Publications by authors named "Maeve A Caldwell"

44 Publications

A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons.

Autophagy 2020 Apr 14:1-17. Epub 2020 Apr 14.

Cell Biology Laboratories, School of Biochemistry, University of Bristol , Bristol, UK.

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions . Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.

Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.
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http://dx.doi.org/10.1080/15548627.2020.1739441DOI Listing
April 2020

Imaging Autophagy in hiPSC-Derived Midbrain Dopaminergic Neuronal Cultures for Parkinson's Disease Research.

Methods Mol Biol 2019 ;1880:257-280

Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, UK.

To appreciate the positive or negative impact of autophagy during the initiation and progression of human diseases, the isolation or de novo generation of appropriate cell types is required to support focused in vitro assays. In human neurodegenerative diseases such as Parkinson's disease (PD), specific subsets of acutely sensitive neurons become susceptible to stress-associated operational decline and eventual cell death, emphasizing the need for functional studies in those vulnerable groups of neurons. In PD, a class of dopaminergic neurons in the ventral midbrain (mDANs) is affected. To study these, human-induced pluripotent stem cells (hiPSCs) have emerged as a valuable tool, as they enable the establishment and study of mDAN biology in vitro. In this chapter, we describe a stepwise protocol for the generation of mDANs from hiPSCs using a monolayer culture system. We then outline how imaging-based autophagy assessment methodologies can be applied to these neurons, thereby providing a detailed account of the application of imaging-based autophagy assays to human iPSC-derived mDANs.
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http://dx.doi.org/10.1007/978-1-4939-8873-0_17DOI Listing
June 2019

Derivation of Neural Stem Cells from the Developing and Adult Human Brain.

Results Probl Cell Differ 2018;66:3-20

Trinity College Institute for Neuroscience, Trinity College Dublin, Dublin, Ireland.

Neural stem cells isolated from the developing and adult brain are an ideal source of cells for use in clinical applications such as cell replacement therapy. The clear advantage of these cells over the more commonly utilised embryonic and pluripotent stem cells is that they are already neurally committed. Of particular importance is the fact that these cells don't require the same level of in vitro culture that can be cost and labour intensive. Foetal neural stem cells can be readily derived from the foetal brain and expand in culture over time. Similarly, adult stem cells have been explored for their potential in vitro and in vivo animal models. In this chapter we identify the progress made in developing these cells as well as the advantages of taking them forward for clinical use.
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http://dx.doi.org/10.1007/978-3-319-93485-3_1DOI Listing
July 2019

Generation of defined neural populations from pluripotent stem cells.

Philos Trans R Soc Lond B Biol Sci 2018 07;373(1750)

Department of Physiology, Trinity College Institute for Neuroscience, Trinity College Dublin, Dublin 2, Ireland

Effective and efficient generation of human neural stem cells and subsequently functional neural populations from pluripotent stem cells has facilitated advancements in the study of human development and disease modelling. This review will discuss the established protocols for the generation of defined neural populations including regionalized neurons and astrocytes, oligodendrocytes and microglia. Early protocols were established in embryonic stem cells (ESC) but the discovery of induced pluripotent stem cells (iPSC) in 2006 provided a new platform for modelling human disorders of the central nervous system (CNS). The ability to produce patient- and disease-specific iPSC lines has created a new age of disease modelling. Human iPSC may be derived from adult somatic cells and subsequently patterned into numerous distinct cell types. The ability to derive defined and regionalized neural populations from iPSC provides a powerful model of CNS disorders.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.
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http://dx.doi.org/10.1098/rstb.2017.0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974438PMC
July 2018

Nanoparticle-induced neuronal toxicity across placental barriers is mediated by autophagy and dependent on astrocytes.

Nat Nanotechnol 2018 05 2;13(5):427-433. Epub 2018 Apr 2.

Trinity College Institute of Neuroscience, Department of Physiology, Trinity College Dublin, Dublin, Ireland.

