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1
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Burbach KF, Wu S, Yoo AS. Notch inhibition enhances morphological reprogramming of microRNA-induced human neurons. Stem Cells 2025; 43:sxae079. [PMID: 39573925 DOI: 10.1093/stmcls/sxae079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2024] [Accepted: 11/04/2024] [Indexed: 11/27/2024]
Abstract
The role of Notch signaling in direct neuronal reprogramming remains unknown despite its importance to brain development in vivo. Here, we use microRNA-induced neurons that are directly reprogrammed from human fibroblasts to determine how Notch signaling contributes to neuronal identity. We found that Notch inhibition during the first week of reprogramming was both necessary and sufficient to enhance neurite outgrowth at a later timepoint, indicating an important role in the erasure of the original cell identity. Accordingly, transcriptomic analysis showed that the effect of Notch inhibition was likely due to improvements in fibroblast fate erasure and silencing of non-neuronal genes. To this effect, we identify MYLIP, whose downregulation in response to Notch inhibition significantly promoted neurite outgrowth. Moreover, Notch inhibition resulted in cells with neuronal transcriptome signatures defined by expressing long genes at a faster rate than the control, demonstrating the effect of accelerated fate erasure on neuronal fate acquisition. Our results demonstrate the antagonistic role of Notch signaling to the pro-neuronal microRNAs 9 and 124 and the benefits of its inhibition to the acquisition of neuronal morphology.
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Affiliation(s)
- Kyle F Burbach
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, United States
- Program in Molecular Genetics and Genomics, Division of Biology & Biomedical Sciences, Washington University in St. Louis, St. Louis, MO 63110, United States
| | - Shanyun Wu
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, United States
- Program in Molecular Genetics and Genomics, Division of Biology & Biomedical Sciences, Washington University in St. Louis, St. Louis, MO 63110, United States
| | - Andrew S Yoo
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, United States
- Center for Regenerative Medicine, Washington University in St. Louis, St. Louis, MO 63110, United States
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2
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Buchan MJ, Gothard G, Mahfooz K, van Rheede JJ, Avery SV, Vourvoukelis A, Demby A, Ellender TJ, Newey SE, Akerman CJ. Higher-order thalamocortical circuits are specified by embryonic cortical progenitor types in the mouse brain. Cell Rep 2024; 43:114157. [PMID: 38678557 DOI: 10.1016/j.celrep.2024.114157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Revised: 02/14/2024] [Accepted: 04/10/2024] [Indexed: 05/01/2024] Open
Abstract
The sensory cortex receives synaptic inputs from both first-order and higher-order thalamic nuclei. First-order inputs relay simple stimulus properties from the periphery, whereas higher-order inputs relay more complex response properties, provide contextual feedback, and modulate plasticity. Here, we reveal that a cortical neuron's higher-order input is determined by the type of progenitor from which it is derived during embryonic development. Within layer 4 (L4) of the mouse primary somatosensory cortex, neurons derived from intermediate progenitors receive stronger higher-order thalamic input and exhibit greater higher-order sensory responses. These effects result from differences in dendritic morphology and levels of the transcription factor Lhx2, which are specified by the L4 neuron's progenitor type. When this mechanism is disrupted, cortical circuits exhibit altered higher-order responses and sensory-evoked plasticity. Therefore, by following distinct trajectories, progenitor types generate diversity in thalamocortical circuitry and may provide a general mechanism for differentially routing information through the cortex.
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Affiliation(s)
| | - Gemma Gothard
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK
| | - Kashif Mahfooz
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK
| | | | - Sophie V Avery
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK
| | | | - Alexander Demby
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK
| | - Tommas J Ellender
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK; Experimental Neurobiology Unit, Universiteitsplein, 2610 Antwerp, Belgium
| | - Sarah E Newey
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK
| | - Colin J Akerman
- Department of Pharmacology, Mansfield Road, OX1 3QT Oxford, UK.
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3
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Hosseini SM, Borys B, Karimi-Abdolrezaee S. Neural stem cell therapies for spinal cord injury repair: an update on recent preclinical and clinical advances. Brain 2024; 147:766-793. [PMID: 37975820 DOI: 10.1093/brain/awad392] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2023] [Revised: 10/22/2023] [Accepted: 11/02/2023] [Indexed: 11/19/2023] Open
Abstract
Traumatic spinal cord injury (SCI) is a leading cause of lifelong disabilities. Permanent sensory, motor and autonomic impairments after SCI are substantially attributed to degeneration of spinal cord neurons and axons, and disintegration of neural network. To date, minimal regenerative treatments are available for SCI with an unmet need for new therapies to reconstruct the damaged spinal cord neuron-glia network and restore connectivity with the supraspinal pathways. Multipotent neural precursor cells (NPCs) have a unique capacity to generate neurons, oligodendrocytes and astrocytes. Due to this capacity, NPCs have been an attractive cell source for cellular therapies for SCI. Transplantation of NPCs has been extensively tested in preclinical models of SCI in the past two decades. These studies have identified opportunities and challenges associated with NPC therapies. While NPCs have the potential to promote neuroregeneration through various mechanisms, their low long-term survival and integration within the host injured spinal cord limit the functional benefits of NPC-based therapies for SCI. To address this challenge, combinatorial strategies have been developed to optimize the outcomes of NPC therapies by enriching SCI microenvironment through biomaterials, genetic and pharmacological therapies. In this review, we will provide an in-depth discussion on recent advances in preclinical NPC-based therapies for SCI. We will discuss modes of actions and mechanism by which engrafted NPCs contribute to the repair process and functional recovery. We will also provide an update on current clinical trials and new technologies that have facilitated preparation of medical-grade human NPCs suitable for transplantation in clinical studies.
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Affiliation(s)
- Seyed Mojtaba Hosseini
- Department of Physiology and Pathophysiology, Spinal Cord Research Centre, Rady Faculty of Health Sciences, University of Manitoba Winnipeg, Manitoba R3E 0J9, Canada
- Manitoba Multiple Sclerosis Research Center, Winnipeg, Manitoba R3E 0J9, Canada
| | - Ben Borys
- Department of Physiology and Pathophysiology, Spinal Cord Research Centre, Rady Faculty of Health Sciences, University of Manitoba Winnipeg, Manitoba R3E 0J9, Canada
| | - Soheila Karimi-Abdolrezaee
- Department of Physiology and Pathophysiology, Spinal Cord Research Centre, Rady Faculty of Health Sciences, University of Manitoba Winnipeg, Manitoba R3E 0J9, Canada
- Manitoba Multiple Sclerosis Research Center, Winnipeg, Manitoba R3E 0J9, Canada
- Children's Hospital Research Institute of Manitoba, Winnipeg, Manitoba R3E 3P4, Canada
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4
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Choi W, Choe MS, Kim SM, Kim SJ, Lee J, Lee Y, Lee SM, Dho SH, Lee MY, Kim LK. RFX4 is an intrinsic factor for neuronal differentiation through induction of proneural genes POU3F2 and NEUROD1. Cell Mol Life Sci 2024; 81:99. [PMID: 38386071 PMCID: PMC10884155 DOI: 10.1007/s00018-024-05129-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2023] [Revised: 12/27/2023] [Accepted: 01/15/2024] [Indexed: 02/23/2024]
Abstract
Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.
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Affiliation(s)
- Wonyoung Choi
- Interdisciplinary Program of Integrated OMICS for Biomedical Science, The Graduate School, Yonsei University, Seoul, Korea
- College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Mu Seog Choe
- College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Su Min Kim
- Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06230, Republic of Korea
| | - So Jin Kim
- College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Jiyeon Lee
- Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06230, Republic of Korea
| | - Yeongun Lee
- Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06230, Republic of Korea
| | - Sun-Min Lee
- Department of Physics, Konkuk University, Seoul, Republic of Korea
| | - So Hee Dho
- Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06230, Republic of Korea
| | - Min-Young Lee
- College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea.
| | - Lark Kyun Kim
- Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06230, Republic of Korea.
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Burbach KF, Yoo AS. Notch Inhibition Enhances Morphological Reprogramming of microRNA-Induced Human Neurons. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.12.575384. [PMID: 38260259 PMCID: PMC10802628 DOI: 10.1101/2024.01.12.575384] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2024]
Abstract
Although the importance of Notch signaling in brain development is well-known, its specific contribution to cellular reprogramming remains less defined. Here, we use microRNA-induced neurons that are directly reprogrammed from human fibroblasts to determine how Notch signaling contributes to neuronal identity. We found that inhibiting Notch signaling led to an increase in neurite extension, while activating Notch signaling had the opposite effect. Surprisingly, Notch inhibition during the first week of reprogramming was both necessary and sufficient to enhance neurite outgrowth at a later timepoint. This timeframe is when the reprogramming miRNAs, miR-9/9* and miR-124, primarily induce a post-mitotic state and erase fibroblast identity. Accordingly, transcriptomic analysis showed that the effect of Notch inhibition was likely due to improvements in fibroblast fate erasure and silencing of anti-neuronal genes. To this effect, we identify MYLIP , whose downregulation in response to Notch inhibition significantly promoted neurite outgrowth. Moreover, Notch inhibition resulted in cells with neuronal transcriptome signature defined by expressing long genes at a faster rate than the control, demonstrating the effect of accelerated fate erasure on neuronal fate acquisition. Our results demonstrate the critical role of Notch signaling in mediating morphological changes in miRNA-based neuronal reprogramming of human adult fibroblasts.
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6
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Kunke M, Knöfler H, Dahlke E, Zanon Rodriguez L, Böttner M, Larionov A, Saudenova M, Ohrenschall GM, Westermann M, Porubsky S, Bernardes JP, Häsler R, Magnin JL, Koepsell H, Jouret F, Theilig F. Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease. Cell Death Dis 2023; 14:562. [PMID: 37626062 PMCID: PMC10457389 DOI: 10.1038/s41419-023-06074-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 08/01/2023] [Accepted: 08/15/2023] [Indexed: 08/27/2023]
Abstract
Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.
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Affiliation(s)
- Madlen Kunke
- Institute of Anatomy, Christian Albrechts-University Kiel, Kiel, Germany
| | - Hannah Knöfler
- Institute of Anatomy, Christian Albrechts-University Kiel, Kiel, Germany
| | - Eileen Dahlke
- Institute of Anatomy, Christian Albrechts-University Kiel, Kiel, Germany
| | | | - Martina Böttner
- Institute of Anatomy, Christian Albrechts-University Kiel, Kiel, Germany
| | - Alexey Larionov
- Institute of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland
| | | | | | | | | | - Joana P Bernardes
- Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Kiel, Germany
| | - Robert Häsler
- Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Kiel, Germany
| | | | - Hermann Koepsell
- Institute of Anatomy and Cell Biology, Julius-Maximilians-University of Würzburg, Würzburg, Germany
| | - François Jouret
- Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA), Cardiovascular Sciences, University of Liège (ULiège), Liège, Belgium
- Division of Nephrology, CHU of Liège, University of Liège (CHU ULiège), Liège, Belgium
| | - Franziska Theilig
- Institute of Anatomy, Christian Albrechts-University Kiel, Kiel, Germany.
