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Ballion B, Bonnet ML, Brot S, Gaillard A. Electrophysiological characterisation of intranigral-grafted hiPSC-derived dopaminergic neurons in a mouse model of Parkinson's disease. Stem Cell Res Ther 2025; 16:232. [PMID: 40346597 PMCID: PMC12065326 DOI: 10.1186/s13287-025-04344-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2024] [Accepted: 04/15/2025] [Indexed: 05/11/2025] Open
Abstract
BACKGROUND Parkinson's disease (PD) is a complex neurological disorder characterized by the progressive degeneration of midbrain dopaminergic (mDA) neurons in the substantia nigra (SN). This degeneration disrupts the basal ganglia loops, leading to both motor and non-motor dysfunctions. Cell therapy for PD aims to replace lost mDA neurons to restore the DA neurotransmission in the denervated forebrain targets. In clinical trials for PD, mDA neurons are implanted into the target area, the striatum, and not in the SN where they are normally located. This ectopic localisation of cells may affect the functionality of transplanted neurons due to the absence of appropriate host afferent regulation. We recently demonstrated that human induced pluripotent stem cells (hiPSCs) derived mDA progenitors grafted into the substantia nigra pars compacta (SNpc) in a mouse model of PD, differentiated into mature mDA neurons, restored the degenerated nigrostriatal pathway, and induced motor recovery. The objective of the present study was to evaluate the long-term functionality of these intranigral-grafted mDA neurons by assessing their electrophysiological properties. METHODS We performed intranigral transplantation of hiPSC-derived mDA progenitors in a 6-hydroxydopamine RAG2-KO mouse model of PD. We recorded in vivo unit extracellular activity of grafted mDA neurons in anesthetised mice from 9 to 12 months post-transplantation. Their electrophysiological properties, including firing rates, patterns and spike characteristics, were analysed and compared with those of native nigral dopaminergic neurons from control mice. RESULTS We demonstrated that these grafted mDA neurons exhibited functional characteristics similar to those of native nigral dopaminergic neurons, such as large bi- or triphasic spike waveforms, low firing rates, pacemaker-like properties, and two single-spike firing patterns. Although grafted mDA neurons also displayed low discharge frequencies below 10 Hz, their mean frequency was significantly lower than that of nigral mDA neurons, with a differential pattern distribution. CONCLUSIONS Our findings indicate that grafted mDA neurons exhibit dopaminergic-like functional properties, including intrinsic membrane potential oscillations leading to regular firing patterns. Additionally, they demonstrated irregular and burst firing patterns, suggesting they receive modulatory inputs. However, grafted mDA neurons displayed distinct properties, potentially related to their human origin or the incomplete maturation one year after transplantation.
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Affiliation(s)
- Bérengère Ballion
- Laboratoire des neurosciences expérimentales et cliniques (LNEC), Université de Poitiers- INSERM 1084, Poitiers Cedex 9, 86073, France.
| | - Marie-Laure Bonnet
- Laboratoire des neurosciences expérimentales et cliniques (LNEC), Université de Poitiers- INSERM 1084, Poitiers Cedex 9, 86073, France
- Centre hospitalier universitaire (CHU) de Poitiers, Poitiers, 86021, France
| | - Sébastien Brot
- Laboratoire des neurosciences expérimentales et cliniques (LNEC), Université de Poitiers- INSERM 1084, Poitiers Cedex 9, 86073, France
| | - Afsaneh Gaillard
- Laboratoire des neurosciences expérimentales et cliniques (LNEC), Université de Poitiers- INSERM 1084, Poitiers Cedex 9, 86073, France.
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Basso V, Döbrössy MD, Thompson LH, Kirik D, Fuller HR, Gates MA. State of the Art in Sub-Phenotyping Midbrain Dopamine Neurons. BIOLOGY 2024; 13:690. [PMID: 39336117 PMCID: PMC11428604 DOI: 10.3390/biology13090690] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 08/27/2024] [Accepted: 08/29/2024] [Indexed: 09/30/2024]
Abstract
Dopaminergic neurons in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNpc) comprise around 75% of all dopaminergic neurons in the human brain. While both groups of dopaminergic neurons are in close proximity in the midbrain and partially overlap, development, function, and impairments in these two classes of neurons are highly diverse. The molecular and cellular mechanisms underlying these differences are not yet fully understood, but research over the past decade has highlighted the need to differentiate between these two classes of dopaminergic neurons during their development and in the mature brain. This differentiation is crucial not only for understanding fundamental circuitry formation in the brain but also for developing therapies targeted to specific dopaminergic neuron classes without affecting others. In this review, we summarize the state of the art in our understanding of the differences between the dopaminergic neurons of the VTA and the SNpc, such as anatomy, structure, morphology, output and input, electrophysiology, development, and disorders, and discuss the current technologies and methods available for studying these two classes of dopaminergic neurons, highlighting their advantages, limitations, and the necessary improvements required to achieve more-precise therapeutic interventions.