The potential for maternal nanoparticle (NP) exposures to cause developmental toxicity in the fetus without the direct passage of NPs has previously been shown, but the mechanism remained elusive. We now demonstrate that exposure of cobalt and chromium NPs to BeWo cell barriers, an in vitro model of the human placenta, triggers impairment of the autophagic flux and release of interleukin-6. This contributes to the altered differentiation of human neural progenitor cells and DNA damage in the derived neurons and astrocytes. Crucially, neuronal DNA damage is mediated by astrocytes. Inhibiting the autophagic degradation in the BeWo barrier by overexpression of the dominant-negative human ATG4B significantly reduces the levels of DNA damage in astrocytes. In vivo, indirect NP toxicity in mice results in neurodevelopmental abnormalities with reactive astrogliosis and increased DNA damage in the fetal hippocampus. Our results demonstrate the potential importance of autophagy to elicit NP toxicity and the risk of indirect developmental neurotoxicity after maternal NP exposure.
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http://dx.doi.org/10.1038/s41565-018-0085-3DOI Listing
May 2018

Loss of MicroRNA-7 Regulation Leads to α-Synuclein Accumulation and Dopaminergic Neuronal Loss In Vivo.

Mol Ther 2017 10 31;25(10):2404-2414. Epub 2017 Aug 31.

Trinity College Institute for Neuroscience, Trinity College Dublin, Dublin 2, Ireland. Electronic address:

Abnormal alpha-synuclein (α-synuclein) expression and aggregation is a key characteristic of Parkinson's disease (PD). However, the exact mechanism(s) linking α-synuclein to the other central feature of PD, dopaminergic neuron loss, remains unclear. Therefore, improved cell and in vivo models are needed to investigate the role of α-synuclein in dopaminergic neuron loss. MicroRNA-7 (miR-7) regulates α-synuclein expression by binding to the 3' UTR of the Synuclein Alpha Non A4 Component of Amyloid Precursor (SNCA) gene and inhibiting its translation. We show that miR-7 is decreased in the substantia nigra of patients with PD and, therefore, may play an essential role in the regulation of α-synuclein expression. Furthermore, we have found that lentiviral-mediated expression of miR-7 complementary binding sites to stably induce a loss of miR-7 function results in an increase in α-synuclein expression in vitro and in vivo. We have also shown that depletion of miR-7 using a miR-decoy produces a loss of nigral dopaminergic neurons accompanied by a reduction of striatal dopamine content. These data suggest that miR-7 has an important role in the regulation of α-synuclein and dopamine physiology and may provide a new paradigm to study the pathology of PD.
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http://dx.doi.org/10.1016/j.ymthe.2017.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628933PMC
October 2017

Induced Pluripotent Stem Cell Neuronal Models for the Study of Autophagy Pathways in Human Neurodegenerative Disease.

Cells 2017 Aug 11;6(3). Epub 2017 Aug 11.

Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.

Human induced pluripotent stem cells (hiPSCs) are invaluable tools for research into the causes of diverse human diseases, and have enormous potential in the emerging field of regenerative medicine. Our ability to reprogramme patient cells to become hiPSCs, and to subsequently direct their differentiation towards those classes of neurons that are vulnerable to stress, is revealing how genetic mutations cause changes at the molecular level that drive the complex pathogeneses of human neurodegenerative diseases. Autophagy dysregulation is considered to be a major contributor in neural decline during the onset and progression of many human neurodegenerative diseases, meaning that a better understanding of the control of non-selective and selective autophagy pathways (including mitophagy) in disease-affected classes of neurons is needed. To achieve this, it is essential that the methodologies commonly used to study autophagy regulation under basal and stressed conditions in standard cell-line models are accurately applied when using hiPSC-derived neuronal cultures. Here, we discuss the roles and control of autophagy in human stem cells, and how autophagy contributes to neural differentiation in vitro. We also describe how autophagy-monitoring tools can be applied to hiPSC-derived neurons for the study of human neurodegenerative disease in vitro.
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http://dx.doi.org/10.3390/cells6030024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617970PMC
August 2017

Astrocytes in a dish: Using pluripotent stem cells to model neurodegenerative and neurodevelopmental disorders.

Brain Pathol 2017 07;27(4):530-544

Trinity College Institute for Neuroscience, Trinity College Dublin 2, Ireland.