- Institute of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.
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7
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Rani R, Nayak M, Nayak B. Exploring the reprogramming potential of B cells and comprehending its clinical and therapeutic perspective. Transpl Immunol 2023; 78:101804. [PMID: 36921730 DOI: 10.1016/j.trim.2023.101804] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 02/08/2023] [Accepted: 02/21/2023] [Indexed: 03/14/2023]
Abstract
Initiating from multipotent progenitors, the lineages extrapolated from hematopoietic stem cells are determined by transcription factors specific to each of them. The commitment factors assist in the differentiation of progenitor cells into terminally differentiated cells. B lymphocytes constitute a population of cells that expresses clonally diverse cell surface immunoglobulin (Ig) receptors specific to antigenic epitopes. B cells are a significant facet of the adaptive immune system. The secreted antibodies corresponding to the B cell recognize the antigens via the B cell receptor (BCR). Following antigen recognition, the B cell is activated and thereafter undergoes clonal expansion and proliferation to become memory B cells. The essence of 'cellular reprogramming' has aided in reliably altering the cells to desired tissue type. The potential of reprogramming has been harnessed to decipher and find solutions for various genetically inherited diseases and degenerative disorders. B lymphocytes can be reprogrammed to their initial naive state from where they get differentiated into any lineage or cell type similar to a pluripotent stem cell which can be accomplished by the deletion of master regulators of the B cell lineage. B cells can be reprogrammed into pluripotent stem cells and also can undergo transdifferentiation at the midway of cell differentiation to other cell types. Mandated expression of C/EBP in specialized B cells corresponds to their fast and effective reprogramming into macrophages, reversing the cell fate of these lymphocytes and allowing them to differentiate freshly into other types of cells. The co-expression of C/EBPα and OKSM (Oct4, Sox2, Klf4, c-Myc) amplified the reprogramming efficiency of B lymphocytes. Various human somatic cells including the immune cells are compliant to reprogramming which paves a path for opportunities like autologous tissue grafts, blood transfusion, and cancer immunotherapy. The ability to reprogram B cells offers an unprecedented opportunity for developing a therapeutic approach for several human diseases. Here, we will focus on all the proteins and transcription factors responsible for the developmental commitment of B lymphocytes and how it is harnessed in various applications.
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Affiliation(s)
- Reetika Rani
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha. 769008, India
| | - Madhusmita Nayak
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha. 769008, India
| | - Bismita Nayak
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha. 769008, India.
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8
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Messerschmidt VL, Chintapula U, Bonetesta F, Laboy-Segarra S, Naderi A, Nguyen KT, Cao H, Mager E, Lee J. In vivo Evaluation of Non-viral NICD Plasmid-Loaded PLGA Nanoparticles in Developing Zebrafish to Improve Cardiac Functions. Front Physiol 2022; 13:819767. [PMID: 35283767 PMCID: PMC8906778 DOI: 10.3389/fphys.2022.819767] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Accepted: 01/07/2022] [Indexed: 12/12/2022] Open
Abstract
In the era of the advanced nanomaterials, use of nanoparticles has been highlighted in biomedical research. However, the demonstration of DNA plasmid delivery with nanoparticles for in vivo gene delivery experiments must be carefully tested due to many possible issues, including toxicity. The purpose of the current study was to deliver a Notch Intracellular Domain (NICD)-encoded plasmid via poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to investigate the toxic environmental side effects for an in vivo experiment. In addition, we demonstrated the target delivery to the endothelium, including the endocardial layer, which is challenging to manipulate gene expression for cardiac functions due to the beating heart and rapid blood pumping. For this study, we used a zebrafish animal model and exposed it to nanoparticles at varying concentrations to observe for specific malformations over time for toxic effects of PLGA nanoparticles as a delivery vehicle. Our nanoparticles caused significantly less malformations than the positive control, ZnO nanoparticles. Additionally, the NICD plasmid was successfully delivered by PLGA nanoparticles and significantly increased Notch signaling related genes. Furthermore, our image based deep-learning analysis approach evaluated that the antibody conjugated nanoparticles were successfully bound to the endocardium to overexpress Notch related genes and improve cardiac function such as ejection fraction, fractional shortening, and cardiac output. This research demonstrates that PLGA nanoparticle-mediated target delivery to upregulate Notch related genes which can be a potential therapeutic approach with minimum toxic effects.
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Affiliation(s)
- Victoria L Messerschmidt
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX, United States.,University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Uday Chintapula
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX, United States.,University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Fabrizio Bonetesta
- Department of Biological Sciences, University of North Texas, Denton, TX, United States
| | - Samantha Laboy-Segarra
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX, United States.,University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Amir Naderi
- Department of Electrical Engineering and Computer Science, University of California, Irvine, Irvine, CA, United States
| | - Kytai T Nguyen
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX, United States.,University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Hung Cao
- Department of Electrical Engineering and Computer Science, University of California, Irvine, Irvine, CA, United States
| | - Edward Mager
- Department of Biological Sciences, University of North Texas, Denton, TX, United States
| | - Juhyun Lee
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX, United States.,University of Texas Southwestern Medical Center, Dallas, TX, United States
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9
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Vasan L, Park E, David LA, Fleming T, Schuurmans C. Direct Neuronal Reprogramming: Bridging the Gap Between Basic Science and Clinical Application. Front Cell Dev Biol 2021; 9:681087. [PMID: 34291049 PMCID: PMC8287587 DOI: 10.3389/fcell.2021.681087] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Accepted: 06/02/2021] [Indexed: 12/15/2022] Open
Abstract
Direct neuronal reprogramming is an innovative new technology that involves the conversion of somatic cells to induced neurons (iNs) without passing through a pluripotent state. The capacity to make new neurons in the brain, which previously was not achievable, has created great excitement in the field as it has opened the door for the potential treatment of incurable neurodegenerative diseases and brain injuries such as stroke. These neurological disorders are associated with frank neuronal loss, and as new neurons are not made in most of the adult brain, treatment options are limited. Developmental biologists have paved the way for the field of direct neuronal reprogramming by identifying both intrinsic cues, primarily transcription factors (TFs) and miRNAs, and extrinsic cues, including growth factors and other signaling molecules, that induce neurogenesis and specify neuronal subtype identities in the embryonic brain. The striking observation that postmitotic, terminally differentiated somatic cells can be converted to iNs by mis-expression of TFs or miRNAs involved in neural lineage development, and/or by exposure to growth factors or small molecule cocktails that recapitulate the signaling environment of the developing brain, has opened the door to the rapid expansion of new neuronal reprogramming methodologies. Furthermore, the more recent applications of neuronal lineage conversion strategies that target resident glial cells in situ has expanded the clinical potential of direct neuronal reprogramming techniques. Herein, we present an overview of the history, accomplishments, and therapeutic potential of direct neuronal reprogramming as revealed over the last two decades.
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Affiliation(s)
- Lakshmy Vasan
- Sunnybrook Research Institute, Biological Sciences Platform, Toronto, ON, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Eunjee Park
- Sunnybrook Research Institute, Biological Sciences Platform, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Luke Ajay David
- Sunnybrook Research Institute, Biological Sciences Platform, Toronto, ON, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Taylor Fleming
- Sunnybrook Research Institute, Biological Sciences Platform, Toronto, ON, Canada
| | - Carol Schuurmans
- Sunnybrook Research Institute, Biological Sciences Platform, Toronto, ON, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
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10
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McIntyre WB, Pieczonka K, Khazaei M, Fehlings MG. Regenerative replacement of neural cells for treatment of spinal cord injury. Expert Opin Biol Ther 2021; 21:1411-1427. [PMID: 33830863 DOI: 10.1080/14712598.2021.1914582] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Introduction: Traumatic Spinal Cord Injury (SCI) results from primary physical injury to the spinal cord, which initiates a secondary cascade of neural cell death. Current therapeutic approaches can attenuate the consequences of the primary and secondary events, but do not address the degenerative aspects of SCI. Transplantation of neural stem/progenitor cells (NPCs) for the replacement of the lost/damaged neural cells is suggested here as a regenerative approach that is complementary to current therapeutics.Areas Covered: This review addresses how neurons, oligodendrocytes, and astrocytes are impacted by traumatic SCI, and how current research in regenerative-NPC therapeutics aims to restore their functionality. Methods used to enhance graft survival, as well as bias progenitor cells towards neuronal, oligodendrogenic, and astroglia lineages are discussed.Expert Opinion: Despite an NPC's ability to differentiate into neurons, oligodendrocytes, and astrocytes in the transplant environment, their potential therapeutic efficacy requires further optimization prior to translation into the clinic. Considering the temporospatial identity of NPCs could promote neural repair in region specific injuries throughout the spinal cord. Moreover, understanding which cells are targeted by NPC-derived myelinating cells can help restore physiologically-relevant myelin patterns. Finally, the duality of astrocytes is discussed, outlining their context-dependent importance in the treatment of SCI.
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Affiliation(s)
- William Brett McIntyre
- Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada.,Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
| | - Katarzyna Pieczonka
- Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada.,Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
| | - Mohamad Khazaei
- Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada
| | - Michael G Fehlings
- Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada.,Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.,Department of Surgery, University of Toronto, Toronto, ON, Canada
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11
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Falcone C, Santo M, Liuzzi G, Cannizzaro N, Grudina C, Valencic E, Peruzzotti-Jametti L, Pluchino S, Mallamaci A. Foxg1 Antagonizes Neocortical Stem Cell Progression to Astrogenesis. Cereb Cortex 2020; 29:4903-4918. [PMID: 30821834 DOI: 10.1093/cercor/bhz031] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Revised: 01/06/2019] [Accepted: 02/09/2019] [Indexed: 12/12/2022] Open
Abstract
Neocortical astrogenesis follows neuronogenesis and precedes oligogenesis. Among key factors dictating its temporal articulation, there are progression rates of pallial stem cells (SCs) towards astroglial lineages as well as activation rates of astrocyte differentiation programs in response to extrinsic gliogenic cues. In this study, we showed that high Foxg1 SC expression antagonizes astrocyte generation, while stimulating SC self-renewal and committing SCs to neuronogenesis. We found that mechanisms underlying this activity are mainly cell autonomous and highly pleiotropic. They include a concerted downregulation of 4 key effectors channeling neural SCs to astroglial fates, as well as defective activation of core molecular machineries implementing astroglial differentiation programs. Next, we found that SC Foxg1 levels specifically decline during the neuronogenic-to-gliogenic transition, pointing to a pivotal Foxg1 role in temporal modulation of astrogenesis. Finally, we showed that Foxg1 inhibits astrogenesis from human neocortical precursors, suggesting that this is an evolutionarily ancient trait.
