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Zhao C, Ikeya M. Novel insights from human induced pluripotent stem cells on origins and roles of fibro/adipogenic progenitors as heterotopic ossification precursors. Front Cell Dev Biol 2024; 12:1457344. [PMID: 39286484 PMCID: PMC11402712 DOI: 10.3389/fcell.2024.1457344] [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: 06/30/2024] [Accepted: 08/21/2024] [Indexed: 09/19/2024] Open
Abstract
Fibro/adipogenic progenitors (FAPs) that reside in muscle tissue are crucial for muscular homeostasis and regeneration as they secrete signaling molecules and components of the extracellular matrix. During injury or disease, FAPs differentiate into different cell types and significantly modulate muscular function. Recent advances in lineage tracing and single-cell transcriptomics have proven that FAPs are heterogeneous both in resting and post-injury or disease states. Their heterogeneity may be owing to the varied tissue microenvironments and their diverse developmental origins. Therefore, understanding FAPs' developmental origins can help predict their characteristics and behaviors under different conditions. FAPs are thought to be the major cell populations in the muscle connective tissue (MCT). During embryogenesis, the MCT directs muscular development throughout the body and serves as a prepattern for muscular morphogenesis. The developmental origins of FAPs as stromal cells in the MCT were studied previously. In adult tissues, FAPs are important precursors for heterotopic ossification, especially in the context of the rare genetic disorder fibrodysplasia ossificans progressiva. A new developmental origin for FAPs have been suggested that differs from conventional developmental perspectives. In this review, we summarize the developmental origins and functions of FAPs as stromal cells of the MCT and present novel insights obtained by using patient-derived induced pluripotent stem cells and mouse models of heterotopic ossification. This review broadens the current understanding of FAPs and suggests potential avenues for further investigation.
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Affiliation(s)
- Chengzhu Zhao
- Laboratory of Skeletal Development and Regeneration, Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, China
| | - Makoto Ikeya
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
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2
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Granados AA, Kanrar N, Elowitz MB. Combinatorial expression motifs in signaling pathways. CELL GENOMICS 2024; 4:100463. [PMID: 38216284 PMCID: PMC10794782 DOI: 10.1016/j.xgen.2023.100463] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 08/02/2023] [Accepted: 11/15/2023] [Indexed: 01/14/2024]
Abstract
In animal cells, molecular pathways often comprise families of variant components, such as ligands or receptors. These pathway components are differentially expressed by different cell types, potentially tailoring pathway function to cell context. However, it has remained unclear how pathway expression profiles are distributed across cell types and whether similar profiles can occur in dissimilar cell types. Here, using single-cell gene expression datasets, we identified pathway expression motifs, defined as recurrent expression profiles that are broadly distributed across diverse cell types. Motifs appeared in core pathways, including TGF-β, Notch, Wnt, and the SRSF splice factors, and involved combinatorial co-expression of multiple components. Motif usage was weakly correlated between pathways in adult cell types and during dynamic developmental transitions. Together, these results suggest a mosaic view of cell type organization, in which different cell types operate many of the same pathways in distinct modes.
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Affiliation(s)
- Alejandro A Granados
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute and Department of Applied Physics, California Institute of Technology, Pasadena, CA 91125, USA
| | - Nivedita Kanrar
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute and Department of Applied Physics, California Institute of Technology, Pasadena, CA 91125, USA
| | - Michael B Elowitz
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute and Department of Applied Physics, California Institute of Technology, Pasadena, CA 91125, USA.
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3
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Mehrotra P, Ikhapoh I, Lei P, Tseropoulos G, Zhang Y, Wang J, Liu S, Bronner ME, Andreadis ST. Wnt/BMP Mediated Metabolic Reprogramming Preserves Multipotency of Neural Crest-Like Stem Cells. Stem Cells 2023; 41:287-305. [PMID: 36617947 PMCID: PMC10020983 DOI: 10.1093/stmcls/sxad001] [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: 07/05/2022] [Accepted: 12/27/2022] [Indexed: 01/10/2023]
Abstract
Neural crest-like stem cells resembling embryonic neural crest cells (NCs) can be derived from adult human tissues such as the epidermis. However, these cells lose their multipotency rapidly in culture limiting their expansion for clinical use. Here, we show that the multipotency of keratinocyte-derived NCs (KC-NCs) can be preserved by activating the Wnt and BMP signaling axis, promoting expression of key NC-specifier genes and ultimately enhancing their differentiation potential. We also show that transcriptional changes leading to multipotency are linked to metabolic reprogramming of KC-NCs to a highly glycolytic state. Specifically, KC-NCs treated with CHIR and BMP2 rely almost exclusively on glycolysis for their energy needs, as seen by increased lactate production, glucose uptake, and glycolytic enzyme activities. This was accompanied by mitochondrial depolarization and decreased mitochondrial ATP production. Interestingly, the glycolytic end-product lactate stabilized β-catenin and further augmented NC-gene expression. Taken together, our study shows that activation of the Wnt/BMP signaling coordinates the metabolic demands of neural crest-like stem cells governing decisions regarding multipotency and differentiation, with possible implications for regenerative medicine.
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Affiliation(s)
- Pihu Mehrotra
- Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA
| | - Izuagie Ikhapoh
- Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA
| | - Pedro Lei
- Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA
| | - Georgios Tseropoulos
- Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA
| | - Yali Zhang
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Jianmin Wang
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Song Liu
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Marianne E Bronner
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
| | - Stelios T Andreadis
- Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA
- Department of Biomedical Engineering, University at Buffalo, NY, Buffalo, NY, USA
- Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, Buffalo, NY, USA
- Center for Cell, Gene and Tissue Engineering (CGTE), University at Buffalo, Buffalo, NY, USA
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Walia B, Li T, Crosio G, Montero A, Huang A. Axin2-lineage cells contribute to neonatal tendon regeneration. Connect Tissue Res 2022; 63:530-543. [PMID: 35180018 PMCID: PMC9491382 DOI: 10.1080/03008207.2022.2036732] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Accepted: 01/17/2022] [Indexed: 02/03/2023]
Abstract
PURPOSE Tendon injuries are a challenging clinical problem with few treatment options. Identifying the molecular regulators of tendon is required for the development of new therapies. While the Wnt pathway is critical for the maintenance and differentiation of many tissues, the role of Wnt signaling in tendon cell biology remains largely unexplored. METHODS The effects of Wnt activation were tested in vitro using neonatal tendon-derived cells cultured in 2D and 3D conditions. The inducible Axin2CreERT2 was then used to label Axin2+ cells in vivo and cells were traced during neonatal tendon regeneration. RESULTS We showed that activation of Wnt signaling results in proliferation of neonatal tendon cells. While tendon marker expression was inhibited by Wnt activation under 2D conditions, Scx expression was not affected under 3D uniaxial tension, suggesting that the microenvironment contextualizes tendon cell response to Wnt signaling. Using an in vivo model of neonatal tendon regeneration, we further showed that Wnt signaling cells comprise a subpopulation of tenocyte and epitenon cells that proliferate after injury and are recruited during regeneration. DISCUSSION Collectively, these studies suggest that Wnt signaling may play a role in tendon cell proliferation, differentiation, and regeneration.
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Affiliation(s)
- B. Walia
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY
| | - T.M. Li
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY
| | - G. Crosio
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY
| | - A.M. Montero
- Department of Orthopedic Surgery, Columbia University, New York, NY
| | - A.H. Huang
- Department of Orthopedic Surgery, Columbia University, New York, NY
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5
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Sun X, Tao B, Wang Y, Hu W, Sun Y. Isolation and Characterization of Germline Stem Cells in Protogynous Hermaphroditic Monopterus albus. Int J Mol Sci 2022; 23:ijms23115861. [PMID: 35682541 PMCID: PMC9180834 DOI: 10.3390/ijms23115861] [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: 04/12/2022] [Revised: 05/22/2022] [Accepted: 05/22/2022] [Indexed: 02/04/2023] Open
Abstract
Germline stem cells (GSCs) are a group of unique adult stem cells in gonads that act as important transmitters for genetic information. Donor GSCs have been used to produce offspring by transplantation in fisheries. In this study, we successfully isolated and enriched GSCs from the ovary, ovotestis, and testis of Monopterus albus, one of the most important breeding freshwater fishes in China. Transcriptome comparison assay suggests that a distinct molecular signature exists in each type of GSC, and that different signaling activities are required for the maintenance of distinct GSCs. Functional analysis shows that fGSCs can successfully colonize and contribute to the germline cell lineage of a host zebrafish gonad after transplantation. Finally, we describe a simple feeder-free method for the isolation and enrichment of GSCs that can contribute to the germline cell lineage of zebrafish embryos and generate the germline chimeras after transplantation.
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Affiliation(s)
- Xiaoyun Sun
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; (X.S.); (B.T.); (Y.W.)
| | - Binbin Tao
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; (X.S.); (B.T.); (Y.W.)
| | - Yongxin Wang
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; (X.S.); (B.T.); (Y.W.)
- University of Chinese Academy of Sciences, Beijing 100049, China;
| | - Wei Hu
- University of Chinese Academy of Sciences, Beijing 100049, China;
- The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan 430072, China
| | - Yuhua Sun
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; (X.S.); (B.T.); (Y.W.)
- University of Chinese Academy of Sciences, Beijing 100049, China;
- The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan 430072, China
- Correspondence:
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Manzari-Tavakoli A, Babajani A, Farjoo MH, Hajinasrollah M, Bahrami S, Niknejad H. The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation. Front Mol Neurosci 2022; 15:827275. [PMID: 35370542 PMCID: PMC8965007 DOI: 10.3389/fnmol.2022.827275] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Accepted: 02/14/2022] [Indexed: 11/21/2022] Open
Abstract
The bone morphogenetic proteins (BMPs) are a group of potent morphogens which are critical for the patterning, development, and function of the central nervous system. The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within in vitro tissue engineering approaches and pre-clinical practices and develop futuristic therapeutic strategies for patients with neurological disease.
