Mirella Dottori

NXY-059, a Failed Stroke Neuroprotectant, Offers No Protection to Stem Cell-Derived Human Neurons

Publisher: Elsevier BV

Date: 08-2018

DOI: 10.1016/J.JSTROKECEREBROVASDIS.2018.03.015

Abstract: Developing new medicines is a complex process where understanding the reasons for both failure and success takes us forward. One gap in our understanding of most candidate stroke drugs before clinical trial is whether they have a protective effect on human tissues. NXY-059 is a spin-trap reagent hypothesized to have activity against the damaging oxidative biology which accompanies ischemic stroke. Re-examination of the preclinical in vivo dataset for this agent in the wake of the failed SAINT-II RCT highlighted the presence of a range of biases leading to overestimation of the magnitude of NXY-059's effects in laboratory animals. Therefore, NXY-059 seemed an ideal candidate to evaluate in human neural tissues to determine whether human tissue testing might improve screening efficiency. The aim of this randomized and blinded study was to assess the effects of NXY-059 on human stem cell-derived neurons in the presence of ischemia-like injury induced by oxygen glucose deprivation or oxidative stress induced by hydrogen peroxide or sodium nitroprusside. In MTT assays of cell survival, lactate dehydrogenase assays of total cell death and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic-like cell death, NXY-059 at concentrations ranging from 1 µm to 1 mm was completely without activity. Conversely an antioxidant cocktail comprising 100 µm each of ascorbate, reduced glutathione, and dithiothreitol used as a positive control provided marked neuronal protection in these assays. These findings support our hypothesis that stroke drug screening in human neural tissues will be of value and provides an explanation for the failure of NXY-059 as a human stroke drug.

Self-Organized Nanostructure Modified Microelectrode for Sensitive Electrochemical Glutamate Detection in Stem Cells-Derived Brain Organoids

Publisher: MDPI AG

Date: 05-02-2018

DOI: 10.3390/BIOS8010014

Phenotypic and Functional Characterization of Peripheral Sensory Neurons derived from Human Embryonic Stem Cells.

Publisher: Springer Science and Business Media LLC

Date: 12-01-2018

DOI: 10.1038/S41598-017-19093-0

Abstract: The dorsal root ganglia (DRG) consist of a multitude of sensory neuronal subtypes that function to relay sensory stimuli, including temperature, pressure, pain and position to the central nervous system. Our knowledge of DRG sensory neurons have been predominantly driven by animal studies and considerably less is known about the human DRG. Human embryonic stem cells (hESC) are valuable resource to help close this gap. Our previous studies reported an efficient system for deriving neural crest and DRG sensory neurons from hESC. Here we show that this differentiation system gives rise to heterogeneous populations of sensory neuronal subtypes as demonstrated by phenotypic and functional analyses. Furthermore, using microelectrode arrays the maturation rate of the hESC-derived sensory neuronal cultures was monitored over 8 weeks in culture, showing their spontaneous firing activities starting at about 12 days post-differentiation and reaching maximum firing at about 6 weeks. These studies are highly valuable for developing an in vitro platform to study the ersity of sensory neuronal subtypes found within the human DRG.

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Publisher: Oxford University Press (OUP)

Date: 18-03-2021

DOI: 10.1002/SCTM.20-0334

Abstract: Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.

Glycogen Synthase Kinase 3β and Activin/Nodal Inhibition in Human Embryonic Stem Cells Induces a Pre‐Neuroepithelial State That Is Required for Specification to a Floor Plate Cell Lineage

Publisher: Oxford University Press (OUP)

Date: 22-10-2012

DOI: 10.1002/STEM.1204

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Publisher: SAGE Publications

Date: 13-04-2020

DOI: 10.1177/1073858420912404

Abstract: Because our beliefs regarding our in iduality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges lified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.”