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Affiliation(s)
- Valentina Basso
- School of Medicine, Keele University, Staffordshire ST5 5BG, UK
| | - Máté D Döbrössy
- Laboratory of Stereotaxy and Interventional Neurosciences, Department of Stereotactic and Functional, Neurosurgery, Medical Center, University of Freiburg, 79106 Freiburg im Breisgau, Germany
- Department of Stereotactic and Functional Neurosurgery, Medical Center, University of Freiburg, 79106 Freiburg im Breisgau, Germany
- Faculty of Biology, University of Freiburg, 79104 Freiburg im Breisgau, Germany
| | - Lachlan H Thompson
- Charles Perkins Centre, Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, Australia
- Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
| | - Deniz Kirik
- Brain Repair and Imaging in Neural Systems (B.R.A.I.N.S) Unit, Department of Experimental Medical Science, Lund University, BMC D11, 22184 Lund, Sweden
| | - Heidi R Fuller
- School of Pharmacy and Bioengineering, Keele University, Staffordshire ST5 5BG, UK
- Wolfson Centre for Inherited Neuromuscular Disease, TORCH Building, RJAH Orthopaedic Hospital, Oswestry SY10 7AG, UK
| | - Monte A Gates
- School of Medicine, Keele University, Staffordshire ST5 5BG, UK
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Björklund A, Parmar M. Dopamine Cell Therapy: From Cell Replacement to Circuitry Repair. JOURNAL OF PARKINSONS DISEASE 2021; 11:S159-S165. [PMID: 33814467 PMCID: PMC8543294 DOI: 10.3233/jpd-212609] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Cell therapy for Parkinson's disease (PD) is aimed to replace the degenerated midbrain dopamine (mDA) neurons and restore DA neurotransmission in the denervated forebrain targets. A limitation of the intrastriatal grafting approach, which is currently used in clinical trials, is that the mDA neurons are implanted into the target area, in most cases the putamen, and not in the ventral midbrain where they normally reside. This ectopic location of the cells may limit their functionality due to the lack of appropriate afferent regulation from the host. Homotopic transplantation, into the substantia nigra, is now being pursued in rodent PD models as a way to achieve more complete circuitry repair. Intranigral grafts of mDA neurons, derived from human embryonic stem cells, have the capacity to re-establish the nigrostriatal and mesolimbic pathways in their entirety and restore dense functional innervations in striatal, limbic and cortical areas. Tracing of host afferent inputs using the rabies tracing technique shows that the afferent connectivity of grafts implanted in the nigra matches closely that of the intrinsic mDA system, suggesting a degree of circuitry reconstruction that exceeds what has been achieved before. This approach holds great promise, but to match the larger size of the human brain, and the 10 times greater distance between substantia nigra and its forebrain targets, it may be necessary to find ways to improve the growth capacity of the grafted mDA neurons, pointing to a combined approach where growth promoting factors are used to enhance the performance of mDA neuron grafts.
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Affiliation(s)
- Anders Björklund
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
| | - Malin Parmar
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
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Adler AF, Cardoso T, Nolbrant S, Mattsson B, Hoban DB, Jarl U, Wahlestedt JN, Grealish S, Björklund A, Parmar M. hESC-Derived Dopaminergic Transplants Integrate into Basal Ganglia Circuitry in a Preclinical Model of Parkinson's Disease. Cell Rep 2020; 28:3462-3473.e5. [PMID: 31553914 PMCID: PMC6899556 DOI: 10.1016/j.celrep.2019.08.058] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2019] [Revised: 04/30/2019] [Accepted: 08/19/2019] [Indexed: 12/20/2022] Open
Abstract
Cell replacement is currently being explored as a therapeutic approach for neurodegenerative disease. Using stem cells as a source, transplantable progenitors can now be generated under conditions compliant with clinical application in patients. In this study, we elucidate factors controlling target-appropriate innervation and circuitry integration of human embryonic stem cell (hESC)-derived grafts after transplantation to the adult brain. We show that cell-intrinsic factors determine graft-derived axonal innervation, whereas synaptic inputs from host neurons primarily reflect the graft location. Furthermore, we provide evidence that hESC-derived dopaminergic grafts transplanted in a long-term preclinical rat model of Parkinson’s disease (PD) receive synaptic input from subtypes of host cortical, striatal, and pallidal neurons that are known to regulate the function of endogenous nigral dopamine neurons. This refined understanding of how graft neurons integrate with host circuitry will be important for the design of clinical stem-cell-based replacement therapies for PD, as well as for other neurodegenerative diseases.
Pattern of graft-derived innervation is determined by phenotype of grafted cells Synaptic inputs from host-to-graft depend on location of graft Intrastriatal dopaminergic grafts receive correct excitatory and inhibitory host inputs Individual host neurons provide inputs to both dopaminergic grafts and the host nigra
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Affiliation(s)
- Andrew F Adler
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Tiago Cardoso
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Sara Nolbrant
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Bengt Mattsson
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Deirdre B Hoban
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Ulla Jarl
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Jenny Nelander Wahlestedt
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Shane Grealish
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Anders Björklund
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Malin Parmar
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden.
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Björklund A, Parmar M. Neuronal Replacement as a Tool for Basal Ganglia Circuitry Repair: 40 Years in Perspective. Front Cell Neurosci 2020; 14:146. [PMID: 32547369 PMCID: PMC7272540 DOI: 10.3389/fncel.2020.00146] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Accepted: 04/30/2020] [Indexed: 01/07/2023] Open
Abstract
The ability of new neurons to promote repair of brain circuitry depends on their capacity to re-establish afferent and efferent connections with the host. In this review article, we give an overview of past and current efforts to restore damaged connectivity in the adult mammalian brain using implants of fetal neuroblasts or stem cell-derived neuronal precursors, with a focus on strategies aimed to repair damaged basal ganglia circuitry induced by lesions that mimic the pathology seen in humans affected by Parkinson’s or Huntington’s disease. Early work performed in rodents showed that neuroblasts obtained from striatal primordia or fetal ventral mesencephalon can become anatomically and functionally integrated into lesioned striatal and nigral circuitry, establish afferent and efferent connections with the lesioned host, and reverse the lesion-induced behavioral impairments. Recent progress in the generation of striatal and nigral progenitors from pluripotent stem cells have provided compelling evidence that they can survive and mature in the lesioned brain and re-establish afferent and efferent axonal connectivity with a remarkable degree of specificity. The studies of cell-based circuitry repair are now entering a new phase. The introduction of genetic and virus-based techniques for brain connectomics has opened entirely new possibilities for studies of graft-host integration and connectivity, and the access to more refined experimental techniques, such as chemo- and optogenetics, has provided new powerful tools to study the capacity of grafted neurons to impact the function of the host brain. Progress in this field will help to guide the efforts to develop therapeutic strategies for cell-based repair in Huntington’s and Parkinson’s disease and other neurodegenerative conditions involving damage to basal ganglia circuitry.