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Affiliation(s)
- Asma Manzari-Tavakoli
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Rayan Center for Neuroscience & Behavior, Department of Biology, Faculty of Science, Ferdowsi University, Mashhad, Iran
| | - Amirhesam Babajani
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Hadi Farjoo
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mostafa Hajinasrollah
- Department of Stem Cells and Developmental Biology, Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Soheyl Bahrami
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in AUVA Research Center, Vienna, Austria
| | - Hassan Niknejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Hong L, Li N, Gasque V, Mehta S, Ye L, Wu Y, Li J, Gewies A, Ruland J, Hirschi KK, Eichmann A, Hendry C, van Dijk D, Mani A. Prdm6 controls heart development by regulating neural crest cell differentiation and migration. JCI Insight 2022; 7:156046. [PMID: 35108221 PMCID: PMC8876496 DOI: 10.1172/jci.insight.156046] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Accepted: 01/13/2022] [Indexed: 11/22/2022] Open
Abstract
The molecular mechanisms that drive the acquisition of distinct neural crest cell (NCC) fates is still poorly understood. Here, we identified Prdm6 as an epigenetic modifier that temporally and spatially regulates the expression of NCC specifiers and determines the fate of a subset of migrating cardiac NCCs (CNCCs). Using transcriptomic analysis and genetic and fate mapping approaches in transgenic mice, we showed that disruption of Prdm6 was associated with impaired CNCC differentiation, delamination, and migration and led to patent ductus arteriosus (DA) and ventricular noncompaction. Bulk and single-cell RNA-Seq analyses of the DA and CNCCs identified Prdm6 as a regulator of a network of CNCC specification genes, including Wnt1, Tfap2b, and Sox9. Loss of Prdm6 in CNCCs diminished its expression in the pre-epithelial–mesenchymal transition (pre-EMT) cluster, resulting in the retention of NCCs in the dorsal neural tube. This defect was associated with diminished H4K20 monomethylation and G1-S progression and augmented Wnt1 transcript levels in pre-EMT and neural tube clusters, which we showed was the major driver of the impaired CNCC migration. Altogether, these findings revealed Prdm6 as a key regulator of CNCC differentiation and migration and identified Prdm6 and its regulated network as potential targets for the treatment of congenital heart diseases.
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Affiliation(s)
- Lingjuan Hong
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | - Na Li
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | - Victor Gasque
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | - Sameet Mehta
- Yale Center for Genome Analysis, Yale University School of Medicine, New Haven, United States of America
| | - Lupeng Ye
- Department of Genetics, Yale University School of Medicine, New Haven, United States of America
| | - Yinyu Wu
- Department of Genetics, Yale University School of Medicine, New Haven, United States of America
| | - Jinyu Li
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | | | | | - Karen K Hirschi
- University of Virginia School of Medicine, Charlottesville, United States of America
| | - Anne Eichmann
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | - Caroline Hendry
- Department of Genetics, Yale University School of Medicine, New Haven, United States of America
| | - David van Dijk
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
| | - Arya Mani
- Cardiovascular Research Center, Yale University School of Medicine, New Haven, United States of America
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8
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Shinozuka T, Takada S. Morphological and Functional Changes of Roof Plate Cells in Spinal Cord Development. J Dev Biol 2021; 9:jdb9030030. [PMID: 34449633 PMCID: PMC8395932 DOI: 10.3390/jdb9030030] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2021] [Revised: 07/27/2021] [Accepted: 07/28/2021] [Indexed: 01/09/2023] Open
Abstract
The most dorsal region, or roof plate, is the dorsal organizing center of developing spinal cord. This region is also involved in development of neural crest cells, which are the source of migratory neural crest cells. During early development of the spinal cord, roof plate cells secrete signaling molecules, such as Wnt and BMP family proteins, which regulate development of neural crest cells and dorsal spinal cord. After the dorso-ventral pattern is established, spinal cord dynamically changes its morphology. With this morphological transformation, the lumen of the spinal cord gradually shrinks to form the central canal, a cavity filled with cerebrospinal fluid that is connected to the ventricular system of the brain. The dorsal half of the spinal cord is separated by a glial structure called the dorsal (or posterior) median septum. However, underlying mechanisms of such morphological transformation are just beginning to be understood. Recent studies reveal that roof plate cells dramatically stretch along the dorso-ventral axis, accompanied by reduction of the spinal cord lumen. During this stretching process, the tips of roof plate cells maintain contact with cells surrounding the shrinking lumen, eventually exposed to the inner surface of the central canal. Interestingly, Wnt expression remains in stretched roof plate cells and activates Wnt/β-catenin signaling in ependymal cells surrounding the central canal. Wnt/β-catenin signaling in ependymal cells promotes proliferation of neural progenitor and stem cells in embryonic and adult spinal cord. In this review, we focus on the role of the roof plate, especially that of Wnt ligands secreted by roof plate cells, in morphological changes occurring in the spinal cord.
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Affiliation(s)
- Takuma Shinozuka
- Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Aichi, Okazaki 444-8787, Japan
- National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Aichi, Okazaki 444-8787, Japan
- Correspondence: (T.S.); (S.T.)
| | - Shinji Takada
- Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Aichi, Okazaki 444-8787, Japan
- National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Aichi, Okazaki 444-8787, Japan
- Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Aichi, Okazaki 444-8787, Japan
- Correspondence: (T.S.); (S.T.)
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9
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Farina AR, Cappabianca LA, Zelli V, Sebastiano M, Mackay AR. Mechanisms involved in selecting and maintaining neuroblastoma cancer stem cell populations, and perspectives for therapeutic targeting. World J Stem Cells 2021; 13:685-736. [PMID: 34367474 PMCID: PMC8316860 DOI: 10.4252/wjsc.v13.i7.685] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 03/09/2021] [Accepted: 04/14/2021] [Indexed: 02/06/2023] Open
Abstract
Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumours that originate from cells of neural crest (NC) origin and in particular neuroblasts committed to the sympathoadrenal progenitor cell lineage. Therapeutic resistance, post-therapeutic relapse and subsequent metastatic NB progression are driven primarily by cancer stem cell (CSC)-like subpopulations, which through their self-renewing capacity, intermittent and slow cell cycles, drug-resistant and reversibly adaptive plastic phenotypes, represent the most important obstacle to improving therapeutic outcomes in unfavourable NBs. In this review, dedicated to NB CSCs and the prospects for their therapeutic eradication, we initiate with brief descriptions of the unique transient vertebrate embryonic NC structure and salient molecular protagonists involved NC induction, specification, epithelial to mesenchymal transition and migratory behaviour, in order to familiarise the reader with the embryonic cellular and molecular origins and background to NB. We follow this by introducing NB and the potential NC-derived stem/progenitor cell origins of NBs, before providing a comprehensive review of the salient molecules, signalling pathways, mechanisms, tumour microenvironmental and therapeutic conditions involved in promoting, selecting and maintaining NB CSC subpopulations, and that underpin their therapy-resistant, self-renewing metastatic behaviour. Finally, we review potential therapeutic strategies and future prospects for targeting and eradication of these bastions of NB therapeutic resistance, post-therapeutic relapse and metastatic progression.
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Affiliation(s)
- Antonietta Rosella Farina
- Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, L'Aquila 67100, AQ, Italy
| | - Lucia Annamaria Cappabianca
- Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, L'Aquila 67100, AQ, Italy
| | - Veronica Zelli
- Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, L'Aquila 67100, AQ, Italy
| | - Michela Sebastiano
- Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, L'Aquila 67100, AQ, Italy
| | - Andrew Reay Mackay
- Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, L'Aquila 67100, AQ, Italy.
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10
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Keuls RA, Parchem RJ. Single-Cell Multiomic Approaches Reveal Diverse Labeling of the Nervous System by Common Cre-Drivers. Front Cell Neurosci 2021; 15:648570. [PMID: 33935652 PMCID: PMC8079645 DOI: 10.3389/fncel.2021.648570] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2020] [Accepted: 03/15/2021] [Indexed: 11/27/2022] Open
Abstract
Neural crest development involves a series of dynamic, carefully coordinated events that result in human disease when not properly orchestrated. Cranial neural crest cells acquire unique multipotent developmental potential upon specification to generate a broad variety of cell types. Studies of early mammalian neural crest and nervous system development often use the Cre-loxP system to lineage trace and mark cells for further investigation. Here, we carefully profile the activity of two common neural crest Cre-drivers at the end of neurulation in mice. RNA sequencing of labeled cells at E9.5 reveals that Wnt1-Cre2 marks cells with neuronal characteristics consistent with neuroepithelial expression, whereas Sox10-Cre predominantly labels the migratory neural crest. We used single-cell mRNA and single-cell ATAC sequencing to profile the expression of Wnt1 and Sox10 and identify transcription factors that may regulate the expression of Wnt1-Cre2 in the neuroepithelium and Sox10-Cre in the migratory neural crest. Our data identify cellular heterogeneity during cranial neural crest development and identify specific populations labeled by two Cre-drivers in the developing nervous system.