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Affiliation(s)
- Carmen Falcone
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
| | - Manuela Santo
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
| | - Gabriele Liuzzi
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
| | - Noemi Cannizzaro
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
| | - Clara Grudina
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
| | - Erica Valencic
- Department of Diagnostics, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Luca Peruzzotti-Jametti
- Dept of Clinical Neurosciences, University of Cambridge, Clifford Allbutt Building -- Cambridge Biosciences Campus, Hills Road, Cambridge, UK
| | - Stefano Pluchino
- Dept of Clinical Neurosciences, University of Cambridge, Clifford Allbutt Building -- Cambridge Biosciences Campus, Hills Road, Cambridge, UK
| | - Antonello Mallamaci
- Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy
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12
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Hwang M, Lee EJ, Chung MJ, Park S, Jeong KS. Five transcriptional factors reprogram fibroblast into myogenic lineage cells via paraxial mesoderm stage. Cell Cycle 2020; 19:1804-1816. [PMID: 32579865 DOI: 10.1080/15384101.2020.1780384] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
It is hard to supply satellite cells as a cell source for therapy of muscle degenerative disease since the sampling of muscle tissue is very invasive to a patient with muscular disease. Direct conversion allows us to get specific cell types by transduction of defined transcriptional factors. To induce myogenic direct conversion, we transduced five transcriptional factors including Pax3, Sox2, Klf4, c-Myc, and Esrrb into mouse embryonic fibroblasts. We found that the transduction of the five transcriptional factors induced myogenic direct conversion of fibroblast. We revealed that the transduced cells with the five transcriptional factors were converted to myogenic lineage cells through a paraxial mesoderm-like stage. The expression level of myogenic-related genes of the transduced cells gradually increased as the passage increased. The induced myogenic lineage cells differentiated into muscle fibers in virto and in vivo. The current study revealed that the five transcription factors generated myogenic lineage cells from fibroblast via a paraxial mesoderm stage. The induced myogenic lineage cells may have a potential being applied as cell source for degenerative muscle disease.
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Affiliation(s)
- Meeyul Hwang
- Department of Pathology, College of Veterinary Medicine, Kyungpook National University , Daegu, Republic of Korea
| | - Eun-Joo Lee
- Department of Pathology, College of Veterinary Medicine, Kyungpook National University , Daegu, Republic of Korea
| | - Myung-Jin Chung
- Department of Pathology, College of Veterinary Medicine, Kyungpook National University , Daegu, Republic of Korea.,Stem Cell Therapeutic Research Center, Kyungpook National University , Daegu, Republic of Korea
| | - SunYoung Park
- Department of Pathology, College of Veterinary Medicine, Kyungpook National University , Daegu, Republic of Korea.,Stem Cell Therapeutic Research Center, Kyungpook National University , Daegu, Republic of Korea
| | - Kyu-Shik Jeong
- Department of Pathology, College of Veterinary Medicine, Kyungpook National University , Daegu, Republic of Korea.,Stem Cell Therapeutic Research Center, Kyungpook National University , Daegu, Republic of Korea
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13
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Reprogramming Fibroblasts to Neural Stem Cells by Overexpression of the Transcription Factor Ptf1a. Methods Mol Biol 2020; 2117:245-263. [PMID: 31960384 DOI: 10.1007/978-1-0716-0301-7_15] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Neural stem cells (NSCs) have the features of both neural progenitors and stem cells, and show great potentials in translational research and regenerative medicine. Studies on NSCs have been greatly accelerated by the introduction of induced neural stem cells (iNSCs). The iNSCs are usually differentiated from induced pluripotent stem cells (iPSCs) or transdifferentiated from somatic cells such as fibroblasts or glial cells. Here, we describe a detailed protocol to reprogram human and mouse fibroblasts into iNSCs by overexpression of a transcription factor Ptf1a delivered by lentiviruses. The obtained iNSC lines have a strong self-renewal ability and are capable of differentiating into various types of neurons, astrocytes, and oligodendrocytes both in vitro and in vivo. The protocol is quite simple but powerful to produce iNSC lines.
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14
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Chen S, Zhang J, Zhang D, Jiao J. Acquisition of functional neurons by direct conversion: Switching the developmental clock directly. J Genet Genomics 2019; 46:459-465. [PMID: 31771824 DOI: 10.1016/j.jgg.2019.10.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Revised: 09/20/2019] [Accepted: 10/08/2019] [Indexed: 01/04/2023]
Abstract
Identifying approaches for treating neurodegeneration is a thorny task but is important for a growing number of patients. Researchers have focused on discovering the underlying molecular mechanisms of reprogramming and optimizing the technologies for acquiring neurons. Direct conversion is one of the most important processes for treating neurological disorders. Induced neurons derived from direct conversion, which bypass the pluripotency stage, are more effective, more quickly obtained, and are safer than those produced via induced pluripotent stem cells (iPSCs). Based on iPSC strategies, scientists have derived methods to obtain functional neurons by direct conversion, such as neuron-related transcriptional factors, small molecules, microRNAs, and epigenetic modifiers. In this review, we discuss the present strategies for direct conversion of somatic cells into functional neurons and the potentials of direct conversion for producing functional neurons and treating neurodegeneration.
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Affiliation(s)
- Shuangquan Chen
- School of Pharmaceutical Sciences, Tsinghua University, Beijing, 100084, China
| | - Juan Zhang
- Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Dongming Zhang
- Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Jianwei Jiao
- Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China; Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
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15
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Zarei-Kheirabadi M, Hesaraki M, Shojaei A, Kiani S, Baharvand H. Generation of neural stem cells from adult astrocytes by using a single reprogramming factor. J Cell Physiol 2019; 234:18697-18706. [PMID: 30912162 DOI: 10.1002/jcp.28510] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Revised: 02/16/2019] [Accepted: 02/20/2019] [Indexed: 01/08/2023]
Abstract
Generating neural stem cells (NSCs) from astroglia as an abundant cell type in the mammalian brain has a promising outlook to be used in cell-replacement therapy for treatment of neurodegenerative disorders and neuronal trauma. However, little is known about a single reprogramming factor that may lead to the generation of induced NSCs (iNSCs) from adult brain-derived astrocytes in the absence of extrinsic inductive signals. Here, we show that zinc-finger nuclear protein Zfp521 alone is sufficient for converting the adult mouse brain-derived astrocytes into iNSCs. In vitro, Zfp521-iNSCs demonstrated long-term self-renewal and multipotency and expressed related markers. Moreover, single-seeded iNSCs were able to produce NSC colonies. These results suggest that application of Zfp521 to generate iNSCs could be regarded as a new approach for conversion of resident astrocytes into iNSCs in cell therapy for in vivo treatment of neural injuries.
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Affiliation(s)
- Masoumeh Zarei-Kheirabadi
- Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mehdi Hesaraki
- Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Amir Shojaei
- Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sahar Kiani
- Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
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16
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The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain. J Neurosci 2018; 39:44-62. [PMID: 30425119 DOI: 10.1523/jneurosci.0688-18.2018] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Revised: 10/16/2018] [Accepted: 10/22/2018] [Indexed: 02/08/2023] Open
Abstract
Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.
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17
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Braccioli L, Vervoort SJ, Puma G, Nijboer CH, Coffer PJ. SOX4 inhibits oligodendrocyte differentiation of embryonic neural stem cells in vitro by inducing Hes5 expression. Stem Cell Res 2018; 33:110-119. [PMID: 30343100 DOI: 10.1016/j.scr.2018.10.005] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Revised: 09/28/2018] [Accepted: 10/02/2018] [Indexed: 12/27/2022] Open
Abstract
SOX4 has been shown to promote neuronal differentiation both in the adult and embryonic neural progenitors. Ectopic SOX4 expression has also been shown to inhibit oligodendrocyte differentiation in mice, however the underlying molecular mechanisms remain poorly understood. Here we demonstrate that SOX4 regulates transcriptional targets associated with neural development in neural stem cells (NSCs), reducing the expression of genes promoting oligodendrocyte differentiation. Interestingly, we observe that SOX4 levels decreased during oligodendrocyte differentiation in vitro. Moreover, we show that SOX4 knockdown induces increased oligodendrocyte differentiation, as the percentage of Olig2-positive/2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase)-positive maturing oligodendrocytes increases, while the number of Olig2-positive oligodendrocyte precursors is unaffected. Conversely, conditional SOX4 overexpression utilizing a doxycycline inducible system decreases the percentage of maturing oligodendrocytes, suggesting that SOX4 inhibits maturation from precursor to mature oligodendrocyte. We identify the transcription factor Hes5 as a direct SOX4 target gene and we show that conditional overexpression of Hes5 rescues the increased oligodendrocyte differentiation mediated by SOX4 depletion in NSCs. Taken together, these observations support a novel role for SOX4 in NSC by controlling oligodendrocyte differentiation through induction of Hes5 expression.
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Affiliation(s)
- Luca Braccioli
- Laboratory of Neuroimmunology and Developmental Origins of Disease (NIDOD), University Medical Center Utrecht, Utrecht University, Utrecht, 3508, AB, the Netherlands; Center for Molecular Medicine and Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CT, the Netherlands
| | - Stephin J Vervoort
- Center for Molecular Medicine and Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CT, the Netherlands
| | - Gianmarco Puma
- Center for Molecular Medicine and Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CT, the Netherlands
| | - Cora H Nijboer
- Laboratory of Neuroimmunology and Developmental Origins of Disease (NIDOD), University Medical Center Utrecht, Utrecht University, Utrecht, 3508, AB, the Netherlands
| | - Paul J Coffer
- Center for Molecular Medicine and Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CT, the Netherlands; Division of Pediatrics, University Medical Center Utrecht, Utrecht University, Utrecht, 3508, AB, the Netherlands.
