First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

The role of ADAM10 in astrocytes: Implications for Alzheimer's disease.

Much of the early research into AD relies on a neuron-centric view of the brain, however, evidence of multiple altered cellular interactions between glial cells and the vasculature early in AD has been demonstrated. As such, alterations in astrocyte function are widely recognized a contributing factor in the pathogenesis of AD. The processes by which astrocytes may be involved in AD make them an interesting target for therapeutic intervention, but in order for this to be most effective, there is a need for the specific mechanisms involving astrocyte dysfunction to be investigated. "α disintegrin and metalloproteinase" 10 (ADAM10) is capable of proteolytic cleavage of the amyloid precursor protein which prevents amyloid-ß generation. As such ADAM10 has been identified as an interesting enzyme in AD pathology. ADAM10 is also known to play a role in a significant number of cellular processes, most notable in notch signaling and in inflammatory processes. There is a growing research base for the involvement of ADAM10 in regulating astrocytic function, primarily from an immune perspective. This review aims to bring together available evidence for ADAM10 activity in astrocytes, and how this relates to AD pathology.

A requirement for astrocyte IP3R2 signaling for whisker experience-dependent depression and homeostatic upregulation in the mouse barrel cortex.

Changes to sensory experience result in plasticity of synapses in the cortex. This experience-dependent plasticity (EDP) is a fundamental property of the brain. Yet, while much is known about neuronal roles in EDP, very little is known about the role of astrocytes. To address this issue, we used the well-described mouse whiskers-to-barrel cortex system, which expresses a number of forms of EDP. We found that all-whisker deprivation induced characteristic experience-dependent Hebbian depression (EDHD) followed by homeostatic upregulation in L2/3 barrel cortex of wild type mice. However, these changes were not seen in mutant animals (IP3R2-/-) that lack the astrocyte-expressed IP3 receptor subtype. A separate paradigm, the single-whisker experience, induced potentiation of whisker-induced response in both wild-type (WT) mice and IP3R2-/- mice. Recordings in ex vivo barrel cortex slices reflected the in vivo results so that long-term depression (LTD) could not be elicited in slices from IP3R2-/- mice, but long-term potentiation (LTP) could. Interestingly, 1 Hz stimulation inducing LTD in WT paradoxically resulted in NMDAR-dependent LTP in slices from IP3R2-/- animals. The LTD to LTP switch was mimicked by acute buffering astrocytic [Ca2+] i in WT slices. Both WT LTD and IP3R2-/- 1 Hz LTP were mediated by non-ionotropic NMDAR signaling, but only WT LTD was P38 MAPK dependent, indicating an underlying mechanistic switch. These results demonstrate a critical role for astrocytic [Ca2+] i in several EDP mechanisms in neocortex.

Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds.

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 µm) after seven days compared to bulk hydrogels (22.90 ± 4.70 µm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.

Trabecular bone organoids: a micron-scale 'humanised' prototype designed to study the effects of microgravity and degeneration.

Bone is a highly responsive organ, which continuously adapts to the environment it is subjected to in order to withstand metabolic demands. These events are difficult to study in this particular tissue in vivo, due to its rigid, mineralised structure and inaccessibility of the cellular component located within. This manuscript presents the development of a micron-scale bone organoid prototype, a concept that can allow the study of bone processes at the cell-tissue interface. The model is constructed with a combination of primary female osteoblastic and osteoclastic cells, seeded onto femoral head micro-trabeculae, where they recapitulate relevant phenotypes and functions. Subsequently, constructs are inserted into a simulated microgravity bioreactor (NASA-Synthecon) to model a pathological state of reduced mechanical stimulation. In these constructs, we detected osteoclastic bone resorption sites, which were different in morphology in the simulated microgravity group compared to static controls. Once encapsulated in human fibrin and exposed to analogue microgravity for 5 days, masses of bone can be observed being lost from the initial structure, allowing to simulate the bone loss process further. Constructs can function as multicellular, organotypic units. Large osteocytic projections and tubular structures develop from the initial construct into the matrix at the millimetre scale. Micron-level fragments from the initial bone structure are detected travelling along these tubules and carried to sites distant from the native structure, where new matrix formation is initiated. We believe this model allows the study of fine-level physiological processes, which can shed light into pathological bone loss and imbalances in bone remodelling.