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Affiliation(s)
- Rachel A. Keuls
- Development, Disease Models & Therapeutics Graduate Program, Baylor College of Medicine, Houston, TX, United States
- Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, United States
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, United States
- Department of Neuroscience, Baylor College of Medicine, Houston, TX, United States
| | - Ronald J. Parchem
- Development, Disease Models & Therapeutics Graduate Program, Baylor College of Medicine, Houston, TX, United States
- Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, United States
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, United States
- Department of Neuroscience, Baylor College of Medicine, Houston, TX, United States
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11
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Höving AL, Windmöller BA, Knabbe C, Kaltschmidt B, Kaltschmidt C, Greiner JFW. Between Fate Choice and Self-Renewal-Heterogeneity of Adult Neural Crest-Derived Stem Cells. Front Cell Dev Biol 2021; 9:662754. [PMID: 33898464 PMCID: PMC8060484 DOI: 10.3389/fcell.2021.662754] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Accepted: 03/18/2021] [Indexed: 12/16/2022] Open
Abstract
Stem cells of the neural crest (NC) vitally participate to embryonic development, but also remain in distinct niches as quiescent neural crest-derived stem cell (NCSC) pools into adulthood. Although NCSC-populations share a high capacity for self-renewal and differentiation resulting in promising preclinical applications within the last two decades, inter- and intrapopulational differences exist in terms of their expression signatures and regenerative capability. Differentiation and self-renewal of stem cells in developmental and regenerative contexts are partially regulated by the niche or culture condition and further influenced by single cell decision processes, making cell-to-cell variation and heterogeneity critical for understanding adult stem cell populations. The present review summarizes current knowledge of the cellular heterogeneity within NCSC-populations located in distinct craniofacial and trunk niches including the nasal cavity, olfactory bulb, oral tissues or skin. We shed light on the impact of intrapopulational heterogeneity on fate specifications and plasticity of NCSCs in their niches in vivo as well as during in vitro culture. We further discuss underlying molecular regulators determining fate specifications of NCSCs, suggesting a regulatory network including NF-κB and NC-related transcription factors like SLUG and SOX9 accompanied by Wnt- and MAPK-signaling to orchestrate NCSC stemness and differentiation. In summary, adult NCSCs show a broad heterogeneity on the level of the donor and the donors' sex, the cell population and the single stem cell directly impacting their differentiation capability and fate choices in vivo and in vitro. The findings discussed here emphasize heterogeneity of NCSCs as a crucial parameter for understanding their role in tissue homeostasis and regeneration and for improving their applicability in regenerative medicine.
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Affiliation(s)
- Anna L. Höving
- Department of Cell Biology, University of Bielefeld, Bielefeld, Germany
- Institute for Laboratory- and Transfusion Medicine, Heart and Diabetes Centre North Rhine-Westphalia (NRW), Ruhr University Bochum, Bad Oeynhausen, Germany
| | - Beatrice A. Windmöller
- Department of Cell Biology, University of Bielefeld, Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld FBMB e.V., Bielefeld, Germany
| | - Cornelius Knabbe
- Institute for Laboratory- and Transfusion Medicine, Heart and Diabetes Centre North Rhine-Westphalia (NRW), Ruhr University Bochum, Bad Oeynhausen, Germany
- Forschungsverbund BioMedizin Bielefeld FBMB e.V., Bielefeld, Germany
| | - Barbara Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld FBMB e.V., Bielefeld, Germany
- Molecular Neurobiology, University of Bielefeld, Bielefeld, Germany
| | - Christian Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld FBMB e.V., Bielefeld, Germany
| | - Johannes F. W. Greiner
- Department of Cell Biology, University of Bielefeld, Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld FBMB e.V., Bielefeld, Germany
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12
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Valek L, Tegeder I. Nucleoredoxin Knockdown in SH-SY5Y Cells Promotes Cell Renewal. Antioxidants (Basel) 2021; 10:antiox10030449. [PMID: 33805811 PMCID: PMC7999887 DOI: 10.3390/antiox10030449] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Revised: 03/04/2021] [Accepted: 03/09/2021] [Indexed: 01/13/2023] Open
Abstract
Nucleoredoxin (NXN) is a redox regulator of Disheveled and thereby of WNT signaling. Deficiency in mice leads to cranial dysmorphisms and defects of heart, brain, and bone, suggesting defects of cell fate determination. We used shRNA-mediated knockdown of NXN in SH-SY5Y neuroblastoma cells to study its impact on neuronal cells. We expected that shNXN cells would easily succumb to redox stress, but there were no differences in viability on stimulation with hydrogen peroxide. Instead, the proliferation of naïve shNXN cells was increased with a higher rate of mitotic cells in cell cycle analyses. In addition, basal respiratory rates were higher, whereas the relative change in oxygen consumption upon mitochondrial stressors was similar to control cells. shNXN cells had an increased expression of redox-sensitive heat shock proteins, Hsc70/HSPA8 and HSP90, and autophagy markers suggested an increase in autophagosome formation upon stimulation with bafilomycin and higher flux under low dose rapamycin. A high rate of self-renewal, autophagy, and upregulation of redox-sensitive chaperones appears to be an attractive anti-aging combination if it were to occur in neurons in vivo for which SH-SY5Y cells are a model.
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13
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Li T, Chen S, Pei M. Contribution of neural crest-derived stem cells and nasal chondrocytes to articular cartilage regeneration. Cell Mol Life Sci 2020; 77:4847-4859. [PMID: 32504256 PMCID: PMC9150440 DOI: 10.1007/s00018-020-03567-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 05/06/2020] [Accepted: 05/27/2020] [Indexed: 12/12/2022]
Abstract
Due to poor self-regenerative potential of articular cartilage, stem cell-based regeneration becomes a hopeful approach for the treatment of articular cartilage defects. Recent studies indicate that neural crest-derived cells (NCDCs) have the potential for repairing articular cartilage with even greater chondrogenic capacity than mesoderm-derived cells (MDCs): a conventional stem cell source for cartilage regeneration. Given that NCDCs originate from a different germ layer in the early embryo compared with MDCs that give rise to articular cartilage, a mystery remains regarding their capacity for articular cartilage regeneration. In this review, we summarize the similarities and differences between MDCs and NCDCs including articular and nasal chondrocytes in cell origin, anatomy, and chondrogenic differentiation and propose that NCDCs might be promising cell origins for articular cartilage regeneration.
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Affiliation(s)
- Tianyou Li
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, 64 Medical Center Drive, PO Box 9196, Morgantown, WV, 26506-9196, USA
- Department of Pediatric Orthopaedics, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China
| | - Song Chen
- Department of Orthopaedics, The General Hospital of Western Theater Command, Chengdu, 610083, Sichuan, China
| | - Ming Pei
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, 64 Medical Center Drive, PO Box 9196, Morgantown, WV, 26506-9196, USA.
- WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA.
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14
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Perera SN, Kerosuo L. On the road again: Establishment and maintenance of stemness in the neural crest from embryo to adulthood. STEM CELLS (DAYTON, OHIO) 2020; 39:7-25. [PMID: 33017496 PMCID: PMC7821161 DOI: 10.1002/stem.3283] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 09/08/2020] [Accepted: 09/11/2020] [Indexed: 12/22/2022]
Abstract
Unique to vertebrates, the neural crest (NC) is an embryonic stem cell population that contributes to a greatly expanding list of derivatives ranging from neurons and glia of the peripheral nervous system, facial cartilage and bone, pigment cells of the skin to secretory cells of the endocrine system. Here, we focus on what is specifically known about establishment and maintenance of NC stemness and ultimate fate commitment mechanisms, which could help explain its exceptionally high stem cell potential that exceeds the "rules set during gastrulation." In fact, recent discoveries have shed light on the existence of NC cells that coexpress commonly accepted pluripotency factors like Nanog, Oct4/PouV, and Klf4. The coexpression of pluripotency factors together with the exceptional array of diverse NC derivatives encouraged us to propose a new term "pleistopotent" (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post-gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC-derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos.
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Affiliation(s)
- Surangi N Perera
- Neural Crest Development and Disease Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
| | - Laura Kerosuo
- Neural Crest Development and Disease Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
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15
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Biniazan F, Manzari-Tavakoli A, Safaeinejad F, Moghimi A, Rajaei F, Niknejad H. The differentiation effect of bone morphogenetic protein (BMP) on human amniotic epithelial stem cells to express ectodermal lineage markers. Cell Tissue Res 2020; 383:751-763. [PMID: 32960356 DOI: 10.1007/s00441-020-03280-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Accepted: 08/12/2020] [Indexed: 11/28/2022]
Abstract
Stem cells are a promising tool for treatment of a variety of degenerative diseases. Human amniotic epithelial stem cells (hAECs) have desirable and unique characteristics that make them a proper candidate for cell therapy. In this study, we have investigated the effects of BMP-4 (bone morphogenetic protein-4) and its inhibition on differentiation of AECs into ectodermal lineages. Analysis of AEC-derived ectodermal lineages (neurons and keratinocytes) was performed by using flow cytometry technique for Map2 and β-tubulin (as neuron markers), Olig2 and MBP (as oligodendrocyte markers), and K14 and K10 (as keratinocyte markers). The results of this study illustrated that noggin (as BMP antagonist), BMP4, and both BMP4 and heparin (together or separately) increased neural and keratinocyte marker expression, respectively. The expression of markers MAP2, olig2, and K14 in hAECs has been significantly decreased 21 days after exposure to differentiation medium (without growth factors) compared with isolation day, which supports the hypothesis that AECs can be dedifferentiated into pluripotent cells. Moreover, activation and inhibition of BMP signaling have no effects on viability of hAECs. The results of this study showed that BMP signaling and its inhibition are the key factors for ectodermal lineage differentiation of amnion-derived stem cells.
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Affiliation(s)
- Felor Biniazan
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.,Cellular and Molecular Research Center, Qazvin University of Medical Science, Qazvin, Iran
| | - Asma Manzari-Tavakoli
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Fahimeh Safaeinejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Ali Moghimi
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
| | - Farzad Rajaei
- Cellular and Molecular Research Center, Qazvin University of Medical Science, Qazvin, Iran.
| | - Hassan Niknejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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16
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Bhattacharya D, Azambuja AP, Simoes-Costa M. Metabolic Reprogramming Promotes Neural Crest Migration via Yap/Tead Signaling. Dev Cell 2020; 53:199-211.e6. [PMID: 32243782 PMCID: PMC7236757 DOI: 10.1016/j.devcel.2020.03.005] [Citation(s) in RCA: 107] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2019] [Revised: 01/05/2020] [Accepted: 03/04/2020] [Indexed: 02/04/2023]
Abstract
The Warburg effect is one of the metabolic hallmarks of cancer cells, characterized by enhanced glycolysis even under aerobic conditions. This physiological adaptation is associated with metastasis , but we still have a superficial understanding of how it affects cellular processes during embryonic development. Here we report that the neural crest, a migratory stem cell population in vertebrate embryos, undergoes an extensive metabolic remodeling to engage in aerobic glycolysis prior to delamination. This increase in glycolytic flux promotes Yap/Tead signaling, which activates the expression of a set of transcription factors to drive epithelial-to-mesenchymal transition. Our results demonstrate how shifts in carbon metabolism can trigger the gene regulatory circuits that control complex cell behaviors. These findings support the hypothesis that the Warburg effect is a precisely regulated developmental mechanism that is anomalously reactivated during tumorigenesis and metastasis.