Generation of Adrenal Chromaffin-like Cells from Human Pluripotent Stem Cells

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.STEMCR.2017.11.003

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics

Publisher: Cold Spring Harbor Laboratory

Date: 22-05-2023

DOI: 10.1101/2023.05.19.541551

Abstract: Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is currently no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging and time consuming it is imperative to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question, we cultured DIPG cells with cortical organoids. We created “mosaic” co-cultures enriched for tumour cell-neuronal cell interactions versus “assembloid” co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This revealed that tumour cell adhesion was reduced, and DNA synthesis and replication were increased, in DIPG cells under either co-culture condition. By contrast, the mosaic co-culture was associated with pathways implicated in dendrite growth. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Generation of Human Embryonic Stem Cell Reporter Knock‐In Lines by Homologous Recombination

Publisher: Wiley

Date: 11-2009

DOI: 10.1002/9780470151808.SC05B01S11

Abstract: This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock‐in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single‐cell deposition flow cytometry. Reporter knock‐in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives. Curr. Protoc. Stem Cell Biol . 11:5B.1.1‐5B.1.34. © 2009 by John Wiley & Sons, Inc.

Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: A systematic analysis for the Global Burden of Disease Study 2013

Publisher: Elsevier BV

Date: 2015

DOI: 10.1016/S0140-6736(14)61682-2

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells

Publisher: Springer Science and Business Media LLC

Date: 04-2011

DOI: 10.1186/SCRT57

Graphene foam as a biocompatible scaffold for culturing human neurons

Publisher: The Royal Society

Date: 03-2018

DOI: 10.1098/RSOS.171364

Abstract: In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation.

Comparison of Transplant Efficiency between Spontaneously Derived and Noggin-Primed Human Embryonic Stem Cell Neural Precursors in the Quinolinic Acid Rat Model of Huntington's Disease

Publisher: SAGE Publications

Date: 08-2010

DOI: 10.3727/096368910X494632

Abstract: Human neural precursors (hNP) derived from embryonic stem cells (hESC) may provide a viable cellular source for transplantation therapy for Huntington's disease (HD). However, developing effective transplantation therapy for the central nervous system (CNS) using hESC relies on optimizing the in vitro production of hNP to control appropriate in vivo posttransplantation neuronal differentiation. The current study provides the first direct in vivo comparison of the transplant efficiency and posttransplantation characteristics of spontaneously derived and noggin-primed hNP following transplantation into the quinolinic acid (QA) rat model of HD. We show that spontaneously derived and noggin-primed hNP both survived robustly up to 8 weeks after transplantation into the QA-lesioned striatum of the adult rat. Transplanted hNP underwent extensive migration and large-scale differentiation towards a predominantly neuronal fate by 8 weeks posttransplantation. Furthermore, in vitro noggin priming of hNP specifically increased the extent of neuronal differentiation at both 4 and 8 weeks posttransplantation when compared to spontaneously derived hNP grafts. The results of this study suggest that in vitro noggin priming provides an effective mechanism by which to enhance hNP transplant efficiency for the treatment of HD.

Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation.

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.STEMCR.2021.11.015

More than a Corepressor: The Role of CoREST Proteins in Neurodevelopment

Publisher: Society for Neuroscience

Date: 19-02-2020

DOI: 10.1523/ENEURO.0337-19.2020

Abstract: The molecular mechanisms governing normal neurodevelopment are tightly regulated by the action of transcription factors. Repressor element 1 (RE1) silencing transcription factor (REST) is widely documented as a regulator of neurogenesis that acts by recruiting corepressor proteins and repressing neuronal gene expression in non-neuronal cells. The REST corepressor 1 (CoREST1), CoREST2, and CoREST3 are best described for their role as part of the REST complex. However, recent evidence has shown the proteins have the ability to repress expression of distinct target genes in a REST-independent manner. These findings indicate that each CoREST paralogue may have distinct and critical roles in regulating neurodevelopment and are more than simply “REST corepressors,” whereby they act as independent repressors orchestrating biological processes during neurodevelopment.