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Affiliation(s)
- Anders Björklund
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
| | - Malin Parmar
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
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6
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Adler AF, Björklund A, Parmar M. Transsynaptic tracing and its emerging use to assess graft-reconstructed neural circuits. Stem Cells 2020; 38:716-726. [PMID: 32101353 DOI: 10.1002/stem.3166] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Revised: 01/20/2020] [Accepted: 02/19/2020] [Indexed: 12/16/2022]
Abstract
Fetal neural progenitor grafts have been evaluated in preclinical animal models of spinal cord injury and Parkinson's disease for decades, but the initial reliance on primary tissue as a cell source limited the scale of their clinical translatability. With the development of robust methods to differentiate human pluripotent stem cells to specific neural subtypes, cell replacement therapy holds renewed promise to treat a variety of neurodegenerative diseases and injuries at scale. As these cell sources are evaluated in preclinical models, new transsynaptic tracing methods are making it possible to study the connectivity between host and graft neurons with greater speed and detail than was previously possible. To date, these studies have revealed that widespread, long-lasting, and anatomically appropriate synaptic contacts are established between host and graft neurons, as well as new aspects of host-graft connectivity which may be relevant to clinical cell replacement therapy. It is not yet clear, however, whether the synaptic connectivity between graft and host neurons is as cell-type specific as it is in the endogenous nervous system, or whether that connectivity is responsible for the functional efficacy of cell replacement therapy. Here, we review evidence suggesting that the new contacts established between host and graft neurons may indeed be cell-type specific, and how transsynaptic tracing can be used in the future to further elucidate the mechanisms of graft-mediated functional recovery in spinal cord injury and Parkinson's disease.
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Affiliation(s)
- Andrew F Adler
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden.,Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Anders Björklund
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
| | - Malin Parmar
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden.,Lund Stem Cell Center, Lund University, Lund, Sweden
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7
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Collier TJ, Sortwell CE, Mercado NM, Steece‐Collier K. Cell therapy for Parkinson's disease: Why it doesn't work every time. Mov Disord 2019; 34:1120-1127. [PMID: 31234239 PMCID: PMC6771700 DOI: 10.1002/mds.27742] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 05/08/2019] [Accepted: 05/20/2019] [Indexed: 11/29/2022] Open
Abstract
The clinical experience with cell replacement therapy for advanced PD has yielded notable successes and failures. A recent autopsy case report of an individual that received implants of fetal dopamine neurons 16 years previously, but at no time experienced clinical benefit despite the best documented survival of grafted neurons and most extensive reinnervation of the striatum, raises sobering issues. With good reason, a great deal of effort in cell replacement science continues to focus on optimizing the cell source and implantation procedure. Here, we describe our preclinical studies in aged rats indicating that despite survival of large numbers of transplanted dopamine neurons and dense reinnervation of the striatum, synaptic connections between graft and host are markedly decreased and behavioral recovery is impaired. This leads us to the hypothesis that the variability in therapeutic response to dopamine neuron grafts may be less about the viability of transplanted neurons and more about the integrity of the aged, dopamine-depleted striatum and its capacity for repair. Replacement of dopamine innervation only can be fully effective if the correct target is present. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
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Affiliation(s)
- Timothy J. Collier
- Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA, and Hauenstein Neuroscience CenterMercy Health/St. Mary'sGrand RapidsMichiganUSA
| | - Caryl E. Sortwell
- Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA, and Hauenstein Neuroscience CenterMercy Health/St. Mary'sGrand RapidsMichiganUSA
| | - Natosha M. Mercado
- Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA, and Hauenstein Neuroscience CenterMercy Health/St. Mary'sGrand RapidsMichiganUSA
| | - Kathy Steece‐Collier
- Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA, and Hauenstein Neuroscience CenterMercy Health/St. Mary'sGrand RapidsMichiganUSA
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Boronat-García A, Guerra-Crespo M, Drucker-Colín R. Historical perspective of cell transplantation in Parkinson’s disease. World J Transplant 2017; 7:179-192. [PMID: 28698835 PMCID: PMC5487308 DOI: 10.5500/wjt.v7.i3.179] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Revised: 04/27/2017] [Accepted: 05/15/2017] [Indexed: 02/05/2023] Open
Abstract
Cell grafting has been considered a therapeutic approach for Parkinson’s disease (PD) since the 1980s. The classical motor symptoms of PD are caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to a decrement in dopamine release in the striatum. Consequently, the therapy of cell-transplantation for PD consists in grafting dopamine-producing cells directly into the brain to reestablish dopamine levels. Different cell sources have been shown to induce functional benefits on both animal models of PD and human patients. However, the observed motor improvements are highly variable between individual subjects, and the sources of this variability are not fully understood. The purpose of this review is to provide a general overview of the pioneering studies done in animal models of PD that established the basis for the first clinical trials in humans, and compare these with the latest findings to identify the most relevant aspects that remain unanswered to date. The main focus of the discussions presented here will be on the mechanisms associated with the survival and functionality of the transplants. These include the role of the dopamine released by the grafts and the capacity of the grafted cells to extend fibers and to integrate into the motor circuit. The complete understanding of these aspects will require extensive research on basic aspects of molecular and cellular physiology, together with neuronal network function, in order to uncover the real potential of cell grafting for treating PD.