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Affiliation(s)
| | - Ana Paula Azambuja
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA
| | - Marcos Simoes-Costa
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA.
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17
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In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like. Cells 2020; 9:cells9041000. [PMID: 32316463 PMCID: PMC7226083 DOI: 10.3390/cells9041000] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 04/13/2020] [Accepted: 04/14/2020] [Indexed: 02/07/2023] Open
Abstract
Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.
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18
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Wnt Signaling in Neural Crest Ontogenesis and Oncogenesis. Cells 2019; 8:cells8101173. [PMID: 31569501 PMCID: PMC6829301 DOI: 10.3390/cells8101173] [Citation(s) in RCA: 44] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 09/23/2019] [Accepted: 09/25/2019] [Indexed: 02/06/2023] Open
Abstract
Neural crest (NC) cells are a temporary population of multipotent stem cells that generate a diverse array of cell types, including craniofacial bone and cartilage, smooth muscle cells, melanocytes, and peripheral neurons and glia during embryonic development. Defective neural crest development can cause severe and common structural birth defects, such as craniofacial anomalies and congenital heart disease. In the early vertebrate embryos, NC cells emerge from the dorsal edge of the neural tube during neurulation and then migrate extensively throughout the anterior-posterior body axis to generate numerous derivatives. Wnt signaling plays essential roles in embryonic development and cancer. This review summarizes current understanding of Wnt signaling in NC cell induction, delamination, migration, multipotency, and fate determination, as well as in NC-derived cancers.
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19
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Shi C, Yuan Y, Guo Y, Jing J, Ho TV, Han X, Li J, Feng J, Chai Y. BMP Signaling in Regulating Mesenchymal Stem Cells in Incisor Homeostasis. J Dent Res 2019; 98:904-911. [PMID: 31136721 DOI: 10.1177/0022034519850812] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Bone morphogenetic protein (BMP) signaling performs multiple essential functions during craniofacial development. In this study, we used the adult mouse incisor as a model to uncover how BMP signaling maintains tissue homeostasis and regulates mesenchymal stem cell (MSC) fate by mediating WNT and FGF signaling. We observed a severe defect in the proximal region of the adult mouse incisor after loss of BMP signaling in the Gli1+ cell lineage, indicating that BMP signaling is required for cell proliferation and odontoblast differentiation. Our study demonstrates that BMP signaling serves as a key regulator that antagonizes WNT and FGF signaling to regulate MSC lineage commitment. In addition, BMP signaling in the Gli1+ cell lineage is also required for the maintenance of quiescent MSCs, suggesting that BMP signaling not only is important for odontoblast differentiation but also plays a crucial role in providing feedback to the MSC population. This study highlights multiple important roles of BMP signaling in regulating tissue homeostasis.
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Affiliation(s)
- C Shi
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA.,2 Department of Orthodontics, The Affiliated Stomatology Hospital of Kunming Medical University, Kunming, China
| | - Y Yuan
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA
| | - Y Guo
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA.,3 Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing, China
| | - J Jing
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA.,4 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - T V Ho
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA
| | - X Han
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA
| | - J Li
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA.,5 Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China
| | - J Feng
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA
| | - Y Chai
- 1 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA
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20
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Etchevers HC, Dupin E, Le Douarin NM. The diverse neural crest: from embryology to human pathology. Development 2019; 146:146/5/dev169821. [PMID: 30858200 DOI: 10.1242/dev.169821] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2018] [Accepted: 02/07/2019] [Indexed: 01/13/2023]
Abstract
We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, paediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, when increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.
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Affiliation(s)
- Heather C Etchevers
- Aix-Marseille Université, INSERM, MMG, U1251, 27 boulevard Jean Moulin 13005 Marseille, France
| | - Elisabeth Dupin
- Sorbonne Universités, UPMC Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
| | - Nicole M Le Douarin
- Sorbonne Universités, UPMC Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
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21
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Wu P, Zhang Y, Xing Y, Xu W, Guo H, Deng F, Ma X, Li Y. The balance of Bmp6 and Wnt10b regulates the telogen-anagen transition of hair follicles. Cell Commun Signal 2019; 17:16. [PMID: 30791955 PMCID: PMC6385416 DOI: 10.1186/s12964-019-0330-x] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2018] [Accepted: 02/15/2019] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND The periodic growth of hair follicles is regulated by the balance of activators and inhibitors. The BMP signaling pathway plays an important role during hair follicle regeneration, but the exact BMP protein that controls this process has not been revealed. METHODS The expression of BMP6 was determined via in situ hybridization and immunofluorescence. The in vivo effect of BMP6 overexpression was studied by using a previously established adenovirus injection model. The hair follicle regeneration was assessed by gross observation, H&E staining and 5-bromo-2-deoxyuridine (BrdU) tracing. The expression patterns of BMP6 signaling and Wnt10b signaling in both AdBMP6-treated and AdWnt10b-treated skins were determined by in situ hybridization and immunofluorescence. RESULTS BMP6 was expressed differently in the stages of hair follicle cycle. The telogen-anagen transition of hair follicles was inhibited by adenovirus-mediated overexpression of BMP6. In the in vivo model, the BMP6 signaling was inhibited by Wnt10b and the Wnt10b signaling was inhibited by BMP6. The activation of hair follicle stem cells (HFSCs) was also competitively regulated by Wnt10b and BMP6. CONCLUSIONS Combined with previously reported data of Wnt10b, our findings indicate that BMP6 and Wnt10b are major inhibitors and activators respectively and their balance regulates the telogen-anagen transition of hair follicles. To the best of our knowledge, our data provide previously unreported insights into the regulation of hair follicle cycling and provide new clues for the diagnosis and therapies of hair loss.
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Affiliation(s)
- Pan Wu
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
| | - Yiming Zhang
- Department of Plastic and Cosmetic Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China
| | - Yizhan Xing
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
| | - Wei Xu
- Department of Dermatology, Chongqing First People’s Hospital and Chongqing Traditional Chinese Medicine Hospital, Chongqing, China
| | - Haiying Guo
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
| | - Fang Deng
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
| | - Xiaogen Ma
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
- Department of Plastic and Cosmetic Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China
| | - Yuhong Li
- Department of Cell Biology, Army Medical University, Gaotanyan street No. 30, Shapingba, Chongqing, 400038 China
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22
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Le Douarin NM, Dupin E. The “beginnings” of the neural crest. Dev Biol 2018; 444 Suppl 1:S3-S13. [DOI: 10.1016/j.ydbio.2018.07.019] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2018] [Revised: 07/20/2018] [Accepted: 07/20/2018] [Indexed: 12/14/2022]
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23
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Dupin E, Calloni GW, Coelho-Aguiar JM, Le Douarin NM. The issue of the multipotency of the neural crest cells. Dev Biol 2018; 444 Suppl 1:S47-S59. [DOI: 10.1016/j.ydbio.2018.03.024] [Citation(s) in RCA: 67] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2017] [Revised: 03/29/2018] [Accepted: 03/30/2018] [Indexed: 12/25/2022]
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24
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Petratou K, Subkhankulova T, Lister JA, Rocco A, Schwetlick H, Kelsh RN. A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest. PLoS Genet 2018; 14:e1007402. [PMID: 30286071 PMCID: PMC6191144 DOI: 10.1371/journal.pgen.1007402] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Revised: 10/16/2018] [Accepted: 08/22/2018] [Indexed: 12/29/2022] Open
Abstract
Multipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells (chromatophores), but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we focus on the iridophore GRN, where mutant phenotypes identify the transcription factors Sox10, Tfec and Mitfa and the receptor tyrosine kinase, Ltk, as key players. Here we present expression data, as well as loss and gain of function results, guiding the derivation of an initial iridophore specification GRN. Moreover, we use an iterative process of mathematical modelling, supplemented with a Monte Carlo screening algorithm suited to the qualitative nature of the experimental data, to allow for rigorous predictive exploration of the GRN dynamics. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. Our study reveals multiple important regulatory features, notably a sox10-dependent positive feedback loop between tfec and ltk driving iridophore specification; the molecular basis of sox10 maintenance throughout iridophore development; and the cooperation between sox10 and tfec in driving expression of pnp4a, a key differentiation gene. We also assess a candidate repressor of mitfa, a melanocyte-specific target of sox10. Surprisingly, our data challenge the reported role of Foxd3, an established mitfa repressor, in iridophore regulation. Our study builds upon our previous systems biology approach, by incorporating physiologically-relevant parameter values and rigorous evaluation of parameter values within a qualitative data framework, to establish for the first time the core GRN guiding specification of the iridophore lineage.
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Affiliation(s)
- Kleio Petratou
- Department of Biology and Biochemistry and Centre for Regenerative Medicine, Faculty of Science, University of Bath, Bath, United Kingdom
| | - Tatiana Subkhankulova
- Department of Biology and Biochemistry and Centre for Regenerative Medicine, Faculty of Science, University of Bath, Bath, United Kingdom
| | - James A. Lister
- Department of Human and Molecular Genetics and Massey Cancer Center, VCU School of Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America
| | - Andrea Rocco
- Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom
| | - Hartmut Schwetlick
- Department of Mathematical Sciences, Faculty of Science, University of Bath, Bath, United Kingdom
| | - Robert N. Kelsh
- Department of Biology and Biochemistry and Centre for Regenerative Medicine, Faculty of Science, University of Bath, Bath, United Kingdom
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25
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Luo L, Hu DH, Yin JQ, Xu RX. Molecular Mechanisms of Transdifferentiation of Adipose-Derived Stem Cells into Neural Cells: Current Status and Perspectives. Stem Cells Int 2018; 2018:5630802. [PMID: 30302094 PMCID: PMC6158979 DOI: 10.1155/2018/5630802] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2018] [Revised: 07/12/2018] [Accepted: 07/19/2018] [Indexed: 12/19/2022] Open
Abstract
Neurological diseases can severely compromise both physical and psychological health. Recently, adult mesenchymal stem cell- (MSC-) based cell transplantation has become a potential therapeutic strategy. However, most studies related to the transdifferentiation of MSCs into neural cells have had disappointing outcomes. Better understanding of the mechanisms underlying MSC transdifferentiation is necessary to make adult stem cells more applicable to treating neurological diseases. Several studies have focused on adipose-derived stromal/stem cell (ADSC) transdifferentiation. The purpose of this review is to outline the molecular characterization of ADSCs, to describe the methods for inducing ADSC transdifferentiation, and to examine factors influencing transdifferentiation, including transcription factors, epigenetics, and signaling pathways. Exploring and understanding the mechanisms are a precondition for developing and applying novel cell therapies.
