In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF.

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens, including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However, these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here, we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary, high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes, resulting in subsequent cholinergic neuron differentiation. Thus, our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells.

Thyroid hormone triggers the developmental loss of axonal regenerative capacity via thyroid hormone receptor α1 and krüppel-like factor 9 in Purkinje cells.

Neurons in the CNS of higher vertebrates lose their ability to regenerate their axons at a stage of development that coincides with peak circulating thyroid hormone (T(3)) levels. Here, we examined whether this peak in T(3) is involved in the loss of axonal regenerative capacity in Purkinje cells (PCs). This event occurs at the end of the first postnatal week in mice. Using organotypic culture, we found that the loss of axon regenerative capacity was triggered prematurely by early exposure of mouse PCs to T(3), whereas it was delayed in the absence of T(3). Analysis of mutant mice showed that this effect was mainly mediated by the T(3) receptor α1. Using gain- and loss-of-function approaches, we also showed that Krüppel-like factor 9 was a key mediator of this effect of T(3). These results indicate that the sudden physiological increase in T(3) during development is involved in the onset of the loss of axon regenerative capacity in PCs. This loss of regenerative capacity might be part of the general program triggered by T(3) throughout the body, which adapts the animal to its postnatal environment.

Mature Purkinje cells require the retinoic acid-related orphan receptor-α (RORα) to maintain climbing fiber mono-innervation and other adult characteristics.

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of RORα in mature PCs, we used Cre-lox mouse genetic tools in vivo that delete it specifically from PCs between postnatal days 10-21. Up to 14 d of age, differences between mutant and control PCs were not detectable: both were mono-innervated by climbing fibers (CFs) extending along their well-developed dendrites with spiny branchlets. By week 4, mutant mice were ataxic, some PCs had died, and remaining PC soma and dendrites were atrophic, with almost complete disappearance of spiny branchlets. The innervation pattern of surviving RORα-deleted PCs was abnormal with several immature characteristics. Notably, multiple functional CF innervation was reestablished on these mature PCs, simultaneously with the relocation of CF contacts to the PC soma and their stem dendrite. This morphological modification of CF contacts could be induced even later, using lentivirus-mediated depletion of rora from adult PCs. These data show that the late postnatal expression of RORα cell-autonomously regulates the maintenance of PC dendritic complexity, and the CF innervation status of the PC (dendritic vs somatic contacts, and mono-innervation vs multi-innervation). Thus, the differentiation state of adult neurons is under the control of transcription factors; and in their absence, adult neurons lose their mature characteristics and acquire some characteristics of an earlier developmental stage.

Pax3 induces target-specific reinnervation through axon collateral expression of PSA-NCAM.

Damaged or dysfunctional neural circuits can be replaced after a lesion by axon sprouting and collateral growth from undamaged neurons. Unfortunately, these new connections are often disorganized and rarely produce clinical improvement. Here we investigate how to promote post-lesion axonal collateral growth, while retaining correct cellular targeting. In the mouse olivocerebellar path, brain-derived neurotrophic factor (BDNF) induces correctly-targeted post-lesion cerebellar reinnervation by remaining intact inferior olivary axons (climbing fibers). In this study we identified cellular processes through which BDNF induces this repair. BDNF injection into the denervated cerebellum upregulates the transcription factor Pax3 in inferior olivary neurons and induces rapid climbing fiber sprouting. Pax3 in turn increases polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the sprouting climbing fiber path, facilitating collateral outgrowth and pathfinding to reinnervate the correct targets, cerebellar Purkinje cells. BDNF-induced reinnervation can be reproduced by olivary Pax3 overexpression, and abolished by olivary Pax3 knockdown, suggesting that Pax3 promotes axon growth and guidance through upregulating PSA-NCAM, probably on the axon's growth cone. These data indicate that restricting growth-promotion to potential reinnervating afferent neurons, as opposed to stimulating the whole circuit or the injury site, allows axon growth and appropriate guidance, thus accurately rebuilding a neural circuit.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

Neurons derived from sporadic Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. METHODS: We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer's disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer's disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods. RESULTS: Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-ß 1-40 (Aß1-40) and amyloid-ß 1-42 (Aß1-42). However, significantly increased Aß1-42/Aß1-40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 ß, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons. CONCLUSIONS: On the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aß1-40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.

Astrocyte Differentiation of Human Pluripotent Stem Cells: New Tools for Neurological Disorder Research.

Astrocytes have a central role in brain development and function, and so have gained increasing attention over the past two decades. Consequently, our knowledge about their origin, differentiation and function has increased significantly, with new research showing that astrocytes cultured alone or co-cultured with neurons have the potential to improve our understanding of various central nervous system diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, or Alexander disease. The generation of astrocytes derived from pluripotent stem cells (PSCs) opens up a new area for studying neurologic diseases in vitro; these models could be exploited to identify and validate potential drugs by detecting adverse effects in the early stages of drug development. However, as it is now known that a range of astrocyte populations exist in the brain, it will be important in vitro to develop standardized protocols for the in vitro generation of astrocyte subsets with defined maturity status and phenotypic properties. This will then open new possibilities for co-cultures with neurons and the generation of neural organoids for research purposes. The aim of this review article is to compare and summarize the currently available protocols and their strategies to generate human astrocytes from PSCs. Furthermore, we discuss the potential role of human-induced PSCs derived astrocytes in disease modeling.

Bmi1 Loss in the Organ of Corti Results in p16ink4a Upregulation and Reduced Cell Proliferation of Otic Progenitors In Vitro.

The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16ink4a in vitro. Viral vector-mediated overexpression of p16ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres in vitro. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16ink4a.

Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation.

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons.

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.