Neuroscience and Neurobiology have historically been neuron biased, yet up to 40% of the cells in the brain are astrocytes. These cells are heterogeneous and regionally diverse but universally essential for brain homeostasis. Astrocytes regulate synaptic transmission as part of the tripartite synapse, provide metabolic and neurotrophic support, recycle neurotransmitters, modulate blood flow and brain blood barrier permeability and are implicated in the mechanisms of neurodegeneration. Using pluripotent stem cells (PSC), it is now possible to study regionalised human astrocytes in a dish and to model their contribution to neurodevelopmental and neurodegenerative disorders. The evidence challenging the traditional neuron-centric view of degeneration within the CNS is reviewed here, with focus on recent findings and disease phenotypes from human PSC-derived astrocytes. In addition we compare current protocols for the generation of regionalised astrocytes and how these can be further refined by our growing knowledge of neurodevelopment. We conclude by proposing a functional and phenotypical characterisation of PSC-derived astrocytic cultures that is critical for reproducible and robust disease modelling.
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http://dx.doi.org/10.1111/bpa.12522DOI Listing
July 2017

Parkinson's disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes.

Nat Med 2016 Jan 30;22(1):54-63. Epub 2015 Nov 30.

Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.

Mitochondrial dysfunction represents a critical step during the pathogenesis of Parkinson's disease (PD), and increasing evidence suggests abnormal mitochondrial dynamics and quality control as important underlying mechanisms. The VPS35 gene, which encodes a key component of the membrane protein-recycling retromer complex, is the third autosomal-dominant gene associated with PD. However, how VPS35 mutations lead to neurodegeneration remains unclear. Here we demonstrate that PD-associated VPS35 mutations caused mitochondrial fragmentation and cell death in cultured neurons in vitro, in mouse substantia nigra neurons in vivo and in human fibroblasts from an individual with PD who has the VPS35(D620N) mutation. VPS35-induced mitochondrial deficits and neuronal dysfunction could be prevented by inhibition of mitochondrial fission. VPS35 mutants showed increased interaction with dynamin-like protein (DLP) 1, which enhanced turnover of the mitochondrial DLP1 complexes via the mitochondria-derived vesicle-dependent trafficking of the complexes to lysosomes for degradation. Notably, oxidative stress increased the VPS35-DLP1 interaction, which we also found to be increased in the brains of sporadic PD cases. These results revealed a novel cellular mechanism for the involvement of VPS35 in mitochondrial fission, dysregulation of which is probably involved in the pathogenesis of familial, and possibly sporadic, PD.
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http://dx.doi.org/10.1038/nm.3983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826611PMC
January 2016

Long-term culture of pluripotent stem-cell-derived human neurons on diamond--A substrate for neurodegeneration research and therapy.

Biomaterials 2015 Aug 20;61:139-49. Epub 2015 May 20.

Regenerative Medicine Laboratory, School of Clinical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom; Trinity College Institute of Neuroscience, Lloyd Institute, Trinity College, Dublin 2, Ireland. Electronic address:

Brain Computer Interfaces (BCI) currently represent a field of intense research aimed both at understanding neural circuit physiology and at providing functional therapy for traumatic or degenerative neurological conditions. Due to its chemical inertness, biocompatibility and stability, diamond is currently being actively investigated as a potential substrate material for culturing cells and for use as the electrically active component of a neural sensor. Here we provide a protocol for the differentiation of mature, electrically active neurons on microcrystalline synthetic thin-film diamond substrates starting from undifferentiated pluripotent stem cells. Furthermore, we investigate the optimal characteristics of the diamond microstructure for long-term neuronal sustainability. We also analyze the effect of boron as a dopant for such a culture. We found that the diamond crystalline structure has a significant influence on the neuronal culture unlike the boron doping. Specifically, small diamond microcrystals promote higher neurite density formation. We find that boron incorporated into the diamond does not influence the neurite density and has no deleterious effect on cell survival.
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http://dx.doi.org/10.1016/j.biomaterials.2015.04.050DOI Listing
August 2015

A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration.

Neurogenetics 2015 Jul 17;16(3):215-221. Epub 2015 Apr 17.

Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center Jerusalem, Jerusalem, Israel.