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Affiliation(s)
- Liang Luo
- Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shanxi 710032, China
- Stem Cell Research Center, Neurosurgery Institute of PLA Army, Beijing 100700, China
- Bayi Brain Hospital, General Hospital of PLA Army, Beijing 100700, China
| | - Da-Hai Hu
- Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shanxi 710032, China
| | - James Q. Yin
- Stem Cell Research Center, Neurosurgery Institute of PLA Army, Beijing 100700, China
- Bayi Brain Hospital, General Hospital of PLA Army, Beijing 100700, China
| | - Ru-Xiang Xu
- Stem Cell Research Center, Neurosurgery Institute of PLA Army, Beijing 100700, China
- Bayi Brain Hospital, General Hospital of PLA Army, Beijing 100700, China
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26
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Liao D, Li X, Dong Y, Sun G. The Role of Wnt/β-Catenin Signaling Pathway in the Transdifferentiation from Periodontal Ligament Stem Cells to Schwann Cells. Cell Reprogram 2018; 19:384-388. [PMID: 29215941 DOI: 10.1089/cell.2017.0017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
This study was aimed to investigate the role of Wnt/β-catenin signaling pathway in the differentiation from periodontal ligament stem cells (PDLSCs) to Schwann cells (SCs) and the possible mechanisms. FzB was applied to inhibit the Wnt/β-catenin signaling pathway of differentiated PDLSCs (dPDLSCs), and then immunofluorescence, polymerase chain reaction (PCR), and Western blotting analysis were performed to detect SC marker genes and proteins such as S100, glial fibrillary acidic protein (GFAP), and P75NTR. Results showed that when the Wnt/β-catenin signaling pathway was inhibited, the expression of S100, GFAP, and P75NTR protein significantly decreased in dPDLSCs, p < 0.05, whereas PCR results showed that expression of SC myelinogenesis-related genes krox-20, Oct-6, P0, and PMP-22 was significantly downregulated at the same time, p < 0.05. These results showed that Wnt/β-catenin signaling pathway participated in the differentiation from PDLSCs to SCs, and inhibiting it could inhibit the differentiation process.
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Affiliation(s)
- Dapeng Liao
- 1 Department of Dentistry, Second Affiliated Hospital, School of Medicine, Zhejiang University , China
| | - Xiaojie Li
- 2 Department of Dentistry, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University , China
| | - Yan Dong
- 1 Department of Dentistry, Second Affiliated Hospital, School of Medicine, Zhejiang University , China
| | - Gang Sun
- 1 Department of Dentistry, Second Affiliated Hospital, School of Medicine, Zhejiang University , China
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27
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da Costa MC, Trentin AG, Calloni GW. FGF8 and Shh promote the survival and maintenance of multipotent neural crest progenitors. Mech Dev 2018; 154:251-258. [PMID: 30075227 DOI: 10.1016/j.mod.2018.07.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Revised: 07/30/2018] [Accepted: 07/31/2018] [Indexed: 02/07/2023]
Abstract
The developmental mechanisms that control the building of the complex head of vertebrates and particularly, facial skeletogenesis, remain poorly known. Progenitor cells derived from the embryonic neural crest (NC) are the major constituents and players of facial tissue development. Deciphering the cellular and molecular machinery that controls NC cell (NCC) differentiation into bone, cartilage, fat and other mesenchymal tissues, is thus a main issue for understanding vertebrate facial variations. In this work, we investigated the effects of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (Shh), two signaling molecules essential for craniofacial development, on the in vitro differentiation and multipotentiality of mesencephalic NCCs (MNCCs) isolated from the quail embryo. Comparison of distinct temporal treatments with FGF8 and/or Shh showed that both promoted chondrogenesis of MNCCs by increasing the amount and size of cartilage nodules. Higher rates of chondrogenesis were observed when MNCCs were treated with FGF8 during the migration phase, thus mimicking the in vivo exposure of migrating NCCs to FGF8 secreted by the isthmic brain signaling center. An in vitro cell cloning assay revealed that, after concomitant treatment with FGF8 and Shh, about 80% of NC progenitors displayed chondrogenic potential, while in untreated cultures, only 18% exhibited this potential. In addition, colony analysis showed for the first time the existence of a highly multipotent progenitor able to clonally give rise to adipocytes in addition to other cephalic NC phenotypes (i.e. glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes) (GNMFCA progenitor). This progenitor was observed only when clonal cultures were treated with both FGF8 and Shh. Several other types of multipotent cells, which generated four, five or six distinct phenotypes, accounted for 55% of the progenitors in FGF8 and Shh treated cultures, versus 13,5% in the untreated ones. Together, these data reveal an essential role for both FGF8 and Shh together in maintenance of MNCC multipotentiality by favoring the development of NC progenitors endowed with a broad array of mesectodermal potentials.
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Affiliation(s)
- Meline Coelho da Costa
- Laboratório de Plasticidade e Diferenciação de Células da Crista Neural, Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário - Trindade, 88040-900 Florianópolis, SC, Brazil; Laboratório de Células Tronco e Regeneração Tecidual, Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário - Trindade, 88040-900 Florianópolis, SC, Brazil
| | - Andréa Gonçalves Trentin
- Laboratório de Células Tronco e Regeneração Tecidual, Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário - Trindade, 88040-900 Florianópolis, SC, Brazil
| | - Giordano Wosgrau Calloni
- Laboratório de Plasticidade e Diferenciação de Células da Crista Neural, Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário - Trindade, 88040-900 Florianópolis, SC, Brazil.
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28
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Debbache J, Parfejevs V, Sommer L. Cre-driver lines used for genetic fate mapping of neural crest cells in the mouse: An overview. Genesis 2018; 56:e23105. [PMID: 29673028 PMCID: PMC6099459 DOI: 10.1002/dvg.23105] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Revised: 03/23/2018] [Accepted: 03/26/2018] [Indexed: 01/01/2023]
Abstract
The neural crest is one of the embryonic structures with the broadest developmental potential in vertebrates. Morphologically, neural crest cells emerge during neurulation in the dorsal folds of the neural tube before undergoing an epithelial‐to‐mesenchymal transition (EMT), delaminating from the neural tube, and migrating to multiple sites in the growing embryo. Neural crest cells generate cell types as diverse as peripheral neurons and glia, melanocytes, and so‐called mesectodermal derivatives that include craniofacial bone and cartilage and smooth muscle cells in cardiovascular structures. In mice, the fate of neural crest cells has been determined mainly by means of transgenesis and genome editing technologies. The most frequently used method relies on the Cre‐loxP system, in which expression of Cre‐recombinase in neural crest cells or their derivatives genetically enables the expression of a Cre‐reporter allele, thus permanently marking neural crest‐derived cells. Here, we provide an overview of the Cre‐driver lines used in the field and discuss to what extent these lines allow precise neural crest stage and lineage‐specific fate mapping.
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Affiliation(s)
- Julien Debbache
- Stem Cell Biology, Institute of Anatomy, University of Zurich, Zurich, CH-8057, Switzerland
| | - Vadims Parfejevs
- Stem Cell Biology, Institute of Anatomy, University of Zurich, Zurich, CH-8057, Switzerland
| | - Lukas Sommer
- Stem Cell Biology, Institute of Anatomy, University of Zurich, Zurich, CH-8057, Switzerland
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29
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Geary L, LaBonne C. FGF mediated MAPK and PI3K/Akt Signals make distinct contributions to pluripotency and the establishment of Neural Crest. eLife 2018; 7:33845. [PMID: 29350613 PMCID: PMC5790379 DOI: 10.7554/elife.33845] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2017] [Accepted: 01/15/2018] [Indexed: 12/12/2022] Open
Abstract
Early vertebrate embryos possess cells with the potential to generate all embryonic cell types. While this pluripotency is progressively lost as cells become lineage restricted, Neural Crest cells retain broad developmental potential. Here, we provide novel insights into signals essential for both pluripotency and neural crest formation in Xenopus. We show that FGF signaling controls a subset of genes expressed by pluripotent blastula cells, and find a striking switch in the signaling cascades activated by FGF signaling as cells lose pluripotency and commence lineage restriction. Pluripotent cells display and require Map Kinase signaling, whereas PI3 Kinase/Akt signals increase as developmental potential is restricted, and are required for transit to certain lineage restricted states. Importantly, retaining a high Map Kinase/low Akt signaling profile is essential for establishing Neural Crest stem cells. These findings shed important light on the signal-mediated control of pluripotency and the molecular mechanisms governing genesis of Neural Crest.
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Affiliation(s)
- Lauren Geary
- Department of Molecular Biosciences, Northwestern University, Evanston, United States
| | - Carole LaBonne
- Department of Molecular Biosciences, Northwestern University, Evanston, United States.,Robert H Lurie Comprehensive Cancer Center, Northwestern University, Evanston, United States
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30
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Schwartzentruber J, Foskolou S, Kilpinen H, Rodrigues J, Alasoo K, Knights AJ, Patel M, Goncalves A, Ferreira R, Benn CL, Wilbrey A, Bictash M, Impey E, Cao L, Lainez S, Loucif AJ, Whiting PJ, Gutteridge A, Gaffney DJ. Molecular and functional variation in iPSC-derived sensory neurons. Nat Genet 2018; 50:54-61. [PMID: 29229984 PMCID: PMC5742539 DOI: 10.1038/s41588-017-0005-8] [Citation(s) in RCA: 159] [Impact Index Per Article: 22.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Accepted: 11/06/2017] [Indexed: 12/20/2022]
Abstract
Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.