Extensive Plasmid Library to Prepare Tau Protein Variants and Study Their Functional Biochemistry.

Tau neurofibrillary tangles are key pathological features of Alzheimer's disease and other tauopathies. Recombinant protein technology is vital for studying the structure and function of tau in physiology and aggregation in pathophysiology. However, open-source and well-characterized plasmids for efficiently expressing and purifying different tau variants are lacking. We generated 44 sequence-verified plasmids including those encoding full length (FL) tau-441, its four-repeat microtubule-binding (K18) fragment, and their respective selected familial pathological variants (N279K, V337M, P301L, C291R, and S356T). Moreover, plasmids for expressing single (C291A), double (C291A/C322A), and triple (C291A/C322A/I260C) cysteine-modified variants were generated to study alterations in cysteine content and locations. Furthermore, protocols for producing representative tau forms were developed. We produced and characterized the aggregation behavior of the triple cysteine-modified tau-K18, often used in real-time cell internalization and aggregation studies because it can be fluorescently labeled on a cysteine outside the microtubule-binding core. Similar to the wild type (WT), triple cysteine-modified tau-K18 aggregated by progressive ß-sheet enrichment, albeit at a slower rate. On prolonged incubation, cysteine-modified K18 formed paired helical filaments similar to those in Alzheimer's disease, sharing morphological phenotypes with WT tau-K18 filaments. Nonetheless, cysteine-modified tau-K18 filaments were significantly shorter (p = 0.002) and mostly wider than WT filaments, explainable by their different principal filament elongation pathways: vertical (end-to-end) and lateral growth for WT and cysteine-modified, respectively. Cysteine rearrangement may therefore induce filament polymorphism. Together, the plasmid library, the protein production methods, and the new insights into cysteine-dependent aggregation should facilitate further studies and the design of antiaggregation agents.

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains.

The inter-cellular propagation of tau aggregates in several neurodegenerative diseases involves, in part, recurring cycles of extracellular tau uptake, initiation of endogenous tau aggregation, and extracellular release of at least part of this protein complex. However, human brain tau extracts from diverse tauopathies exhibit variant or "strain" specificity in inducing inter-cellular propagation in both cell and animal models. It is unclear if these distinctive properties are affected by disease-specific differences in aggregated tau conformation and structure. We have used a combined structural and cell biological approach to study if two frontotemporal dementia (FTD)-associated pathologic mutations, V337M and N279K, affect the aggregation, conformation and cellular internalization of the tau four-repeat domain (K18) fragment. In both heparin-induced and native-state aggregation experiments, each FTD variant formed soluble and fibrillar aggregates with remarkable morphological and immunological distinctions from the wild type (WT) aggregates. Exogenously applied oligomers of the FTD tau-K18 variants (V337M and N279K) were significantly more efficiently taken up by SH-SY5Y neuroblastoma cells than WT tau-K18, suggesting mutation-induced changes in cellular internalization. However, shared internalization mechanisms were observed: endocytosed oligomers were distributed in the cytoplasm and nucleus of SH-SY5Y cells and the neurites and soma of human induced pluripotent stem cell-derived neurons, where they co-localized with endogenous tau and the nuclear protein nucleolin. Altogether, evidence of conformational and aggregation differences between WT and disease-mutated tau K18 is demonstrated, which may explain their distinct cellular internalization potencies. These findings may account for critical aspects of the molecular pathogenesis of tauopathies involving WT and mutated tau.

Preparation of stable tau oligomers for cellular and biochemical studies.

Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer's disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an efficient method which maintains the stability and globular conformation of preformed oligomers. This study demonstrates that labeling a single-cysteine form of the pro-aggregant tau four-repeat region (K18) with either Alexa Fluor 488-C5-maleimide or N-ethylmaleimide in reducing conditions stabilizes oligomers by impeding their further aggregation. Furthermore, the use of this approach to study the propagation of labeled extracellular tau K18 oligomers into human neuroblastoma cells and human stem cell-derived neurons is described. This method is potentially applicable for preparing stabilized oligomers of tau for diagnostic and biomarker tests, as well as for in vitro structure-activity relationship assays.

In vitro Models for Seizure-Liability Testing Using Induced Pluripotent Stem Cells.

The brain is the most complex organ in the body, controlling our highest functions, as well as regulating myriad processes which incorporate the entire physiological system. The effects of prospective therapeutic entities on the brain and central nervous system (CNS) may potentially cause significant injury, hence, CNS toxicity testing forms part of the "core battery" of safety pharmacology studies. Drug-induced seizure is a major reason for compound attrition during drug development. Currently, the rat ex vivo hippocampal slice assay is the standard option for seizure-liability studies, followed by primary rodent cultures. These models can respond to diverse agents and predict seizure outcome, yet controversy over the relevance, efficacy, and cost of these animal-based methods has led to interest in the development of human-derived models. Existing platforms often utilize rodents, and so lack human receptors and other drug targets, which may produce misleading data, with difficulties in inter-species extrapolation. Current electrophysiological approaches are typically used in a low-throughput capacity and network function may be overlooked. Human-derived induced pluripotent stem cells (iPSCs) are a promising avenue for neurotoxicity testing, increasingly utilized in drug screening and disease modeling. Furthermore, the combination of iPSC-derived models with functional techniques such as multi-electrode array (MEA) analysis can provide information on neuronal network function, with increased sensitivity to neurotoxic effects which disrupt different pathways. The use of an in vitro human iPSC-derived neural model for neurotoxicity studies, combined with high-throughput techniques such as MEA recordings, could be a suitable addition to existing pre-clinical seizure-liability testing strategies.

Downregulation of aquaporin 3 inhibits cellular proliferation, migration and invasion in the MDA-MB-231 breast cancer cell line.

Aquaporins are membrane proteins that regulate cellular water flow. Recently, aquaporins have been proposed as mediators of cancer cell biology. A subset of aquaporins, referred to as aquaglyceroporins are known to facilitate the transport of glycerol. The present study describes the effect of gene knockdown of the aquaglyceroporin AQP3 on MDA-MB-231 breast cancer cell proliferation, migration, invasion, adherence and response to the chemotherapeutic agent 5-fluorouracil. shRNA mediated AQP3 gene knockdown induced a 28% reduction in cellular proliferation (P<0.01), a 39% decrease in migration (P<0.0001), a 24% reduction in invasion (P<0.05) and a 25% increase in cell death at 100 µM 5-FU (P<0.01). Analysis of cell permeability to water and glycerol revealed that MDA-MB-231 cells with knocked down AQP3 demonstrated a modest decrease in water permeability (17%; P<0.05) but a more marked decrease in glycerol permeability (77%; P<0.001). These results suggest that AQP3 has a role in multiple aspects of breast cancer cell pathophysiology and therefore represents a novel target for therapeutic intervention.

Characterization of Extractable Species from Polypropylene Microplates.

The use of microplates (for bioassays, immunoassays, and general research) that are manufactured from plastic materials has proved problematic due to issues with accuracy, repeatability, and specificity of the results generated. The cause of these issues has been identified as leachables present in the plastic materials. This article presents an extractables study performed with available microplates manufactured with plastic. Common microplates from five different vendors were obtained, including plates from SiO2 Medical Products (SIO) containing a plasma treatment designed to produce an ultra-low protein-binding surface. The microplates were solvent extracted, and the resulting extracts were analyzed for organic extractables. The extractables profiles were examined and compared among the five different plate types. Detected extractables were identified in each of the extracts, and the potential effect on protein binding is discussed.