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Hodges H, Pollock K, Stroemer P, Patel S, Stevanato L, Reuter I, Sinden J. Making Stem Cell Lines Suitable for Transplantation. Cell Transplant 2017. [DOI: 10.3727/000000007783464605] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Human stem cells, progenitor cells, and cell lines have been derived from embryonic, fetal, and adult sources in the search for graft tissue suitable for the treatment of CNS disorders. An increasing number of experimental studies have shown that grafts from several sources survive, differentiate into distinct cell types, and exert positive functional effects in experimental animal models, but little attention has been given to developing cells under conditions of good manufacturing practice (GMP) that can be scaled up for mass treatment. The capacity for continued division of stem cells in culture offers the opportunity to expand their production to meet the widespread clinical demands posed by neurodegenerative diseases. However, maintaining stem cell division in culture long term, while ensuring differentiation after transplantation, requires genetic and/or oncogenetic manipulations, which may affect the genetic stability and in vivo survival of cells. This review outlines the stages, selection criteria, problems, and ultimately the successes arising in the development of conditionally immortal clinical grade stem cell lines, which divide in vitro, differentiate in vivo, and exert positive functional effects. These processes are specifically exemplified by the murine MHP36 cell line, conditionally immortalized by a temperature-sensitive mutant of the SV40 large T antigen, and cell lines transfected with the c-myc protein fused with a mutated estrogen receptor (c-mycERTAM), regulated by a tamoxifen metabolite, but the issues raised are common to all routes for the development of effective clinical grade cells.
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Affiliation(s)
- Helen Hodges
- Department of Psychology, Institute of Psychiatry, Kings College, London, UK
- ReNeuron Ltd., Guildford, Surrey, UK
| | | | | | | | | | - Iris Reuter
- Department of Psychology, Institute of Psychiatry, Kings College, London, UK
- Department of Neurology, University of Giessen and Marburg, Germany
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Dunnett SB, Björklund A. Mechanisms and use of neural transplants for brain repair. PROGRESS IN BRAIN RESEARCH 2017; 230:1-51. [PMID: 28552225 DOI: 10.1016/bs.pbr.2016.11.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Under appropriate conditions, neural tissues transplanted into the adult mammalian brain can survive, integrate, and function so as to influence the behavior of the host, opening the prospect of repairing neuronal damage, and alleviating symptoms associated with neuronal injury or neurodegenerative disease. Alternative mechanisms of action have been postulated: nonspecific effects of surgery; neurotrophic and neuroprotective influences on disease progression and host plasticity; diffuse or locally regulated pharmacological delivery of deficient neurochemicals, neurotransmitters, or neurohormones; restitution of the neuronal and glial environment necessary for proper host neuronal support and processing; promoting local and long-distance host and graft axon growth; formation of reciprocal connections and reconstruction of local circuits within the host brain; and up to full integration and reconstruction of fully functional host neuronal networks. Analysis of neural transplants in a broad range of anatomical systems and disease models, on simple and complex classes of behavioral function and information processing, have indicated that all of these alternative mechanisms are likely to contribute in different circumstances. Thus, there is not a single or typical mode of graft function; rather grafts can and do function in multiple ways, specific to each particular context. Consequently, to develop an effective cell-based therapy, multiple dimensions must be considered: the target disease pathogenesis; the neurodegenerative basis of each type of physiological dysfunction or behavioral symptom; the nature of the repair required to alleviate or remediate the functional impairments of particular clinical relevance; and identification of a suitable cell source or delivery system, along with the site and method of implantation, that can achieve the sought for repair and recovery.
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11
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Takahashi J. Strategies for bringing stem cell-derived dopamine neurons to the clinic: The Kyoto trial. PROGRESS IN BRAIN RESEARCH 2017; 230:213-226. [PMID: 28552230 DOI: 10.1016/bs.pbr.2016.11.004] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Concerted efforts are realizing cell-based therapy for Parkinson's disease (PD). In this chapter, I describe efforts at the Center for iPS Cell Research and Application (CiRA), Kyoto University. These efforts use induced pluripotent stem cells (iPSCs) as donor cells. The iPSCs were established as human leukocyte antigen homozygous at CiRA and are intended for allogeneic transplantation. Our manufacturing protocol includes a feeder-free cell culture with laminin fragment LM511-E8 and the sorting of CORIN+ cells. Animal experiments, including those with monkey PD models, proved that the grafted cells survive and function as dopaminergic neurons in the brain without forming any tumors. Furthermore, I emphasize that not only the donor cells but also the host brain environment is critical for successful transplantation. To achieve optimization of the host environment, drug administration, gene modification, and rehabilitation are recommended. Based on these results, researchers plan to start a clinical trial at Kyoto University Hospital in the near future.
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Affiliation(s)
- Jun Takahashi
- Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
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12
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Zhao Z, Ma Y, Chen Z, Liu Q, Li Q, Kong D, Yuan K, Hu L, Wang T, Chen X, Peng Y, Jiang W, Yu Y, Liu X. Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells. Front Cell Neurosci 2016; 10:291. [PMID: 28066186 PMCID: PMC5168467 DOI: 10.3389/fncel.2016.00291] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2016] [Accepted: 12/05/2016] [Indexed: 01/30/2023] Open
Abstract
Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.