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Affiliation(s)
- Jeremy Schwartzentruber
- Wellcome Trust Sanger Institute, Hinxton, UK.
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, UK.
| | - Stefanie Foskolou
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | - Helena Kilpinen
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, UK
| | | | - Kaur Alasoo
- Wellcome Trust Sanger Institute, Hinxton, UK
| | | | - Minal Patel
- Wellcome Trust Sanger Institute, Hinxton, UK
| | | | - Rita Ferreira
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | | | - Anna Wilbrey
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | - Magda Bictash
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | - Emma Impey
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | - Lishuang Cao
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | - Sergio Lainez
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
| | | | - Paul John Whiting
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK
- AR-UK Drug Discovery Institute, Institute of Neurology, University College London, London, UK
| | - Alex Gutteridge
- Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK.
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31
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Münst S, Koch P, Kesavan J, Alexander-Mays M, Münst B, Blaess S, Brüstle O. In vitro segregation and isolation of human pluripotent stem cell-derived neural crest cells. Methods 2017; 133:65-80. [PMID: 29037816 DOI: 10.1016/j.ymeth.2017.09.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Revised: 09/24/2017] [Accepted: 09/27/2017] [Indexed: 01/17/2023] Open
Abstract
The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.
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Affiliation(s)
- Sabine Münst
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Philipp Koch
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Jaideep Kesavan
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Michael Alexander-Mays
- Institute of Human Genetics, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Bernhard Münst
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Sandra Blaess
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany.
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32
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Kjolby RAS, Harland RM. Genome-wide identification of Wnt/β-catenin transcriptional targets during Xenopus gastrulation. Dev Biol 2017; 426:165-175. [PMID: 27091726 PMCID: PMC6288011 DOI: 10.1016/j.ydbio.2016.03.021] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Revised: 03/16/2016] [Accepted: 03/16/2016] [Indexed: 10/21/2022]
Abstract
The canonical Wnt/β-catenin signaling pathway plays multiple roles during Xenopus gastrulation, including posteriorization of the neural plate, patterning of the mesoderm, and induction of the neural crest. Wnt signaling stabilizes β-catenin, which then activates target genes. However, few targets of this signaling pathway that mediate early developmental processes are known. Here we sought to identify transcriptional targets of the Wnt/β-catenin signaling pathway using a genome-wide approach. We selected putative targets using the criteria of reduced expression upon zygotic Wnt knockdown, β-catenin binding within 50kb of the gene, and expression in tissues that receive Wnt signaling. Using these criteria, we found 21 novel direct transcriptional targets of Wnt/β-catenin signaling during gastrulation and in addition have identified putative regulatory elements for further characterization in future studies.
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Affiliation(s)
- Rachel A S Kjolby
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | - Richard M Harland
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
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33
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Ogawa R, Fujita K, Ito K. Mouse embryonic dorsal root ganglia contain pluripotent stem cells that show features similar to embryonic stem cells and induced pluripotent stem cells. Biol Open 2017; 6:602-618. [PMID: 28373172 PMCID: PMC5450311 DOI: 10.1242/bio.021758] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
In the present study, we showed that the dorsal root ganglion (DRG) in the mouse embryo contains pluripotent stem cells (PSCs) that have developmental capacities equivalent to those of embryonic stem (ES) cells and induced pluripotent stem cells. Mouse embryonic DRG cells expressed pluripotency-related transcription factors [octamer-binding transcription factor 4, SRY (sex determining region Y)-box containing gene (Sox) 2, and Nanog] that play essential roles in maintaining the pluripotency of ES cells. Furthermore, the DRG cells differentiated into ectoderm-, mesoderm- and endoderm-derived cells. In addition, these cells produced primordial germ cell-like cells and embryoid body-like spheres. We also showed that the combination of leukemia inhibitor factor/bone morphogenetic protein 2/fibroblast growth factor 2 effectively promoted maintenance of the pluripotency of the PSCs present in DRGs, as well as that of neural crest-derived stem cells (NCSCs) in DRGs, which were previously shown to be present there. Furthermore, the expression of pluripotency-related transcription factors in the DRG cells was regulated by chromodomain helicase DNA-binding protein 7 and Sox10, which are indispensable for the formation of NCSCs, and vice versa. These findings support the possibility that PSCs in mouse embryonic DRGs are NCSCs.
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Affiliation(s)
- Ryuhei Ogawa
- Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
| | - Kyohei Fujita
- Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
| | - Kazuo Ito
- Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
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34
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Xu J, Ueno H, Xu CY, Chen B, Weissman IL, Xu PX. Identification of mouse cochlear progenitors that develop hair and supporting cells in the organ of Corti. Nat Commun 2017; 8:15046. [PMID: 28492243 PMCID: PMC5437288 DOI: 10.1038/ncomms15046] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 02/23/2017] [Indexed: 01/20/2023] Open
Abstract
The adult mammalian cochlear sensory epithelium houses two major types of cells, mechanosensory hair cells and underlying supporting cells, and lacks regenerative capacity. Recent evidence indicates that a subset of supporting cells can spontaneously regenerate hair cells after ablation only within the first week postparturition. Here in vivo clonal analysis of mouse inner ear cells during development demonstrates clonal relationship between hair and supporting cells in sensory organs. We report the identification in mouse of a previously unknown population of multipotent stem/progenitor cells that are capable of not only contributing to the hair and supporting cells but also to other cell types, including glia, in cochlea undergoing development, maturation and repair in response to damage. These multipotent progenitors originate from Eya1-expressing otic progenitors. Our findings also provide evidence for detectable regenerative potential in the postnatal cochlea beyond 1 week of age.
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Affiliation(s)
- Jinshu Xu
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Hiroo Ueno
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA
- Ludwig Center, Stanford University, Stanford, California 94305, USA
- Department of Pathology, Stanford University, Stanford, California 94305, USA
| | - Chelsea Y. Xu
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Binglai Chen
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Irving L. Weissman
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA
- Ludwig Center, Stanford University, Stanford, California 94305, USA
- Department of Pathology, Stanford University, Stanford, California 94305, USA
| | - Pin-Xian Xu
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
- Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
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35
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Maternal separation induces hippocampal changes in cadherin-1 ( CDH-1 ) mRNA and recognition memory impairment in adolescent mice. Neurobiol Learn Mem 2017; 141:157-167. [DOI: 10.1016/j.nlm.2017.04.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Revised: 03/16/2017] [Accepted: 04/17/2017] [Indexed: 01/09/2023]
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36
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Breitfeld J, Scholl C, Steffens M, Laje G, Stingl JC. Gene expression and proliferation biomarkers for antidepressant treatment resistance. Transl Psychiatry 2017; 7:e1061. [PMID: 28291260 PMCID: PMC5416664 DOI: 10.1038/tp.2017.16] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Revised: 12/09/2016] [Accepted: 12/30/2016] [Indexed: 02/07/2023] Open
Abstract
The neurotrophic hypothesis of depression suggests an association between effects on neuroplasticity and clinical response to antidepressant drug therapy. We studied individual variability in antidepressant drug effects on cell proliferation in lymphoblastoid cell lines (LCLs) from n=25 therapy-resistant patients versus n=25 first-line therapy responders from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. Furthermore, the variability in gene expression of genes associated with cell proliferation was analyzed for tentative candidate genes for prediction of individual LCL donor's treatment response. Cell proliferation was quantified by EdU (5-ethynyl-2'-deoxyuridine) assays after 21-day incubation of LCLs with fluoxetine (0.5 ng μl-1) and citalopram (0.3 ng μl-1) as developed and described earlier. Gene expression of a panel of candidate genes derived from genome-wide expression analyses of antidepressant effects on cell proliferation of LCLs from the Munich Antidepressant Response Signature (MARS) study was analyzed by real-time PCR. Significant differences in in vitro cell proliferation effects were detected between the group of LCLs from first-line therapy responders and LCLs from treatment-resistant patients. Gene expression analysis of the candidate gene panel revealed and confirmed influence of the candidate genes ABCB1, FZD7 and WNT2B on antidepressant drug resistance. The potential of these genes as tentative biomarkers for antidepressant drug resistance was confirmed. In vitro cell proliferation testing may serve as functional biomarker for individual neuroplasticity effects of antidepressants.
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Affiliation(s)
- J Breitfeld
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - C Scholl
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - M Steffens
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - G Laje
- Washington Behavioral Medicine Associates, LLC, Chevy Chase, MD, USA
| | - J C Stingl
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
- Centre for Translational Medicine, University Bonn Medical Faculty, Bonn, Germany
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37
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Olmsted-Davis EA, Salisbury EA, Hoang D, Davis EL, Lazard Z, Sonnet C, Davis TA, Forsberg JA, Davis AR. Progenitors in Peripheral Nerves Launch Heterotopic Ossification. Stem Cells Transl Med 2017; 6:1109-1119. [PMID: 28198109 PMCID: PMC5442844 DOI: 10.1002/sctm.16-0347] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 10/31/2016] [Indexed: 12/23/2022] Open
Abstract
Studies presented here, using a murine model of bone morphogenetic protein type 2 (BMP2)-induced heterotopic ossification (HO) show that the protein initiates HO by signaling through progenitors in the endoneurium of peripheral nerves. In the mouse, these cells were identified in the endoneurium one day after BMP2 induction using antibody against phosphoSMAD (PS) 1, 5, and 8. Studies conducted in a tracking mouse that contains a tamoxifen-regulated Wnt1-Cre recombinase crossed with a td Tomato red (TR) reporter (Wnt1CreErt :Ai9Tm) confirmed their neural origin. In this model both BMP2 induction and tamoxifen are absolutely required to induce TR. SP7+ (osterix+ )TR+ cells were found in the endoneurium on day 1 and associated with bone on day 7. Quantification of TR+ and TR- cells isolated by fluorescence-activated cell sorting showed that all SP7+ cells were found in the TR+ population, whereas only about 80% of the TR+ cells expressed SP7. Pre-chondrocytes (Sox 9+ ) and transient brown fat (tBAT, UCP1+ ) also coexpressed TR, suggesting that the progenitor in nerves is multi-potential. The endoneurium of human nerves near the site of HO contained many PS+ cells, and SP7+ cells were found in nerves and on bone in tissue from patients with HO. Control tissues and nerves did not contain these PS+ and SP7+ cells. Some osteoblasts on bone from patients with HO were positive for PS, suggesting the continued presence of BMP during bone formation. The data suggests that the progenitors for HO are derived from the endoneurium in both the mouse model of HO and in humans with HO. Stem Cells Translational Medicine 2017;6:1109-1119.
