1
|
Redi G, Del Piano F, Cappellini S, Paladino M, den Breejen A, Fens MHAM, Caiazzo M. Delivery Systems in Neuronal Direct Cell Reprogramming. Cell Reprogram 2025. [PMID: 40372965 DOI: 10.1089/cell.2025.0008] [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: 05/17/2025] Open
Abstract
Neuronal direct cell reprogramming approach allows direct conversion of somatic cells into neurons via forced expression of neuronal cell-lineage transcription factors (TFs). These so-called induced neuronal cells have significant potential as research tools and for therapeutic applications, such as in cell replacement therapy. However, the optimization of TF delivery strategies is crucial to reach clinical practice. In this review, we outlined the currently explored delivery technologies in neuronal direct cell reprogramming and their limitations and advantages. The first employed delivery strategies were mainly integrating viral systems, such as lentiviruses that exert consistently high transgene expression in most cell types. On the other hand, viral systems cause major safety concerns, including the risk for insertional mutagenesis and inflammation. More recently, several safer nonviral delivery systems have been investigated as well; however, these systems generally exert inferior reprogramming efficiency compared with viral systems. Emerging delivery technologies could provide new opportunities in the achievement of safe and effective delivery for neuronal direct cell reprogramming.
Collapse
Affiliation(s)
- Giulia Redi
- Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy
| | - Filomena Del Piano
- Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy
| | - Sara Cappellini
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
| | - Martina Paladino
- Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
| | - Anne den Breejen
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
| | - Marcel H A M Fens
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
| | - Massimiliano Caiazzo
- Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
| |
Collapse
|
2
|
Soliman Y, Al-Khodor J, Yildirim Köken G, Mustafaoglu N. A guide for blood-brain barrier models. FEBS Lett 2025; 599:599-644. [PMID: 39533665 DOI: 10.1002/1873-3468.15053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Revised: 10/18/2024] [Accepted: 10/20/2024] [Indexed: 11/16/2024]
Abstract
Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.
Collapse
Affiliation(s)
- Yomna Soliman
- Faculty of Engineering and Natural Sciences, Sabancı University, Istanbul, Turkey
- Faculty of Pharmacy, Mansoura University, Egypt
| | - Jana Al-Khodor
- Faculty of Engineering and Natural Sciences, Sabancı University, Istanbul, Turkey
| | | | - Nur Mustafaoglu
- Faculty of Engineering and Natural Sciences, Sabancı University, Istanbul, Turkey
- Sabancı University Nanotechnology Research and Application Center, Istanbul, Turkey
| |
Collapse
|
3
|
Di Matteo F, Bonrath R, Pravata V, Schmidt H, Ayo Martin AC, Di Giaimo R, Menegaz D, Riesenberg S, de Vrij FMS, Maccarrone G, Holzapfel M, Straub T, Kushner SA, Robertson SP, Eder M, Cappello S. Neuronal hyperactivity in neurons derived from individuals with gray matter heterotopia. Nat Commun 2025; 16:1737. [PMID: 39966398 PMCID: PMC11836124 DOI: 10.1038/s41467-025-56998-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2024] [Accepted: 02/05/2025] [Indexed: 02/20/2025] Open
Abstract
Periventricular heterotopia (PH), a common form of gray matter heterotopia associated with developmental delay and drug-resistant seizures, poses a challenge in understanding its neurophysiological basis. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 mimic PH features. However, neuronal activity in these 3D models has not yet been investigated. Here we show that silicon probe recordings reveal exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs, suggesting functional changes in neuronal networks. Transcriptome and proteome analyses identify changes in neuronal morphology and synaptic function. Furthermore, patch-clamp recordings reveal a decreased spike threshold specifically in DCHS1 neurons, likely due to increased somatic voltage-gated sodium channels. Additional analyses reveal increased morphological complexity of PH neurons and synaptic alterations contributing to hyperactivity, with rescue observed in DCHS1 neurons by wild-type DCHS1 expression. Overall, we provide new comprehensive insights into the cellular changes underlying symptoms of gray matter heterotopia.
Collapse
Affiliation(s)
- Francesco Di Matteo
- Division of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
- Max Planck Institute of Psychiatry, Munich, Germany
| | - Rebecca Bonrath
- Division of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany
| | - Veronica Pravata
- Division of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany
| | | | - Ane Cristina Ayo Martin
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
- Max Planck Institute of Psychiatry, Munich, Germany
| | - Rossella Di Giaimo
- Division of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany
- Max Planck Institute of Psychiatry, Munich, Germany
- Department of Biology, University Federico II, Naples, Italy
| | | | | | - Femke M S de Vrij
- Department of Psychiatry, Erasmus MC University Medical Center, Rotterdam, The Netherlands
- ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | | | | | - Tobias Straub
- Bioinformatics Core, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany
| | - Steven A Kushner
- Department of Psychiatry, Erasmus MC University Medical Center, Rotterdam, The Netherlands
- Department of Psychiatry, Columbia University Medical Center, New York, NY, USA
| | - Stephen P Robertson
- Department of Women's and Children's Health, University of Otago, Dunedin, New Zealand
| | - Matthias Eder
- Max Planck Institute of Psychiatry, Munich, Germany.
| | - Silvia Cappello
- Division of Physiological Genomics, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany.
- Max Planck Institute of Psychiatry, Munich, Germany.
| |
Collapse
|
4
|
Berg LJ, Lee CK, Matsumura H, Leinhaas A, Konang R, Shaib AH, Royero P, Schlee J, Sheng C, Beck H, Schwarz MK, Brose N, Rhee JS, Brüstle O. Human neural stem cells directly programmed from peripheral blood show functional integration into the adult mouse brain. Stem Cell Res Ther 2024; 15:488. [PMID: 39707492 DOI: 10.1186/s13287-024-04110-7] [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: 06/10/2024] [Accepted: 12/10/2024] [Indexed: 12/23/2024] Open
Abstract
Transplantation of induced pluripotent stem cell-derived neural cells represents a promising strategy for treating neurodegenerative diseases. However, reprogramming of somatic cells and their subsequent neural differentiation is complex and time-consuming, thereby impeding autologous applications. Recently, direct transcription factor-based conversion of blood cells into induced neural stem cells (iNSCs) has emerged as a potential alternative. However, little is known about the functionality of iNSC-derived neurons upon in vivo transplantation. Here, we grafted human iNSCs derived from adult peripheral blood by temporary overexpression of the transcription factors SOX2 and cMYC into the hippocampus or striatum of adult unlesioned immunodeficient Rag2tm1FwaIl2rgtm1Wjl mice of both sexes. Engrafted cells gave rise to stable transplants composed of mature neurons displaying extensive neurite outgrowth and dendritic spine formation. Functional analyses of acute slices using patch clamp recordings revealed that already after 12 weeks of in vivo maturation, most of iNSC-derived cells possess unique properties exclusive to neurons and exhibit voltage-dependent ion channel currents as well as action potential firing. Moreover, the formation of spontaneous inhibitory and excitatory postsynaptic currents, along with Rabies virus-based retrograde monosynaptic tracing data, strongly supports the structural and functional integration of graft-derived neurons. Taken together, our data demonstrate that iNSCs directly derived from peripheral blood cells have the inherent capacity to achieve full functional maturation in vivo, qualifying them as an alternative potential donor source for restorative applications and deserving further investigation.
Collapse
Affiliation(s)
- Lea Jessica Berg
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Chung Ku Lee
- Department of Molecular Neurobiology, Max-Planck-Institute for Multidisciplinary Sciences, City Campus, Hermann-Rein-Str. 3, Göttingen, 37075, Germany
| | - Hideaki Matsumura
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Anke Leinhaas
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Rachel Konang
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Ali H Shaib
- Department of Molecular Neurobiology, Max-Planck-Institute for Multidisciplinary Sciences, City Campus, Hermann-Rein-Str. 3, Göttingen, 37075, Germany
- Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Humboldtallee 23, Göttingen, 37073, Germany
| | - Pedro Royero
- Institute of Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Julia Schlee
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Chao Sheng
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Heinz Beck
- Institute of Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Martin Karl Schwarz
- Institute of Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany
- Cellomics Unit, LIFE & BRAIN GmbH, Venusberg-Campus 1, Bonn, 53127, Germany
| | - Nils Brose
- Department of Molecular Neurobiology, Max-Planck-Institute for Multidisciplinary Sciences, City Campus, Hermann-Rein-Str. 3, Göttingen, 37075, Germany
| | - Jeong Seop Rhee
- Department of Molecular Neurobiology, Max-Planck-Institute for Multidisciplinary Sciences, City Campus, Hermann-Rein-Str. 3, Göttingen, 37075, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Bonn, 53127, Germany.
| |
Collapse
|
5
|
Shiri H, Javan M. Sox2-mediated transdifferentiation of hAT-MSCs into induced neural progenitor-like cells for remyelination therapies. Tissue Cell 2024; 91:102553. [PMID: 39255744 DOI: 10.1016/j.tice.2024.102553] [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: 06/11/2024] [Revised: 08/03/2024] [Accepted: 09/03/2024] [Indexed: 09/12/2024]
Abstract
Mesenchymal stem cells (MSCs) are converted to neural cells using growth factors and chemicals. Although these neural cells are effective at modulating disease symptoms, they are less effective at replacing lost neural cells. Direct transdifferentiation seems to be a promising method for generating the required cells for regenerative medicine applications. Sox2 is a key transcription factor in neural progenitor (NP) fate determination and has been frequently used for transdifferentiating different cell types to NPs. Here, we demonstrated that the overexpression of a single transcription factor, Sox2, in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) led to the generation of induced NPs-like cells that were clonogenic, proliferative and passageable, and showed the potential to differentiate into three neural lineages. NPs are known as progenitors with the potential to differentiate into oligodendrocytes. In vivo, following transplantation into demyelinated adult mouse brains, they survived, differentiated and integrated into the adult brain while participating in the remyelination process and behavioral improvement. This report introduces a beneficial, low-cost and effective approach for generating NPs from an accessible adult source for autologous applications in treating neurodegenerative diseases, including remyelination therapies for multiple sclerosis and other demyelinating diseases.
