In vitro models for human neurological diseases

Abstract

The present invention relates to a method for the preparation of an in vitro model for a human neurological disorder. Moreover, the invention relates to the in vitro model, produced according to the inventive method disclosed herein. In addition, the invention relates to the use of the in vitro model of the invention in drug testing. In addition, the invention relates to methods for testing the efficacy of drugs in the prevention, delay and amelioration of a human neurological disorder.

Description

[0001] IN VITRO MODELS FOR HUMAN NEUROLOGICAL DISEASES

[0002] Technical field

[0003] The inventions belong to the technical field of developing in vitro models for neurodegenerative diseases, especially proteinopathies and the use these models for studying disease initiation and development as well as the use these models for drug testing, i.e. the finding of effective medicaments and diagnostic tools for very specific proteinopathies.

[0004] Background

[0005] Including Alzheimer's disease (AD), abundant filamentous protein deposits are the main pathological characteristic of many neurodegenerative disorders collectively termed proteinopathies, which are clinically characterized by dementia and / or motor dysfunction. Many studies including our pioneer ones, supports the hypothesis that cell-to-cell transfer coupled to a seeding activity is the pathogenic mechanism (1). Proteinopathies present different neuropathological features regarding the type of protein filaments involved, the type of cell or the brain region they affect and disease propagation leading to specific clinical observations (2). Transgenic mouse models have been of great help, but they remain complex, and they do not recapitulate all characteristics of human proteinopathies, such as tauopathies. Additionally, due to the lack of specific biomarkers, final AD and tauopathy diagnostics is so far only possible after analysis of postmortem brain tissue. In addition, there is to date no treatment available that effectively triggers or slows the progression of AD and tauopathies. The same holds true for other proteinopathies, i.e. neurodegenerative disorders that are characterized by the accumulation of specific proteins within neurons or in the brain parenchyma. Parkinson's disease as well as prion disease are examples of other proteinopathies. To overcome these situations, robust in vitro proteinopathy models that recapitulate human phenotypes and that allow for the analysis of the development of a certain neurodegenerative disease are needed.

[0006] The derivation of human induced pluripotent stem cells (HiPSCs) with the ability to develop into any cell type has become a potential tool to study neurodegenerative disorders as well as for screening drugs (3-6).

[0007] The present invention provides solutions to understand, analyze and characterize proteinopathies, including filament strain propagation in vitro by inducing specific proteinopathies in neural cells derived from healthy HiPSCs. With this invention, proteinopathy-specific 3D cerebral models are provided which help to understand the pathophysiological mechanisms leading to certain disease induction and propagation, to improve clinical diagnostic and to screen for therapeutic options.

[0008] Summary of the invention

[0009] In a first aspect, the present invention relates to a method for the preparation of an in vitro model for human neurological disorders, the method comprising

[0010] (a) providing human induced pluripotent stem cells (HiPSCs),

[0011] (b) developing neural progenitor cells (NPCs) from the HiPSCs,

[0012] (c) expanding NPCs

[0013] (d) preparing an oriented three-dimensional neural network based on the NPCs of step (c), comprising the steps of

[0014] (i) preparing monolayers of spheres

[0015] (ii) seeding the NPCs of step (c) on each of the monolayers of spheres,

[0016] (ii) transferring to one of these monolayers the spheres of another monolayer, wherein the transferred spheres comprise attached NPCs,

[0017] (iii) repeating step (ii) for at least one more time in order to form a three- dimensional neural network,

[0018] (iv) allowing NPCs to differentiate into neural cells, wherein the spheres have a size of 40-60 pm and wherein the subsequently added spheres are layered above the initial monolayer;

[0019] (e) treating the oriented neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder, or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0020] In an embodiment, step (d)(ii) is performed around 3-7 days after step (i).

[0021] In an embodiment, step (d)(iii) is performed around 1-2 days after step (ii).

[0022] In an embodiment, the neurological disorder is a neurodegenerative disorder.

[0023] In an embodiment, the neurodegenerative disorder is a proteinopathy. In an embodiment, the proteinopathy is selected from the list comprising Alzheimer's disease, cerebral p-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, prion diseases, Parkinson's disease (RD), frontotemporal lobar degeneration (FTLD), FTLD-FUS (fused in sarcoma), Huntington's disease and other trinucleotide repeat disorders, familial British dementia, familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHWA-I), CADASIL, Alexander disease, Pelizaeus-Merzbacher disease, familial amyloidotic neuropathy, senile systemic amyloidosis, synucleinopathies, and tauopathies.