The composition of the neuronal cell surface dictates synaptic plasticity and thereby cognitive development. This remodeling of the synapses is governed by the endocytic network which internalize transmembrane proteins, then sort them back to the cell surface or carry them to the lysosome for degradation. The multi-protein retromer complex is central to this selection, capturing specific transmembrane proteins and remodeling the cell membrane to form isolated cargo-enriched transport carriers. We investigated a consanguineous family with four patients who presented in infancy with intractable myoclonic epilepsy and lack of psychomotor development. Using exome analysis, we identified a homozygous deleterious mutation in SNX27, which encodes sorting nexin 27, a retromer cargo adaptor. In western analysis of patient fibroblasts, the encoded mutant protein was expressed at an undetectable level when compared with a control sample. The patients' presentation and clinical course recapitulate that reported for the SNX27 knock-out mouse. Since the cargo proteins for SNX27-mediated sorting include subunits of ionotropic glutamate receptors and endosome-to-cell surface synaptic insertion of AMPA receptors is severely perturbed in SNX27(-/-) neurons, it is proposed that at least part of the neurological aberrations observed in the patients is attributed to defective sorting of ionotropic glutamate receptors. SNX27 deficiency is now added to the growing list of neurodegenerative disorders associated with retromer dysfunction.
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http://dx.doi.org/10.1007/s10048-015-0446-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962907PMC
July 2015

Enhanced efficacy of the CDNF/MANF family by combined intranigral overexpression in the 6-OHDA rat model of Parkinson's disease.

Mol Ther 2015 Feb 5;23(2):244-54. Epub 2014 Nov 5.

1] School of Clinical Sciences, Medical Sciences Building, University Walk, Bristol, UK [2] Regenerative Medicine Laboratory, Medical Sciences Building, University Walk, Bristol, UK.

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.
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http://dx.doi.org/10.1038/mt.2014.206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445614PMC
February 2015

Retromer Binding to FAM21 and the WASH Complex Is Perturbed by the Parkinson Disease-Linked VPS35(D620N) Mutation.

Curr Biol 2014 Jul 21;24(14):1678. Epub 2014 Jul 21.

The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK. Electronic address:

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http://dx.doi.org/10.1016/j.cub.2014.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628949PMC
July 2014

Retromer binding to FAM21 and the WASH complex is perturbed by the Parkinson disease-linked VPS35(D620N) mutation.

Curr Biol 2014 Jul 3;24(14):1670-1676. Epub 2014 Jul 3.

The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK. Electronic address:

Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.
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http://dx.doi.org/10.1016/j.cub.2014.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110399PMC
July 2014

Galanin promotes neuronal differentiation from neural progenitor cells in vitro and contributes to the generation of new olfactory neurons in the adult mouse brain.

Exp Neurol 2014 Jun 12;256:93-104. Epub 2014 Apr 12.

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, School of Clinical Sciences, University of Bristol, BS1 3NY, UK; School of Clinical Sciences, University of Bristol, Medical Sciences Building, University Walk, BS8 1TD, UK. Electronic address:

Galanin is a pleiotropic neuropeptide widely expressed in the nervous system. It plays a role in many diverse physiological functions - including nociception, cognition and metabolism regulation - and acts as neurotrophic/neuroprotective factor for several neuronal populations. In this article we sought to determine the role of galanin on neural stem cell function and its contribution to the plasticity of the nervous system. Here we show that galanin and its receptors are expressed in neural progenitor cells (NPCs) isolated from the developing striatum. Stimulation with galanin results in upregulation of Bcl-Xl, Bcl-2, Mash-1 and Olig-2 that are part of well known pro-survival/pro-neuronal signalling pathways. Accordingly, treatment with galanin increases the number of neurons upon differentiation from these progenitors. We then show that these effects are recapitulated in NPCs isolated from the adult subventricular zone (SVZ), where galanin increases the total number of neurons and the number of newly-generated neurons upon differentiation in vitro. The significance of these findings is highlighted in the adult brain where loss of galanin leads to a marked decrease in the rate of adult SVZ neurogenesis and a reduction in the number of newly generated cells in the olfactory bulb. Interestingly, Gal-KO mice display normal performances in simple tasks of olfactory detection and discrimination, which points to the existence of a certain degree of redundancy in SVZ neurogenesis. Our findings establish the role of galanin as a modulator of neural stem cell function and support the importance of galanin for brain plasticity and repair.
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http://dx.doi.org/10.1016/j.expneurol.2014.04.001DOI Listing
June 2014

Modeling astrocytic contribution toward neurodegeneration with pluripotent stem cells: focus on Alzheimer's and Parkinson's diseases.