Neural differentiation medium for human pluripotent stem cells to model physiological glucose levels in human brain

Publisher: Elsevier BV

Date: 08-2021

DOI: 10.1016/J.BRAINRESBULL.2021.05.016

Abstract: Cortical neurospheres (NSPs) derived from human pluripotent stem cells (hPSC), have proven to be a successful platform to investigate human brain development and neuro-related diseases. Currently, many of the standard hPSC neural differentiation media, use concentrations of glucose (approximately 17.5-25 mM) and insulin (approximately 3.2 μM) that are much greater than the physiological concentrations found in the human brain. These culture conditions make it difficult to analyse perturbations of glucose or insulin on neuronal development and differentiation. We established a new hPSC neural differentiation medium that incorporated physiological brain concentrations of glucose (2.5 mM) and significantly reduced insulin levels (0.86 μM). This medium supported hPSC neural induction and formation of cortical NSPs. The revised hPSC neural differentiation medium, may provide an improved platform to model brain development and to investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels.

Neural Differentiation of Human Embryonic Stem Cells

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-59745-133-8_3

Abstract: Embryonic stem cells (ESCs) are pluripotent and capable of indefinite self-renewal in vitro. These features make them a highly advantageous source for deriving any cell type of the central and peripheral nervous system. We describe neural induction of human (h)ESCs, by using the bone morphogenic protein inhibitor protein noggin. Neural progenitors derived from noggin-treated hESCs can be propagated as neurospheres and further differentiated in vitro and in vivo to mature neurons and glia. This complete protocol of neural differentiation, from hESCs to mature neuronal cells, can be used as an in vitro model to study human neurogenesis and neurodegeneration.

Abnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.NBD.2021.105370

Abstract: CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by pathogenic variants in the Cyclin-dependent kinase-like 5 (CDKL5) gene, resulting in dysfunctional CDKL5 protein. It predominantly affects females and causes seizures in the first few months of life, ultimately resulting in severe intellectual disability. In the absence of targeted therapies, treatment is currently only symptomatic. CDKL5 is a serine/threonine kinase that is highly expressed in the brain, with a critical role in neuronal development. Evidence of mitochondrial dysfunction in CDD is gathering, but has not been studied extensively. We used human patient-derived induced pluripotent stem cells with a pathogenic truncating mutation (p.Arg59*) and CRISPR/Cas9 gene-corrected isogenic controls, differentiated into neurons, to investigate the impact of CDKL5 mutation on cellular function. Quantitative proteomics indicated mitochondrial defects in CDKL5 p.Arg59* neurons, and mitochondrial bioenergetics analysis confirmed decreased activity of mitochondrial respiratory chain complexes. Additionally, mitochondrial trafficking velocity was significantly impaired, and there was a higher percentage of stationary mitochondria. We propose mitochondrial dysfunction is contributing to CDD pathology, and should be a focus for development of targeted treatments for CDD.

Abstracts from the Spectra 2018 Symposium Part 5: Contexts

Publisher: MIT Press - Journals

Date: 2021

DOI: 10.1162/LEON_A_02153

Distinct Subdomains of the EphA3 Receptor Mediate Ligand Binding and Receptor Dimerization

Publisher: Elsevier BV

Date: 08-1998

DOI: 10.1074/JBC.273.32.20228

Differential susceptibility of human neural progenitors and neurons to ischaemic injury

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.BRAINRESBULL.2019.12.005

Abstract: Neuroprotection for stroke has shown great promise but has had little translational success. Developing drugs for humans logically requires human tissue evaluation. Human embryonic stem cell (hESC)-derived neuronal cultures at different developmental stages were subject to oxygen glucose deprivation (OGD) to determine how developing maturity altered response to ischemic injury. H9 hESCs were induced by Noggin to generate neural progenitors (NPs) and highly arbourised structurally complex neurons. They were both subjected to OGD or OGD with reoxygenation (OGD-R) for 1-6 h.Outcome was assessed by measures of cell death, survival and morphology. NPs did not die after OGD but experienced progressive loss of metabolic activity. Highly arbourised neurons showed minimal cell death initially but 44 % and 78 % died after 4 and 6 h OGD. Metabolic dysfunction was greater in these more mature neurons (∼70 %) than in NPs and evident after 1 h OGD, before detection of neuronal death at 4 h. OGD-R salvaged metabolic activity but not cell death in mature neurons. In NPs there was little metabolic salvage and cell death was induced (50 % and 65 % at 4 and 6 h OGD-R, respectively). Highly arbourised neurons are more sensitive to ischaemic injury than NPs which did however develop marked vulnerability to prolonged injury with reoxygenation. These observations imply that therapeutic potential may be highly dependent of the developmental state of the neurons we aim to protect.

