Differentiation of SH-SY5Y neuroblastoma cells using retinoic acid and BDNF: a model for neuronal and synaptic differentiation in neurodegeneration.

There has been much interest in the use of cell culture models of neurones, to avoid the animal welfare and cost issues of using primary and human-induced pluripotent stem cell (hiPSC)-derived neurones respectively. The human neuroblastoma cell line, SH-SY5Y, is extensively used in laboratories as they can be readily expanded, are of low cost and can be differentiated into neuronal-like cells. However, much debate remains as to their phenotype once differentiated, and their ability to recapitulate the physiology of bona fide neurones. Here, we characterise a differentiation protocol using retinoic acid and BDNF, which results in extensive neurite outgrowth/branching within 10 days, and expression of key neuronal and synaptic markers. We propose that these differentiated SH-SY5Y cells may be a useful substitute for primary or hiPSC-derived neurones for cell biology studies, in order to reduce costs and animal usage. We further propose that this characterised differentiation timecourse could be used as an in vitro model for neuronal differentiation, for proof-of principle studies on neurogenesis, e.g. relating to neurodegenerative diseases. Finally, we demonstrate profound changes in Tau phosphorylation during differentiation of these cells, suggesting that they should not be used for neurodegeneration studies in their undifferentiated state.

Human stem cell-derived ventral midbrain astrocytes exhibit a region-specific secretory profile.

This scientific commentary refers to 'Human stem cell-derived astrocytes exhibit region-specific heterogeneity in their secretory profiles', by Clarke et al. (https://doi.org/10.1093/brain/awaa258) in Brain.

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells.

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required. We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

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

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 in vitro. 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 in vitro 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.

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

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 ATG4BC74A 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.

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

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.

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

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.

Nodal dependent differential localisation of dishevelled-2 demarcates regions of differing cell behaviour in the visceral endoderm.

The anterior visceral endoderm (AVE), a signalling centre within the simple epithelium of the visceral endoderm (VE), is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Little is known about the underlying basis for this barrier and how surrounding cells in the VE respond to or influence AVE migration. We use high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy to show that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct patterns of F-actin localisation, in cortical rings and an apical shroud, respectively. We genetically perturb PCP signalling and show that this disrupts the localisation pattern of Dvl2 and F-actin and the normal migration of AVE cells. In Nodal null embryos, membrane localisation of Dvl2 is reduced, while in mutants for the Nodal inhibitor Lefty1, Dvl2 is ectopically membrane localised, establishing a role for Nodal in modulating PCP signalling. These results show that the limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. In addition to coordinating global cell movements across epithelia (such as during convergence extension), PCP signalling in interplay with TGFß signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them.

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

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.

Early embryonic expression patterns of the mouse Flamingo and Prickle orthologues.

The Drosophila melanogaster proteins Flamingo and Prickle act in the planar cell polarity (PCP) pathway, which is required for acquisition of epithelial polarity in the wing, eye, and epidermis. In mammals, PCP signaling has been shown to regulate cell movements and polarity in a variety of tissues. Here, we show that the murine Flamingo orthologues Celsr1-3 and the Prickle orthologues Prickle1, Prickle2, and Testin have dynamic patterns of expression during pregastrulation and gastrulation stages. Celsr1 is expressed in the anterior visceral endoderm and nascent mesoderm, Celsr2 and Celsr3 mark the prospective neuroectoderm, Prickle1 is expressed in the primitive streak and mesoderm, Prickle2 in the node, and Testin in the anterior visceral endoderm, the extraembryonic ectoderm, primitive streak, and mesoderm. Analysis of a gene-trap mutation in Testin indicates that this gene is not required for embryogenesis; therefore, other Prickle homologues may compensate for its function during development.

BMP signalling inhibits premature neural differentiation in the mouse embryo.

The specification of a subset of epiblast cells to acquire a neural fate constitutes the first step in the generation of the nervous system. Little is known about the signals required for neural induction in the mouse. We have analysed the role of BMP signalling in this process. We demonstrate that prior to gastrulation, Bmp2/4 signalling via Bmpr1a maintains epiblast pluripotency and prevents precocious neural differentiation of this tissue, at least in part by maintaining Nodal signalling. We find that during gastrulation, BMPs of the 60A subgroup cooperate with Bmp2/4 to maintain pluripotency. The inhibition of neural fate by BMPs is independent of FGF signalling, as inhibition of FGF signalling between 5.5 and 7.5 days post-coitum does not block neural differentiation in the mouse embryo. Together, our results demonstrate that inhibition of BMP signalling has a central role during neural induction in mammals and suggest that FGFs do not act as neural inducers in the post-implantation mouse embryo.

Global hypomethylation of the genome in XX embryonic stem cells.

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.