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18
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Spinal cord organogenesis model reveals role of Flk1 + cells in self-organization of neural progenitor cells into complex spinal cord tissue. Stem Cell Res 2018; 33:156-165. [PMID: 30368192 DOI: 10.1016/j.scr.2018.09.001] [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: 07/26/2017] [Revised: 08/02/2018] [Accepted: 09/05/2018] [Indexed: 12/15/2022] Open
Abstract
A platform for studying spinal cord organogenesis in vivo where embryonic stem cell (ESC)-derived neural progenitor cells (NPC) self-organize into spinal cord-like tissue after transplantation in subarachnoid space of the spinal cord has been described. We advance the applicability of this platform by imaging in vivo the formed graft through T2w magnetic resonance imaging (MRI). Furthermore, we used diffusion tensor imaging (DTI) to verify the stereotypical organization of the graft showing that it mimics the host spinal cord. Within the graft white matter (WM) we identified astrocytes that form glial limitans, myelinating oligodendrocytes, and myelinated axons with paranodes. Within the graft grey matter (GM) we identified cholinergic, glutamatergic, serotonergic and dopaminergic neurons. Furthermore, we demonstrate the presence of ESC-derived complex vasculature that includes the presence of blood brain barrier. In addition to the formation of mature spinal cord tissue, we describe factors that drive this process. Specifically, we identify Flk1+ cells as necessary for spinal cord formation, and synaptic connectivity with the host spinal cord and formation of host-graft chimeric vasculature as contributing factors. This model can be used to study spinal cord organogenesis, and as an in vivo drug discovery platform for screening potential therapeutic compounds and their toxicity.
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19
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Vieira MS, Santos AK, Vasconcellos R, Goulart VAM, Parreira RC, Kihara AH, Ulrich H, Resende RR. Neural stem cell differentiation into mature neurons: Mechanisms of regulation and biotechnological applications. Biotechnol Adv 2018; 36:1946-1970. [PMID: 30077716 DOI: 10.1016/j.biotechadv.2018.08.002] [Citation(s) in RCA: 126] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 07/31/2018] [Accepted: 08/01/2018] [Indexed: 02/07/2023]
Abstract
The abilities of stem cells to self-renew and form different mature cells expand the possibilities of applications in cell-based therapies such as tissue recomposition in regenerative medicine, drug screening, and treatment of neurodegenerative diseases. In addition to stem cells found in the embryo, various adult organs and tissues have niches of stem cells in an undifferentiated state. In the central nervous system of adult mammals, neurogenesis occurs in two regions: the subventricular zone and the dentate gyrus in the hippocampus. The generation of the different neural lines originates in adult neural stem cells that can self-renew or differentiate into astrocytes, oligodendrocytes, or neurons in response to specific stimuli. The regulation of the fate of neural stem cells is a finely controlled process relying on a complex regulatory network that extends from the epigenetic to the translational level and involves extracellular matrix components. Thus, a better understanding of the mechanisms underlying how the process of neurogenesis is induced, regulated, and maintained will provide elues for development of novel for strategies for neurodegenerative therapies. In this review, we focus on describing the mechanisms underlying the regulation of the neuronal differentiation process by transcription factors, microRNAs, and extracellular matrix components.
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Affiliation(s)
- Mariana S Vieira
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Instituto Nanocell, Divinopólis, MG, Brazil
| | - Anderson K Santos
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
| | - Rebecca Vasconcellos
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Instituto Nanocell, Divinopólis, MG, Brazil
| | - Vânia A M Goulart
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
| | - Ricardo C Parreira
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Instituto Nanocell, Divinopólis, MG, Brazil
| | - Alexandre H Kihara
- Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Bernardo do Campo, SP, Brazil
| | - Henning Ulrich
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil.
| | - Rodrigo R Resende
- Departamento de Bioquímica e Imunologia, Instituto de Ciência Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Instituto Nanocell, Divinopólis, MG, Brazil.
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20
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Nikolakopoulou P, Chatzigeorgiou A, Kourtzelis I, Toutouna L, Masjkur J, Arps-Forker C, Poser SW, Rozman J, Rathkolb B, Aguilar-Pimentel JA, Wolf E, Klingenspor M, Ollert M, Schmidt-Weber C, Fuchs H, Gailus-Durner V, Hrabe de Angelis M, Tsata V, Monasor LS, Troullinaki M, Witt A, Anastasiou V, Chrousos G, Yi CX, García-Cáceres C, Tschöp MH, Bornstein SR, Androutsellis-Theotokis A. Streptozotocin-induced β-cell damage, high fat diet, and metformin administration regulate Hes3 expression in the adult mouse brain. Sci Rep 2018; 8:11335. [PMID: 30054579 PMCID: PMC6063949 DOI: 10.1038/s41598-018-29434-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Accepted: 07/09/2018] [Indexed: 12/18/2022] Open
Abstract
Diabetes mellitus is a group of disorders characterized by prolonged high levels of circulating blood glucose. Type 1 diabetes is caused by decreased insulin production in the pancreas whereas type 2 diabetes may develop due to obesity and lack of exercise; it begins with insulin resistance whereby cells fail to respond properly to insulin and it may also progress to decreased insulin levels. The brain is an important target for insulin, and there is great interest in understanding how diabetes affects the brain. In addition to the direct effects of insulin on the brain, diabetes may also impact the brain through modulation of the inflammatory system. Here we investigate how perturbation of circulating insulin levels affects the expression of Hes3, a transcription factor expressed in neural stem and progenitor cells that is involved in tissue regeneration. Our data show that streptozotocin-induced β-cell damage, high fat diet, as well as metformin, a common type 2 diabetes medication, regulate Hes3 levels in the brain. This work suggests that Hes3 is a valuable biomarker helping to monitor the state of endogenous neural stem and progenitor cells in the context of diabetes mellitus.
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Affiliation(s)
| | - Antonios Chatzigeorgiou
- Department of Clinical Pathobiochemistry, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
| | - Ioannis Kourtzelis
- Department of Clinical Pathobiochemistry, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
| | - Louiza Toutouna
- Department of Medicine, Technische Universität Dresden, Dresden, Germany
| | - Jimmy Masjkur
- Department of Medicine, Technische Universität Dresden, Dresden, Germany
| | - Carina Arps-Forker
- Department of Medicine, Technische Universität Dresden, Dresden, Germany
| | - Steven W Poser
- Department of Medicine, Technische Universität Dresden, Dresden, Germany
| | - Jan Rozman
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
| | - Birgit Rathkolb
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
- Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-University Munich, Feodor-Lynen Str. 25, 81377, Munich, Germany
| | - Juan Antonio Aguilar-Pimentel
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
| | - Eckhard Wolf
- Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-University Munich, Feodor-Lynen Str. 25, 81377, Munich, Germany
| | - Martin Klingenspor
- Chair of Molecular Nutritional Medicine, Technical University Munich, EKFZ - Else Kröner Fresenius Center for Nutritional Medicine, Gregor-Mendel-Str. 2, 85350, Freising-Weihenstephan, Germany
- ZIEL - Institute for Food and Health, Technical University Munich, Gregor-Mendel-Str. 2, 85350, Freising-Weihenstephan, Germany
| | - Markus Ollert
- Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg
- Department of Dermatology and Allergy Center, Odense Research Center for Anaphylaxis, University of Southern Denmark, Odense, Denmark
| | - Carsten Schmidt-Weber
- Center of Allergy & Environment (ZAUM), Technische Universität München, and Helmholtz Zentrum München, Ingolstädter Landstr.1, 85764, Neuherberg, Germany
| | - Helmut Fuchs
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
| | - Valerie Gailus-Durner
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
| | - Martin Hrabe de Angelis
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany
- Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Alte Akademie 8, 85354, Freising, Germany
| | - Vasiliki Tsata
- DFG-Center for Regenerative Therapies Dresden, Cluster of Excellence, Technische Universität Dresden, Dresden, Germany
| | | | - Maria Troullinaki
- Department of Clinical Pathobiochemistry, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
| | - Anke Witt
- Department of Clinical Pathobiochemistry, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
| | - Vivian Anastasiou
- DZD/Paul Langerhans Institute Dresden of Helmholtz Centre Munich, Faculty of Medicine, Technische Universität Dresden, Dresden, Germany
| | - George Chrousos
- First Department of Pediatrics, National and Kapodistrian University of Athens Medical School, Athens, Greece
- Aghia Sophia Children's Hospital, Athens, Greece
| | - Chun-Xia Yi
- Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
- Helmholtz Diabetes Center (HDC) & German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 85764, Neuherberg, Germany
| | - Cristina García-Cáceres
- Helmholtz Diabetes Center (HDC) & German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 85764, Neuherberg, Germany
| | - Matthias H Tschöp
- Helmholtz Diabetes Center (HDC) & German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 85764, Neuherberg, Germany
- Division of Metabolic Diseases, Technische Universität München, 80333, Munich, Germany
| | - Stefan R Bornstein
- Department of Medicine, Technische Universität Dresden, Dresden, Germany
| | - Andreas Androutsellis-Theotokis
- Department of Medicine, Technische Universität Dresden, Dresden, Germany.
- DFG-Center for Regenerative Therapies Dresden, Cluster of Excellence, Technische Universität Dresden, Dresden, Germany.
- Division of Cancer and Stem Cells, University of Nottingham, Nottingham, NG7 2RD, UK.
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Golas MM. Human cellular models of medium spiny neuron development and Huntington disease. Life Sci 2018; 209:179-196. [PMID: 30031060 DOI: 10.1016/j.lfs.2018.07.030] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Revised: 06/22/2018] [Accepted: 07/17/2018] [Indexed: 12/24/2022]
Abstract
The loss of gamma-aminobutyric acid (GABA)-ergic medium spiny neurons (MSNs) in the striatum is the hallmark of Huntington disease (HD), an incurable neurodegenerative disorder characterized by progressive motor, psychiatric, and cognitive symptoms. Transplantation of MSNs or their precursors represents a promising treatment strategy for HD. In initial clinical trials in which HD patients received fetal neurografts directly into the striatum without a pretransplant cell-differentiation step, some patients exhibited temporary benefits. Meanwhile, major challenges related to graft overgrowth, insufficient survival of grafted cells, and limited availability of donated fetal tissue remain. Thus, the development of approaches that allow modeling of MSN differentiation and HD development in cell culture platforms may improve our understanding of HD and translate, ultimately, into HD treatment options. Here, recent advances in the in vitro differentiation of MSNs derived from fetal neural stem cells/progenitor cells (NSCs/NPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and induced NSCs (iNSCs) as well as advances in direct transdifferentiation are reviewed. Progress in non-allele specific and allele specific gene editing of HTT is presented as well. Cell characterization approaches involving phenotyping as well as in vitro and in vivo functional assays are also discussed.