Plasma irisin is elevated in type 2 diabetes and is associated with increased E-selectin levels.

BACKGROUND: Irisin is a hormone released mainly from skeletal muscle after exercise which increases adipose tissue energy expenditure. Adipocytes can also release irisin after exercise, acting as a local adipokine to induce white adipose tissue to take on a brown adipose tissue-like phenotype, suggesting that irisin and its receptor may represent a novel molecular target for the treatment of obesity and obesity-related diabetes. Previous reports provide conflicting evidence regarding circulating irisin levels in patients with type 2 diabetes (T2DM). METHODS: This study investigated plasma irisin concentrations in 79 T2DM individuals, assessing potential associations with measures of segmental body composition, markers of endothelial dysfunction and peripheral blood mononuclear cell telomere length (TL). RESULTS: Resting, overnight-fasted plasma irisin levels were significantly higher in this group of T2DM patients compared with levels we previously reported in healthy volunteers (p < 0.001). Moreover, plasma irisin displayed a positive correlation with body mass index (p = 0.04), body fat percentage (p = 0.03), HbA1c (p = 0.03) and soluble E-selectin (p < 0.001). A significant negative association was observed between plasma irisin and visceral adiposity (p = 0.006) in T2DM patients. Multiple regression analysis revealed that circulating soluble E-selectin levels could be predicted by plasma irisin (p = 0.004). Additionally, cultured human umbilical vein endothelial cells (HUVEC) exposed to 200 ng/ml irisin for 4 h showed a significant fourfold increase in E-selectin and 2.5-fold increase in ICAM-1 gene expression (p = 0.001 and p = 0.015 respectively), and there was a 1.8-fold increase in soluble E-selectin in conditioned media (p < 0.05). CONCLUSION: These data suggest that elevated plasma irisin in T2DM is associated with indices of adiposity, and that irisin may be involved in pro-atherogenic endothelial disturbances that accompany obesity and T2DM. Accordingly, irisin may constitute a potentially novel therapeutic opportunity in the field of obesity and cardiovascular diabetology.

Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag.

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

Amyloid ß 1-42 induces hypometabolism in human stem cell-derived neuron and astrocyte networks.

Alzheimer's disease (AD) is the most common form of dementia, affecting more than 35 million people worldwide. Brain hypometabolism is a major feature of AD, appearing decades before cognitive decline and pathologic lesions. To date, the majority of studies on hypometabolism in AD have used transgenic animal models or imaging studies of the human brain. As it is almost impossible to validate these findings using human tissue, alternative models are required. In this study, we show that human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta 1-42 (Aß1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose, pyruvate, lactate, and glutamate. In addition, a significant increase in the glycogen content of cells was also observed. These changes were accompanied by changes in NAD(+)/NADH, ATP, and glutathione levels, suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aß-induced hypometabolism. Further research using this model may elucidate the mechanisms associated with Aß-induced hypometabolism.

A predictive in vitro model of the impact of drugs with anticholinergic properties on human neuronal and astrocytic systems.