Collapse
Affiliation(s)
- Hamed Shiri
- Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mohammad Javan
- Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran; Institute for Brain and Cognition, Tarbiat Modares University, Tehran, Iran; Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| |
Collapse
|
6
|
Amirbekyan M, Adhikarla V, Cheng JP, Moschonas EH, Bondi CO, Rockne RC, Kline AE, Gutova M. Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury. Sci Rep 2023; 13:17874. [PMID: 37857701 PMCID: PMC10587115 DOI: 10.1038/s41598-023-44426-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 10/08/2023] [Indexed: 10/21/2023] Open
Abstract
Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this hypothesis, immunocompetent adult female rats received either controlled cortical impact injury or sham surgery. LMNSC008 cells or a vehicle were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. Our proof-of-concept studies, although preliminary, support the rationale of using intranasal delivery of LMNSC008 cells for functional studies in preclinical models of TBI and provide support for potential translatability in TBI patients.
Collapse
Affiliation(s)
- Mari Amirbekyan
- Department of Stem Cell Biology and Regenerative Medicine, Beckman Research Institute, City of Hope, 1500 East Duarte Road, Duarte, CA, 91010, USA
| | - Vikram Adhikarla
- Division of Mathematical Oncology and Computational Systems Biology, Department of Computational and Quantitative Medicine, Beckman Research Institute, City of Hope, Duarte, CA, USA
| | - Jeffrey P Cheng
- Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15224, USA
- Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA
| | - Eleni H Moschonas
- Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15224, USA
- Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA
- Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA
| | - Corina O Bondi
- Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15224, USA
- Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA
- Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA
- Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Russell C Rockne
- Division of Mathematical Oncology and Computational Systems Biology, Department of Computational and Quantitative Medicine, Beckman Research Institute, City of Hope, Duarte, CA, USA
| | - Anthony E Kline
- Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15224, USA.
- Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA.
- Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA.
- Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, PA, USA.
- Critical Care Medicine, and Psychology, University of Pittsburgh, Pittsburgh, PA, USA.
- Psychology, University of Pittsburgh, Pittsburgh, PA, USA.
| | - Margarita Gutova
- Department of Stem Cell Biology and Regenerative Medicine, Beckman Research Institute, City of Hope, 1500 East Duarte Road, Duarte, CA, 91010, USA.
| |
Collapse
|
7
|
Amirbekyan M, Cheng JP, Adhikarla V, Moschonas EH, Bondi CO, Rockne RC, Kline AE, Gutova M. Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury. RESEARCH SQUARE 2023:rs.3.rs-3242570. [PMID: 37720043 PMCID: PMC10503851 DOI: 10.21203/rs.3.rs-3242570/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/19/2023]
Abstract
Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this, immunocompetent adult female rats received a controlled cortical impact injury (CCI) or sham surgery. LMNSC008 cells or a vehicle (VEH) were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. The data demonstrate a robust proof-of-concept for LMNSC008 therapy for TBI and provides a strong rationale for IN delivery for translation in TBI patients.
Collapse
Affiliation(s)
| | - Jeffrey P Cheng
- University of Pittsburgh School of Medicine Children's Hospital of Pittsburgh John G. Rangos Research Center - Room 6126
| | | | - Eleni H Moschonas
- University of Pittsburgh School of Medicine Children's Hospital of Pittsburgh John G. Rangos Research Center - Room 6126
| | - Corina O Bondi
- University of Pittsburgh School of Medicine Children's Hospital of Pittsburgh John G. Rangos Research Center - Room 6126
| | | | - Anthony E Kline
- University of Pittsburgh School of Medicine Children's Hospital of Pittsburgh John G. Rangos Research Center - Room 6126
| | | |
Collapse
|
8
|
Chen J, Huang L, Yang Y, Xu W, Qin Q, Qin R, Liang X, Lai X, Huang X, Xie M, Chen L. Somatic Cell Reprogramming for Nervous System Diseases: Techniques, Mechanisms, Potential Applications, and Challenges. Brain Sci 2023; 13:brainsci13030524. [PMID: 36979334 PMCID: PMC10046178 DOI: 10.3390/brainsci13030524] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2023] [Revised: 03/14/2023] [Accepted: 03/20/2023] [Indexed: 03/30/2023] Open
Abstract
Nervous system diseases present significant challenges to the neuroscience community due to ethical and practical constraints that limit access to appropriate research materials. Somatic cell reprogramming has been proposed as a novel way to obtain neurons. Various emerging techniques have been used to reprogram mature and differentiated cells into neurons. This review provides an overview of somatic cell reprogramming for neurological research and therapy, focusing on neural reprogramming and generating different neural cell types. We examine the mechanisms involved in reprogramming and the challenges that arise. We herein summarize cell reprogramming strategies to generate neurons, including transcription factors, small molecules, and microRNAs, with a focus on different types of cells.. While reprogramming somatic cells into neurons holds the potential for understanding neurological diseases and developing therapeutic applications, its limitations and risks must be carefully considered. Here, we highlight the potential benefits of somatic cell reprogramming for neurological disease research and therapy. This review contributes to the field by providing a comprehensive overview of the various techniques used to generate neurons by cellular reprogramming and discussing their potential applications.
Collapse
Affiliation(s)
- Jiafeng Chen
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Lijuan Huang
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Yue Yang
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Wei Xu
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Qingchun Qin
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Rongxing Qin
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Xiaojun Liang
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Xinyu Lai
- Key Laboratory of Longevity and Aging-Related Diseases of Chinese Ministry of Education, Nanning 530021, China
| | - Xiaoying Huang
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Minshan Xie
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
| | - Li Chen
- Department of Neurology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
- Key Laboratory of Longevity and Aging-Related Diseases of Chinese Ministry of Education, Nanning 530021, China
| |
Collapse
|
9
|
Berg LJ, Brüstle O. Stem cell programming - prospects for perinatal medicine. J Perinat Med 2023:jpm-2022-0575. [PMID: 36809086 DOI: 10.1515/jpm-2022-0575] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Accepted: 12/23/2022] [Indexed: 02/23/2023]
Abstract
Recreating human cell and organ systems in vitro has tremendous potential for disease modeling, drug discovery and regenerative medicine. The aim of this short overview is to recapitulate the impressive progress that has been made in the fast-developing field of cell programming during the past years, to illuminate the advantages and limitations of the various cell programming technologies for addressing nervous system disorders and to gauge their impact for perinatal medicine.
Collapse
Affiliation(s)
- Lea J Berg
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| |
Collapse
|
10
|
Migliorini F, Schenker H, Maffulli N, Hildebrand F, Eschweiler J. Histomorphometry of Ossification in Functionalised Ceramics with Tripeptide Arg-Gly-Asp (RGD): An In Vivo Study. Life (Basel) 2022; 12:life12050761. [PMID: 35629427 PMCID: PMC9146276 DOI: 10.3390/life12050761] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 05/16/2022] [Accepted: 05/16/2022] [Indexed: 12/31/2022] Open
Abstract
The present study investigated the osseointegration promoted by functionalised ceramics with peptide Arg-Gly-Asp (RGD) in a rabbit model in vivo. Histomorphometry of the RGD functionalised ceramic implants was conducted by a trained pathologist to quantify the amount of mature and immature ossification at the bone interface, and then compared to titanium alloy implants. The region of interest was the area surrounding the implant. The percentage of ROI covered by osteoid implant contact and mature bone implant contact were assessed. The presence of bone resorption, necrosis, and/or inflammation in the areas around the implant were quantitatively investigated. All 36 rabbits survived the experimental period of 6 and 12 weeks. All implants remained in situ. No necrosis, bone resorption, or inflammation were identified. At 12 weeks follow-up, the overall mean bone implant contact (p = 0.003) and immature osteoid contact (p = 0.03) were improved compared to the mean values evidenced at 6 weeks. At 6 weeks follow-up, the overall osteoid implant contact was greater in the RGD enhanced group compared to the titanium implant (p = 0.01). The other endpoints of interest were similar between the two implants at all follow-up points (p ≥ 0.05). Functionalised ceramics with peptide RGD promoted ossification in vivo. The overall osteoid and bone implant contact improved significantly from 6 to 12 weeks. Finally, RGD enhanced ceramic promoted faster osteoid implant contact in vivo than titanium implants. Overall, the amount of ossification at 12 weeks is comparable with the titanium implants. No necrosis, bone resorption, or inflammation were observed in any sample.
Collapse
Affiliation(s)
- Filippo Migliorini
- Department of Orthopaedic, Trauma and Reconstructive Surgery, RWTH University Hospital, 52074 Aachen, Germany; (F.M.); (H.S.); (F.H.); (J.E.)
| | - Hanno Schenker
- Department of Orthopaedic, Trauma and Reconstructive Surgery, RWTH University Hospital, 52074 Aachen, Germany; (F.M.); (H.S.); (F.H.); (J.E.)
| | - Nicola Maffulli
- Department of Medicine, Surgery and Dentistry, University of Salerno, 84081 Baronissi, Italy
- School of Pharmacy and Bioengineering, Keele University Faculty of Medicine, Stoke on Trent ST4 7QB, UK
- Barts and The London School of Medicine and Dentistry, Centre for Sports and Exercise Medicine, Mile End Hospital, Queen Mary University of London, London E1 4DG, UK
- Correspondence:
| | - Frank Hildebrand
- Department of Orthopaedic, Trauma and Reconstructive Surgery, RWTH University Hospital, 52074 Aachen, Germany; (F.M.); (H.S.); (F.H.); (J.E.)
| | - Jörg Eschweiler
- Department of Orthopaedic, Trauma and Reconstructive Surgery, RWTH University Hospital, 52074 Aachen, Germany; (F.M.); (H.S.); (F.H.); (J.E.)
| |
Collapse
|
11
|
Barak M, Fedorova V, Pospisilova V, Raska J, Vochyanova S, Sedmik J, Hribkova H, Klimova H, Vanova T, Bohaciakova D. Human iPSC-Derived Neural Models for Studying Alzheimer's Disease: from Neural Stem Cells to Cerebral Organoids. Stem Cell Rev Rep 2022; 18:792-820. [PMID: 35107767 PMCID: PMC8930932 DOI: 10.1007/s12015-021-10254-3] [Citation(s) in RCA: 44] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/28/2021] [Indexed: 12/05/2022]
Abstract
During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer’s disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery.