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Affiliation(s)
- Zhenqiang Zhao
- Department of Neurology, Jinling Hospital, Southern Medical UniversityNanjing, China; Department of Neurology, First Affiliated Hospital, Hainan Medical UniversityHaikou, China
| | - Yanlin Ma
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical UniversityGuangzhou, China; Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, First Affiliated Hospital, Hainan Medical UniversityHaikou, China
| | - Zhibin Chen
- Department of Neurology, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Qian Liu
- Department of Neurology, Jinling Hospital, Southern Medical University Nanjing, China
| | - Qi Li
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Deyan Kong
- Department of Neurology, Jinling Hospital, Southern Medical UniversityNanjing, China; Department of Neurology, Affiliated Ruikang Hospital, Guangxi Traditional Chinese Medical UniversityNanning, China
| | - Kunxiong Yuan
- Department of Neurology, Jinling Hospital, Southern Medical UniversityNanjing, China; Department of Neurology, Central HospitalShenzhen, China
| | - Lan Hu
- Department of Laboratory Medicines, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Tan Wang
- Department of Neurology, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Xiaowu Chen
- Department of Neurology, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Yanan Peng
- Department of Neurology, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Weimin Jiang
- Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, First Affiliated Hospital, Hainan Medical University Haikou, China
| | - Yanhong Yu
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University Guangzhou, China
| | - Xinfeng Liu
- Department of Neurology, Jinling Hospital, Southern Medical University Nanjing, China
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Nishimura K, Doi D, Samata B, Murayama S, Tahara T, Onoe H, Takahashi J. Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5β1. Stem Cell Reports 2016; 6:511-524. [PMID: 26997644 PMCID: PMC4834042 DOI: 10.1016/j.stemcr.2016.02.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Revised: 02/11/2016] [Accepted: 02/12/2016] [Indexed: 12/11/2022] Open
Abstract
For cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5β1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5β1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.
Integrin α5 is expressed in striatal neurons innervated by nigral DA neurons Administration of E2B activates integrin α5β1 in the rat striatum E2B facilitates integration of grafted iPSC-derived DA neurons into host striatum
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Affiliation(s)
- Kaneyasu Nishimura
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Daisuke Doi
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Bumpei Samata
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Shigeo Murayama
- Department of Neuropathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan
| | - Tsuyoshi Tahara
- Bio-function Imaging Team, RIKEN Center for Life Science Technologies, Kobe 650-0047, Japan
| | - Hirotaka Onoe
- Bio-function Imaging Team, RIKEN Center for Life Science Technologies, Kobe 650-0047, Japan
| | - Jun Takahashi
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
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Daadi MM, Klausner JQ, Bajar B, Goshen I, Lee-Messer C, Lee SY, Winge MCG, Ramakrishnan C, Lo M, Sun G, Deisseroth K, Steinberg GK. Optogenetic Stimulation of Neural Grafts Enhances Neurotransmission and Downregulates the Inflammatory Response in Experimental Stroke Model. Cell Transplant 2015; 25:1371-80. [PMID: 26132738 DOI: 10.3727/096368915x688533] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Compelling evidence suggests that transplantation of neural stem cells (NSCs) from multiple sources ameliorates motor deficits after stroke. However, it is currently unknown to what extent the electrophysiological activity of grafted NSC progeny participates in the improvement of motor deficits and whether excitatory phenotypes of the grafted cells are beneficial or deleterious to sensorimotor performances. To address this question, we used optogenetic tools to drive the excitatory outputs of the grafted NSCs and assess the impact on local circuitry and sensorimotor performance. We genetically engineered NSCs to express the Channelrhodopsin-2 (ChR2), a light-gated cation channel that evokes neuronal depolarization and initiation of action potentials with precise temporal control to light stimulation. To test the function of these cells in a stroke model, rats were subjected to an ischemic stroke and grafted with ChR2-NSCs. The grafted NSCs identified with a human-specific nuclear marker survived in the peri-infarct tissue and coexpressed the ChR2 transgene with the neuronal markers TuJ1 and NeuN. Gene expression analysis in stimulated versus vehicle-treated animals showed a differential upregulation of transcripts involved in neurotransmission, neuronal differentiation, regeneration, axonal guidance, and synaptic plasticity. Interestingly, genes involved in the inflammatory response were significantly downregulated. Behavioral analysis demonstrated that chronic optogenetic stimulation of the ChR2-NSCs enhanced forelimb use on the stroke-affected side and motor activity in an open field test. Together these data suggest that excitatory stimulation of grafted NSCs elicits beneficial effects in experimental stroke model through cell replacement and non-cell replacement, anti-inflammatory/neurotrophic effects.
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Affiliation(s)
- Marcel M Daadi
- Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA
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15
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Grealish S, Heuer A, Cardoso T, Kirkeby A, Jönsson M, Johansson J, Björklund A, Jakobsson J, Parmar M. Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons. Stem Cell Reports 2015; 4:975-83. [PMID: 26004633 PMCID: PMC4471831 DOI: 10.1016/j.stemcr.2015.04.011] [Citation(s) in RCA: 83] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2014] [Revised: 04/22/2015] [Accepted: 04/22/2015] [Indexed: 01/19/2023] Open
Abstract
Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson’s disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models.
Monosynaptic tracing is used to uncover circuit integration of hESC-derived neurons Afferent and efferent synaptic contacts of grafted hESC-derived neurons are revealed Network integration in a rat model of Parkinson’s disease is stable for 6 months
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Affiliation(s)
- Shane Grealish
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Andreas Heuer
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Tiago Cardoso
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Agnete Kirkeby
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Marie Jönsson
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden
| | - Jenny Johansson
- Department of Experimental Medical Science, Molecular Neurogenetics, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Anders Björklund
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Johan Jakobsson
- Lund Stem Cell Center, Lund University, 22184 Lund, Sweden; Department of Experimental Medical Science, Molecular Neurogenetics, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden
| | - Malin Parmar
- Department of Experimental Medical Science, Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden.