EphA4 is not required for Purkinje cell compartmentation

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1016/S0165-3806(02)00278-X

Abstract: The Purkinje cells of both the adult and the developing cerebellar cortex are organized into parasagittal stripes or 'segments' expressing a variety of biochemical markers. We show that in the developing mouse cerebellar cortex, members of the Eph receptor gene family are expressed in mediolaterally alternating Purkinje cell segments. Since members of the Eph receptors family have been shown to play a role in hindbrain segmentation and boundary formation (Philos. Trans. R. Soc. Lond. B: Biol. Sci. 355 (2000) 993), we analyzed the effect of a null mutation of the EphA4 gene on Purkinje cell compartmentation. Using well characterized markers of Purkinje cell compartmentation in both the developing and the adult cerebellum, we observed no significant alteration in the banding pattern of these markers between the EphA4 knockout mice and their wild type controls. The ribboned pattern of migrating granule cells in the developing cerebellum also appears unaltered. The expression of other members of this gene family, including ephrin-B2, EphA2, and ephrin-A1, in a compartmentalized pattern within the Purkinje cell layer suggests a possible redundancy and/or a compensation of EphA4 function in the segmental patterning of cerebellar Purkinje cells.

Generation of Neural Crest Progenitors from Human Pluripotent Stem Cells

Publisher: Springer New York

Date: 2019

DOI: 10.1007/978-1-4939-9412-0_3

Abstract: There are a vast range of diseases and disorders that are neurocristopathic in origin, including Hirschsprung's disease, pheochromocytoma, familial dysautonomia, craniofacial disorders, and melanomas. Having a source of human neural crest cells is highly valuable for investigating potential treatments for such diseases. This chapter describes a robust and well-characterized protocol for deriving neural crest from human pluripotent stem cells (hPSCs), which can then be differentiated to neuronal and non-neuronal lineages. The protocol is adapted to suit hPSC maintenance as a monolayer bulk culture or as manual-passaged colonies, which makes it widely applicable to researchers that may use different systems for hPSC maintenance.

A reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells

Publisher: Springer Science and Business Media LLC

Date: 08-05-2014

DOI: 10.1186/SCRT453

Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.JNEUMETH.2018.07.005

Abstract: Differentiation of human embryonic stem cells (hESCs) into distinct neural lineages has been widely studied. However, preparation of mixed yet neurochemically mature populations, for the study of neurological diseases involving mixed cell types has received less attention. We combined two commonly used differentiation methods to provide robust and reproducible cultures in which a mixture of primarily GABAergic and Glutamatergic neurons was obtained. Detailed characterisation by immunocytochemistry (ICC) and quantitative real-time PCR (qPCR) assessed the neurochemical phenotype, and the maturation state of these neurons. We found that once neurospheres (NSs) had attached to the culture plates, proliferation of neural stem cell was suppressed. Neuronal differentiation and synaptic development then occurred after 21 days in vitro (DIV). By 49DIV, there were large numbers of neurochemically and structurally mature neurons. The qPCR studies indicated that expression of GABAergic genes increased the most (93.3-fold increase), followed by glutamatergic (51-fold increase), along with smaller changes in expression of cholinergic (3-fold increase) and dopaminergic genes (6-fold increase), as well as a small change in glial cell marker expression (5-fold increase). Existing methods isolate hESC-derived neural progenitors for onward differentiation to mature neurons using either migration or dissociative paradigms. These give poor survival or yield. By combining these approaches, we obtain high yields of morphologically and neurochemically mature neurons. These can be maintained in culture for extended periods. Our method provides a novel, effective and robust neural culture system with structurally and neurochemically mature cell populations and neural networks, suitable for studying a range of neurological diseases from a human perspective.