Collapse
Affiliation(s)
- Martin Barak
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Veronika Fedorova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Veronika Pospisilova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Jan Raska
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Simona Vochyanova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Jiri Sedmik
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
- International Clinical Research Center, St. Anne's Faculty Hospital Brno, Brno, Czech Republic
| | - Hana Hribkova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Hana Klimova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
| | - Tereza Vanova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic
- International Clinical Research Center, St. Anne's Faculty Hospital Brno, Brno, Czech Republic
| | - Dasa Bohaciakova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic.
- International Clinical Research Center, St. Anne's Faculty Hospital Brno, Brno, Czech Republic.
| |
Collapse
|
12
|
Samoilova EM, Belopasov VV, Baklaushev VP. Transcription Factors of Direct Neuronal Reprogramming in Ontogenesis and Ex Vivo. Mol Biol 2021; 55:645-669. [DOI: 10.1134/s0026893321040087] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2020] [Revised: 12/14/2020] [Accepted: 12/15/2020] [Indexed: 03/07/2025]
|
13
|
Xiang Z, Xu L, Liu M, Wang Q, Li W, Lei W, Chen G. Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex. Neural Regen Res 2021; 16:750-756. [PMID: 33063738 PMCID: PMC8067918 DOI: 10.4103/1673-5374.295925] [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] [Indexed: 12/12/2022] Open
Abstract
Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss. Glial cells, on the other hand, can divide and repopulate themselves under injury or diseased conditions. We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons. Here, using astrocytic lineage-tracing reporter mice (Aldh1l1-CreERT2 mice crossing with Ai14 mice), we demonstrate that lineage-traced astrocytes can be successfully converted into NeuN-positive neurons after expressing NeuroD1 through adeno-associated viruses. Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons. Importantly, we demonstrate that for in vivo cell conversion study, using a safe level of adeno-associated virus dosage (1010–1012 gc/mL, 1 µL) in the rodent brain is critical to avoid artifacts caused by toxic dosage, such as that used in a recent bioRxiv study (2 × 1013 gc/mL, 1 µL, mouse cortex). For therapeutic purpose under injury or diseased conditions, or for non-human primate studies, adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments. Moreover, for future in vivo glia-to-neuron conversion studies, we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions. The study was approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. IACUC-20180330-06) on March 30, 2018.
Collapse
Affiliation(s)
- Zongqin Xiang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration; Department of Neurosurgery, the First Affiliated Hospital, Jinan University, Guangzhou, Guangdong Province, China
| | - Liang Xu
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| | - Minhui Liu
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| | - Qingsong Wang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| | - Wen Li
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| | - Wenliang Lei
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| | - Gong Chen
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
| |
Collapse
|
14
|
Flitsch LJ, Laupman KE, Brüstle O. Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration. Front Cell Neurosci 2020; 14:121. [PMID: 32508594 PMCID: PMC7251072 DOI: 10.3389/fncel.2020.00121] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Accepted: 04/14/2020] [Indexed: 12/11/2022] Open
Abstract
Traditionally, in vitro generation of donor cells for brain repair has been dominated by the application of extrinsic growth factors and morphogens. Recent advances in cell engineering strategies such as reprogramming of somatic cells into induced pluripotent stem cells and direct cell fate conversion have impressively demonstrated the feasibility to manipulate cell identities by the overexpression of cell fate-determining transcription factors. These strategies are now increasingly implemented for transcription factor-guided differentiation of neural precursors and forward programming of pluripotent stem cells toward specific neural subtypes. This review covers major achievements, pros and cons, as well as future prospects of transcription factor-based cell fate specification and the applicability of these approaches for the generation of donor cells for brain repair.
Collapse
Affiliation(s)
- Lea J Flitsch
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| | - Karen E Laupman
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| |
Collapse
|
15
|
Di Matteo F, Pipicelli F, Kyrousi C, Tovecci I, Penna E, Crispino M, Chambery A, Russo R, Ayo-Martin AC, Giordano M, Hoffmann A, Ciusani E, Canafoglia L, Götz M, Di Giaimo R, Cappello S. Cystatin B is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy. EMBO Mol Med 2020; 12:e11419. [PMID: 32378798 PMCID: PMC7278547 DOI: 10.15252/emmm.201911419] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2019] [Revised: 04/01/2020] [Accepted: 04/02/2020] [Indexed: 12/18/2022] Open
Abstract
Progressive myoclonus epilepsy (PME) of Unverricht–Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors’ proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell‐non‐autonomous mechanisms. Similarly, in patient‐derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients’ hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1.
Collapse
Affiliation(s)
- Francesco Di Matteo
- Max Planck Institute of Psychiatry, Munich, Germany.,International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
| | - Fabrizia Pipicelli
- Max Planck Institute of Psychiatry, Munich, Germany.,International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
| | | | - Isabella Tovecci
- Max Planck Institute of Psychiatry, Munich, Germany.,Department of Biology, University Federico II, Naples, Italy
| | - Eduardo Penna
- Department of Biology, University Federico II, Naples, Italy
| | | | - Angela Chambery
- Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy
| | - Rosita Russo
- Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy
| | - Ane Cristina Ayo-Martin
- Max Planck Institute of Psychiatry, Munich, Germany.,International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
| | | | | | - Emilio Ciusani
- Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
| | | | - Magdalena Götz
- Physiological Genomics, Biomedical Center (BMC), Ludwig-Maximilians-Universitaet (LMU), Planegg/Martinsried, Germany.,Helmholtz Center Munich, Biomedical Center (BMC), Institute of Stem Cell Research, Planegg/Martinsried, Germany.,SyNergy Excellence Cluster, Munich, Germany
| | - Rossella Di Giaimo
- Max Planck Institute of Psychiatry, Munich, Germany.,Department of Biology, University Federico II, Naples, Italy
| | | |
Collapse
|
16
|
Pouya A, Rassouli H, Rezaei-Larijani M, Salekdeh GH, Baharvand H. SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells. Biochem Biophys Res Commun 2020; 525:S0006-291X(20)30315-6. [PMID: 32070492 DOI: 10.1016/j.bbrc.2020.02.047] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Accepted: 02/07/2020] [Indexed: 01/26/2023]
Abstract
Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient's somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.
Collapse
Affiliation(s)
- Alireza Pouya
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hassan Rassouli
- Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Medical Laser, Medical Laser Research Center, Yara Institute, ACECR, Tehran, Iran
| | - Mehran Rezaei-Larijani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ghasem Hosseini Salekdeh
- Department of Molecular Systems 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.
| |
Collapse
|
17
|
Flitsch LJ, Brüstle O. Evolving principles underlying neural lineage conversion and their relevance for biomedical translation. F1000Res 2019; 8. [PMID: 31559012 PMCID: PMC6743253 DOI: 10.12688/f1000research.18926.1] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/23/2019] [Indexed: 12/19/2022] Open
Abstract
Scientific and technological advances of the past decade have shed light on the mechanisms underlying cell fate acquisition, including its transcriptional and epigenetic regulation during embryonic development. This knowledge has enabled us to purposefully engineer cell fates
in vitro by manipulating expression levels of lineage-instructing transcription factors. Here, we review the state of the art in the cell programming field with a focus on the derivation of neural cells. We reflect on what we know about the mechanisms underlying fate changes in general and on the degree of epigenetic remodeling conveyed by the distinct reprogramming and direct conversion strategies available. Moreover, we discuss the implications of residual epigenetic memory for biomedical applications such as disease modeling and neuroregeneration. Finally, we cover recent developments approaching cell fate conversion in the living brain and define questions which need to be addressed before cell programming can become an integral part of translational medicine.
Collapse
Affiliation(s)
- Lea Jessica Flitsch
- Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, North Rhine Wesphalia, 53127, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, North Rhine Wesphalia, 53127, Germany
| |
Collapse
|
18
|
Carbonell AU, Cho CH, Tindi JO, Counts PA, Bates JC, Erdjument-Bromage H, Cvejic S, Iaboni A, Kvint I, Rosensaft J, Banne E, Anagnostou E, Neubert TA, Scherer SW, Molholm S, Jordan BA. Haploinsufficiency in the ANKS1B gene encoding AIDA-1 leads to a neurodevelopmental syndrome. Nat Commun 2019; 10:3529. [PMID: 31388001 PMCID: PMC6684583 DOI: 10.1038/s41467-019-11437-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 07/13/2019] [Indexed: 12/23/2022] Open
Abstract
Neurodevelopmental disorders, including autism spectrum disorder, have complex polygenic etiologies. Single-gene mutations in patients can help define genetic factors and molecular mechanisms underlying neurodevelopmental disorders. Here we describe individuals with monogenic heterozygous microdeletions in ANKS1B, a predicted risk gene for autism and neuropsychiatric diseases. Affected individuals present with a spectrum of neurodevelopmental phenotypes, including autism, attention-deficit hyperactivity disorder, and speech and motor deficits. Neurons generated from patient-derived induced pluripotent stem cells demonstrate loss of the ANKS1B-encoded protein AIDA-1, a brain-specific protein highly enriched at neuronal synapses. A transgenic mouse model of Anks1b haploinsufficiency recapitulates a range of patient phenotypes, including social deficits, hyperactivity, and sensorimotor dysfunction. Identification of the AIDA-1 interactome using quantitative proteomics reveals protein networks involved in synaptic function and the etiology of neurodevelopmental disorders. Our findings formalize a link between the synaptic protein AIDA-1 and a rare, previously undefined genetic disease we term ANKS1B haploinsufficiency syndrome.