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Thompson LH, Björklund A. Reconstruction of brain circuitry by neural transplants generated from pluripotent stem cells. Neurobiol Dis 2015; 79:28-40. [PMID: 25913029 DOI: 10.1016/j.nbd.2015.04.003] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Revised: 04/09/2015] [Accepted: 04/15/2015] [Indexed: 12/15/2022] Open
Abstract
Pluripotent stem cells (embryonic stem cells, ESCs, and induced pluripotent stem cells, iPSCs) have the capacity to generate neural progenitors that are intrinsically patterned to undergo differentiation into specific neuronal subtypes and express in vivo properties that match the ones formed during normal embryonic development. Remarkable progress has been made in this field during recent years thanks to the development of more refined protocols for the generation of transplantable neuronal progenitors from pluripotent stem cells, and the access to new tools for tracing of neuronal connectivity and assessment of integration and function of grafted neurons. Recent studies in brains of neonatal mice or rats, as well as in rodent models of brain or spinal cord damage, have shown that ESC- or iPSC-derived neural progenitors can be made to survive and differentiate after transplantation, and that they possess a remarkable capacity to extend axons over long distances and become functionally integrated into host neural circuitry. Here, we summarize these recent developments in the perspective of earlier studies using intracerebral and intraspinal transplants of primary neurons derived from fetal brain, with special focus on the ability of human ESC- and iPSC-derived progenitors to reconstruct damaged neural circuitry in cortex, hippocampus, the nigrostriatal system and the spinal cord, and we discuss the intrinsic and extrinsic factors that determine the growth properties of the grafted neurons and their capacity to establish target-specific long-distance axonal connections in the damaged host brain.
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Affiliation(s)
- Lachlan H Thompson
- Florey Institute for Neuroscience and Mental Health, University of Melbourne, Royal Parade, Parkville, Victoria 3010, Australia
| | - Anders Björklund
- Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, S-22184 Lund, Sweden.
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Optogenetics enables functional analysis of human embryonic stem cell-derived grafts in a Parkinson's disease model. Nat Biotechnol 2015; 33:204-9. [PMID: 25580598 DOI: 10.1038/nbt.3124] [Citation(s) in RCA: 227] [Impact Index Per Article: 22.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2014] [Accepted: 12/15/2014] [Indexed: 12/12/2022]
Abstract
Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease. However, little is known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.
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Hohmann M, Rumpel R, Fischer M, Donert M, Ratzka A, Klein A, Wesemann M, Effenberg A, Fahlke C, Grothe C. Electrophysiological Characterization of eGFP-Labeled Intrastriatal Dopamine Grafts. Cell Transplant 2014; 24:1451-67. [PMID: 25199117 DOI: 10.3727/096368914x683034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Substitution of degenerated dopaminergic (DA) neurons by intrastriatally transplanted ventral mesencephalon (VM)-derived progenitor cells has been shown to improve motor functions in parkinsonian patients and animal models, whereas investigations of electrophysiological properties of the grafted DA neurons have been rarely performed. Here we show electrophysiological properties of grafted VM progenitor cells at different time intervals up to 12 weeks after transplantation measured in acute brain slices using eGFP-Flag transfection to identify the graft. We were able to classify typical DA neurons according to the biphasic progression (voltage "sag") to hyperpolarizing current injections. Two types of DA-like neurons were classified. Whereas type 1 neurons were characterized by delayed action potentials after hyperpolarization and irregular spontaneous firing, type 2 neurons displayed burst firing after hyperpolarization, spontaneous bursts, and regular firing. Comparison to identified DA neurons in vitro indicates a high integration of the intrastriatally grafted neurons, since in vitro cultures displayed regular firing spontaneously, whereas grafted identified DA neurons showed irregular firing. Additionally, type 1 and type 2 neurons exhibited a slight increase in the spontaneous firing frequency over time intervals after grafting, which might reflect a progressive integration of the grafted DA neurons. Our results provide evidence of the differentiation of grafted VM progenitor cells into mature integrated DA neurons, which are shown to replace the missing DA neurons functionally early after grafting.
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Affiliation(s)
- Meltem Hohmann
- Institute of Neuroanatomy, Hannover Medical School, Hannover, Germany
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Tønnesen J, Kokaia M. Electrophysiological investigations of synaptic connectivity between host and graft neurons. PROGRESS IN BRAIN RESEARCH 2013. [PMID: 23195416 DOI: 10.1016/b978-0-444-59575-1.00005-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/21/2023]
Abstract
The functional synaptic integration of grafted stem cell-derived neurons is one of the key aspects of neural cell replacement therapies for neurological disorders such as Parkinson's disease. However, little is currently known about the synaptic connectivity between graft and host cells after transplantation, not only in the settings of clinical trials but also in experimental studies. This knowledge gap is primarily due to the lack of experimental electrophysiological approaches allowing interrogation of synaptic connectivity between prospectively identified host and graft neurons and hampers our understanding of the mechanisms underlying functional integration of stem cell-derived neurons in the host brain, as well as the optimization of protocols for deriving stem cells for neural cell replacement therapy. Recent optogenetic tools allow for direct investigation of connectivity between host and graft neural populations and have already been applied to show bidirectional integration of dopaminergic neurons in a host tissue. These new tools have potential to advance our understanding of functional integration in the near future. Here, we provide an overview of the current literature addressing functional integration of stem cell-derived neurons in the settings of Parkinson's disease models and discuss some experimental paradigms to approach this issue.