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Affiliation(s)
- Monika M Golas
- Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 3, Building 1233, DK-8000 Aarhus C, Denmark; Department of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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22
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Omrani MR, Yaqubi M, Mohammadnia A. Transcription Factors in Regulatory and Protein Subnetworks during Generation of Neural Stem Cells and Neurons from Direct Reprogramming of Non-fibroblastic Cell Sources. Neuroscience 2018; 380:63-77. [PMID: 29653196 DOI: 10.1016/j.neuroscience.2018.03.033] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2017] [Revised: 03/19/2018] [Accepted: 03/20/2018] [Indexed: 12/31/2022]
Abstract
Direct reprogramming of non-fibroblastic cells to the neuronal cell types including induced neurons (iNs) and induced neural stem cells (iNSCs) has provided an alternative approach for the direct reprogramming of fibroblasts to those cells. However, to increase the efficiency of the reprogramming process the underlying mechanisms should be clarified. In the current study, we analyzed the gene expression profiles of five different cellular conversions to understand the most significant molecular mechanisms and transcription factors (TFs) underlying each conversion. For each conversion, we found the list of differentially expressed genes (DEGs) and the list of differentially expressed TFs (DE-TFs) which regulate expression of DEGs. Moreover, we constructed gene regulatory networks based on the TF-binding sites' data and found the most central regulators and the most active part of the networks. Furthermore, protein complexes were identified from constructed protein-protein interaction networks for DE-TFs. Finally, we proposed a list of main regulators for each conversion; for example, in the direct conversion of epithelial-like cells (ECs) to iNSCs, combination of centrality with active modules or protein complex analyses highlighted the role of POU3F2, BACH1, AR, PBX1, SOX2 and NANOG genes in this conversion. To the best of our knowledge, this study is the first one that analyzed the direct conversion of non-fibroblastic cells toward iNs and iNSCs and we believe that the expression manipulation of identified genes may increase efficiency of these processes.
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Affiliation(s)
- Mohammad Reza Omrani
- National Institute of Genetics Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Moein Yaqubi
- Department of Psychiatry, Ludmer Centre for Neuroinformatics and Mental Health, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada.
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23
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Asfahani RI, Tahoun MM, Miller-Hodges EV, Bellerby J, Virasami AK, Sampson RD, Moulding D, Sebire NJ, Hohenstein P, Scambler PJ, Waters AM. Activation of podocyte Notch mediates early Wt1 glomerulopathy. Kidney Int 2018; 93:903-920. [PMID: 29398135 PMCID: PMC6169130 DOI: 10.1016/j.kint.2017.11.014] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2017] [Revised: 11/07/2017] [Accepted: 11/09/2017] [Indexed: 01/26/2023]
Abstract
The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis.
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Affiliation(s)
- Rowan I Asfahani
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK
| | - Mona M Tahoun
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK; Clinical and Chemical Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt
| | - Eve V Miller-Hodges
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland
| | - Jack Bellerby
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK
| | - Alex K Virasami
- Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Robert D Sampson
- Institute of Ophthalmology, University College of London, London, UK
| | - Dale Moulding
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK
| | - Neil J Sebire
- Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | | | - Peter J Scambler
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK
| | - Aoife M Waters
- Programme of Developmental Biology of Birth Defects, Great Ormond Street Institute of Child Health, University College of London, London, UK; Great Ormond Street Hospital NHS Foundation Trust, London, UK.
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24
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Samoilova EM, Kalsin VA, Kushnir NM, Chistyakov DA, Troitskiy AV, Baklaushev VP. Adult Neural Stem Cells: Basic Research and Production Strategies for Neurorestorative Therapy. Stem Cells Int 2018; 2018:4835491. [PMID: 29760724 PMCID: PMC5901847 DOI: 10.1155/2018/4835491] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 02/01/2018] [Indexed: 12/24/2022] Open
Abstract
Over many decades, constructing genetically and phenotypically stable lines of neural stem cells (NSC) for clinical purposes with the aim of restoring irreversibly lost functions of nervous tissue has been one of the major goals for multiple research groups. The unique ability of stem cells to maintain their own pluripotent state even in the adult body has made them into the choice object of study. With the development of the technology for induced pluripotent stem cells (iPSCs) and direct transdifferentiation of somatic cells into the desired cell type, the initial research approaches based on the use of allogeneic NSCs from embryonic or fetal nervous tissue are gradually becoming a thing of the past. This review deals with basic molecular mechanisms for maintaining the pluripotent state of embryonic/induced stem and reprogrammed somatic cells, as well as with currently existing reprogramming strategies. The focus is on performing direct reprogramming while bypassing the stage of iPSCs which is known for genetic instability and an increased risk of tumorigenesis. A detailed description of various protocols for obtaining reprogrammed neural cells used in the therapy of the nervous system pathology is also provided.
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Affiliation(s)
- E. M. Samoilova
- Federal Research Clinical Center of the Federal Biomedical Agency of Russian Federation, 28 Orekhovy Blvd, Moscow 115682, Russia
| | - V. A. Kalsin
- Federal Research Clinical Center of the Federal Biomedical Agency of Russian Federation, 28 Orekhovy Blvd, Moscow 115682, Russia
| | - N. M. Kushnir
- Federal Research Clinical Center of the Federal Biomedical Agency of Russian Federation, 28 Orekhovy Blvd, Moscow 115682, Russia
| | - D. A. Chistyakov
- Department of Basic and Applied Neurobiology, V.P. Serbsky Federal Medical Research Center for Psychiatry and Narcology, Moscow, Russia
| | - A. V. Troitskiy
- Federal Research Clinical Center of the Federal Biomedical Agency of Russian Federation, 28 Orekhovy Blvd, Moscow 115682, Russia
- Institute for Advanced Studies, Federal Biomedical Agency of Russian Federation, Moscow, Russia
| | - V. P. Baklaushev
- Federal Research Clinical Center of the Federal Biomedical Agency of Russian Federation, 28 Orekhovy Blvd, Moscow 115682, Russia
- Institute for Advanced Studies, Federal Biomedical Agency of Russian Federation, Moscow, Russia
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25
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Shahbazi E, Mirakhori F, Ezzatizadeh V, Baharvand H. Reprogramming of somatic cells to induced neural stem cells. Methods 2018; 133:21-28. [PMID: 28939501 DOI: 10.1016/j.ymeth.2017.09.007] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2017] [Revised: 09/02/2017] [Accepted: 09/12/2017] [Indexed: 12/26/2022] Open
Abstract
Recent investigations have demonstrated that defined sets of exogenous factors (chemical and/or biochemical) can convert human and mouse somatic cells into induced neural stem cells (iNSCs). Considering the self-renewal and multi-potential differentiation capabilities of iNSCs, generation of these cells has considerably enhanced cell therapy for treatment of neurodegenerative disorders. These cells can also serve as models for investigation of the mechanism(s) underlying neurodegenerative diseases and as an asset in drug discovery. Meanwhile, using the process of direct conversion/transdifferentiation, by bypassing pluripotent state and consequently reducing tumorigenesis and genetic instability risks, establishment of several desired cells are feasible. In this review, we describe the pros and cons of different methods employed to directly reprogram somatic cells to iNSCs along with the progress of iNSCs applications and the future challenges.
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Affiliation(s)
- Ebrahim Shahbazi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Fahimeh Mirakhori
- Institute for Cell Engineering, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Vahid Ezzatizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
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26
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Guo Y, Zhang K, Cheng C, Ji Z, Wang X, Wang M, Chu M, Tang DG, Zhu HH, Gao WQ. Numb -/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling. Clin Cancer Res 2017; 23:6744-6756. [PMID: 28751447 DOI: 10.1158/1078-0432.ccr-17-0913] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2017] [Revised: 06/09/2017] [Accepted: 07/18/2017] [Indexed: 11/16/2022]
Abstract
Purpose: To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb-/low prostate cancer cells in castration resistance.Experimental Design: The expression of Numb was assessed using multiple Oncomine datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated in vitro and in vivo In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter-based lentiviral reporter system. The difference between Numb-/low and Numbhigh prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR.Results: We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a Numb promoter-based lentiviral reporter system, we were able to distinguish Numb-/low prostate cancer cells from Numbhigh cells. Numb-/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell-associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb-/low cells in response to ADT.Conclusions: Numb-/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. Clin Cancer Res; 23(21); 6744-56. ©2017 AACR.
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Affiliation(s)
- Yanjing Guo
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Kai Zhang
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
| | - Chaping Cheng
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
| | - Zhongzhong Ji
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
| | - Xue Wang
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
| | - Minglei Wang
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Mingliang Chu
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Dean G Tang
- Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Carlton and Elm Streets, Buffalo, New York
| | - Helen He Zhu
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
| | - Wei-Qiang Gao
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
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27
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Khazaei N, Rastegar-Pouyani S, O'Toole N, Wee P, Mohammadnia A, Yaqubi M. Regulating the transcriptomes that mediate the conversion of fibroblasts to various nervous system neural cell types. J Cell Physiol 2017; 233:3603-3614. [PMID: 29044560 DOI: 10.1002/jcp.26221] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Accepted: 10/05/2017] [Indexed: 12/31/2022]
Abstract
Our understanding of the mechanism of cell fate transition during the direct reprogramming of fibroblasts into various central nervous system (CNS) neural cell types has been limited by the lack of a comprehensive analysis on generated cells, independently and in comparison with other CNS neural cell types. Here, we applied an integrative approach on 18 independent high throughput expression data sets to gain insight into the regulation of the transcriptome during the conversion of fibroblasts into induced neural stem cells, induced neurons (iNs), induced astrocytes, and induced oligodendrocyte progenitor cells (iOPCs). We found common down-regulated genes to be mostly related to fibroblast-specific functions, and suggest their potential as markers for screening of the silencing of the fibroblast-specific program. For example, Tagln was significantly down-regulated across all considered data sets. In addition, we identified specific profiles of up-regulated genes for each CNS neural cell types, which could be potential markers for maturation and efficiency screenings. Furthermore, we identified the main TFs involved in the regulation of the gene expression program during direct reprogramming. For example, in the generation of iNs from fibroblasts, the Rest TF was the main regulator of this reprogramming. In summary, our computational approach for meta-analyzing independent expression data sets provides significant details regarding the molecular mechanisms underlying the regulation of the gene expression program, and also suggests potentially useful candidate genes for screening down-regulation of fibroblast gene expression profile, maturation, and efficiency, as well as candidate TFs for increasing the efficiency of the reprogramming process.
