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

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.

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

Neurodegenerative diseases are generally characterized by a progressive loss of neuronal subpopulations, with no available cure to date. One of the main reasons for the limited clinical outcomes of new drug formulations is the lack of appropriate in vitro human cell models for research and validation. Stem cell technologies provide an opportunity to address this challenge by using patient-derived cells as a platform to test various drug formulations, including particle-based drug carriers. The therapeutic efficacy of drug delivery systems relies on efficient cellular uptake of the carrier and can be dependent on its size, shape, and surface chemistry. Although considerable efforts have been made to understand the effects of the physiochemical properties of particles on two-dimensional cell culture models, little is known of their effect in three-dimensional (3D) cell models of neurodegenerative diseases. Herein, we investigated the role of particle size (235-1000 nm), charge (cationic and anionic), and density (1.05 and 1.8 g cm-3) on the interactions of particles with human embryonic stem cell-derived 3D cell cultures of sensory neurons, called sensory neurospheres (sNSP). Templated layer-by-layer particles, with silica or polystyrene cores, and self-assembled glycogen/DNA polyplexes were used. Particles with sizes <280 nm effectively penetrated sNSP. Additionally, effective plasmid DNA delivery was observed up to 6 days post-transfection with glycogen/DNA polyplexes. The findings provide guidance in nanoparticle design for therapies aimed at neurodegenerative diseases, in particular Friedreich's ataxia, whereby sensory neurons are predominantly affected. They also demonstrate the application of 3D models of human sensory neurons in preclinical drug development.

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

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

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

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.

Vertical Nanowire Electrode Arrays as Novel Electrochemical Label-Free Immunosensors.

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.

Generation of genetically modified Friedreich's ataxia induced pluripotent stem cell lines and isogenic control lines carrying an inducible neurogenin-2 expression cassette.

Friedreich's ataxia (FRDA) is a rare neurodegenerative disease caused by an expansion of a GAA repeat sequence within the Frataxin (FXN) gene. Prominent regions of neurodegeneration include sensory neurons within the dorsal root ganglia. Here we present a set of genetically modified FRDA induced pluripotent stem cell (iPSC) lines that carry an inducible neurogenin-2 (NGN2) expression cassette. Exogenous expression of NGN2 in iPSC derived neural crest progenitors efficiently generates functionally mature sensory neurons. These cell lines will provide a streamlined source of FRDA iPSC sensory neurons for studying both disease mechanism and screening potential therapeutics.

Generation of a gene-edited H9 embryonic stem cell line carrying a DOX-inducible NGN2 expression cassette in the CLYBL locus.

The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models.

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.

Touch sensation requires the mechanically gated ion channel ELKIN1.

Touch perception is enabled by mechanically activated ion channels, the opening of which excites cutaneous sensory endings to initiate sensation. In this study, we identify ELKIN1 as an ion channel likely gated by mechanical force, necessary for normal touch sensitivity in mice. Touch insensitivity in Elkin1-/- mice was caused by a loss of mechanically activated currents (MA currents) in around half of all sensory neurons activated by light touch (low-threshold mechanoreceptors). Reintroduction of Elkin1 into sensory neurons from Elkin1-/- mice restored MA currents. Additionally, small interfering RNA-mediated knockdown of ELKIN1 from induced human sensory neurons substantially reduced indentation-induced MA currents, supporting a conserved role for ELKIN1 in human touch. Our data identify ELKIN1 as a core component of touch transduction in mice and potentially in humans.

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid.

We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.

Glycogen synthase kinase 3ß 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.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3ß and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6- expression, we describe as "pre-neuroepithelial." Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage.

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.

Transgene and Chemical Transdifferentiation of Somatic Cells for Rapid and Efficient Neurological Disease Cell Models.

For neurological diseases, molecular and cellular research relies on the use of model systems to investigate disease processes and test potential therapeutics. The last decade has witnessed an increase in the number of studies using induced pluripotent stem cells to generate disease relevant cell types from patients. The reprogramming process permits the generation of a large number of cells but is potentially disadvantaged by introducing variability in clonal lines and the removal of phenotypes of aging, which are critical to understand neurodegenerative diseases. An under-utilized approach to disease modeling involves the transdifferentiation of aged cells from patients, such as fibroblasts or blood cells, into various neural cell types. In this review we discuss techniques used for rapid and efficient direct conversion to neural cell types. We examine the limitations and future perspectives of this rapidly advancing field that could improve neurological disease modeling and drug discovery.

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

Directed neuronal differentiation of human pluripotent stem cells (hPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2 (NGN2) has been widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modeling, drug screening, and neuronal replacement therapies. Could NGN2 be a "one-glove-fits-all" approach for neuronal differentiations? This review summarizes the cellular roles of NGN2 and describes the applications and limitations of using NGN2 for the rapid and directed differentiation of neurons.

Cellular pathophysiology of Friedreich's ataxia cardiomyopathy.

Friedreich's ataxia (FRDA) is a hereditary neuromuscular disorder. Cardiomyopathy is the leading cause of premature death in FRDA. FRDA cardiomyopathy is a complex and progressive disease with no cure or treatment to slow its progression. At the cellular level, cardiomyocyte hypertrophy, apoptosis and fibrosis contribute to the cardiac pathology. However, the heart is composed of multiple cell types and several clinical studies have reported the involvement of cardiac non-myocytes such as vascular cells, autonomic neurons, and inflammatory cells in the pathogenesis of FRDA cardiomyopathy. In fact, several of the cardiac pathologies associated with FRDA including cardiomyocyte necrosis, fibrosis, and arrhythmia, could be contributed to by a diseased vasculature and autonomic dysfunction. Here, we review available evidence regarding the current understanding of cellular mechanisms for, and the involvement of, cardiac non-myocytes in the pathogenesis of FRDA cardiomyopathy.

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

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.

Gli1 is an inducing factor in generating floor plate progenitor cells from human embryonic stem cells.

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.

Development of Functional Thyroid C Cell-like Cells from Human Pluripotent Cells in 2D and in 3D Scaffolds.

Medullary thyroid carcinoma contributes to about 3-4% of thyroid cancers and affects C cells rather than follicular cells. Thyroid C cell differentiation from human pluripotent stem cells has not been reported. We report the stepwise differentiation of human embryonic stem cells into thyroid C cell-like cells through definitive endoderm and anterior foregut endoderm and ultimobranchial body-like intermediates in monolayer and 3D Matrigel culture conditions. The protocol involved sequential treatment with interferon/transferrin/selenium/pyruvate, foetal bovine serum, and activin A, then IGF-1 (Insulin-like growth factor 1), on the basis of embryonic thyroid developmental sequence. As well as expressing C cell lineage relative to follicular-lineage markers by qPCR (quantitative polymerase chain reaction) and immunolabelling, these cells by ELISA (enzyme-linked immunoassay) exhibited functional properties in vitro of calcitonin storage and release of calcitonin on calcium challenge. This method will contribute to developmental studies of the human thyroid gland and facilitate in vitro modelling of medullary thyroid carcinoma and provide a valuable platform for drug screening.

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

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.