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Affiliation(s)
- Jan Tønnesen
- Synaptic Plasticity and Superresolution Microscopy Group, Interdisciplinary Institute for Neurosciences, Université de Bordeaux Segalen, Bordeaux, France
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20
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Survival, differentiation, and connectivity of ventral mesencephalic dopamine neurons following transplantation. PROGRESS IN BRAIN RESEARCH 2012. [DOI: 10.1016/b978-0-444-59575-1.00004-1] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
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Lepski G, Jannes CE, Wessolleck J, Kobayashi E, Nikkhah G. Equivalent neurogenic potential of wild-type and GFP-labeled fetal-derived neural progenitor cells before and after transplantation into the rodent hippocampus. Transplantation 2011; 91:390-7. [PMID: 21169879 DOI: 10.1097/tp.0b013e3182063083] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
INTRODUCTION The hippocampal formation is a specific structure in the brain where neurogenesis occurs throughout adulthood and in which the neuronal cell loss causes various demential states. The main goal of this study was to verify whether fetal neural progenitor cells (NPCs) from transgenic rats expressing green fluorescent protein (GFP) retain the ability to differentiate into neuronal cells and to integrate into the hippocampal circuitry after transplantation. METHODS NPCs were isolated from E14 (gestational age: 14 days postconception) transgenic-Lewis and wild-type Sprague-Dawley rat embryos. Wild-type and transgenic cells were expanded and induced to differentiate into a neuronal lineage in vitro. Immunocytochemical and electrophysiological analysis were performed in both groups. GFP-expressing cells were implanted into the hippocampus and recorded electrophysiologically 3 months thereafter. Immunohistochemical analysis confirmed neuronal differentiation, and the yield of neuronal cells was determined stereologically. RESULTS NPCs derived from wild-type and transgenic animals are similar regarding their ability to generate neuronal cells in vitro. Neuronal maturity was confirmed by immunocytochemistry and electrophysiology, with demonstration of voltage-gated ionic currents, firing activity, and spontaneous synaptic currents. GFP-NPCs were also able to differentiate into mature neurons after implantation into the hippocampus, where they formed functional synaptic contacts. CONCLUSIONS GFP-transgenic cells represent an important tool in transplantation studies. Herein, we demonstrate their ability to generate functional neurons both in vitro and in vivo conditions. Neurons derived from fetal NPCs were able to integrate into the normal hippocampal circuitry. The high yield of mature neurons generated render these cells important candidates for restorative approaches based on cell therapy.
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Affiliation(s)
- Guilherme Lepski
- Department of Stereotactic and Functional Neurosurgery, University of Freiburg, Freiburg, Germany.
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Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model. PLoS One 2011; 6:e17560. [PMID: 21394212 PMCID: PMC3048875 DOI: 10.1371/journal.pone.0017560] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2010] [Accepted: 01/19/2011] [Indexed: 12/11/2022] Open
Abstract
Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D2 autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.
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23
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Delayed functional maturation of human neuronal progenitor cells in vitro. Mol Cell Neurosci 2011; 47:36-44. [PMID: 21362477 DOI: 10.1016/j.mcn.2011.02.011] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2010] [Revised: 02/19/2011] [Accepted: 02/21/2011] [Indexed: 01/19/2023] Open
Abstract
INTRODUCTION Differentiation of neuronal progenitor cells (NPCs) in vitro into functional neurons is dependent on a complex cascade of molecular signaling pathways, many of which remain unknown. More specifically, in human NPCs the relationship between the expression of typical neuronal marker proteins and functional properties, such as firing action potential and synaptic transmission, is not well understood. In the present report, the immunocytochemical, morphological and electrophysiological changes that human NPCs undergo during neuronal differentiation in vitro were investigated. METHODS Human NPCs were differentiated toward a neuronal phenotype. The time course of the expression of neuronal markers and morphological cell changes was mapped and passive and active electrophysiological membrane properties assessed, throughout the neuronal maturation process. RESULTS The acquisition of neuronal markers preceded functional physiological maturation by several weeks. Cell input resistance decreased in the first 2 weeks as cells became less sensitive to input current, while cell capacitance progressively increased with continued neuronal process growth. Functional maturation was observed only by the fifth/sixth week, preceded by a marked increase in Na+ and K+ currents. In contrast, electrophysiological maturation of rodent precursor cells was observed at the end of the first week in vitro. Functionally, human neuronal cells became capable of firing action potentials and forming active synaptic contacts. Many features of the firing pattern however remained immature. CONCLUSIONS The results showed that human NPCs develop remarkably slowly and retain immature neuronal features for a prolonged period. The importance of Na-dependent activity for proper neuronal maturation is emphasized.
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Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. Proc Natl Acad Sci U S A 2009; 106:7613-8. [PMID: 19383789 DOI: 10.1073/pnas.0902396106] [Citation(s) in RCA: 163] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%-95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.
