spherical cell clumps, while the p75low cell population generated a 2D monolayer. We detected substantial differences in gene expression profiles between the two cell groups and showed that both p75high and p75low cells differentiated into mesenchymal stem cells (MSCs), while only p75high cells had the ability to become peripheral neurons. CONCLUSIONS This study will provide a framework for the generation and isolation of NCSC populations for effective cell therapy for peripheral neuropathies and MSC-based cell therapy.
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Affiliation(s)
- Hyun‐Mun Kim
- Department of Biomedical ScienceGraduate School of CHA UniversitySungnamKorea
| | - Hye Bin Noh
- Department of Biomedical ScienceGraduate School of CHA UniversitySungnamKorea
| | - Sang‐Hyuk Lee
- Department of Biomedical ScienceGraduate School of CHA UniversitySungnamKorea
| | - Kun‐Gu Lee
- Department of Biomedical ScienceGraduate School of CHA UniversitySungnamKorea
| | - Bomi Chang
- Center for Cognition and SocialityInstitute for Basic ScienceDaejeonKorea
- Brain Science InstituteKorea Institute of Science and TechnologySeoulKorea
- Department of Biotechnology, College of Life Science and Biotechnology, Translational Research Center for Protein Function ControlYonsei UniversitySeoulKorea
| | - Eunji Cheong
- Department of Biotechnology, College of Life Science and Biotechnology, Translational Research Center for Protein Function ControlYonsei UniversitySeoulKorea
| | - C. Justin Lee
- Center for Cognition and SocialityInstitute for Basic ScienceDaejeonKorea
| | - Dong‐Youn Hwang
- Department of Biomedical ScienceGraduate School of CHA UniversitySungnamKorea
- Department of Microbiology, School of MedicineCHA UniversitySungnamKorea
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46
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Lai X, Liu J, Zou Z, Wang Y, Wang Y, Liu X, Huang W, Ma Y, Chen Q, Li F, Wu G, Li W, Wang W, Yuan Y, Jiang B. SOX10 ablation severely impairs the generation of postmigratory neural crest from human pluripotent stem cells. Cell Death Dis 2021; 12:814. [PMID: 34453037 PMCID: PMC8397771 DOI: 10.1038/s41419-021-04099-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 08/05/2021] [Accepted: 08/16/2021] [Indexed: 12/12/2022]
Abstract
Animal studies have indicated that SOX10 is one of the key transcription factors regulating the proliferation, migration and differentiation of multipotent neural crest (NC), and mutation of SOX10 in humans may lead to type 4 Waardenburg syndrome (WS). However, the exact role of SOX10 in human NC development and the underlying molecular mechanisms of SOX10-related human diseases remain poorly understood due to the lack of appropriate human model systems. In this study, we successfully generated SOX10-knockout human induced pluripotent stem cells (SOX10-/- hiPSCs) by the CRISPR-Cas9 gene editing tool. We found that loss of SOX10 significantly inhibited the generation of p75highHNK1+/CD49D+ postmigratory neural crest stem cells (NCSCs) and upregulated the cell apoptosis rate during NC commitment from hiPSCs. Moreover, we discovered that both the neuronal and glial differentiation capacities of SOX10-/- NCSCs were severely compromised. Intriguingly, we showed that SOX10-/- hiPSCs generated markedly more TFAP2C+nonneural ectoderm cells (NNE) than control hiPSCs during neural crest differentiation. Our results indicate that SOX10 is crucial for the transition of premigratory cells to migrating NC and is vital for NC survival. Taken together, these results provide new insights into the function of SOX10 in human NC development, and the SOX10-knockout hiPSC lines may serve as a valuable cell model to study the pathogenesis of SOX10-related human neurocristopathies.