Collapse
Affiliation(s)
- Abigail U Carbonell
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Chang Hoon Cho
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Jaafar O Tindi
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Pamela A Counts
- Department of Pediatrics, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Juliana C Bates
- Department of Pediatrics, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Hediye Erdjument-Bromage
- Department of Cell Biology and Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, 10016, NY, USA
| | - Svetlana Cvejic
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Alana Iaboni
- Autism Research Centre, Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, M46 1R8, ON, Canada
| | - Ifat Kvint
- Pediatric Neurology Clinic, Kaplan Medical Center, Hebrew University Hadassah Medical School, Rehovot, 76100, Israel
| | - Jenny Rosensaft
- Genetics Institute, Kaplan Medical Center, Hebrew University Hadassah Medical School, Rehovot, 76100, Israel
| | - Ehud Banne
- Genetics Institute, Kaplan Medical Center, Hebrew University Hadassah Medical School, Rehovot, 76100, Israel
| | - Evdokia Anagnostou
- Autism Research Centre, Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, M46 1R8, ON, Canada
| | - Thomas A Neubert
- Department of Cell Biology and Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, 10016, NY, USA
- Department of Pharmacology, New York University School of Medicine, New York, 10016, NY, USA
| | - Stephen W Scherer
- Centre for Applied Genomics and McLaughlin Centre, Hospital for Sick Children and University of Toronto, Toronto, M56 0A4, ON, Canada
| | - Sophie Molholm
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
- Department of Pediatrics, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
- Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, 10461, NY, USA
| | - Bryen A Jordan
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA.
- Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, 10461, NY, USA.
| |
Collapse
|
19
|
Fetahu IS, Ma D, Rabidou K, Argueta C, Smith M, Liu H, Wu F, Shi YG. Epigenetic signatures of methylated DNA cytosine in Alzheimer's disease. SCIENCE ADVANCES 2019; 5:eaaw2880. [PMID: 31489368 PMCID: PMC6713504 DOI: 10.1126/sciadv.aaw2880] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Accepted: 07/18/2019] [Indexed: 05/23/2023]
Abstract
Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common untreatable form of dementia. Identifying molecular biomarkers that allow early detection remains a key challenge in the diagnosis, treatment, and prognostic evaluation of the disease. Here, we report a novel experimental and analytical model characterizing epigenetic alterations during AD onset and progression. We generated the first integrated base-resolution genome-wide maps of the distribution of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), and 5-formyl/carboxy-cytosine (5fC/caC) in normal and AD neurons. We identified 27 AD region-specific and 39 CpG site-specific epigenetic signatures that were independently validated across our familial and sporadic AD models, and in an independent clinical cohort. Thus, our work establishes a new model and strategy to study the epigenetic alterations underlying AD onset and progression and provides a set of highly reliable AD-specific epigenetic signatures that may have early diagnostic and prognostic implications.
Collapse
Affiliation(s)
- Irfete S. Fetahu
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
- Harvard Medical School, Boston, MA 02115, USA
| | - Dingailu Ma
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
- Laboratory of Epigenetics, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China
| | - Kimberlie Rabidou
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
| | - Christian Argueta
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
- Harvard Medical School, Boston, MA 02115, USA
| | - Michael Smith
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
| | - Hang Liu
- Laboratory of Epigenetics, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China
| | - Feizhen Wu
- Laboratory of Epigenetics, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China
- Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
| | - Yujiang G. Shi
- Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
- Harvard Medical School, Boston, MA 02115, USA
| |
Collapse
|
20
|
Vojnits K, Mahammad S, Collins TJ, Bhatia M. Chemotherapy-Induced Neuropathy and Drug Discovery Platform Using Human Sensory Neurons Converted Directly from Adult Peripheral Blood. Stem Cells Transl Med 2019; 8:1180-1191. [PMID: 31347791 PMCID: PMC6811699 DOI: 10.1002/sctm.19-0054] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Accepted: 06/07/2019] [Indexed: 12/11/2022] Open
Abstract
Chemotherapy‐induced peripheral neuropathy (PN) is a disorder damaging the peripheral nervous system (PNS) and represents one of the most common side effects of chemotherapy, negatively impacting the quality of life of patients to the extent of withdrawing life‐saving chemotherapy dose or duration. Unfortunately, the pathophysiological effects of PN are poorly understood, in part due to the lack of availability of large numbers of human sensory neurons (SNs) for study. Previous reports have demonstrated that human SNs can be directly converted from primitive CD34+ hematopoietic cells, but was limited to a small‐scale product of SNs and derived exclusively from less abundant allogenic sources of cord or drug mobilized peripheral blood (PB). To address this shortcoming, we have developed and report detailed procedures toward the generation of human SN directly converted from conventionally drawn PB of adults that can be used in a high‐content screening platform for discovery‐based studies of chemotherapy agents on neuronal biology. In the absence of mobilization drugs, cryogenically preserved adult human PB could be induced to (i)SN via development through expandable neural precursor differentiation. iSNs could be transferable to high‐throughput procedures suitable for high‐content screening applicable to neuropathy for example, alterations in neurite morphology in response to chemotherapeutics. Our study provides the first reported platform using adult PB‐derived iSNs to study peripheral nervous system‐related neuropathies as well as target and drug screening potential for the ability to prevent, block, or repair chemotherapy‐induced PN damage. stem cells translational medicine2019;8:1180–1191
Collapse
Affiliation(s)
- Kinga Vojnits
- Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada
| | - Saleemulla Mahammad
- Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada
| | - Tony J Collins
- Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada
| | - Mickie Bhatia
- Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.,Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
| |
Collapse
|
21
|
Cord blood research, banking, and transplantation: achievements, challenges, and perspectives. Bone Marrow Transplant 2019; 55:48-61. [PMID: 31089283 DOI: 10.1038/s41409-019-0546-9] [Citation(s) in RCA: 83] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2019] [Revised: 04/15/2019] [Accepted: 04/24/2019] [Indexed: 12/13/2022]
Abstract
The first hematopoietic transplant in which umbilical cord blood (UCB) was used as the source of hematopoietic cells was performed in October 1988. Since then, significant achievements have been reported in terms of our understanding of the biology of UCB-derived hematopoietic stem (HSCs) and progenitor (HPCs) cells. Over 40,000 UCB transplants (UCBTs) have been performed, in both children and adults, for the treatment of many different diseases, including hematologic, metabolic, immunologic, neoplastic, and neurologic disorders. In addition, cord blood banking has been developed to the point that around 800,000 units are being stored in public banks and more than 4 million units in private banks worldwide. During these 30 years, research in the UCB field has transformed the hematopoietic transplantation arena. Today, scientific and clinical teams are still working on different ways to improve and expand the use of UCB cells. A major effort has been focused on enhancing engraftment to potentially reduce risk of infection and cost. To that end, we have to understand in detail the molecular mechanisms controlling stem cell self-renewal that may lead to the development of ex vivo systems for HSCs expansion, characterize the mechanisms regulating the homing of HSCs and HPCs, and determine the relative place of UCBTs, as compared to other sources. These challenges will be met by encouraging innovative research on the basic biology of HSCs and HPCs, developing novel clinical trials, and improving UCB banking both in the public and private arenas.
Collapse
|
22
|
Gancheva MR, Kremer KL, Gronthos S, Koblar SA. Using Dental Pulp Stem Cells for Stroke Therapy. Front Neurol 2019; 10:422. [PMID: 31110489 PMCID: PMC6501465 DOI: 10.3389/fneur.2019.00422] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 04/08/2019] [Indexed: 12/26/2022] Open
Abstract
Stroke is a leading cause of permanent disability world-wide, but aside from rehabilitation, there is currently no clinically-proven pharmaceutical or biological agent to improve neurological disability. Cell-based therapies using stem cells, such as dental pulp stem cells, are a promising alternative for treatment of neurological diseases, including stroke. The ischaemic environment in stroke affects multiple cell populations, thus stem cells, which act through cellular and molecular mechanisms, are promising candidates. The most common stem cell population studied in the neurological setting has been mesenchymal stem cells due to their accessibility. However, it is believed that neural stem cells, the resident stem cell of the adult brain, would be most appropriate for brain repair. Using reprogramming strategies, alternative sources of neural stem and progenitor cells have been explored. We postulate that a cell of closer origin to the neural lineage would be a promising candidate for reprogramming and modification towards a neural stem or progenitor cell. One such candidate population is dental pulp stem cells, which reside in the root canal of teeth. This review will focus on the neural potential of dental pulp stem cells and their investigations in the stroke setting to date, and include an overview on the use of different sources of neural stem cells in preclinical studies and clinical trials of stroke.
Collapse
Affiliation(s)
- Maria R. Gancheva
- Stroke Research Programme Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia
| | - Karlea L. Kremer
- Stroke Research Programme Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia
| | - Stan Gronthos
- Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia
- South Australian Health and Medical Research Institute, Adelaide, SA, Australia
| | - Simon A. Koblar
- Stroke Research Programme Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia
- South Australian Health and Medical Research Institute, Adelaide, SA, Australia
- Central Adelaide Local Health Network, Adelaide, SA, Australia
| |
Collapse
|
23
|
Lei W, Li W, Ge L, Chen G. Non-engineered and Engineered Adult Neurogenesis in Mammalian Brains. Front Neurosci 2019; 13:131. [PMID: 30872991 PMCID: PMC6401632 DOI: 10.3389/fnins.2019.00131] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2018] [Accepted: 02/05/2019] [Indexed: 12/31/2022] Open
Abstract
Adult neurogenesis has been extensively studied in rodent animals, with distinct niches found in the hippocampus and subventricular zone (SVZ). In non-human primates and human postmortem samples, there has been heated debate regarding adult neurogenesis, but it is largely agreed that the rate of adult neurogenesis is much reduced comparing to rodents. The limited adult neurogenesis may partly explain why human brains do not have self-repair capability after injury or disease. A new technology called “in vivo cell conversion” has been invented to convert brain internal glial cells in the injury areas directly into functional new neurons to replenish the lost neurons. Because glial cells are abundant throughout the brain and spinal cord, such engineered glia-to-neuron conversion technology can be applied throughout the central nervous system (CNS) to regenerate new neurons. Thus, compared to cell transplantation or the non-engineered adult neurogenesis, in vivo engineered neuroregeneration technology can provide a large number of functional new neurons in situ to repair damaged brain and spinal cord.