Neural development in human embryonic stem cells-applications of lentiviral vectors

Publisher: Wiley

Date: 12-07-2011

DOI: 10.1002/JCB.23116

Abstract: The derivation of neural lineages from human embryonic stem cells (hESCs) in vitro is based largely on exposure of hESCs to exogenous signals and substrates, designed to mimic conditions in the developing embryo. However, selection of specific lineages and the discovery of gene function in human neural development may be enhanced by the ability to intrinsically regulate gene expression. Recombinant lentiviral vectors provide an efficient method to stably introduce genes into hESC and their differentiating derivatives. Here we review the methods used to derive neural cells from hESCs, transduction of these cells with lentiviral vectors, and improvements that have been made to the vectors to enhance viral integration and transgene expression. Finally, we explore prospects for future uses of lentiviral vectors in hESC research, including their applications in library screening for drug development, zinc finger nucleases for gene editing and optogenetics to interrogate cellular pathways and function.

Long-Distance Axonal Growth and Protracted Functional Maturation of Neurons Derived from Human Induced Pluripotent Stem Cells After Intracerebral Transplantation

Publisher: Oxford University Press (OUP)

Date: 15-02-2017

DOI: 10.1002/SCTM.16-0198

Abstract: The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re-construction in animal models of brain injury.

The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation

Publisher: Springer Science and Business Media LLC

Date: 15-08-2015

DOI: 10.1007/S00439-015-1591-0

Abstract: GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.

The Convergence of Cochlear Implantation with Induced Pluripotent Stem Cell Therapy

Publisher: Springer Science and Business Media LLC

Date: 29-09-2011

DOI: 10.1007/S12015-011-9320-0

Abstract: According to 2010 estimates from The National Institute on Deafness and other Communication Disorders, approximately 17% (36 million) American adults have reported some degree of hearing loss. Currently, the only clinical treatment available for those with severe-to-profound hearing loss is a cochlear implant, which is designed to electrically stimulate the auditory nerve in the absence of hair cells. Whilst the cochlear implant has been revolutionary in terms of providing hearing to the severe-to-profoundly deaf, there are variations in cochlear implant performance which may be related to the degree of degeneration of auditory neurons following hearing loss. Hence, numerous experimental studies have focused on enhancing the efficacy of cochlear implants by using neurotrophins to preserve the auditory neurons, and more recently, attempting to replace these dying cells with new neurons derived from stem cells. As a result, several groups are now investigating the potential for both embryonic and adult stem cells to replace the degenerating sensory elements in the deaf cochlea. Recent advances in our knowledge of stem cells and the development of induced pluripotency by Takahashi and Yamanaka in 2006, have opened a new realm of science focused on the use of induced pluripotent stem (iPS) cells for therapeutic purposes. This review will provide a broad overview of the potential benefits and challenges of using iPS cells in combination with a cochlear implant for the treatment of hearing loss, including differentiation of iPS cells into an auditory neural lineage and clinically relevant transplantation approaches.

A method for genetic modification of human embryonic stem cells using electroporation

Publisher: Springer Science and Business Media LLC

Date: 04-2007

DOI: 10.1038/NPROT.2007.105

Abstract: The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell lification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.

Abstracts from the Spectra 2018 Symposium: Contexts

Publisher: MIT Press

Date: 16-09-2020

DOI: 10.1162/LEON_A_01972

Modelling the dorsal root ganglia using human pluripotent stem cells: A platform to study peripheral neuropathies

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.BIOCEL.2018.05.005

Abstract: Sensory neurons of the dorsal root ganglia (DRG) are the primary responders to stimuli inducing feelings of touch, pain, temperature, vibration, pressure and muscle tension. They consist of multiple subpopulations based on their morphology, molecular and functional properties. Our understanding of DRG sensory neurons has been predominantly driven by rodent studies and using transformed cell lines, whereas less is known about human sensory DRG neurons simply because of limited availability of human tissue. Although these previous studies have been fundamental for our understanding of the sensory system, it is imperative to profile human DRG subpopulations as it is becoming evident that human sensory neurons do not share the identical molecular and functional properties found in other species. Furthermore, there are wide range of diseases and disorders that directly/indirectly cause sensory neuronal degeneration or dysfunctionality. Having an in vitro source of human DRG sensory neurons is paramount for studying their development, unique neuronal properties and for accelerating regenerative therapies to treat sensory neuropathies. Here we review the major studies describing generation of DRG sensory neurons from human pluripotent stem cells and fibroblasts and the gaps that need to be addressed for using in vitro-generated human DRG neurons to model human DRG tissue.