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Affiliation(s)
- Niusha Khazaei
- Meakins-Christie Laboratories, Department of Medicine, McGill University and McGill University Health Centre Research Institute, Montréal, Canada
| | | | - Nicholas O'Toole
- Douglas Mental Health University Institute, McGill University, Ludmer Centre for Neuroinformatics and Mental Health Montreal, Quebec, Canada
| | - Ping Wee
- Faculty of Medicine and Dentistry, Department of Medical Genetics and Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada
| | | | - Moein Yaqubi
- Department of Psychiatry, Sackler Program for Epigenetics and Psychobiology at McGill University, Ludmer Centre for Neuroinformatics and Mental Health, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada
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28
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Tang Y, Yu P, Cheng L. Current progress in the derivation and therapeutic application of neural stem cells. Cell Death Dis 2017; 8:e3108. [PMID: 29022921 PMCID: PMC5682670 DOI: 10.1038/cddis.2017.504] [Citation(s) in RCA: 132] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Revised: 08/28/2017] [Accepted: 08/31/2017] [Indexed: 12/13/2022]
Abstract
Neural stem cells (NSCs) have a unique role in neural regeneration. Cell therapy based on NSC transplantation is a promising tool for the treatment of nervous system diseases. However, there are still many issues and controversies associated with the derivation and therapeutic application of these cells. In this review, we summarize the different sources of NSCs and their derivation methods, including direct isolation from primary tissues, differentiation from pluripotent stem cells and transdifferentiation from somatic cells. We also review the current progress in NSC implantation for the treatment of various neural defects and injuries in animal models and clinical trials. Finally, we discuss potential optimization strategies for NSC derivation and propose urgent challenges to the clinical translation of NSC-based therapies in the near future.
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Affiliation(s)
- Yuewen Tang
- National Research Center for Translational Medicine, State Key Laboratory of Medical Genomics, Shanghai Institute of Haematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Pei Yu
- Department of Orthopaedics, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Lin Cheng
- National Research Center for Translational Medicine, State Key Laboratory of Medical Genomics, Shanghai Institute of Haematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
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29
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Abstract
Cellular heterogeneity in cancer represents a significant challenge. In order to develop effective and lasting therapies, it is essential to understand the source of this heterogeneity, and its role in tumor progression and therapy resistance. Here, we consider not only genetic and epigenetic mechanisms, but also inflammation and cell state reprogramming in creating tumor heterogeneity. We discuss similarities between normal mammary epithelial developmental states and various breast cancer molecular sub-types, and the cells that are thought to propagate them. We emphasize that while stem cell phenotypes and mesenchymal character have often been conflated, existing data suggest that the combination of intrinsic genetic and epigenetic changes, and microenvironmental influences generate multiple types of tumor propagating cells distinguishable by their positions along a continuum of epithelial to mesenchymal, stem to differentiated and embryonic to mature cell states. Consequently, in addition to the prospect of stem cell-directed tumor therapies, there is a need to understand interrelationships between stem cell, epithelial–mesenchymal, and tumor-associated reprogramming events to develop new therapies that mitigate cell state plasticity and minimize the evolution of tumor heterogeneity.
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30
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Xu Z, Chu X, Jiang H, Schilling H, Chen S, Feng J. Induced dopaminergic neurons: A new promise for Parkinson's disease. Redox Biol 2017; 11:606-612. [PMID: 28110217 PMCID: PMC5256671 DOI: 10.1016/j.redox.2017.01.009] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2017] [Revised: 01/06/2017] [Accepted: 01/11/2017] [Indexed: 12/28/2022] Open
Abstract
Motor symptoms that define Parkinson’s disease (PD) are caused by the selective loss of nigral dopaminergic (DA) neurons. Cell replacement therapy for PD has been focused on midbrain DA neurons derived from human fetal mesencephalic tissue, human embryonic stem cells (hESC) or human induced pluripotent stem cells (iPSC). Recent development in the direct conversion of human fibroblasts to induced dopaminergic (iDA) neurons offers new opportunities for transplantation study and disease modeling in PD. The iDA neurons are generated directly from human fibroblasts in a short period of time, bypassing lengthy differentiation process from human pluripotent stem cells and the concern for potentially tumorigenic mitotic cells. They exhibit functional dopaminergic neurotransmission and relieve locomotor symptoms in animal models of Parkinson’s disease. In this review, we will discuss this recent development and its implications to Parkinson’s disease research and therapy.
Fibroblasts can be directly converted to induced dopaminergic neurons by transcription factors. Many different types of cells can be converted to induced neurons in vitro and in vivo. Appropriate cell culture conditions enhance the direct conversion to induced neurons. The conversion to induced neurons is enhanced by G1 arrest and p53 attenuation. iDA neurons is a promising tool for PD research and therapy.
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Affiliation(s)
- Zhimin Xu
- Veterans Affairs Western New York Healthcare System, Buffalo, NY 14215, USA; Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA; Laboratory of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Science and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Xingkun Chu
- Laboratory of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Science and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Houbo Jiang
- Veterans Affairs Western New York Healthcare System, Buffalo, NY 14215, USA; Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA
| | - Haley Schilling
- Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA
| | - Shengdi Chen
- Laboratory of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Science and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Jian Feng
- Veterans Affairs Western New York Healthcare System, Buffalo, NY 14215, USA; Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA.
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31
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Gao R, Xiu W, Zhang L, Zang R, Yang L, Wang C, Wang M, Wang M, Yi L, Tang Y, Gao Y, Wang H, Xi J, Liu W, Wang Y, Wen X, Yu Y, Zhang Y, Chen L, Chen J, Gao S. Direct induction of neural progenitor cells transiently passes through a partially reprogrammed state. Biomaterials 2016; 119:53-67. [PMID: 28006658 DOI: 10.1016/j.biomaterials.2016.12.007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2016] [Revised: 12/07/2016] [Accepted: 12/07/2016] [Indexed: 02/09/2023]
Abstract
The generation of functional neural progenitor cells (NPCs) holds great promise for both research and clinical applications in neurodegenerative diseases. Traditionally, NPCs are derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or NPCs can be directly converted from somatic cells by sets of transcription factors or by a combination of chemical cocktails and/or hypoxia. However, the ethical issues of ESCs, the risk of tumorigenesis from iPSCs and transgenic integration from exogenous genes as well as complicated manipulation and time-consuming of chemical induced NPCs (ciNPCs) limit the applications of these strategies. Here, we describe a novel method for generating growth factor-induced neural progenitor cells (giNPCs) from mouse embryonic and adult fibroblasts by using inductive and/or permissive signaling culture conditions. These giNPCs closely resemble brain-derived NPCs in terms of transcription networks and neural lineage differentiation potentials. Moreover, this somatic cell to NPC induction is a gradual process that includes initiation, intermediate, maturation and stabilization stages. Importantly, gene expression and histone modification analyses further indicate a partially reprogrammed state during the generation process of induced NPCs, in which lineage specific genes and pluripotency associated genes are transiently activated. Our study therefore describes the potential safety problems that also exist in the transgene-free direct induction strategy and highlights the importance of excluding the possibility of residual partially reprogrammed and/or teratoma-like cells from the generated NPCs for future clinical trials.
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Affiliation(s)
- Rui Gao
- School of Life Sciences, Tsinghua University, Beijing, 100084, China; National Institute of Biological Sciences, NIBS, Beijing, 102206, China
| | - Wenchao Xiu
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Linfeng Zhang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Ruge Zang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Lei Yang
- Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, 200092, China
| | - Chenfei Wang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Min Wang
- Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200032, China
| | - Mingzhu Wang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Li Yi
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Yuanyuan Tang
- Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, 200092, China
| | - Yawei Gao
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Hong Wang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Jiajie Xi
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Wenqiang Liu
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Yixuan Wang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Xuejun Wen
- The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute of Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai, 200092, China
| | - Yongchun Yu
- Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200032, China
| | - Yong Zhang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Liang Chen
- School of Life Sciences, Tsinghua University, Beijing, 100084, China; National Institute of Biological Sciences, NIBS, Beijing, 102206, China.
| | - Jiayu Chen
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China.
| | - Shaorong Gao
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China.
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32
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Engineering cell fate: Spotlight on cell-activation and signaling-directed lineage conversion. Tissue Cell 2016; 48:475-87. [DOI: 10.1016/j.tice.2016.07.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Revised: 06/13/2016] [Accepted: 07/25/2016] [Indexed: 12/23/2022]
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33
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Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair. Biomaterials 2016; 102:148-61. [DOI: 10.1016/j.biomaterials.2016.06.020] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 06/07/2016] [Indexed: 11/22/2022]
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34
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Tian E, Sun G, Sun G, Chao J, Ye P, Warden C, Riggs AD, Shi Y. Small-Molecule-Based Lineage Reprogramming Creates Functional Astrocytes. Cell Rep 2016; 16:781-92. [PMID: 27396343 PMCID: PMC9228989 DOI: 10.1016/j.celrep.2016.06.042] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2015] [Revised: 04/07/2016] [Accepted: 06/08/2016] [Indexed: 12/28/2022] Open
Abstract
Growing evidence indicates important roles for astrocytes in neurodevelopment and diseases. However, astrocytes and their roles in these processes remain poorly understood. Despite recent progress in reprogramming somatic cells into different types of neural cells, reprogramming to astrocytes has lagged. Here, we show that functional astrocytes can be generated from mammalian fibroblasts using only small molecules. Induced mouse astrocytes resemble primary astrocytes in astrocytic gene expression and epigenomic status and exhibit functional properties in promoting neuronal maturation, glutamate uptake, and calcium signaling. Moreover, these cells can recapitulate the Alexander disease phenotype of protein aggregation when expressing Gfap with a disease-causing mutation. The same compounds can also reprogram human fibroblasts into astroglial progenitor cells that can further mature into functional astrocytes. These chemically induced astrocytes may provide cellular models to uncover roles of astrocytes in normal neurodevelopment and pathogenesis of neurological diseases.
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Affiliation(s)
- E Tian
- Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Guoqiang Sun
- Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Guihua Sun
- Diabetes and Metabolism Research Institute, City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Jianfei Chao
- Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Peng Ye
- Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Charles Warden
- Integrative Genomics Core, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Arthur D Riggs
- Diabetes and Metabolism Research Institute, City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Yanhong Shi
- Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA.