Neuroreport 2013 Dec;24(18):1053-7

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Bristol, UK.

Astrocytes are an exceptionally complex type of cell observed throughout the brain and the spinal cord. They reveal heterogeneous identities and largely participate in brain activity. In this article we review astrocyte heterogeneity and function in the brain. We have also focused on their involvement in neurodegenerative diseases including Parkinson's and Alzheimer's diseases, discussing the contribution of this type of glial cell toward mechanisms that can lead to neuronal death, which was previously attributed exclusively to neurons. Furthermore, we also highlight the latest progress with stem cell technology for the generation of astrocytes from embryonic and induced pluripotent stem cells. We allude to the need for developing new models that better recapitulate astrocyte heterogeneity and their diverse function within the brain. Those models will greatly contribute in understanding astrocytes' contribution toward molecular mechanisms occurring in neurodegenerative pathologies affecting specific brain regions such as in Parkinson's and Alzheimer's diseases.
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http://dx.doi.org/10.1097/WNR.0000000000000064DOI Listing
December 2013

Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling.

Stem Cell Res 2013 Nov 9;11(3):1206-21. Epub 2013 Aug 9.

Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK.

Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75(NTR), the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development.
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http://dx.doi.org/10.1016/j.scr.2013.08.002DOI Listing
November 2013

Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts.

Regen Med 2012 Sep;7(5):675-83

Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

Aim: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate.

Materials & Methods: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A.

Results: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers.

Conclusion: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.
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http://dx.doi.org/10.2217/rme.12.55DOI Listing
September 2012

Quantitative evaluation of the human subventricular zone.

Brain 2012 Aug 25;135(Pt 8):e221, 1-4; author reply e222, 1-6. Epub 2012 Apr 25.

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http://dx.doi.org/10.1093/brain/aws087DOI Listing
August 2012

Connexin 36 expression regulates neuronal differentiation from neural progenitor cells.

PLoS One 2011 Mar 9;6(3):e14746. Epub 2011 Mar 9.

Henry Wellcome Laboratories for Integrative Neuroscience & Endocrinology, University of Bristol, Bristol, United Kingdom.

Background: Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate inter- and intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development.

Methodology/principal Findings: To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the number of neurons derived from the transduced neurospheres. The number of oligodendrocytes was also significantly increased following transduction with Cx36 indicating they may support neuronal differentiation.

Conclusions/significance: Our data suggests that astrocytic and neuronal differentiation during development are governed by mechanisms that include the differential expression of Cx36.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014746PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052311PMC
March 2011

FGF but not EGF induces phosphorylation of the cAMP response element binding protein in olfactory mucosa-derived cell cultures.

Exp Cell Res 2010 May 6;316(9):1489-99. Epub 2010 Mar 6.

MRC Cambridge Centre for Stem Cell Biology and Regenerative Medicine, Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.

The stem/progenitor cells of the olfactory epithelium are potentially useful cells for autologous cell-based therapy because of their relative accessibility compared to other sources of neural stem cells. However, they have very limited potential to self-renew in vitro under growth factor stimulation compared to central nervous system-derived stem/progenitor cells. Using a sphere-forming assay and immunocytochemistry to identify cells that contained phosphorylated cAMP response element binding protein (pCREB) as an indicator of cell responsiveness to growth factor activation, we found that olfactory-spheres primed with FGF2 responded to FGF2 and EGF stimulation. In contrast, olfactory-spheres primed with EGF failed to respond to FGF2 or EGF stimulation despite the detection of FGFR1 and EGFR and their transcripts. These data demonstrate that FGF2 but not EGF permit the maintenance of a subset of cells responsive to FGF2 and EGF, whereas EGF induces unresponsive to either growth factor possibly via intrinsic mechanisms of regulation.
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http://dx.doi.org/10.1016/j.yexcr.2010.03.001DOI Listing
May 2010

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase.

Mutat Res 2010 Jan;683(1-2):1-8

Bristol Implant Research Centre, University of Bristol, Bristol, UK.

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.
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http://dx.doi.org/10.1016/j.mrfmmm.2009.09.012DOI Listing
January 2010

Dopaminergic modulation of neurogenesis in the subventricular zone of the adult brain.

Cell Cycle 2009 Sep 14;8(18):2888-94. Epub 2009 Sep 14.

Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.

Dopaminergic receptors are expressed on neural precursor cells (NPCs) in the subventricular zone (SVZ) and are known to regulate NPC proliferation and differentiation fate in this region. We now report that this optimally requires the simultaneous activation of both D1-like and D2-like dopaminergic receptors with the agonists Bromocriptine, SKF-38393 and 7-OH-pipat maleate (BSP) in vitro. This is consistent with our previous findings that dopamine stimulates NPC proliferation through an EGF paracrine mechanism within the SVZ. Furthermore this combined dopamine agonist therapy rescues NPC proliferation in the SVZ in the 6-OHDA animal model of PD and importantly significantly increases neuronal differentiation in the olfactory bulb to a greater extent than we showed previously with levodopa. This result has implications for the use of dopaminergic therapies in PD and in the development of such therapies focusing on upregulating SVZ neurogenesis.
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http://dx.doi.org/10.4161/cc.8.18.9512DOI Listing
September 2009

Dopamine-induced proliferation of adult neural precursor cells in the mammalian subventricular zone is mediated through EGF.

Proc Natl Acad Sci U S A 2009 May 11;106(21):8754-9. Epub 2009 May 11.

Department of Clinical Neurosciences, Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, United Kingdom.

A reduction in dopaminergic innervation of the subventricular zone (SVZ) is responsible for the impaired proliferation of its resident precursor cells in this region in Parkinson's disease (PD). Here, we show that this effect involves EGF, but not FGF2. In particular, we demonstrate that dopamine increases the proliferation of SVZ-derived cells by releasing EGF in a PKC-dependent manner in vitro and that activation of the EGF receptor (EGFR) is required for this effect. We also show that dopamine selectively expands the GFAP(+) multipotent stem cell population in vitro by promoting their self-renewal. Furthermore, in vivo dopamine depletion leads to a decrease in precursor cell proliferation in the SVZ concomitant with a reduction in local EGF production, which is reversed through the administration of the dopamine precursor levodopa (L-DOPA). Finally, we show that EGFR(+) cells are depleted in the SVZ of human PD patients compared with age-matched controls. We have therefore demonstrated a unique role for EGF as a mediator of dopamine-induced precursor cell proliferation in the SVZ, which has potential implications for future therapies in PD.
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http://dx.doi.org/10.1073/pnas.0803955106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689002PMC
May 2009

Graft outcomes influenced by co-expression of Pax7 in graft and host tissue.

J Anat 2009 Mar;214(3):396-405

School of Biomedical Science, Edith Cowan University, Joondalup Drive, Western Australia, Australia.

Cell replacement therapies offer promise in the treatment of neurotrauma and neurodegenerative disorders and have concentrated on the use of primary fetal brain tissue. However, there is a growing promise of using neural stem cells, in which case other factors may be important in their successful engraftment. We therefore investigated whether the co-expression of the major developmental transcription factor (Pax7 in this study) of donor tissue to graft site influences transplant survival and differentiation in the rat midbrain. Neural progenitor cells were prepared from either the Pax7-expressing dorsal (DM) or non-Pax7-expressing ventral mesencephalon (VM) of embryonic EGFP(+/+) rats. Cells were dissociated and grafted into the adult rat superior colliculus (SC) lesioned with quinolinic acid 3 days previously, a time shown to be associated with the up-regulation of Pax7. Grafts were then examined 4 weeks later. Our results suggest the origin of the graft tissue did not alter graft survival in the SC; however, dorsal grafts appear to have a higher incidence of neuronal survival, whereas ventral grafts have a higher incidence of astrocytic survivors.
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http://dx.doi.org/10.1111/j.1469-7580.2009.01049.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673790PMC
March 2009

Twist-1 regulates the miR-199a/214 cluster during development.

Nucleic Acids Res 2009 Jan 23;37(1):123-8. Epub 2008 Nov 23.

The Henry-Wellcome Laboratories for Integrated Neuroscience and Endocrinology, University of Bristol, Bristol, UK.

MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations.
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http://dx.doi.org/10.1093/nar/gkn920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615617PMC
January 2009

Laminin enhances the growth of human neural stem cells in defined culture media.

BMC Neurosci 2008 Jul 23;9:71. Epub 2008 Jul 23.