Hypothermia protects human neurons

Publisher: SAGE Publications

Date: 03-01-2014

DOI: 10.1111/IJS.12224

Abstract: Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury. Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H 2 O 2 -induced oxidative stress were used to induce relevant injury. Hypothermia to 33°C protected these human neurons against H 2 O 2 -induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% ( P ≤ 0·0001 95% confidence interval 34·8–71·04) and 42% ( P ≤ 0·0001 95% confidence interval 27·5–56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3–71·3 and 26%, P ≤ 0·001 95% confidence interval 12·4–52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H 2 O 2 -induced oxidative stress even when hypothermia was initiated six-hours after onset of injury ( P ≤ 0·05 95% confidence interval 0·57–43·1). We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H 2 O 2 -induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.

Generation of Pluripotent Stem Cells and their Developmental Potential

Publisher: BENTHAM SCIENCE PUBLISHERS

Date: 19-05-2012

DOI: 10.2174/978160805289911201010041

Neural Stem Cells Express Non-Neural Markers During Embryoid Body Coculture

Publisher: Oxford University Press (OUP)

Date: 04-2006

DOI: 10.1634/STEMCELLS.2005-0151

Abstract: The capacity of neural stem cells (NSC) to transdifferentiate into a wide range of non-neuronal lineages is the subject of debate. One approach to test NSC plasticity is to ectopically place NSCs in permissive or instructive microenvironments in which the signals driving differentiation of multiple cell types are being elicited. Here we produce embryoid body neurosphere aggregates by combining neurosphere derivatives from fetal mice constitutively expressing green fluorescent protein with embryonic stem (ES) cells isolated from Zin40 mice constitutively expressing nuclear β-galacosidase. Under these conditions, we assess neurosphere-derivative–immunoreactivity to anti-neurofilament heavy chain, anti-pan-cytokeratin, anti-smooth muscle α-actinin and anti-α-fetoprotein–specific antibodies. Furthermore, we determine lineage-specific transgene expression and undertake fluorescence in situ hybridization to assess ES cell–neural stem cell–fusion indices. Our data demonstrate that following coculture in hanging drops with ES cells, neurosphere derivatives display immunoreactivity to non-neural markers, in particular smooth muscle, which is not dependent upon cell–cell fusion. These results suggest that given an appropriate environment, NSC may lose their in vivo restrictions and display non-neuronal phenotypes.

Cell and Gene Therapy for Friedreich Ataxia: Progress to Date

Publisher: Mary Ann Liebert Inc

Date: 08-2014

DOI: 10.1089/HUM.2013.180

Abstract: Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since in iduals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease.

Kinase-Dependent and Kinase-Independent Functions of EphA4 Receptors in Major Axon Tract Formation In Vivo

Publisher: Elsevier BV

Date: 2001

DOI: 10.1016/S0896-6273(01)00181-7

Abstract: The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal lobes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

Development and reorganization of corticospinal projections in EphA4 deficient mice

Publisher: Wiley

Date: 2001

DOI: 10.1002/CNE.1064

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.BBRC.2006.03.127

Abstract: We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.

Distribution of Particles in Human Stem Cell-Derived 3D Neuronal Cell Models: Effect of Particle Size, Charge, and Density

Publisher: American Chemical Society (ACS)

Date: 29-07-2020

DOI: 10.1021/ACS.BIOMAC.0C00626

Neurons derived from human embryonic stem cells extend long-distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation

Publisher: Frontiers Media SA

Date: 2012

DOI: 10.3389/FNCEL.2012.00011

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/C9BM01757G

Abstract: Multilayered particles in gene therapy for Friedreich's ataxia induce a 27 000-fold increase in frataxin gene expression in a patient-derived cell model.

Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.SCR.2013.10.011

Abstract: In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-cl recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

Friedreich’s ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency

Publisher: Impact Journals, LLC

Date: 30-05-2017

DOI: 10.18632/AGING.101247

Stem Cells and Their Developmental Potential

Publisher: WORLD SCIENTIFIC

Date: 07-2005

DOI: 10.1142/9789812569370_0004

EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract

Publisher: Proceedings of the National Academy of Sciences

Date: 27-10-1998

DOI: 10.1073/PNAS.95.22.13248

Abstract: Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.