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Toutouna L, Nikolakopoulou P, Poser SW, Masjkur J, Arps-Forker C, Troullinaki M, Grossklaus S, Bosak V, Friedrich U, Ziemssen T, Bornstein SR, Chavakis T, Androutsellis-Theotokis A. Hes3 expression in the adult mouse brain is regulated during demyelination and remyelination. Brain Res 2016; 1642:124-130. [DOI: 10.1016/j.brainres.2016.03.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Revised: 03/14/2016] [Accepted: 03/16/2016] [Indexed: 11/24/2022]
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36
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Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nat Rev Neurosci 2016; 17:424-37. [PMID: 27194476 DOI: 10.1038/nrn.2016.46] [Citation(s) in RCA: 224] [Impact Index Per Article: 24.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The scarcity of live human brain cells for experimental access has for a long time limited our ability to study complex human neurological disorders and elucidate basic neuroscientific mechanisms. A decade ago, the development of methods to reprogramme somatic human cells into induced pluripotent stem cells enabled the in vitro generation of a wide range of neural cells from virtually any human individual. The growth of methods to generate more robust and defined neural cell types through reprogramming and direct conversion into induced neurons has led to the establishment of various human reprogramming-based neural disease models.
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Shetty AK, Hattiangady B. Grafted Subventricular Zone Neural Stem Cells Display Robust Engraftment and Similar Differentiation Properties and Form New Neurogenic Niches in the Young and Aged Hippocampus. Stem Cells Transl Med 2016; 5:1204-15. [PMID: 27194744 PMCID: PMC4996439 DOI: 10.5966/sctm.2015-0270] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 04/01/2016] [Indexed: 12/30/2022] Open
Abstract
The engraftment and differentiation of alkaline phosphatase-positive neural stem cells (NSCs) expanded from the postnatal subventricular zone (SVZ), 3 months after grafting into the intact young or aged rat hippocampus, were examined. Both young and aged hippocampi supported robust engraftment and similar differentiation of SVZ-NSC graft-derived cells. As clinical application of neural stem cell (NSC) grafting into the brain would also encompass aged people, critical evaluation of engraftment of NSC graft-derived cells in the aged hippocampus has significance. We examined the engraftment and differentiation of alkaline phosphatase-positive NSCs expanded from the postnatal subventricular zone (SVZ), 3 months after grafting into the intact young or aged rat hippocampus. Graft-derived cells engrafted robustly into both young and aged hippocampi. Although most graft-derived cells pervasively migrated into different hippocampal layers, the graft cores endured and contained graft-derived neurons expressing neuron-specific nuclear antigen (NeuN) and γ-amino butyric acid in both groups. A fraction of migrated graft-derived cells in the neurogenic subgranular zone-granule cell layer also expressed NeuN. Neuronal differentiation was, however, occasionally seen amid graft-derived cells that had migrated into non-neurogenic regions, where substantial fractions differentiated into S-100β+ astrocytes, NG2+ oligodendrocyte progenitors, or Olig2+ putative oligodendrocytes. In both age groups, graft cores located in non-neurogenic regions displayed many doublecortin-positive (DCX+) immature neurons at 3 months after grafting. Analyses of cells within graft cores using birth dating and putative NSC markers revealed that DCX+ neurons were newly born neurons derived from engrafted cells and that putative NSCs persisted within the graft cores. Thus, both young and aged hippocampi support robust engraftment and similar differentiation of SVZ-NSC graft-derived cells. Furthermore, some grafted NSCs retain the “stemness” feature and produce new neurons even at 3 months after grafting, implying that grafting of SVZ-NSCs into the young or aged hippocampus leads to establishment of new neurogenic niches in non-neurogenic regions. Significance The results demonstrate that advanced age of the host at the time of grafting has no major adverse effects on engraftment, migration, and differentiation of grafted subventricular zone-neural stem cells (SVZ-NSCs) in the intact hippocampus, as both young and aged hippocampi promoted excellent engraftment, migration, and differentiation of SVZ-NSC graft-derived cells in the present study. Furthermore, SVZ-NSC grafts showed ability for establishing neurogenic niches in non-neurogenic regions, generating new neurons for extended periods after grafting. This phenomenon will be beneficial if these niches can continuously generate new neurons and glia in the grafted hippocampus, as newly generated neurons and glia are expected to improve, not only the microenvironment, but also the plasticity and function of the aged hippocampus. Overall, these results have significance because the potential application of NSC grafting for treatment of neurodegenerative disorders at early stages of disease progression and age-related impairments would mostly involve aged persons as recipients.
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Affiliation(s)
- Ashok K Shetty
- Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, Texas, USAResearch Service, Olin E. Teague Veterans' Medical Center, Central Texas Veterans Health Care System, Temple, Texas, USADepartment of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, Texas, USADivision of Neurosurgery, Duke University Medical Center, Durham, North Carolina, USAResearch and Surgery Services, Durham Veterans Affairs Medical Center, Durham, North Carolina, USA
| | - Bharathi Hattiangady
- Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, Texas, USAResearch Service, Olin E. Teague Veterans' Medical Center, Central Texas Veterans Health Care System, Temple, Texas, USADepartment of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, Texas, USADivision of Neurosurgery, Duke University Medical Center, Durham, North Carolina, USAResearch and Surgery Services, Durham Veterans Affairs Medical Center, Durham, North Carolina, USA
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Kim SM, Kim JW, Kwak TH, Park SW, Kim KP, Park H, Lim KT, Kang K, Kim J, Yang JH, Han H, Lee I, Hyun JK, Bae YM, Schöler HR, Lee HT, Han DW. Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts. J Biol Chem 2016; 291:14199-14212. [PMID: 27189941 DOI: 10.1074/jbc.m115.713578] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2015] [Indexed: 01/10/2023] Open
Abstract
The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.
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Affiliation(s)
- Sung Min Kim
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea; Department of Animal Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Jong-Wan Kim
- Department of Nanobiomedical Science, Dankook University Graduate School, Cheonan 31116, Republic of Korea
| | - Tae Hwan Kwak
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Sang Woong Park
- Department of Physiology, School of Medicine, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea
| | - Kee-Pyo Kim
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany
| | - Hyunji Park
- Department of Physiology, School of Medicine, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea
| | - Kyung Tae Lim
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Kyuree Kang
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Jonghun Kim
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Ji Hun Yang
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany
| | - Heonjong Han
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 04056, Korea
| | - Insuk Lee
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 04056, Korea
| | - Jung Keun Hyun
- Department of Nanobiomedical Science, Dankook University Graduate School, Cheonan 31116, Republic of Korea
| | - Young Min Bae
- Department of Physiology, School of Medicine, Konkuk University, Chungju, Chungbuk 27478, Republic of Korea
| | - Hans R Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany,; University of Münster, Medical Faculty, Domagkstraße 3, 48149 Münster, Germany
| | - Hoon Taek Lee
- Department of Animal Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea
| | - Dong Wook Han
- Department of Stem Cell Biology, School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea; KU Open-Innovation Center, Institute of Biomedical Science & Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Republic of Korea.
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39
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Zhang M, Lin YH, Sun YJ, Zhu S, Zheng J, Liu K, Cao N, Li K, Huang Y, Ding S. Pharmacological Reprogramming of Fibroblasts into Neural Stem Cells by Signaling-Directed Transcriptional Activation. Cell Stem Cell 2016; 18:653-67. [PMID: 27133794 DOI: 10.1016/j.stem.2016.03.020] [Citation(s) in RCA: 151] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 03/19/2016] [Accepted: 03/25/2016] [Indexed: 02/09/2023]
Abstract
Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small-molecule approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts toward a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study provides an effective chemical approach for generating neural stem cells from mouse fibroblasts and reveals mechanistic insights into underlying reprogramming processes.
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Affiliation(s)
- Mingliang Zhang
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yuan-Hung Lin
- Gladstone Institute of Neurological Disease, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yujiao Jennifer Sun
- Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Saiyong Zhu
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jiashun Zheng
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Kai Liu
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Nan Cao
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Ke Li
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yadong Huang
- Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Gladstone Institute of Neurological Disease, San Francisco, CA 94158, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Sheng Ding
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
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40
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Hallmann AL, Araúzo-Bravo MJ, Zerfass C, Senner V, Ehrlich M, Psathaki OE, Han DW, Tapia N, Zaehres H, Schöler HR, Kuhlmann T, Hargus G. Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain. Stem Cell Res 2016; 16:776-81. [PMID: 27153350 DOI: 10.1016/j.scr.2016.04.015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/21/2015] [Revised: 02/21/2016] [Accepted: 04/15/2016] [Indexed: 12/16/2022] Open
Abstract
Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.
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Affiliation(s)
- Anna-Lena Hallmann
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany; Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
| | - Marcos J Araúzo-Bravo
- Group of Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute, 20014 San Sebastián, Spain; IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain
| | - Christina Zerfass
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany
| | - Volker Senner
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany
| | - Marc Ehrlich
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany; Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
| | | | - Dong Wook Han
- Department of Stem Cell Biology, School of Medicine, Konkuk University, 143701 Seoul, Republic of Korea
| | - Natalia Tapia
- Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany; Institute of Biomedicine of Valencia, Spanish National Research Council (IBV-CSIC), Jaime Roig 11, 46010 Valencia, Spain
| | - Holm Zaehres
- Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
| | - Hans R Schöler
- Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
| | - Tanja Kuhlmann
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany
| | - Gunnar Hargus
- Institute of Neuropathology, University Hospital Münster, 48149 Münster, Germany; Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany; Department of Pathology and Cell Biology, Columbia University Medical Center, 10032 New York, USA.
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41
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Shahbazi E, Moradi S, Nemati S, Satarian L, Basiri M, Gourabi H, Zare Mehrjardi N, Günther P, Lampert A, Händler K, Hatay FF, Schmidt D, Molcanyi M, Hescheler J, Schultze JL, Saric T, Baharvand H. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor. Stem Cell Reports 2016; 6:539-551. [PMID: 27052315 PMCID: PMC4834053 DOI: 10.1016/j.stemcr.2016.02.013] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2015] [Revised: 02/19/2016] [Accepted: 02/22/2016] [Indexed: 01/08/2023] Open
Abstract
Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.
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Affiliation(s)
- Ebrahim Shahbazi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| | - Sharif Moradi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| | - Shiva Nemati
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| | - Leila Satarian
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| | - Mohsen Basiri
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| | - Hamid Gourabi
- Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran 1665659911, Iran
| | - Narges Zare Mehrjardi
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Patrick Günther
- Department for Genomics and Immunoregulation, Life and Medical Sciences Institute, University of Bonn, Bonn 53115, Germany
| | - Angelika Lampert
- Institute of Physiology and Pathophysiology, Friedrich-Alexander-University of Erlangen- Nürnberg, Erlangen 91054, Germany; Institute of Physiology, RWTH, Aachen University, Aachen 52074, Germany
| | - Kristian Händler
- Department for Genomics and Immunoregulation, Life and Medical Sciences Institute, University of Bonn, Bonn 53115, Germany
| | - Firuze Fulya Hatay
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Diana Schmidt
- Institute of Physiology and Pathophysiology, Friedrich-Alexander-University of Erlangen- Nürnberg, Erlangen 91054, Germany; IZKF Junior Research Group and BMBF Research Group Neuroscience, IZKF, Friedrich-Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Marek Molcanyi
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Jürgen Hescheler
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany
| | - Joachim L Schultze
- Department for Genomics and Immunoregulation, Life and Medical Sciences Institute, University of Bonn, Bonn 53115, Germany
| | - Tomo Saric
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne 50931, Germany.