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Affiliation(s)
- Elizabeth A Olmsted-Davis
- Center for Cell and Gene Therapy.,Departments of Pediatrics and Orthopedic Surgery, Baylor College of Medicine, Houston, Texas, USA
| | | | | | | | | | | | - Thomas A Davis
- Department of Surgery, Uniformed Services University of the Health Sciences & the Walter Reed National Military Medical Center, Bethesda, Maryland, USA.,Regenerative Medicine Department, Naval Medical Research Center, Silver Spring, Maryland, USA
| | - Jonathan A Forsberg
- Department of Surgery, Uniformed Services University of the Health Sciences & the Walter Reed National Military Medical Center, Bethesda, Maryland, USA.,Regenerative Medicine Department, Naval Medical Research Center, Silver Spring, Maryland, USA
| | - Alan R Davis
- Center for Cell and Gene Therapy.,Departments of Pediatrics and Orthopedic Surgery, Baylor College of Medicine, Houston, Texas, USA
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38
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Abstract
Cytokines of the transforming growth factor β (TGF-β) family, including TGF-βs, bone morphogenic proteins (BMPs), activins, and Nodal, play crucial roles in embryonic development and adult tissue homeostasis by regulating cell proliferation, survival, and differentiation, as well as stem-cell self-renewal and lineage-specific differentiation. Smad proteins are critical downstream mediators of these signaling activities. In addition to regulating the transcription of direct target genes of TGF-β, BMP, activin, or Nodal, Smad proteins also participate in extensive cross talk with other signaling pathways, often in a cell-type- or developmental stage-specific manner. These combinatorial signals often produce context-, time-, and location-dependent biological outcomes that are critical for development. This review discusses recent progress in our understanding of the cross talk between Smad proteins and signaling pathways of Wnt, Notch, Hippo, Hedgehog (Hh), mitogen-activated protein (MAP), kinase, phosphoinositide 3-kinase (PI3K)-Akt, nuclear factor κB (NF-κB), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways.
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Affiliation(s)
- Kunxin Luo
- Department of Molecular and Cell Biology, University of California, Berkeley, and Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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39
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Luo K. Signaling Cross Talk between TGF-β/Smad and Other Signaling Pathways. Cold Spring Harb Perspect Biol 2017. [PMID: 27836834 DOI: 10.1101/cshperspect] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Cytokines of the transforming growth factor β (TGF-β) family, including TGF-βs, bone morphogenic proteins (BMPs), activins, and Nodal, play crucial roles in embryonic development and adult tissue homeostasis by regulating cell proliferation, survival, and differentiation, as well as stem-cell self-renewal and lineage-specific differentiation. Smad proteins are critical downstream mediators of these signaling activities. In addition to regulating the transcription of direct target genes of TGF-β, BMP, activin, or Nodal, Smad proteins also participate in extensive cross talk with other signaling pathways, often in a cell-type- or developmental stage-specific manner. These combinatorial signals often produce context-, time-, and location-dependent biological outcomes that are critical for development. This review discusses recent progress in our understanding of the cross talk between Smad proteins and signaling pathways of Wnt, Notch, Hippo, Hedgehog (Hh), mitogen-activated protein (MAP), kinase, phosphoinositide 3-kinase (PI3K)-Akt, nuclear factor κB (NF-κB), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways.
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Affiliation(s)
- Kunxin Luo
- Department of Molecular and Cell Biology, University of California, Berkeley, and Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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40
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Breitfeld J, Scholl C, Steffens M, Brandenburg K, Probst-Schendzielorz K, Efimkina O, Gurwitz D, Ising M, Holsboer F, Lucae S, Stingl JC. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy. Transl Psychiatry 2016; 6:e950. [PMID: 27845776 PMCID: PMC5314111 DOI: 10.1038/tp.2016.185] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2016] [Revised: 08/02/2016] [Accepted: 08/03/2016] [Indexed: 12/25/2022] Open
Abstract
The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin-a gene involved in neuronal stem cell regeneration-were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.
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Affiliation(s)
- J Breitfeld
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - C Scholl
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - M Steffens
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - K Brandenburg
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
| | - K Probst-Schendzielorz
- Institute of Clinical Pharmacology and Pharmacology of Natural Products, University of Ulm, Ulm, Germany
| | - O Efimkina
- Institute of Clinical Pharmacology and Pharmacology of Natural Products, University of Ulm, Ulm, Germany
| | - D Gurwitz
- Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - M Ising
- Max Planck Institute of Psychiatry, Munich, Germany
| | - F Holsboer
- Max Planck Institute of Psychiatry, Munich, Germany,HMNC Holding GmbH, Munich, Germany
| | - S Lucae
- Max Planck Institute of Psychiatry, Munich, Germany
| | - J C Stingl
- Research Division, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany,Center for Translational Medicine, Bonn University Medical School, Bonn, Germany,Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany. E-mail:
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41
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Fujita K, Ogawa R, Ito K. CHD7, Oct3/4, Sox2, and Nanog control FoxD3 expression during mouse neural crest-derived stem cell formation. FEBS J 2016; 283:3791-3806. [PMID: 27579714 DOI: 10.1111/febs.13843] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Revised: 08/02/2016] [Accepted: 08/30/2016] [Indexed: 11/29/2022]
Abstract
Neural crest-derived stem cells (NCSCs) are tissue-specific stem cells derived from multipotent neural crest cells. NCSCs are present in some adult tissues such as dorsal root ganglia, sciatic nerve, and bone marrow. However, little is known about the formation mechanisms of these cells. We have shown that BMP2/Wnt3a signaling and a chromatin remodeler, CHD7, in mice help to maintain the multipotency of neural crest cells and lead to the formation of NCSCs. In the present study, we analyzed a regulatory gene cascade in the formation of mouse NCSCs. The inhibition of FoxD3 expression significantly suppressed the expression of Sox10, which is an indispensable transcription factor for mouse NCSC formation, in the presence of BMP2/Wnt3a. CHD7, Oct3/4, Sox2, and Nanog occupied multiple conserved regions of mouse FoxD3, mE1, mE2, and mE3, in a BMP2/Wnt3a-dependent manner. Furthermore, siRNA of CHD7, Oct3/4, Sox2, and Nanog significantly suppressed FoxD3 expression. The inhibition of histone H3K4 mono- or trimethylation also repressed FoxD3 expression. The present data suggest that CHD7, Oct3/4, Sox2, and Nanog directly induce FoxD3 expression when stimulated by BMP2/Wnt3a signaling, that FoxD3 promotes Sox10 expression, and that histone H3K4 methylation plays important roles in this process of mouse NCSC formation.
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Affiliation(s)
- Kyohei Fujita
- Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan
| | - Ryuhei Ogawa
- Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan
| | - Kazuo Ito
- Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
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Mašek J, Machoň O, Kořínek V, Taketo MM, Kozmik Z. Tcf7l1 protects the anterior neural fold from adopting the neural crest fate. Development 2016; 143:2206-16. [DOI: 10.1242/dev.132357] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2015] [Accepted: 04/21/2016] [Indexed: 12/11/2022]
Abstract
The neural crest (NC) is crucial for the evolutionary diversification of vertebrates. NC cells are induced at the neural plate border by the coordinated action of several signaling pathways, including Wnt/β-catenin. NC cells are normally generated in the posterior neural plate border, whereas the anterior neural fold is devoid of NC cells. Using the mouse model, we show here that active repression of Wnt/β-catenin signaling is required for maintenance of neuroepithelial identity in the anterior neural fold and for inhibition of NC induction. Conditional inactivation of Tcf7l1, a transcriptional repressor of Wnt target genes, leads to aberrant activation of Wnt/β-catenin signaling in the anterior neuroectoderm and its conversion into NC. This reduces the developing prosencephalon without affecting the anterior-posterior neural character. Thus, Tcf7l1 defines the border between the NC and the prospective forebrain via restriction of the Wnt/β-catenin signaling gradient.
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Affiliation(s)
- Jan Mašek
- Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague 142 20, Czech Republic
| | - Ondřej Machoň
- Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague 142 20, Czech Republic
| | - Vladimír Kořínek
- Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague 142 20, Czech Republic
| | - M. Mark Taketo
- Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Zbyněk Kozmik
- Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague 142 20, Czech Republic
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43
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Maintenance of Skin Epithelial Stem Cells by Wnt-3a In Vitro. Methods Mol Biol 2016. [PMID: 27032938 DOI: 10.1007/7651_2016_320] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
CD49f+ CD34+ cells, a population rich in skin epithelial stem cells (EpSCs), were obtained from adult mouse skin and cultured with Wnt-3a for 10 days. On day 10, CD49f+ CD34+ cells were sorted and subjected to a second 10-day culture with Wnt-3a. The same procedures were repeated until fifteenth 10-day culture. CD49f+ CD34+ cells obtained from each 10-day culture retained the same EpSC-characteristics as seen in the original EpSCs from adult mouse skin. Here, wedescribe the culture protocol using Wnt-3a for successful maintenance of EpSCs.