Wnt3a regulates survival, expansion, and maintenance of neural progenitors derived from human embryonic stem cells

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.MCN.2007.07.013

Abstract: Many reports describe the efficient derivation and expansion of neural progenitors (NP) from human embryonic stem cells (hESC). However, little is known about the signaling factors found within the neurosphere microenvironment that regulate NP maintenance and differentiation. We show that Wnt ligand and receptor transcripts are endogenously upregulated within neurospheres derived from noggin-primed hESC. In addition, neurosphere formation and size were significantly greater in the presence of exogenous Wnt3a compared to control conditions. Inhibition of endogenous Wnt signaling resulted in a significant reduction in the efficiency of neurosphere formation and overall size, due to effects on both NP proliferation and apoptosis. These findings demonstrate a requirement of Wnt signaling for maintenance, proliferation, and survival of NP when cultured in neurosphere conditions.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons

Publisher: Frontiers Media SA

Date: 04-12-2020

DOI: 10.3389/FNCEL.2020.600895

Abstract: Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons ( NGN2 iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2 iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2 iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Generation of RAB39B knockout isogenic human embryonic stem cell lines to model RAB39B-mediated Parkinson's disease

Publisher: Elsevier BV

Date: 04-2018

DOI: 10.1016/J.SCR.2018.02.015

Abstract: Mutations in RAB39B are a known cause of X-linked early onset Parkinson's disease. Isogenic human embryonic stem cell lines carrying two independent deletions of RAB39B were generated using CRISPR/Cas9 genome editing tool. The deletions were confirmed by PCR and direct sequence analysis in two edited stem cell lines. Both cell lines showed pluripotency and displayed a normal karyotype. Further, they were able to form embryoid bodies in vitro, and express markers indicative of differentiation to the three germ layers.

Signals Involved in Neural Differentiation of Human Embryonic Stem Cells

Publisher: S. Karger AG

Date: 2009

DOI: 10.1159/000231890

Abstract: Neural differentiation from embryonic stem cells involves progressive stages of neural induction, expansion and maintenance of neural stem rogenitor cells, and differentiation to neurons and glia. Our understanding of the signals involved in each of these processes is primarily based on our knowledge of neural development during embryogenesis. This review will focus on the signalling pathways that have been identified to play a role in neural differentiation of human embryonic stem cells (hESCs), including their induction to neuroectoderm, maintenance and expansion of hESC-derived neurospheres, differentiation to neurons and specification to specific neuronal lineages. Understanding the signals involved in each of these stages is important for optimising methods to derive specific cell types for transplantation therapies, as well as for providing insight into the mechanisms of human neurogenesis.

Human embryonic stem cell models of Huntington disease

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/S1472-6483(10)60053-3

Abstract: Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.

Characterization of forebrain neurons derived from late-onset Huntington's disease human embryonic stem cell lines

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FNCEL.2013.00037

The hESC line Envy expresses high levels of GFP in all differentiated progeny

Publisher: Springer Science and Business Media LLC

Date: 23-03-2005

DOI: 10.1038/NMETH748

Abstract: Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.

The Role of WD40-Repeat Protein 62 (MCPH2) in Brain Growth: Diverse Molecular and Cellular Mechanisms Required for Cortical Development

Publisher: Springer Science and Business Media LLC

Date: 22-09-2017

DOI: 10.1007/S12035-017-0778-X

Abstract: Genetic disruptions of spindle/centrosome-associated WD40-repeat protein 62 (WDR62) are causative for autosomal recessive primary microcephaly (MCPH) and a broader range of cortical malformations. Since the identification of WDR62 as encoded by the MCPH2 locus in 2010, recent studies that have deleted/depleted WDR62 in various animal models of cortical development have highlighted conserved functions in brain growth. Here, we provide a timely review of our current understanding of WDR62 contributions in the self-renewal, expansion and fate specification of neural stem and progenitor cells that are critical for neocortical development. Recent studies have revealed multiple functions for WDR62 in the regulation of spindle organization, mitotic progression and the duplication and biased inheritance of centrosomes during asymmetric isions. We also discuss recently elaborated WDR62 interaction partners that include Aurora and c-Jun N-terminal kinases as part of complex signalling mechanisms that may define its neural functions. These studies provide new insights into the molecular and cellular processes that are required for brain formation and implicated in the genesis of primary microcephaly.