Collapse
Affiliation(s)
- Wenliang Lei
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China
| | - Wen Li
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China
| | - Longjiao Ge
- Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
| | - Gong Chen
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China.,Department of Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, United States
| |
Collapse
|
24
|
Multipotent Systems: Combining Planning, Self-Organization, and Reconfiguration in Modular Robot Ensembles. SENSORS 2018; 19:s19010017. [PMID: 30577565 PMCID: PMC6338923 DOI: 10.3390/s19010017] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 11/30/2018] [Accepted: 12/17/2018] [Indexed: 11/26/2022]
Abstract
Mobile multirobot systems play an increasing role in many disciplines. Their capabilities can be used, e.g., to transport workpieces in industrial applications or to support operational forces in search and rescue scenarios, among many others. Depending on the respective application, the hardware design and accompanying software of mobile robots are of various forms, especially for integrating different sensors and actuators. Concerning this design, robots of one system compared to each other can be classified to exclusively be either homogeneous or heterogeneous, both resulting in different system properties. While homogeneously configured systems are known to be robust against failures through redundancy but are highly specialized for specific use cases, heterogeneously designed systems can be used for a broad range of applications but suffer from their specialization, i.e., they can only hardly compensate for the failure of one specialist. Up to now, there has been no known approach aiming to unify the benefits of both these types of system. In this paper, we present our approach to filling this gap by introducing a reference architecture for mobile robots that defines the interplay of all necessary technologies for achieving this goal. We introduce the class of robot systems implementing this architecture as multipotent systems that bring together the benefits of both system classes, enabling homogeneously designed robots to become heterogeneous specialists at runtime. When many of these robots work together, we call the structure of this cooperation an ensemble. To achieve multipotent ensembles, we also integrate reconfigurable and self-descriptive hardware (i.e., sensors and actuators) in this architecture, which can be freely combined to change the capabilities of robots at runtime. Because typically a high degree of autonomy in such systems is a prerequisite for their practical usage, we also present the integration of necessary mechanisms and algorithms for achieving the systems’ multipotency. We already achieved the first results with robots implementing our approach of multipotent systems in real-world experiments as well as in a simulation environment, which we present in this paper.
Collapse
|
25
|
Daekee K, Mi-Jung H, Minjun J, Hee-Jin A, Kwang-Won S, Kyung-Sun K. Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression. MOLECULAR THERAPY. NUCLEIC ACIDS 2018; 14:388-397. [PMID: 30731320 PMCID: PMC6365637 DOI: 10.1016/j.omtn.2018.12.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Revised: 12/13/2018] [Accepted: 12/13/2018] [Indexed: 12/02/2022]
Abstract
As the human lifespan has increased due to developments in medical technology, the number of patients with neurological diseases has rapidly increased. Therefore, studies on effective treatments for neurological diseases are becoming increasingly important. To perform these studies, it is essential to obtain a large number of patient-derived neural cells. The purpose of the present study was to establish a technology that allows the high-efficiency generation of genetically stable, direct-conversion-derived neural stem cells (dcNSCs) through the expression of a new combination of reprogramming factors, including a proto-oncogene. Specifically, human c-MYC proto-oncogene and the human SOX2 gene were overexpressed in a precisely controlled manner in various human somatic cells. As a result, the direct conversion into multipotent dcNSCs occurred only when the cells were treated with an MOI of 1 of hc-MYC proto-oncogene and hSOX2 retrovirus. When MOIs of 5 or 10 were utilized, distinct results were obtained. In addition, the pluripotency was bypassed during this process. Notably, as the MOI used to treat the cells increased, expression of the p53 tumor suppressor gene, which is typically a reprogramming hurdle, increased proportionately. Interestingly, p53 was genetically stable in dcNSCs generated through direct conversion into a low p53 expression state. In the present study, generation of genetically stable dcNSCs using direct conversion was optimized by precisely controlling the overexpression of a proto-oncogene. This method could be utilized in future studies, such as in vitro drug screening using generated dcNSCs. In addition, this method could be effectively utilized in studies on direct conversion into other types of target cells.
Collapse
Affiliation(s)
- Kwon Daekee
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea
| | - Han Mi-Jung
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea
| | - Ji Minjun
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea
| | - Ahn Hee-Jin
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea
| | - Seo Kwang-Won
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea
| | - Kang Kyung-Sun
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Biomedical Science Building, #81 Seoul National University, Seoul 08826, South Korea; Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, South Korea.
| |
Collapse
|
26
|
A stably self-renewing adult blood-derived induced neural stem cell exhibiting patternability and epigenetic rejuvenation. Nat Commun 2018; 9:4047. [PMID: 30279449 PMCID: PMC6168501 DOI: 10.1038/s41467-018-06398-5] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2017] [Accepted: 08/24/2018] [Indexed: 12/20/2022] Open
Abstract
Recent reports suggest that induced neurons (iNs), but not induced pluripotent stem cell (iPSC)-derived neurons, largely preserve age-associated traits. Here, we report on the extent of preserved epigenetic and transcriptional aging signatures in directly converted induced neural stem cells (iNSCs). Employing restricted and integration-free expression of SOX2 and c-MYC, we generated a fully functional, bona fide NSC population from adult blood cells that remains highly responsive to regional patterning cues. Upon conversion, low passage iNSCs display a profound loss of age-related DNA methylation signatures, which further erode across extended passaging, thereby approximating the DNA methylation age of isogenic iPSC-derived neural precursors. This epigenetic rejuvenation is accompanied by a lack of age-associated transcriptional signatures and absence of cellular aging hallmarks. We find iNSCs to be competent for modeling pathological protein aggregation and for neurotransplantation, depicting blood-to-NSC conversion as a rapid alternative route for both disease modeling and neuroregeneration. Induced neurons, but not induced pluripotent stem cell (iPSC)-derived neurons, preserve age-related traits. Here, the authors demonstrate that blood-derived induced neural stem cells (iNSCs), despite lacking a pluripotency transit, lose age-related signatures.
Collapse
|
27
|
Kim BE, Choi SW, Shin JH, Kim JJ, Kang I, Lee BC, Lee JY, Kook MG, Kang KS. Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA. Cell Transplant 2018; 27:1154-1167. [PMID: 29909688 PMCID: PMC6158546 DOI: 10.1177/0963689718771885] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Revised: 02/20/2018] [Accepted: 02/27/2018] [Indexed: 12/28/2022] Open
Abstract
Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.
Collapse
Affiliation(s)
- Bo-Eun Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- College of Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Soon Won Choi
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Ji-Hee Shin
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Jae-Jun Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- College of Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Insung Kang
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Byung-Chul Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Jin Young Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Myoung Geun Kook
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Kyung-Sun Kang
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
- Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| |
Collapse
|
28
|
Transdifferentiation of human adult peripheral blood T cells into neurons. Proc Natl Acad Sci U S A 2018; 115:6470-6475. [PMID: 29866841 DOI: 10.1073/pnas.1720273115] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.
Collapse
|
29
|
González-Garza MT, Cruz-Vega DE, Cárdenas-Lopez A, de la Rosa RM, Moreno-Cuevas JE. Comparing stemness gene expression between stem cell subpopulations from peripheral blood and adipose tissue. AMERICAN JOURNAL OF STEM CELLS 2018; 7:38-47. [PMID: 29938124 PMCID: PMC6013721] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Accepted: 04/15/2018] [Indexed: 06/08/2023]
Abstract
Cell therapy presents a promising alternative for the treatment of degenerative diseases. The main sources of adult stem cells are bone marrow, adipose tissue and peripheral blood. Within those tissues, there are cell subpopulations that share pluripotential characteristics. Nevertheless, there is insufficient data to determine which of these stem cell subtypes would have a better possibility to differentiate to a specific tissue. The objective of this research was to analyze and compare the stemness genes expression from peripheral blood and adipose tissue of plastic adherent cells, and those immune-selected by the CD133+ and CD271+ membrane markers. On all cell subpopulation groups, self-renew capacity, the membranes markers CD73, CD90 and CD105, as well as the stemness genes NANOG, OCT4, SOX2, REX1, NOTCH1 and, NESTIN expression were analyzed. Results showed that all samples presented the minimal criteria to define them as human stem cells. All cell subpopulation were capable of self-renewal. Nevertheless, the subpopulation cell types showed differences on the time needed to reach confluence. The slowest doubling times were for those cells bearing the CD133 marker from both sources. Surface markers determined by flow cytometry were positive for CD73, CD90 and, CD105, and negative for CD45. The stemness gene expression was positive in all subpopulation. However, there were significant differences in the amount and pattern of expression among them. Those differences could be advantageous in finding the best option for their application on cell therapy. Cells with high expression of OCT4 gene could be a better opportunity for neuron differentiation like CD133+ blood cells. On the other hand, lowest expression of NOTCH1 on CD271+ cells from the same source could be a better possibility for myoblast differentiation. The observed differences could be used as an advantage to find which cell type and from the different source; this represents the best option for its application on cell therapy. Experiments focused on the best response to specific differentiation, are conducted in order to confirm those possibilities.
Collapse
Affiliation(s)
- Maria Teresa González-Garza
- Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud Morones Prieto 3000 Pte. Monterrey NL. CP64710, México
| | - Delia E Cruz-Vega
- Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud Morones Prieto 3000 Pte. Monterrey NL. CP64710, México
| | | | - Rosa Maria de la Rosa
- Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud Morones Prieto 3000 Pte. Monterrey NL. CP64710, México
| | - Jorge E Moreno-Cuevas
- Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud Morones Prieto 3000 Pte. Monterrey NL. CP64710, México
| |
Collapse
|
30
|
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.
Collapse
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.
| | | |
Collapse
|
31
|
Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation. Nat Commun 2017; 8:2214. [PMID: 29263426 PMCID: PMC5738441 DOI: 10.1038/s41467-017-02363-w] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 11/21/2017] [Indexed: 02/01/2023] Open
Abstract
Tankyrase 1 and 2 are poly(ADP-ribose) polymerases that function in pathways critical to cancer cell growth. Tankyrase-mediated PARylation marks protein targets for proteasomal degradation. Here, we generate human knockout cell lines to examine cell function and interrogate the proteome. We show that either tankyrase 1 or 2 is sufficient to maintain telomere length, but both are required to resolve telomere cohesion and maintain mitotic spindle integrity. Quantitative analysis of the proteome of tankyrase double knockout cells using isobaric tandem mass tags reveals targets of degradation, including antagonists of the Wnt/β-catenin signaling pathway (NKD1, NKD2, and HectD1) and three (Notch 1, 2, and 3) of the four Notch receptors. We show that tankyrases are required for Notch2 to exit the plasma membrane and enter the nucleus to activate transcription. Considering that Notch signaling is commonly activated in cancer, tankyrase inhibitors may have therapeutic potential in targeting this pathway. Tankyrase 1 and 2 are poly(ADP-ribose) polymerases that mark proteins for degradation, but there is a current lack of knowledge about their distinct functions and substrates. Here, the authors elucidate the cellular roles and substrates of these polymerases using comparative functional and proteomics analyses of tankyrase knockout cell lines.