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Affiliation(s)
- Xingqiang Lai
- Department of Cardiology, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong, China
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
| | - Jia Liu
- VIP Medical Service Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Zhengwei Zou
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
- Center for Stem Cell Clinical Translation, First Affiliated Hospital, Gannan Medical University, Ganzhou, Jiangxi, China
| | - Yina Wang
- VIP Medical Service Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Ye Wang
- Fetal Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Xiao Liu
- Department of Laboratory Medicine, Zhongshan People's Hospital, Zhongshan, Guangdong, China
| | - Weijun Huang
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
| | - Yuanchen Ma
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
| | - Qian Chen
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
| | - Fugui Li
- Department of Laboratory Medicine, Zhongshan People's Hospital, Zhongshan, Guangdong, China
| | - Guifu Wu
- Department of Cardiology, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong, China
- NHC Key Laboratory of Assisted Circulation, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Weiqiang Li
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China
| | - Weijia Wang
- Department of Laboratory Medicine, Zhongshan People's Hospital, Zhongshan, Guangdong, China.
| | - Yong Yuan
- Department of Cardiovascular Center, Zhongshan People's Hospital, Zhongshan, Guangdong, China.
| | - Boxiong Jiang
- VIP Medical Service Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
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Wagstaff EL, Heredero Berzal A, Boon CJF, Quinn PMJ, ten Asbroek ALMA, Bergen AA. The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development. Int J Mol Sci 2021; 22:7081. [PMID: 34209272 PMCID: PMC8268497 DOI: 10.3390/ijms22137081] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 06/23/2021] [Accepted: 06/26/2021] [Indexed: 12/12/2022] Open
Abstract
Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.
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Affiliation(s)
- Ellie L. Wagstaff
- Department of Human Genetics, Amsterdam UMC, University of Amsterdam (UvA), 1105 AZ Amsterdam, The Netherlands;
| | - Andrea Heredero Berzal
- Department of Ophthalmology, Amsterdam UMC, University of Amsterdam (UvA), 1105 AZ Amsterdam, The Netherlands; (A.H.B.); (C.J.F.B.)
| | - Camiel J. F. Boon
- Department of Ophthalmology, Amsterdam UMC, University of Amsterdam (UvA), 1105 AZ Amsterdam, The Netherlands; (A.H.B.); (C.J.F.B.)
- Department of Ophthalmology, Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands
| | - Peter M. J. Quinn
- Jonas Children’s Vision Care and Bernard & Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Departments of Ophthalmology, Pathology & Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA; Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center—New York-Presbyterian Hospital, New York, NY 10032, USA;
| | | | - Arthur A. Bergen
- Department of Human Genetics, Amsterdam UMC, University of Amsterdam (UvA), 1105 AZ Amsterdam, The Netherlands;
- Department of Ophthalmology, Amsterdam UMC, University of Amsterdam (UvA), 1105 AZ Amsterdam, The Netherlands; (A.H.B.); (C.J.F.B.)
- Netherlands Institute for Neuroscience (NIN-KNAW), 1105 BA Amsterdam, The Netherlands
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48
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Pan SH, Zhao N, Feng X, Jie Y, Jin ZB. Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules. SCIENCE ADVANCES 2021; 7:7/23/eabg5749. [PMID: 34088673 PMCID: PMC8177713 DOI: 10.1126/sciadv.abg5749] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Accepted: 04/20/2021] [Indexed: 05/06/2023]
Abstract
Reprogramming of somatic cells into desired functional cell types by small molecules has vast potential for developing cell replacement therapy. Here, we developed a stepwise strategy to generate chemically induced neural crest cells (ciNCCs) and chemically induced corneal endothelial cells (ciCECs) from mouse fibroblasts using defined small molecules. The ciNCCs exhibited typical NCC features and could differentiate into ciCECs using another chemical combination in vitro. The resulting ciCECs showed consistent gene expression profiles and self-renewal capacity to those of primary CECs. Notably, these ciCECs could be cultured for as long as 30 passages and still retain the CEC features in defined medium. Transplantation of these ciCECs into an animal model reversed corneal opacity. Our chemical approach for direct reprogramming of mouse fibroblasts into ciNCCs and ciCECs provides an alternative cell source for regeneration of corneal endothelia and other tissues derived from neural crest.
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Affiliation(s)
- Shao-Hui Pan
- Institute of Stem Cell Research, The Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China
| | - Ning Zhao
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, Beijing 100730, China
- Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University and Capital Medical University, Beijing Tongren Hospital, Beijing 100730, China
| | - Xiang Feng
- Institute of Stem Cell Research, The Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China
| | - Ying Jie
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, Beijing 100730, China
| | - Zi-Bing Jin
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, Beijing 100730, China.
- Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University and Capital Medical University, Beijing Tongren Hospital, Beijing 100730, China
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49
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Hirano M, Huang Y, Vela Jarquin D, De la Garza Hernández RL, Jodat YA, Luna Cerón E, García-Rivera LE, Shin SR. 3D bioprinted human iPSC-derived somatosensory constructs with functional and highly purified sensory neuron networks. Biofabrication 2021; 13. [PMID: 33962404 DOI: 10.1088/1758-5090/abff11] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2021] [Accepted: 05/07/2021] [Indexed: 12/20/2022]
Abstract
Engineering three-dimensional (3D) sensible tissue constructs, along with the complex microarchitecture wiring of the sensory nervous system, has been an ongoing challenge in the tissue engineering field. By combining 3D bioprinting and human pluripotent stem cell (hPSC) technologies, sensible tissue constructs could be engineered in a rapid, precise, and controllable manner to replicate 3D microarchitectures and mechanosensory functionalities of the native sensory tissue (e.g. response to external stimuli). Here, we introduce a biofabrication approach to create complex 3D microarchitecture wirings. We develop an hPSC-sensory neuron (SN) laden bioink using highly purified and functional SN populations to 3D bioprint microarchitecture wirings that demonstrate responsiveness to warm/cold sense-inducing chemicals and mechanical stress. Specifically, we tailor a conventional differentiation strategy to our purification method by utilizing p75 cell surface marker and DAPT treatment along with neuronal growth factors in order to selectively differentiate neural crest cells into SNs. To create spatial resolution in 3D architectures and grow SNs in custom patterns and directions, an induced pluripotent stem cell (iPSC)-SN-laden gelatin bioink was printed on laminin-coated substrates using extrusion-based bioprinting technique. Then the printed constructs were covered with a collagen matrix that guided SNs growing in the printed micropattern. Using a sacrificial bioprinting technique, the iPSC-SNs were seeded into the hollow microchannels created by sacrificial gelatin ink printed in the gelatin methacryloyl supporting bath, thereby demonstrating controllability over axon guidance in curved lines up to several tens of centimeters in length on 2D substrates and in straight microchannels in 3D matrices. Therefore, this biofabrication approach could be amenable to incorporate sensible SN networks into the engineered skin equivalents, regenerative skin implants, and augmented somatosensory neuro-prosthetics that have the potential to regenerate sensible functions by connecting host neuron systems in injured areas.
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Affiliation(s)
- Minoru Hirano
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Future Vehicle Research Department, Toyota Research Institute North America, Toyota Motor North America Inc., 1555 Woodridge Ave, Ann Arbor, MI 48105, United States of America
| | - Yike Huang
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 100730, People's Republic of China
| | - Daniel Vela Jarquin
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Instituto Tecnológico y de Estudios Superiores de Monterrey, Calle del Puente 222, Ejidos de Huipulco, Tlalpan, Ciudad de México, CDMX 14380, Mexico
| | - Rosakaren Ludivina De la Garza Hernández
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Instituto Tecnológico y de Estudios Superiores de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Tecnológico, 64849 Monterrey, NL, Mexico
| | - Yasamin A Jodat
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America
| | - Eder Luna Cerón
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Instituto Tecnológico y de Estudios Superiores de Monterrey, Calle del Puente 222, Ejidos de Huipulco, Tlalpan, Ciudad de México, CDMX 14380, Mexico
| | - Luis Enrique García-Rivera
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.,Instituto Tecnológico y de Estudios Superiores de Monterrey, Calle del Puente 222, Ejidos de Huipulco, Tlalpan, Ciudad de México, CDMX 14380, Mexico
| | - Su Ryon Shin
- Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America
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50
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Lee MS, Stebbins MJ, Jiao H, Huang HC, Leiferman EM, Walczak BE, Palecek SP, Shusta EV, Li WJ. Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration. SCIENCE ADVANCES 2021; 7:eabf0907. [PMID: 34138734 PMCID: PMC8133756 DOI: 10.1126/sciadv.abf0907] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 03/29/2021] [Indexed: 05/12/2023]
Abstract
Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.
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Affiliation(s)
- Ming-Song Lee
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Matthew J Stebbins
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Hongli Jiao
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Hui-Ching Huang
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Ellen M Leiferman
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Brian E Walczak
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Sean P Palecek
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Eric V Shusta
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
- Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI 53792, USA
| | - Wan-Ju Li
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA.
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
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