Department of Pathology, University of Cambridge, Cambridge, UK.

Background: Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth.

Results: To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin beta1-dependent manner.

Conclusion: The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production.
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http://dx.doi.org/10.1186/1471-2202-9-71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2496909PMC
July 2008

Secreted factors from olfactory mucosa cells expanded as free-floating spheres increase neurogenesis in olfactory bulb neurosphere cultures.

BMC Neurosci 2008 Feb 18;9:24. Epub 2008 Feb 18.

Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.

Background: The olfactory epithelium is a neurogenic tissue comprising a population of olfactory receptor neurons that are renewed throughout adulthood by a population of stem and progenitor cells. Because of their relative accessibility compared to intra-cranially located neural stem/progenitor cells, olfactory epithelium stem and progenitor cells make attractive candidates for autologous cell-based therapy. However, olfactory stem and progenitor cells expand very slowly when grown as free-floating spheres (olfactory-spheres) under growth factor stimulation in a neurosphere assay.

Results: In order to address whether olfactory mucosa cells extrinsically regulate proliferation and/or differentiation of immature neural cells, we cultured neural progenitor cells derived from mouse neonatal olfactory bulb or subventricular zone (SVZ) in the presence of medium conditioned by olfactory mucosa-derived spheres (olfactory-spheres). Our data demonstrated that olfactory mucosa cells produced soluble factors that affect bulbar neural progenitor cell differentiation but not their proliferation when compared to control media. In addition, olfactory mucosa derived soluble factors increased neurogenesis, especially favouring the generation of non-GABAergic neurons. Olfactory mucosa conditioned medium also contained several factors with neurotrophic/neuroprotective properties. Olfactory-sphere conditioned medium did not affect proliferation or differentiation of SVZ-derived neural progenitors.

Conclusion: These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the olfactory epithelial stem/progenitor cell population.
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http://dx.doi.org/10.1186/1471-2202-9-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275736PMC
February 2008

Induction of A9 dopaminergic neurons from neural stem cells improves motor function in an animal model of Parkinson's disease.

Brain 2008 Mar 17;131(Pt 3):630-41. Epub 2008 Jan 17.

Cambridge Center for Brain Repair and Department of Clinical Neurosciences, Forvie Site, Robinsons Way, Cambridge CB2 2PY, UK.

Neural stem cells (NSCs) are widely endorsed as a cell source for replacement strategies in neurodegenerative disease. However, their usefulness is currently limited by the inability to induce specific neurotransmitter phenotypes in these cells. In order to direct dopaminergic neuronal fate, we overexpressed Pitx3 in NSCs that were then exposed to E11 developing ventral mesencephalon (VM) in explant culture. This resulted in a significant potentiation of dopaminergic differentiation of the cells. When transplanted into the 6-hydroxydopamine lesioned Parkinsonian rats, these cografts of VM and Pitx3 overexpressing NSCs resulted in a significant restitution of motor function. In addition, there were greater numbers of Girk2 positive A9 neurons in the periphery of the transplants that were NSC derived. This demonstrates that given the correct signals, NSCs can be induced to become dopaminergic neurons that can differentiate into the correct nigrastriatal phenotype required for the treatment of Parkinson's disease.
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http://dx.doi.org/10.1093/brain/awm340DOI Listing
March 2008

Contrasting effects of basic fibroblast growth factor and epidermal growth factor on mouse neonatal olfactory mucosa cells.

Eur J Neurosci 2007 Dec;26(12):3345-57

Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) affect proliferation and survival of many cell types, but their role in the maintenance of olfactory mucosa cells remains unclear. In the neonatal mouse olfactory mucosa, cell proliferation mainly occurs in the neuroepithelium and, to a lesser extent, in the lamina propria. To establish whether bFGF and EGF affect proliferation and/or survival of these cells, we isolated olfactory mucosa cells from the neonatal mouse and cultured them as free-floating spheres under bFGF or EGF stimulation. Our data demonstrate that bFGF is a mitogen for the rapidly dividing cells (olfactory neuronal precursors and olfactory ensheathing cells), and also a survival factor for both slowly and rapidly dividing cells of the olfactory mucosa. In contrast, EGF appears to be primarily a survival factor for both the olfactory stem and precursor cells.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05950.xDOI Listing
December 2007