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Andres RH, Choi R, Steinberg GK, Guzman R. Potential of adult neural stem cells in stroke therapy. Regen Med 2008; 3:893-905. [DOI: 10.2217/17460751.3.6.893] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Despite state-of-the-art therapy, clinical outcome after stroke remains poor, with many patients left permanently disabled and dependent on care. Stem cell therapy has evolved as a promising new therapeutic avenue for the treatment of stroke in experimental studies, and recent clinical trials have proven its feasibility and safety in patients. Replacement of damaged cells and restoration of function can be accomplished by transplantation of different cell types, such as embryonic, fetal or adult stem cells, human fetal tissue and genetically engineered cell lines. Adult neural stem cells offer the advantage of avoiding the ethical problems associated with embryonic or fetal stem cells and can be harvested as autologous grafts from the individual patients. Furthermore, stimulation of endogenous adult stem cell-mediated repair mechanisms in the brain might offer new avenues for stroke therapy without the necessity of transplantation. However, important scientific issues need to be addressed to advance our understanding of the molecular mechanisms underlying the critical steps in cell-based repair to allow the introduction of these experimental techniques into clinical practice. This review describes up-to-date experimental concepts using adult neural stem cells for the treatment of stroke.
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Affiliation(s)
- Robert H Andres
- Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, R211, Stanford, CA 94305-5327, USA
| | - Raymond Choi
- Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, R211, Stanford, CA 94305-5327, USA
| | - Gary K Steinberg
- Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, R211, Stanford, CA 94305-5327, USA
| | - Raphael Guzman
- Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, R211, Stanford, CA 94305-5327, USA
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Parish CL, Castelo-Branco G, Rawal N, Tonnesen J, Sorensen AT, Salto C, Kokaia M, Lindvall O, Arenas E. Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice. J Clin Invest 2008; 118:149-60. [PMID: 18060047 DOI: 10.1172/jci32273] [Citation(s) in RCA: 124] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2007] [Accepted: 10/03/2007] [Indexed: 12/23/2022] Open
Abstract
Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.
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Affiliation(s)
- Clare L Parish
- Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
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Mukhida K, Hong M, Miles G, Phillips T, Baghbaderani B, McLeod M, Kobayashi N, Sen A, Behie L, Brownstone R, Mendez I. A multitarget basal ganglia dopaminergic and GABAergic transplantation strategy enhances behavioural recovery in parkinsonian rats. Brain 2008; 131:2106-26. [DOI: 10.1093/brain/awn149] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
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Korecka JA, Verhaagen J, Hol EM. Cell-replacement and gene-therapy strategies for Parkinson's and Alzheimer's disease. Regen Med 2007; 2:425-46. [PMID: 17635050 DOI: 10.2217/17460751.2.4.425] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Parkinson's disease and Alzheimer's disease are the most common neurodegenerative diseases in the elderly population. Given that age is the most important risk factor in these diseases, the number of patients is expected to rise dramatically in the coming years. Therefore, an effective therapy for these diseases is highly sought. Current treatment brings only temporary symptomatic relief and does not result in halting the progression of these diseases. The increasing knowledge on the molecular mechanisms that underlie these diseases enables the design of novel therapies, targeted at degenerating neurons by creating an optimal regenerative cellular environment. Here, we review the progress made in the field of cell-replacement and gene-therapy strategies. New developments in the application of embryonic stem cells and adult neuronal progenitors are discussed. We also discuss the use of genetically engineered cells in neuronal rescuing strategies that have recently advanced into the clinic. The first trials for the treatment of Alzheimer's disease and Parkinson's disease with this approach are ongoing.
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Affiliation(s)
- Joanna A Korecka
- Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA, Amsterdam, The Netherlands
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Brunelli S, Rovere-Querini P, Sciorati C, Manfredi AA, Clementi E. Nitric oxide: emerging concepts about its use in cell-based therapies. Expert Opin Investig Drugs 2006; 16:33-43. [PMID: 17155852 DOI: 10.1517/13543784.16.1.33] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Regenerative medicine is an emerging clinical discipline in which cell-based therapies are used to restore the functions of damaged or defective tissues and organs. Along with the well-established use of cells derived from bone marrow or pancreatic islets, novel approaches of cell therapy have recently emerged that appear particularly promising; that is, those using cell-based vaccines and stem cells. This review focuses on the recent developments of these experimental therapeutic approaches and their drawbacks, with specific focus on dendritic cell vaccines in tumours and mesoangioblasts in muscular dystrophies. The authors discuss how the unique properties of a gaseous messenger, NO, may be exploited to overcome some of the drawbacks of these cell-based approaches in combined therapies based on NO-releasing drugs and cell delivery.
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Affiliation(s)
- Silvia Brunelli
- University of Milano-Bicocca, Department of Experimental, Environmental Medicine and Medical Biotechnology, 20052 Monza, Italy
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Cesnulevicius K, Timmer M, Wesemann M, Thomas T, Barkhausen T, Grothe C. Nucleofection is the most efficient nonviral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate. Stem Cells 2006; 24:2776-91. [PMID: 16902196 DOI: 10.1634/stemcells.2006-0176] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Neuronal progenitor cells (NPCs) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, such as Parkinson disease. However, survival of transplanted cells is, as yet, limited, and the identification of grafted cells in situ remains difficult. The use of NPCs could be more effective with regard to a better survival and maturation when transfected with one or more neurotrophic factors. Therefore, we investigated the possibility of transfecting mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters enhanced green fluorescence protein (EGFP) and red fluorescent protein (DsRed). Different techniques for transfection were compared, and the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure; 6 hours after transfection, viability was approximately 40%, and the transfected cells differentiated into, for example, tyrosine hydroxylase-positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. To provide the progenitor cells with a neurotrophic factor, different isoforms of fibroblast growth factor-2 were introduced. To follow the behavior of the transfected cells in vitro, functional tests such as the cell viability assay (water-soluble tetrazolium salt assay [WST-1]) and the cell proliferation assay (5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay) were performed. In addition, these transfected NPCs were viable after transplantation, expressed tyrosine hydroxylase in vivo, and could easily be detected within the host striatum because of their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases.
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