Collapse
|
32
|
Moghaddam SA, Yousefi B, Sanooghi D, Faghihi F, Hayati Roodbari N, Bana N, Joghataei MT, Pooyan P, Arjmand B. Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro. J Chem Neuroanat 2017; 86:35-40. [PMID: 28754612 DOI: 10.1016/j.jchemneu.2017.07.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Revised: 06/12/2017] [Accepted: 07/19/2017] [Indexed: 01/15/2023]
Abstract
Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133+ hematopoietic stem cells (UCB- CD133+ HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133+ HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133+ cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133+ HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro.
Collapse
Affiliation(s)
| | - Behnam Yousefi
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Davood Sanooghi
- Department of Genetics, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
| | - Faezeh Faghihi
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
| | - Nasim Hayati Roodbari
- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Nikoo Bana
- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Mohammad Taghi Joghataei
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; Department of Neuroscience, School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran; Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
| | - Paria Pooyan
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Babak Arjmand
- Department of Neurosurgery and Iranian Tissue Bank, Tehran University of Medical Sciences/Tehran University, Tehran, Iran
| |
Collapse
|
33
|
Julian LM, McDonald AC, Stanford WL. Direct reprogramming with SOX factors: masters of cell fate. Curr Opin Genet Dev 2017; 46:24-36. [PMID: 28662445 DOI: 10.1016/j.gde.2017.06.005] [Citation(s) in RCA: 57] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2017] [Revised: 04/25/2017] [Accepted: 06/09/2017] [Indexed: 12/13/2022]
Abstract
Over the last decade significant advances have been made toward reprogramming the fate of somatic cells, typically by overexpression of cell lineage-determinant transcription factors. As key regulators of cell fate, the SOX family of transcription factors has emerged as potent drivers of direct somatic cell reprogramming into multiple lineages, in some cases as the sole overexpressed factor. The vast capacity of SOX factors, especially those of the SOXB1, E and F subclasses, to reprogram cell fate is enlightening our understanding of organismal development, cancer and disease, and offers tremendous potential for regenerative medicine and cell-based therapies. Understanding the molecular mechanisms through which SOX factors reprogram cell fate is essential to optimize the development of novel somatic cell transdifferentiation strategies.
Collapse
Affiliation(s)
- Lisa M Julian
- Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1L8L6, Canada
| | - Angela Ch McDonald
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, Ontario M5G0A4, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S3G9, Canada
| | - William L Stanford
- Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1L8L6, Canada; Department of Cellular and Molecular Medicine, Faulty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada; Department of Biochemistry, Microbiology and Immunology, Faulty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada; Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada.
| |
Collapse
|
34
|
Doerr J, Schwarz MK, Wiedermann D, Leinhaas A, Jakobs A, Schloen F, Schwarz I, Diedenhofen M, Braun NC, Koch P, Peterson DA, Kubitscheck U, Hoehn M, Brüstle O. Whole-brain 3D mapping of human neural transplant innervation. Nat Commun 2017; 8:14162. [PMID: 28102196 PMCID: PMC5253698 DOI: 10.1038/ncomms14162] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Accepted: 12/05/2016] [Indexed: 11/22/2022] Open
Abstract
While transplantation represents a key tool for assessing in vivo functionality of neural stem cells and their suitability for neural repair, little is known about the integration of grafted neurons into the host brain circuitry. Rabies virus-based retrograde tracing has developed into a powerful approach for visualizing synaptically connected neurons. Here, we combine this technique with light sheet fluorescence microscopy (LSFM) to visualize transplanted cells and connected host neurons in whole-mouse brain preparations. Combined with co-registration of high-precision three-dimensional magnetic resonance imaging (3D MRI) reference data sets, this approach enables precise anatomical allocation of the host input neurons. Our data show that the same neural donor cell population grafted into different brain regions receives highly orthotopic input. These findings indicate that transplant connectivity is largely dictated by the circuitry of the target region and depict rabies-based transsynaptic tracing and LSFM as efficient tools for comprehensive assessment of host–donor cell innervation. Transplantation of cells into the central nervous system has developed into a major avenue for replacing neurons lost to neurodegenerative disease. Here the authors develop an approach combining viral-based transynaptic tracing labeling and whole brain imaging to trace synaptic innervation of human neurons transplanted into a mouse background.
Collapse
Affiliation(s)
- Jonas Doerr
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.,Life&Brain GmbH, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Martin Karl Schwarz
- Life&Brain GmbH, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.,Department of Epileptology, Functional Neuroconnectomics Group, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Dirk Wiedermann
- Max Planck Institute for Metabolism Research, In-vivo-NMR Laboratory, Gleuelerstrasse 50, 50931 Cologne, Germany
| | - Anke Leinhaas
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Alina Jakobs
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Florian Schloen
- Institute of Physical and Theoretical Chemistry, University of Bonn, Wegeler Strasse 12, 53115 Bonn, Germany
| | - Inna Schwarz
- Department of Epileptology, Functional Neuroconnectomics Group, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Michael Diedenhofen
- Max Planck Institute for Metabolism Research, In-vivo-NMR Laboratory, Gleuelerstrasse 50, 50931 Cologne, Germany
| | - Nils Christian Braun
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Philipp Koch
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| | - Daniel A Peterson
- Center for Stem Cell and Regenerative Medicine, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, 60064 North Chicago, Illinois, USA
| | - Ulrich Kubitscheck
- Institute of Physical and Theoretical Chemistry, University of Bonn, Wegeler Strasse 12, 53115 Bonn, Germany
| | - Mathias Hoehn
- Max Planck Institute for Metabolism Research, In-vivo-NMR Laboratory, Gleuelerstrasse 50, 50931 Cologne, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.,Life&Brain GmbH, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
| |
Collapse
|
35
|
Bruzos-Cidon C, Castaño J, Torrecilla M, Sanchez-Pernaute R, Giorgetti A. Fast and Efficient Neural Conversion of Human Hematopoietic Cells. ACTA ACUST UNITED AC 2016; 39:1F.15.1-1F.15.20. [PMID: 31816186 DOI: 10.1002/cpsc.16] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
A major challenge in regenerative medicine is the generation of functionally effective target cells to replace or repair damaged tissues. The finding that most somatic cells can be directly converted into cells of another lineage by the expression of specific transcription factors has paved the way to novel applications. Induced neurons (iNs) represent an alternative source of neurons for disease modeling, drug screening, and potentially, for cell replacement therapy. This unit describes methods for the efficient conversion of blood cells into iNs, including protocols to isolate cord blood CD133+ cells, infect them with Sendai virus vectors that express SOX2 and c-MYC, and differentiate the infected cells (PB-MNCs) into mature neurons. A method to reprogram peripheral blood mononuclear cells into iNs is also described. Support protocols describe how to culture rat astrocytes and characterize the electrophysiology of iNs. © 2016 by John Wiley & Sons, Inc.
Collapse
Affiliation(s)
- Cristina Bruzos-Cidon
- Department of Pharmacology, Faculty of Medicine, University of the Basque Country (UPV/EHU), Spain
| | - Julio Castaño
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Maria Torrecilla
- Department of Pharmacology, Faculty of Medicine, University of the Basque Country (UPV/EHU), Spain
| | | | | |
Collapse
|
36
|
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.
Collapse
|
37
|
Velasco I, Salazar P, Giorgetti A, Ramos-Mejía V, Castaño J, Romero-Moya D, Menendez P. Concise review: Generation of neurons from somatic cells of healthy individuals and neurological patients through induced pluripotency or direct conversion. Stem Cells 2015; 32:2811-7. [PMID: 24989459 PMCID: PMC4282532 DOI: 10.1002/stem.1782] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2014] [Revised: 05/31/2014] [Accepted: 06/04/2014] [Indexed: 12/14/2022]
Abstract
Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron-like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the last years. The combination of genome-editing strategies and patient-specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening. Stem Cells2014;32:2811–2817
Collapse
Affiliation(s)
- Iván Velasco
- Instituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de México, México, D.F, México; Centro GENYO, Granada, Spain
| | | | | | | | | | | | | |
Collapse
|
38
|
Péron S, Berninger B. Reawakening the sleeping beauty in the adult brain: neurogenesis from parenchymal glia. Curr Opin Genet Dev 2015; 34:46-53. [PMID: 26296150 DOI: 10.1016/j.gde.2015.07.004] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2015] [Accepted: 07/19/2015] [Indexed: 12/12/2022]
Abstract
Life-long neurogenesis is highly restricted to specialized niches in the adult mammalian brain and therefore the brain's capacity for spontaneous regeneration is extremely limited. However, recent work has demonstrated that under certain circumstances parenchymal astrocytes and NG2 glia can generate neuronal progeny. In the striatum, stroke or excitotoxic lesions can reawaken in astrocytes a latent neurogenic program resulting in the genesis of new neurons. By contrast, in brain areas that fail to mount a neurogenic response following injury, such as the cerebral cortex, forced expression of neurogenic reprogramming factors can lineage convert local glia into induced neurons. Yet, injury-induced and reprogramming-induced neurogenesis exhibit intriguing commonalities, suggesting that they may converge on similar mechanisms.
Collapse
Affiliation(s)
- Sophie Péron
- Laboratory "Adult Neurogenesis and Cellular Reprogramming", Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Hanns-Dieter-Hüsch Weg 19, D-55128 Mainz, Germany; Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany
| | - Benedikt Berninger
- Laboratory "Adult Neurogenesis and Cellular Reprogramming", Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Hanns-Dieter-Hüsch Weg 19, D-55128 Mainz, Germany; Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany.
| |
Collapse
|
39
|
Zhao Z, Xu M, Wu M, Tian X, Zhang C, Fu X. Transdifferentiation of Fibroblasts by Defined Factors. Cell Reprogram 2015; 17:151-9. [PMID: 26053515 DOI: 10.1089/cell.2014.0089] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Cellular differentiation is usually considered to be an irreversible process during development due to robust lineage commitment. Feedback and feed-forward loops play a significant role in maintaining lineage-specific gene expression processes in various cell types, and, in turn, factors secreted by cells may regulate the homeostatic balance of these cycles during development and differentiation. The output of biological responses is controlled by such mechanisms in many regulatory pathways through gene networks involved in transcription, RNA metabolism, signal transduction, micromolecular synthesis, and degradation. The pluripotent stage during cellular conversion may be avoided through ectopic expression of lineage-specific factors. Lineage-specific transcription factors produced during development may strengthen cell type-specific gene expression patterns. Cellular phenotypes are further stabilized by epigenetic modifications. This reprogramming approach could have important implications for disease modeling and regenerative and personalized medicine.