The link between off-target anticholinergic effects of medications and acute cognitive impairment in older adults requires urgent investigation. We aimed to determine whether a relevant in vitro model may aid the identification of anticholinergic responses to drugs and the prediction of anticholinergic risk during polypharmacy. In this preliminary study we employed a co-culture of human-derived neurons and astrocytes (NT2.N/A) derived from the NT2 cell line. NT2.N/A cells possess much of the functionality of mature neurons and astrocytes, key cholinergic phenotypic markers and muscarinic acetylcholine receptors (mAChRs). The cholinergic response of NT2 astrocytes to the mAChR agonist oxotremorine was examined using the fluorescent dye fluo-4 to quantitate increases in intracellular calcium [Ca2+]i. Inhibition of this response by drugs classified as severe (dicycloverine, amitriptyline), moderate (cyclobenzaprine) and possible (cimetidine) on the Anticholinergic Cognitive Burden (ACB) scale, was examined after exposure to individual and pairs of compounds. Individually, dicycloverine had the most significant effect regarding inhibition of the astrocytic cholinergic response to oxotremorine, followed by amitriptyline then cyclobenzaprine and cimetidine, in agreement with the ACB scale. In combination, dicycloverine with cyclobenzaprine had the most significant effect, followed by dicycloverine with amitriptyline. The order of potency of the drugs in combination frequently disagreed with predicted ACB scores derived from summation of the individual drug scores, suggesting current scales may underestimate the effect of polypharmacy. Overall, this NT2.N/A model may be appropriate for further investigation of adverse anticholinergic effects of multiple medications, in order to inform clinical choices of suitable drug use in the elderly.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines.

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48h of incubation with increasing concentrations of pesticides ranging from 1 to 1000µM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure.

Plasma irisin levels predict telomere length in healthy adults.

The ageing process is strongly influenced by nutrient balance, such that modest calorie restriction (CR) extends lifespan in mammals. Irisin, a newly described hormone released from skeletal muscles after exercise, may induce CR-like effects by increasing adipose tissue energy expenditure. Using telomere length as a marker of ageing, this study investigates associations between body composition, plasma irisin levels and peripheral blood mononuclear cell telomere length in healthy, non-obese individuals. Segmental body composition (by bioimpedance), telomere length and plasma irisin levels were assessed in 81 healthy individuals (age 43 ± 15.8 years, BMI 24.3 ± 2.9 kg/m(2)). Data showed significant correlations between log-transformed relative telomere length and the following: age (p < 0.001), height (p = 0.045), total body fat percentage (p = 0.031), abdominal fat percentage (p = 0.038), visceral fat level (p < 0.001), plasma leptin (p = 0.029) and plasma irisin (p = 0.011), respectively. Multiple regression analysis using backward elimination revealed that relative telomere length can be predicted by age (b = -0.00735, p = 0.001) and plasma irisin levels (b = 0.04527, p = 0.021). These data support the view that irisin may have a role in the modulation of both energy balance and the ageing process.

Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network.

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.

Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development.

Mood stabilising drugs such as lithium (LiCl) and valproic acid (VPA) are the first line agents for treating conditions such as Bipolar disorder and Epilepsy. However, these drugs have potential developmental effects that are not fully understood. This study explores the use of a simple human neurosphere-based in vitro model to characterise the pharmacological and toxicological effects of LiCl and VPA using gene expression changes linked to phenotypic alterations in cells. Treatment with VPA and LiCl resulted in the differential expression of 331 and 164 genes respectively. In the subset of VPA targeted genes, 114 were downregulated whilst 217 genes were upregulated. In the subset of LiCl targeted genes, 73 were downregulated and 91 were upregulated. Gene ontology (GO) term enrichment analysis was used to highlight the most relevant GO terms associated with a given gene list following toxin exposure. In addition, in order to phenotypically anchor the gene expression data, changes in the heterogeneity of cell subtype populations and cell cycle phase were monitored using flow cytometry. Whilst LiCl exposure did not significantly alter the proportion of cells expressing markers for stem cells/undifferentiated cells (Oct4, SSEA4), neurons (Neurofilament M), astrocytes (GFAP) or cell cycle phase, the drug caused a 1.4-fold increase in total cell number. In contrast, exposure to VPA resulted in significant upregulation of Oct4, SSEA, Neurofilament M and GFAP with significant decreases in both G2/M phase cells and cell number. This neurosphere model might provide the basis of a human-based cellular approach for the regulatory exploration of developmental impact of potential toxic chemicals.