Lbx1 Specifies Somatosensory Association Interneurons in the Dorsal Spinal Cord

Publisher: Elsevier BV

Date: 05-2002

DOI: 10.1016/S0896-6273(02)00690-6

Abstract: Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Lbx1, therefore, plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch.

WDR62 Regulates Early Neural and Glial Progenitor Specification of Human Pluripotent Stem Cells

Publisher: Hindawi Limited

Date: 2017

DOI: 10.1155/2017/7848932

Abstract: Mutations in WD40-repeat protein 62 ( WDR62 ) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker, TBR2, and also glial marker, S100 β . In contrast, inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation.

An In Vitro Model of Developmental Synaptogenesis Using Cocultures of Human Neural Progenitors and Cochlear Explants

Publisher: Mary Ann Liebert Inc

Date: 15-03-2013

DOI: 10.1089/SCD.2012.0082

Generation of Neural Organoids from Human Embryonic Stem Cells Using the Rotary Cell Culture System: Effects of Microgravity on Neural Progenitor Cell Fate

Publisher: Mary Ann Liebert Inc

Date: 15-06-2018

DOI: 10.1089/SCD.2018.0012

Vertical Nanowire Electrode Arrays as Novel Electrochemical Label‐Free Immunosensors

Publisher: Wiley

Date: 11-02-2015

DOI: 10.1002/SMLL.201403540

Abstract: A new method for the fabrication of a label-free electrochemical immunosensor based on vertical nanowires (VNWs) is proposed. The VNWs are functionalized to detect antibodies against a major astrocytic structural protein component, glial fibrillary acidic protein (GFAP). It is revealed that the interaction of GFAP-antibody with functionalized VNWs leads to a clear change in device conductance and the corresponding capacitance.

Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices

Publisher: Hindawi Limited

Date: 20-11-2019

DOI: 10.1155/2019/8419493

Abstract: Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo . Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.

Functional Characterization of Friedreich Ataxia iPS-Derived Neuronal Progenitors and Their Integration in the Adult Brain

Publisher: Public Library of Science (PLoS)

Date: 07-07-2014

DOI: 10.1371/JOURNAL.PONE.0101718

Gli1 Is an Inducing Factor in Generating Floor Plate Progenitor Cells from Human Embryonic Stem Cells  

Publisher: Oxford University Press (OUP)

Date: 10-2010

DOI: 10.1002/STEM.510

Abstract: Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer.

Neural Differentiation of Induced Pluripotent Stem Cells

Publisher: Humana Press

Date: 2011

DOI: 10.1007/978-1-61779-328-8_7

Abstract: The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of deriving pluripotent stem cells from any human patient. Generation of patient-derived stem cells serves as a great source for developing cell replacement therapies and also for creating human cellular model systems of specific diseases or disorders. This is only of benefit if there are well-established differentiation assay systems to generate the cell types of interest. This chapter describes robust and well-characterized protocols for differentiating iPS cells to neural progenitors, neurons, glia and neural crest cells. These established assays can be applied to iPS cell lines derived from patients with neurodegenerative disorders to study cellular mechanisms associated with neurodegeneration as well as investigating the regenerative potential of patient derived stem cells.

University Of Wollongong Illawarra Health And Medical Research Institute

Location: Australia

View Organisation

Discovery Projects - Grant ID: DP230101369

Start Date: 2023

End Date: 12-2025

Amount: $488,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP190101139

Start Date: 11-2020

End Date: 11-2025

Amount: $704,384.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT130100514

Start Date: 02-2014

End Date: 02-2018

Amount: $755,320.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC170100016

Start Date: 05-2018

End Date: 12-2024

Amount: $3,123,492.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR1101002

Start Date: 07-2011

End Date: 12-2019

Amount: $21,000,000.00

Funder: Australian Research Council