Collapse
Affiliation(s)
- Zhiliang Zhao
- 1 Would Healing and Cell Biology Laboratory, Institute of Basic Medical Science, General Hospital of PLA , Beijing 10853, PR China .,2 Department of Plastic Surgery, General Hospital of The Second Artillery Corps , Beijing 100088, PR China .,6 These authors contributed equally to this work
| | - Mengyao Xu
- 3 Department of Gynecology and Obstetrics, General Hospital of Shenyang Military Region , Shenyang 110000, PR China .,6 These authors contributed equally to this work
| | - Meng Wu
- 4 Department of Plastic Surgery, General Hospital of PLA , Beijing 100853, PR China
| | - Xiaocheng Tian
- 2 Department of Plastic Surgery, General Hospital of The Second Artillery Corps , Beijing 100088, PR China
| | - Cuiping Zhang
- 5 Key Laboratory of Wound Repair and Regeneration of PLA, The First Affiliated Hospital, General Hospital of PLA , Beijing 100048, PR China
| | - Xiaobing Fu
- 5 Key Laboratory of Wound Repair and Regeneration of PLA, The First Affiliated Hospital, General Hospital of PLA , Beijing 100048, PR China
| |
Collapse
|
40
|
Nakagomi T, Nakano-Doi A, Kawamura M, Matsuyama T. Do Vascular Pericytes Contribute to Neurovasculogenesis in the Central Nervous System as Multipotent Vascular Stem Cells? Stem Cells Dev 2015; 24:1730-9. [PMID: 25900222 DOI: 10.1089/scd.2015.0039] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Increasing evidence suggests that multipotent stem cells are harbored within a vascular niche inside various organs. Although a precise phenotype of resident vascular stem cells (VSCs) that can function as multipotent stem cells remains unclear, accumulating evidence shows that multipotent VSCs are likely vascular pericytes (PCs) that localize within blood vessels. These PCs are multipotent, possessing the ability to differentiate into various cell types, including vascular lineage cells. In addition, brain PCs are unique: They are derived from neural crest and can differentiate into neural lineage cells. Because PCs in the central nervous system (CNS) can contribute to both neurogenesis and vasculogenesis, they may mediate the reparative process of neurovascular units that are constructed by neural and vascular cells. Here, we describe the activity of PCs when viewed as multipotent VSCs, primarily regarding their neurogenic and vasculogenic potential in the CNS. We also discuss similarities between PCs and other candidates for multipotent VSCs, including perivascular mesenchymal stem cells, neural crest-derived stem cells, adventitial progenitor cells, and adipose-derived stem cells.
Collapse
Affiliation(s)
- Takayuki Nakagomi
- 1 Institute for Advanced Medical Sciences, Hyogo College of Medicine , Hyogo, Japan
| | - Akiko Nakano-Doi
- 1 Institute for Advanced Medical Sciences, Hyogo College of Medicine , Hyogo, Japan
| | - Miki Kawamura
- 1 Institute for Advanced Medical Sciences, Hyogo College of Medicine , Hyogo, Japan .,2 Department of Neurology, Osaka University Graduate School of Medicine , Osaka, Japan
| | - Tomohiro Matsuyama
- 1 Institute for Advanced Medical Sciences, Hyogo College of Medicine , Hyogo, Japan
| |
Collapse
|
41
|
Liao W, Huang N, Yu J, Jares A, Yang J, Zieve G, Avila C, Jiang X, Zhang XB, Ma Y. Direct Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells by OCT4. Stem Cells Transl Med 2015; 4:755-63. [PMID: 25972144 DOI: 10.5966/sctm.2014-0289] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2014] [Accepted: 04/08/2015] [Indexed: 12/12/2022] Open
Abstract
UNLABELLED : Cellular reprogramming or conversion is a promising strategy to generate desired stem cell types from somatic cells. Neural stem cells (NSCs) have the potential to regenerate central nervous system tissue and repair damage in response to injury. However, NSCs are difficult to isolate from human tissues and expand in sufficient quantities for therapy. Here, we report a method to generate neural stem cells from cord blood CD34-positive cells by ectopic expression of OCT4 in a feeder-free system. The induced cells (iNSCs) show a characteristic NSC-like morphology and can be expanded in vitro for more than 20 passages. In addition, the iNSCs are positive for neural stem cell-specific markers such as Nestin and Musashi-1 and are similar in gene expression patterns to a human neural stem cell line. The iNSCs express distinct transcriptional factors for forebrain, hindbrain, and spinal cord regions. Upon differentiation, the iNSCs are able to commit into multilineage mature neural cells. Following in vivo introduction into NOD/SCID mice, iNSCs can survive and differentiate in the mouse brain 3 months post-transplantation. Alternatively, we were also able to derive iNSCs with an episomal vector expressing OCT4. Our results suggest a novel, efficient approach to generate neural precursor cells that can be potentially used in drug discovery or regenerative medicine for neurological disease and injury. SIGNIFICANCE This study describes a novel method to generate expandable induced neural stem cells from human cord blood cells in a feeder-free system by a single factor, OCT4. The data are promising for future applications that require the generation of large amounts of autologous neural stem cells in disease modeling and regenerative medicine.
Collapse
Affiliation(s)
- Wenbin Liao
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Nick Huang
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Jingxia Yu
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Alexander Jares
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Jianchang Yang
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Gary Zieve
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Cecilia Avila
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Xun Jiang
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Xiao-Bing Zhang
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| | - Yupo Ma
- Departments of Pathology, Surgery, and Obstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; Department of Medicine, Loma Linda University, Loma Linda, California, USA
| |
Collapse
|
42
|
Nakagomi T, Kubo S, Nakano-Doi A, Sakuma R, Lu S, Narita A, Kawahara M, Taguchi A, Matsuyama T. Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells. Stem Cells 2015; 33:1962-74. [PMID: 25694098 DOI: 10.1002/stem.1977] [Citation(s) in RCA: 176] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2014] [Accepted: 01/27/2015] [Indexed: 01/01/2023]
Abstract
Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.
Collapse
Affiliation(s)
| | - Shuji Kubo
- Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
| | - Akiko Nakano-Doi
- Institute for Advanced Medical Sciences, Nishinomiya, Hyogo, Japan
| | - Rika Sakuma
- Institute for Advanced Medical Sciences, Nishinomiya, Hyogo, Japan
| | - Shan Lu
- Institute for Advanced Medical Sciences, Nishinomiya, Hyogo, Japan.,Department of Neurology of Hangzhou First People's Hospital, Hangzhou, People's Republic of China
| | - Aya Narita
- Institute for Advanced Medical Sciences, Nishinomiya, Hyogo, Japan
| | - Maiko Kawahara
- Institute for Advanced Medical Sciences, Nishinomiya, Hyogo, Japan.,Graduate School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo, Japan
| | - Akihiko Taguchi
- Department of Regenerative Medicine Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | | |
Collapse
|
43
|
Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc Natl Acad Sci U S A 2015; 112:E2725-34. [PMID: 25870293 DOI: 10.1073/pnas.1504393112] [Citation(s) in RCA: 280] [Impact Index Per Article: 28.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.
Collapse
|
44
|
Yu KR, Shin JH, Kim JJ, Koog MG, Lee JY, Choi SW, Kim HS, Seo Y, Lee S, Shin TH, Jee MK, Kim DW, Jung SJ, Shin S, Han DW, Kang KS. Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b. Cell Rep 2015; 10:441-452. [PMID: 25600877 DOI: 10.1016/j.celrep.2014.12.038] [Citation(s) in RCA: 96] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2014] [Revised: 10/13/2014] [Accepted: 12/16/2014] [Indexed: 12/13/2022] Open
Abstract
A recent study has suggested that fibroblasts can be converted into mouse-induced neural stem cells (miNSCs) through the expression of defined factors. However, successful generation of human iNSCs (hiNSCs) has proven challenging to achieve. Here, using microRNA (miRNA) expression profile analyses, we showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2, a let-7-targeting gene, enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs. Interestingly, HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2, whereas other combinations or SOX2 alone showed a limited conversion ability. Taken together, these findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal, providing a strategy for the clinical treatment of neurological diseases.
Collapse
Affiliation(s)
- Kyung-Rok Yu
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Ji-Hee Shin
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Jae-Jun Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Myung Guen Koog
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Jin Young Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Soon Won Choi
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Hyung-Sik Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Yoojin Seo
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - SeungHee Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; Institute for Stem Cell and Regenerative Medicine in Kang Stem Biotech, Biotechnology Incubating Center, Seoul National University, Seoul 151-742, Korea
| | - Tae-Hoon Shin
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Min Ki Jee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
| | - Dong-Wook Kim
- Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea
| | - Sung Jun Jung
- Department of Physiology, School of Medicine, Hanyang University, Seoul 133-791, Korea
| | - Sue Shin
- Department of Laboratory Medicine, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul 156-707, Korea; Seoul Metropolitan Public Cord Blood Bank, Allcord, Seoul 156-707, Korea
| | - Dong Wook Han
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701, Korea
| | - Kyung-Sun Kang
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
| |
Collapse
|
45
|
Brändl B, Schneider SA, Loring JF, Hardy J, Gribbon P, Müller FJ. Stem cell reprogramming: basic implications and future perspective for movement disorders. Mov Disord 2014; 30:301-12. [PMID: 25546831 DOI: 10.1002/mds.26113] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2013] [Revised: 10/03/2014] [Accepted: 10/29/2014] [Indexed: 12/14/2022] Open
Abstract
The introduction of stem cell-associated molecular factors into human patient-derived cells allows for their reprogramming in the laboratory environment. As a result, human induced pluripotent stem cells (hiPSC) can now be reprogrammed epigenetically without disruption of their overall genomic integrity. For patients with neurodegenerative diseases characterized by progressive loss of functional neurons, the ability to reprogram any individual's cells and drive their differentiation toward susceptible neuronal subtypes holds great promise. Apart from applications in regenerative medicine and cell replacement-based therapy, hiPSCs are increasingly used in preclinical research for establishing disease models and screening for drug toxicities. The rapid developments in this field prompted us to review recent progress toward the applications of stem cell technologies for movement disorders. We introduce reprogramming strategies and explain the critical steps in the differentiation of hiPSCs to clinical relevant subtypes of cells in the context of movement disorders. We summarize and discuss recent discoveries in this field, which, based on the rapidly expanding basic science literature as well as upcoming trends in personalized medicine, will strongly influence the future therapeutic options available to practitioners working with patients suffering from such disorders.