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Di Liddo R, Bertalot T, Schuster A, Schrenk S, Müller O, Apfel J, Reischmann P, Rajendran S, Sfriso R, Gasparella M, Parnigotto PP, Conconi MT, Schäfer KH. Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation. Am J Physiol Gastrointest Liver Physiol 2016; 310:G337-46. [PMID: 26767983 DOI: 10.1152/ajpgi.00191.2015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2015] [Accepted: 12/29/2015] [Indexed: 01/31/2023]
Abstract
In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of β-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote β-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated β-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen β-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total β-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.
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Affiliation(s)
- Rosa Di Liddo
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
| | - Thomas Bertalot
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Anne Schuster
- Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany
| | - Sandra Schrenk
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Oliver Müller
- Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany
| | - Johanna Apfel
- Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany
| | - Patricia Reischmann
- Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany
| | - Senthilkumar Rajendran
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Riccardo Sfriso
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Marco Gasparella
- Department of Woman and Child Health, University of Padova, Padova, Italy
| | - Pier Paolo Parnigotto
- Tissue Engineering and Signaling-Onlus, Caselle di Selvazzano Dentro, Padova, Italy; and
| | - Maria Teresa Conconi
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Karl Herbert Schäfer
- Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany; Medical Faculty Mannheim, Department of Pediatric Surgery, University of Heidelberg, Mannheim, Germany
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Le Douarin NM, Dupin E. The Pluripotency of Neural Crest Cells and Their Role in Brain Development. Curr Top Dev Biol 2016; 116:659-78. [PMID: 26970647 DOI: 10.1016/bs.ctdb.2015.10.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The neural crest (NC) is, in the Chordate phylum, an innovation of vertebrates, which exhibits several original characteristics: its component cells are pluripotent and give rise to both ectodermal and mesodermal cell types. Moreover, during the early stages of neurogenesis, the NC cells exert a paracrine stimulating effect on the development of the preotic brain.
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Affiliation(s)
- Nicole M Le Douarin
- Collège de France, 3 rue d'Ulm, Paris, France; INSERM U968, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR S 968, Institut de la Vision, Paris, France; CNRS, UMR 7210, Paris, France.
| | - Elisabeth Dupin
- INSERM U968, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR S 968, Institut de la Vision, Paris, France; CNRS, UMR 7210, Paris, France
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Ilexonin A Promotes Neuronal Proliferation and Regeneration via Activation of the Canonical Wnt Signaling Pathway after Cerebral Ischemia Reperfusion in Rats. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2016; 2016:9753189. [PMID: 27057202 PMCID: PMC4739464 DOI: 10.1155/2016/9753189] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 09/28/2015] [Indexed: 01/08/2023]
Abstract
Aims. Ilexonin A (IA), a component of the Chinese medicine Ilex pubescens, has been shown to be neuroprotective during ischemic injury. However, the specific mechanism underlying this neuroprotective effect remains unclear. Methods. In this study, we employed a combination of immunofluorescence staining, western blotting, RT-PCR, and behavioral tests, to investigate the molecular mechanisms involved in IA regulation of neuronal proliferation and regeneration after cerebral ischemia and reperfusion in rodents. Results. Increases in β-catenin protein and LEF1 mRNA and decreases in GSK3β protein and Axin mRNA observed in IA-treated compared to control rodents implicated the canonical Wnt pathway as a key signaling mechanism activated by IA treatment. Furthermore, rodents in the IA treatment group showed less neurologic impairment and a corresponding increase in the number of Brdu/nestin and Brdu/NeuN double positive neurons in the parenchymal ischemia tissue following middle cerebral artery occlusion compared to matched controls. Conclusion. Altogether, our data indicate that IA can significantly diminish neurological deficits associated with cerebral ischemia reperfusion in rats as a result of increased neuronal survival via modulation of the canonical Wnt pathway.
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Shakhova O, Sommer L. In Vitro Derivation of Melanocytes from Embryonic Neural Crest Stem Cells. Methods Mol Biol 2015. [PMID: 26659800 DOI: 10.1007/7651_2015_305] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/05/2023]
Abstract
During development, melanocyte progenitors originate from the neural crest, a transient embryonic structure in vertebrates that gives rise to a variety of cell types including neurons and glia of the peripheral nervous system, smooth muscle cells of the cardiovascular system, chondrocytes and osteoblasts of the craniofacial elements, and pigment cells in the skin. In this chapter, we describe a method for the differentiation of multipotent embryonic neural crest stem cells into differentiated pigmented melanocytes by using in vitro explant culture system. This protocol allows the dissection of genetic and cellular mechanisms regulating neural crest stem cell and melanocyte development. Based on this knowledge it is possible to make predictions about processes that might also be implicated in melanoma initiation and progression.
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Affiliation(s)
- Olga Shakhova
- Department of Oncology, University Hospital Zurich, Wagistrasse 14, 8952, Schlieren, Switzerland.
- Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
| | - Lukas Sommer
- Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
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48
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Rollo BN, Zhang D, Stamp LA, Menheniott TR, Stathopoulos L, Denham M, Dottori M, King SK, Hutson JM, Newgreen DF. Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon. Cell Mol Gastroenterol Hepatol 2015; 2:92-109. [PMID: 28174705 PMCID: PMC4980742 DOI: 10.1016/j.jcmgh.2015.09.007] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2015] [Accepted: 09/17/2015] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells. METHODS Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2'-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed. RESULTS ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia. CONCLUSIONS NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.
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Key Words
- Aganglionosis
- CHIR-99021, 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile
- Cell Therapy
- ENC, enteric neural crest
- ENS, enteric nervous system
- EdU, ethynyl-2′-deoxyuridine
- Enteric Nervous System
- FBS, fetal bovine serum
- GFAP, glial fibrillary acidic protein
- GSK3, glycogen synthase kinase 3
- HNK1, human natural killer-1
- HSCR, Hirschsprung disease
- Hirschsprung Disease
- MTR, MitoTracker Red
- Megacolon
- NC, neural crest
- PBS, phosphate-buffered saline
- PFA, paraformaldehyde
- RCH, Royal Children’s Hospital
- SMA, smooth muscle actin
- SOX10, sex-determining region Y–box 10
- TUJ1, neuron-specific class III β-tubulin
- eGFP, enhanced green fluorescent protein
- nNOS, neuronal nitric oxide synthase
- nTCM, neural tissue culture medium
- qRT-PCR, quantitative reverse transcription and polymerase chain reaction
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Affiliation(s)
- Benjamin N. Rollo
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia,Correspondence Address correspondence to: Benjamin N. Rollo, PhD, Murdoch Children’s Research Institute, The Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia. fax: +61-3-9348-1391.Murdoch Children’s Research InstituteThe Royal Children’s HospitalFlemington RoadParkvilleVictoria 3052Australia
| | - Dongcheng Zhang
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia
| | - Lincon A. Stamp
- Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Victoria, Australia
| | - Trevelyan R. Menheniott
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia,Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia
| | - Lefteris Stathopoulos
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia
| | - Mark Denham
- Stem Cell Laboratory, Department of Biomedicine, Danish Research Institute of Translational Neuroscience, Aarhus University, Aarhus, Denmark
| | - Mirella Dottori
- Centre for Neural Engineering, NICTA, University of Melbourne, Australia
| | - Sebastian K. King
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia,Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia,Royal Children’s Hospital, Parkville, Victoria, Australia
| | - John M. Hutson
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia,Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia,Royal Children’s Hospital, Parkville, Victoria, Australia
| | - Donald F. Newgreen
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia
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Lazard ZW, Olmsted-Davis EA, Salisbury EA, Gugala Z, Sonnet C, Davis EL, Beal E, Ubogu EE, Davis AR. Osteoblasts Have a Neural Origin in Heterotopic Ossification. Clin Orthop Relat Res 2015; 473:2790-806. [PMID: 25944403 PMCID: PMC4523517 DOI: 10.1007/s11999-015-4323-9] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
BACKGROUND Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves. QUESTIONS/PURPOSES We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation? METHODS HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control). RESULTS Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRα, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval [CI], 0.59-1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01-6.94; p < 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers. CONCLUSIONS The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site. CLINICAL RELEVANCE The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in part, a neurological disorder. This could result in a major shift in orthopaedic methodologies to prevent or treat this disease. The fact that nerves are intimately involved in the process may also provide clues that will lead to an explanation of the clinical fact that HO often occurs as a result of traumatic brain injury.
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Affiliation(s)
- ZaWaunyka W. Lazard
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
| | - Elizabeth A. Olmsted-Davis
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA , />Departments of Hematology-Oncology and Orthopedic Surgery, Baylor College of Medicine, Houston, TX USA
| | - Elizabeth A. Salisbury
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
| | - Zbigniew Gugala
- />Department of Orthopedic Surgery and Rehabilitation, University of Texas Medical Branch, Galveston, TX USA
| | - Corrine Sonnet
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
| | - Eleanor L. Davis
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
| | - Eric Beal
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
| | - Eroboghene E. Ubogu
- />Department of Neurology, The University of Alabama at Birmingham, Birmingham, AL USA
| | - Alan R. Davis
- />Department of Pediatrics, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA , />Departments of Hematology-Oncology and Orthopedic Surgery, Baylor College of Medicine, Houston, TX USA
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Sato S, Yajima H, Furuta Y, Ikeda K, Kawakami K. Activation of Six1 Expression in Vertebrate Sensory Neurons. PLoS One 2015; 10:e0136666. [PMID: 26313368 PMCID: PMC4551851 DOI: 10.1371/journal.pone.0136666] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 08/05/2015] [Indexed: 12/31/2022] Open
Abstract
SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG). The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8) conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre) that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development.
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Affiliation(s)
- Shigeru Sato
- Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan
- * E-mail:
| | - Hiroshi Yajima
- Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan
| | - Yasuhide Furuta
- Animal Resource Development Unit and Genetic Engineering Team, Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies (CLST), Kobe, Hyogo, Japan
| | - Keiko Ikeda
- Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
| | - Kiyoshi Kawakami
- Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan
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