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran 1461968151, Iran.
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Hou S, Lu P. Direct reprogramming of somatic cells into neural stem cells or neurons for neurological disorders. Neural Regen Res 2016; 11:28-31. [PMID: 26981072 PMCID: PMC4774217 DOI: 10.4103/1673-5374.169602] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cells in vitro and in vivo and their potential treatments of neurological disorders.
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Affiliation(s)
- Shaoping Hou
- Spinal Cord Research Center, Department of Neurobiology & Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA
| | - Paul Lu
- Veterans Administration Medical Center, San Diego, CA, USA; Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA
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Petersen GF, Strappe PM. Generation of diverse neural cell types through direct conversion. World J Stem Cells 2016; 8:32-46. [PMID: 26981169 PMCID: PMC4766249 DOI: 10.4252/wjsc.v8.i2.32] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 11/18/2015] [Accepted: 01/04/2016] [Indexed: 02/06/2023] Open
Abstract
A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace, thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost. The process of neural direct conversion, in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency, shows great potential, with evidence of the generation of a range of functional neural cell types both in vitro and in vivo, through viral and non-viral delivery of exogenous factors, as well as chemical induction methods. Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells, with prospective roles in the investigation of neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications, however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option. In this review, we describe the generation of diverse neural cell types via direct conversion of somatic cells, with comparison against stem cell-based approaches, as well as discussion of their potential research and clinical applications.
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Masjkur J, Poser SW, Nikolakopoulou P, Chrousos G, McKay RD, Bornstein SR, Jones PM, Androutsellis-Theotokis A. Endocrine Pancreas Development and Regeneration: Noncanonical Ideas From Neural Stem Cell Biology. Diabetes 2016; 65:314-30. [PMID: 26798118 DOI: 10.2337/db15-1099] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Loss of insulin-producing pancreatic islet β-cells is a hallmark of type 1 diabetes. Several experimental paradigms demonstrate that these cells can, in principle, be regenerated from multiple endogenous sources using signaling pathways that are also used during pancreas development. A thorough understanding of these pathways will provide improved opportunities for therapeutic intervention. It is now appreciated that signaling pathways should not be seen as "on" or "off" but that the degree of activity may result in wildly different cellular outcomes. In addition to the degree of operation of a signaling pathway, noncanonical branches also play important roles. Thus, a pathway, once considered as "off" or "low" may actually be highly operational but may be using noncanonical branches. Such branches are only now revealing themselves as new tools to assay them are being generated. A formidable source of noncanonical signal transduction concepts is neural stem cells because these cells appear to have acquired unusual signaling interpretations to allow them to maintain their unique dual properties (self-renewal and multipotency). We discuss how such findings from the neural field can provide a blueprint for the identification of new molecular mechanisms regulating pancreatic biology, with a focus on Notch, Hes/Hey, and hedgehog pathways.
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Affiliation(s)
- Jimmy Masjkur
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | - Steven W Poser
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | | | - George Chrousos
- First Department of Pediatrics, University of Athens Medical School and Aghia Sophia Children's Hospital, Athens, Greece
| | | | - Stefan R Bornstein
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | - Peter M Jones
- Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King's College London, London, U.K
| | - Andreas Androutsellis-Theotokis
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany Center for Regenerative Therapies Dresden, Dresden, Germany Department of Stem Cell Biology, Centre for Biomolecular Sciences, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, U.K.
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An Overview of Direct Somatic Reprogramming: The Ins and Outs of iPSCs. Int J Mol Sci 2016; 17:ijms17010141. [PMID: 26805822 PMCID: PMC4730380 DOI: 10.3390/ijms17010141] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Revised: 01/12/2016] [Accepted: 01/13/2016] [Indexed: 02/07/2023] Open
Abstract
Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application.
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Zeltner N, Studer L. Pluripotent stem cell-based disease modeling: current hurdles and future promise. Curr Opin Cell Biol 2015; 37:102-10. [PMID: 26629748 DOI: 10.1016/j.ceb.2015.10.008] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 10/18/2015] [Indexed: 12/14/2022]
Abstract
Human induced pluripotent stem cells (hiPSCs) can yield unlimited numbers of patient-specific cells of any type and may be an important tool in efforts to overcome current shortcomings in biomedical research. In vitro disease models based on the use of hiPSCs have been proposed for various applications. Those include drug discovery and validation, efficacy, safety and toxicity assays, the elucidation of previously unknown disease mechanisms, the enhancement of animal based assays, the promise of conducting clinical trials in the dish and the identification of cell types and stages suitable for cell replacement therapies. Here, we provide an overview of the current state of hiPSC-based disease modeling and discuss recent progress and remaining challenges on the road to realizing the full potential of this novel technology.
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Affiliation(s)
- Nadja Zeltner
- Developmental Biology, Sloan Kettering Institute, New York, USA; Center for Stem Cell Biology, Sloan Kettering Institute, New York, USA
| | - Lorenz Studer
- Developmental Biology, Sloan Kettering Institute, New York, USA; Center for Stem Cell Biology, Sloan Kettering Institute, New York, USA.
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Gopalakrishnan S, Hor P, Ichida JK. New approaches for direct conversion of patient fibroblasts into neural cells. Brain Res 2015; 1656:2-13. [PMID: 26475975 DOI: 10.1016/j.brainres.2015.10.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Revised: 09/21/2015] [Accepted: 10/05/2015] [Indexed: 01/05/2023]
Abstract
Recent landmark studies have demonstrated the production of disease-relevant human cell types by two different methods; differentiation of stem cells using external morphogens or lineage conversion using genetic factors. Directed differentiation changes embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into a desired cell type by providing developmental cues in an in vitro environment. Direct reprogramming is achieved by the introduction of exogenous lineage specific transcription factors to convert any somatic cell type into another, thereby bypassing an intermediate pluripotent stage. A variety of somatic cell types such as blood, keratinocytes and fibroblasts can be used to derive iPSC cells. However, the process is time consuming,laborious, expensive and gives rise to cells with reported epigenetic heterogeneity even amongst different iPSC lines from same patient which could propagate phenotypic variability. A major concern with the use of pluripotent cells as starting material for cell replacement therapy is their incomplete differentiation and their propensity to form tumors following transplantation. In comparison, transcription factor mediated reprogramming offers a direct route to target cell types. This could allow for rapid comparison of large cohorts of patient and control samples at a given time for disease modeling. Additionally, transcription factors that drive maturation may yield more functionally mature cells than directed differentiation. Several studies have demonstrated the feasibility of generating of cell types such as cardiomyocytes, hepatocytes, and neurons from fibroblasts. Here, we will discuss recent advances and key challenges regarding direct reprogramming of somatic cell types into diverse neural cells. This article is part of a Special Issue entitled SI: Exploiting human neurons.
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Affiliation(s)
- Suhasni Gopalakrishnan
- University of Southern California, Department of Stem Cells and Regenerative Medicine, Eli and Edythe Broad, CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, CA 90033, USA
| | - Pooja Hor
- University of Southern California, Department of Stem Cells and Regenerative Medicine, Eli and Edythe Broad, CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, CA 90033, USA
| | - Justin K Ichida
- University of Southern California, Department of Stem Cells and Regenerative Medicine, Eli and Edythe Broad, CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, CA 90033, USA
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Poser SW, Chenoweth JG, Colantuoni C, Masjkur J, Chrousos G, Bornstein SR, McKay RD, Androutsellis-Theotokis A. Concise Review: Reprogramming, Behind the Scenes: Noncanonical Neural Stem Cell Signaling Pathways Reveal New, Unseen Regulators of Tissue Plasticity With Therapeutic Implications. Stem Cells Transl Med 2015; 4:1251-7. [PMID: 26371344 DOI: 10.5966/sctm.2015-0105] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2015] [Accepted: 07/08/2015] [Indexed: 01/06/2023] Open
Abstract
UNLABELLED Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to β-like cells). Specifically for acinar-to-β-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research. SIGNIFICANCE Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations.
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Affiliation(s)
- Steven W Poser
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | - Josh G Chenoweth
- Lieber Institute for Brain Development, Baltimore, Maryland, USA
| | - Carlo Colantuoni
- Lieber Institute for Brain Development, Baltimore, Maryland, USA
| | - Jimmy Masjkur
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | - George Chrousos
- First Department of Pediatrics, University of Athens Medical School, Athens, Greece Aghia Sophia Children's Hospital, Athens, Greece
| | - Stefan R Bornstein
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany
| | - Ronald D McKay
- Lieber Institute for Brain Development, Baltimore, Maryland, USA
| | - Andreas Androutsellis-Theotokis
- Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany Center for Regenerative Therapies Dresden, Dresden, Germany
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Meyer S, Wörsdörfer P, Günther K, Thier M, Edenhofer F. Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells. J Vis Exp 2015:e52831. [PMID: 26275015 DOI: 10.3791/52831] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Generation of induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.
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Affiliation(s)
- Sandra Meyer
- Institute of Anatomy and Cell Biology, University of Würzburg; Institute of Reconstructive Neurobiology, University of Bonn
| | | | | | - Marc Thier
- Institute of Reconstructive Neurobiology, University of Bonn; German Cancer Research Center, Heidelberg
| | - Frank Edenhofer
- Institute of Anatomy and Cell Biology, University of Würzburg; Institute of Reconstructive Neurobiology, University of Bonn;
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Affiliation(s)
- Taketaro Sadahiro
- From the Department of Cardiology, Keio University School of Medicine, Japan Science and Technology CREST, Tokyo, Japan (T.S., M.I.); Japan Science and Technology CREST, Tokyo, Japan (M.I.); Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (S.Y.); and Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.)
| | - Shinya Yamanaka
- From the Department of Cardiology, Keio University School of Medicine, Japan Science and Technology CREST, Tokyo, Japan (T.S., M.I.); Japan Science and Technology CREST, Tokyo, Japan (M.I.); Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (S.Y.); and Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.)
| | - Masaki Ieda
- From the Department of Cardiology, Keio University School of Medicine, Japan Science and Technology CREST, Tokyo, Japan (T.S., M.I.); Japan Science and Technology CREST, Tokyo, Japan (M.I.); Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (S.Y.); and Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.)
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