Collapse
Affiliation(s)
- Björn Brändl
- Center for Psychiatry, University Hospital Schleswig Holstein, Campus Kiel, Germany
| | | | | | | | | | | |
Collapse
|
46
|
Castaño J, Menendez P, Bruzos-Cidon C, Straccia M, Sousa A, Zabaleta L, Vazquez N, Zubiarrain A, Sonntag KC, Ugedo L, Carvajal-Vergara X, Canals JM, Torrecilla M, Sanchez-Pernaute R, Giorgetti A. Fast and efficient neural conversion of human hematopoietic cells. Stem Cell Reports 2014; 3:1118-1131. [PMID: 25458894 PMCID: PMC4264063 DOI: 10.1016/j.stemcr.2014.10.008] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2014] [Revised: 10/14/2014] [Accepted: 10/14/2014] [Indexed: 01/15/2023] Open
Abstract
Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.
Collapse
Affiliation(s)
- Julio Castaño
- Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Barcelona 08036, Spain
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Barcelona 08036, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain
| | - Cristina Bruzos-Cidon
- Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa 48940, Spain
| | - Marco Straccia
- Department of Cell Biology, Immunology and Neurosciences, Faculty of Medicine, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona 08036, Spain; Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona 08036, Spain
| | - Amaia Sousa
- Laboratory of Stem Cells and Neural Repair, Inbiomed, San Sebastian 20009, Spain
| | - Lorea Zabaleta
- Cell Reprogramming and Differentiation Platform, Inbiomed, San Sebastian 20009, Spain
| | - Nerea Vazquez
- Laboratory of Stem Cells and Neural Repair, Inbiomed, San Sebastian 20009, Spain
| | - Amaia Zubiarrain
- Laboratory of Stem Cells and Neural Repair, Inbiomed, San Sebastian 20009, Spain; Cell Reprogramming and Differentiation Platform, Inbiomed, San Sebastian 20009, Spain
| | - Kai-Christian Sonntag
- Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, MA 02478, USA
| | - Luisa Ugedo
- Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa 48940, Spain
| | | | - Josep Maria Canals
- Department of Cell Biology, Immunology and Neurosciences, Faculty of Medicine, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona 08036, Spain; Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona 08036, Spain
| | - Maria Torrecilla
- Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa 48940, Spain
| | | | - Alessandra Giorgetti
- Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Barcelona 08036, Spain.
| |
Collapse
|
47
|
Heinrich C, Bergami M, Gascón S, Lepier A, Viganò F, Dimou L, Sutor B, Berninger B, Götz M. Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex. Stem Cell Reports 2014; 3:1000-14. [PMID: 25458895 PMCID: PMC4264057 DOI: 10.1016/j.stemcr.2014.10.007] [Citation(s) in RCA: 240] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 10/16/2014] [Accepted: 10/16/2014] [Indexed: 02/07/2023] Open
Abstract
The adult cerebral cortex lacks the capacity to replace degenerated neurons following traumatic injury. Conversion of nonneuronal cells into induced neurons has been proposed as an innovative strategy toward brain repair. Here, we show that retrovirus-mediated expression of the transcription factors Sox2 and Ascl1, but strikingly also Sox2 alone, can induce the conversion of genetically fate-mapped NG2 glia into induced doublecortin (DCX)+ neurons in the adult mouse cerebral cortex following stab wound injury in vivo. In contrast, lentiviral expression of Sox2 in the unlesioned cortex failed to convert oligodendroglial and astroglial cells into DCX+ cells. Neurons induced following injury mature morphologically and some acquire NeuN while losing DCX. Patch-clamp recording of slices containing Sox2- and/or Ascl1-transduced cells revealed that a substantial fraction of these cells receive synaptic inputs from neurons neighboring the injury site. Thus, NG2 glia represent a potential target for reprogramming strategies toward cortical repair.
Sox2 or Sox2/Ascl1 can convert glia into induced DCX+ neurons in the injured cortex Sox10-iCreERT2-mediated fate mapping shows that induced neurons are NG2 glia derived Induced neurons receive (or retain) synapses from preexisting neurons Without prior injury, Sox2 does not convert cortical macroglia into neurons
Collapse
Affiliation(s)
- Christophe Heinrich
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany; INSERM U836, University Grenoble Alpes, Grenoble Institute of Neurosciences, 38000 Grenoble, France
| | - Matteo Bergami
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany
| | - Sergio Gascón
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany; Institute for Stem Cell Research, National Research Center for Environment and Health, 85764 Neuherberg, Germany
| | - Alexandra Lepier
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany
| | - Francesca Viganò
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany
| | - Leda Dimou
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany; Institute for Stem Cell Research, National Research Center for Environment and Health, 85764 Neuherberg, Germany
| | - Bernd Sutor
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany
| | - Benedikt Berninger
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany; Institute of Physiological Chemistry, University Medical Center, Johannes Gutenberg University Mainz, 55128 Mainz, Germany; Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz, 55131 Mainz, Germany.
| | - Magdalena Götz
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, 80336 Munich, Germany; Institute for Stem Cell Research, National Research Center for Environment and Health, 85764 Neuherberg, Germany; Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany.
| |
Collapse
|
48
|
Adult-Derived Pluripotent Stem Cells. World Neurosurg 2014; 82:500-8. [DOI: 10.1016/j.wneu.2013.08.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2012] [Revised: 08/07/2013] [Accepted: 08/09/2013] [Indexed: 01/27/2023]
|
49
|
Yokoyama KD, Zhang Y, Ma J. Tracing the evolution of lineage-specific transcription factor binding sites in a birth-death framework. PLoS Comput Biol 2014; 10:e1003771. [PMID: 25144359 PMCID: PMC4140645 DOI: 10.1371/journal.pcbi.1003771] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2014] [Accepted: 06/27/2014] [Indexed: 11/24/2022] Open
Abstract
Changes in cis-regulatory element composition that result in novel patterns of gene expression are thought to be a major contributor to the evolution of lineage-specific traits. Although transcription factor binding events show substantial variation across species, most computational approaches to study regulatory elements focus primarily upon highly conserved sites, and rely heavily upon multiple sequence alignments. However, sequence conservation based approaches have limited ability to detect lineage-specific elements that could contribute to species-specific traits. In this paper, we describe a novel framework that utilizes a birth-death model to trace the evolution of lineage-specific binding sites without relying on detailed base-by-base cross-species alignments. Our model was applied to analyze the evolution of binding sites based on the ChIP-seq data for six transcription factors (GATA1, SOX2, CTCF, MYC, MAX, ETS1) along the lineage toward human after human-mouse common ancestor. We estimate that a substantial fraction of binding sites (∼58–79% for each factor) in humans have origins since the divergence with mouse. Over 15% of all binding sites are unique to hominids. Such elements are often enriched near genes associated with specific pathways, and harbor more common SNPs than older binding sites in the human genome. These results support the ability of our method to identify lineage-specific regulatory elements and help understand their roles in shaping variation in gene regulation across species. Recent experimental studies showed that the evolution of transcription factor binding sites (TFBS) is highly dynamic, with sites differing a great deal even between closely related mammalian species. Despite the substantial experimental evidence for rapid divergence of regulatory protein-binding events across species, computational methods designed to analyze regulatory elements evolution have focused primarily on phylogenetic footprinting approaches, in which putative functional regulatory elements are identified according to strong sequence conservation. Cross-species comparisons of non-coding sequences are limited in their ability to fully understand the evolution of regulatory sequences, particularly in cases where the elements are selected for novelty or species-specific. We have developed a novel framework to reconstruct the history of lineage-specific TFBS and showed that large amount of TFBS in human were born after human-mouse divergence. These elements also have distinct biological implications as compared to more ancient ones. This method can help understand the roles of lineage-specific TFBS in shaping gene regulation across different species.
Collapse
Affiliation(s)
- Ken Daigoro Yokoyama
- Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
| | - Yang Zhang
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
| | - Jian Ma
- Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
- * E-mail:
| |
Collapse
|
50
|
Xu XL, Yang JP, Fu LN, Ren RT, Yi F, Suzuki K, Liu K, Ding ZC, Qu J, Zhang WQ, Li Y, Yuan TT, Yuan GH, Sui LN, Guan D, Duan SL, Pan HZ, Wang P, Zhu XP, Montserrat N, Li M, Bai RJ, Liu L, Izpisua Belmonte JC, Liu GH. Direct reprogramming of porcine fibroblasts to neural progenitor cells. Protein Cell 2014; 5:4-7. [PMID: 24492924 PMCID: PMC3938843 DOI: 10.1007/s13238-013-0015-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Affiliation(s)
- Xiu-Ling Xu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Ji-Ping Yang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Li-Na Fu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Ruo-Tong Ren
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Fei Yi
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Keiichiro Suzuki
- Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037 USA
| | - Kai Liu
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071 China
| | - Zhi-Chao Ding
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Jing Qu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Wei-Qi Zhang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Ying Li
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Ting-Ting Yuan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Guo-Hong Yuan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Li-Na Sui
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Di Guan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Shun-Lei Duan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Hui-Ze Pan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Ping Wang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Xi-Ping Zhu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Nuria Montserrat
- Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
| | - Ming Li
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Rui-Jun Bai
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Lin Liu
- State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071 China
| | - Juan Carlos Izpisua Belmonte
- Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037 USA
| | - Guang-Hui Liu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
- Beijing Institute for Brain Disorders, Beijing, 100069 China
| |
Collapse
|