[0024] In an embodiment, the human neurodegenerative disorder is a tauopathy. In an embodiment, the tauopathy the is selected from the group comprising Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), tangle-only dementia (TD), chronic traumatic encephalopathy (CTE), argyrophilic grain disease (AGD), and corticobasal degeneration (CBD).

[0025] In an embodiment, the neurodegenerative disorder is Alzheimer's disease.

[0026] In an embodiment, the spheres are transparent spheres.

[0027] In an embodiment, the spheres are glass spheres or silica spheres.

[0028] In an embodiment, the spheres are lime-soda glass spheres.

[0029] In an embodiment, the spheres are coated.

[0030] In an embodiment, the spheres are coated with Matrigel®, collagen, laminin, polyornithin or poly(L-lysine), preferably the spheres are coated with Matrigel®.

[0031] In an embodiment, the spheres are coated with Geltrex®.

[0032] In an embodiment, the spheres are Matrigel®-coated glass spheres.

[0033] In an embodiment, the spheres have a diameter size of 40-50 pm.

[0034] In an embodiment, the spheres have a diameter size of 45-50 pm.

[0035] In an embodiment, the spheres have a diameter size of 50 pm.

[0036] In an embodiment, the oriented three-dimensional neural network comprises neurons, astrocytes, oligodendrocytes and microglia.

[0037] In an embodiment, the brain homogenate comprises soluble and / or insoluble filaments.

[0038] In an embodiment, the brain homogenate comprises soluble and / or insoluble tau filaments.

[0039] In an embodiment, step (a) is performed on day 0 of cell culture.

[0040] In an embodiment, step (b) is performed on days 0-7 of cell culture.

[0041] In an embodiment, step (c) is performed on days 7-14 of cell culture. In an embodiment, step (d) is performed on days 14-28 of cell culture.

[0042] In an embodiment, step (e) may be performed on days 28-42 of culture.

[0043] In an embodiment, the brain homogenate is incubated on the three-dimensional neural network for around 6 to 72 hours, preferably for around 24 hours.

[0044] In an embodiment, the treated three-dimensional neural network is cultured for another 4-10 days, preferably 7-10 days, preferably in neuronal differentiation medium.

[0045] In an embodiment, the in vitro model can be analyzed using confocal imaging.

[0046] In another aspect, the invention refers to an in vitro model for a human neurological disorder, wherein the in vitro model has been produced according to the method disclosed herein.

[0047] In another aspect, the invention refers to an in vitro model for a human neurological disorder, wherein the in vitro model is a model of a sporadic form of the human neurological disorder.

[0048] In an embodiment, the in vitro model for a human neurological disorder is an in vitro model of a sporadic form of Alzheimer's Disease.

[0049] In another aspect, the invention refers to the use of the in vitro model as disclosed herein for drug candidate analysis.

[0050] In another aspect, the invention refers to a method for analyzing drug candidates for their efficacy in treating human neurological disorders, the method comprising providing an in vitro model for human neurological disorders as disclosed herein, treating the model with a drug candidate, and analyzing the effect of the drug candidate on the diseased neural cells of the in vitro model.

[0051] In an embodiment, the method for analyzing drug candidate further comprises determining the suitability of the tested drug candidate to treat the neurological disorder of the cells in the in vitro model.

[0052] In an embodiment, the method for analyzing drug candidate further comprises determining the suitability of the tested drug candidate to treat the neurological disorder of the cells in the in vitro model.

[0053] In another aspect, the invention refers to a method for analyzing drug candidates for their efficacy in preventing or delaying the onset of human neurological disorders, the method comprising providing a healthy oriented 3D neural network by performing following consecutive steps: (a) providing human induced pluripotent stem cells (HiPSCs),

[0054] (b) developing neural progenitor cells (NPCs) from the HiPSCs,

[0055] (c) expanding NPCs

[0056] (d) preparing an oriented three-dimensional neural network based on the NPCs of step (c), comprising the steps of

[0057] (i) preparing monolayers of spheres

[0058] (ii) seeding the NPCs of step (c) on each of the monolayers of spheres,

[0059] (ii) transferring to one of these monolayers the spheres of another monolayer, wherein the transferred spheres comprise attached NPCs,

[0060] (iii) repeating step (ii) for at least one more time in order to form a three- dimensional neural network,

[0061] (iv) allowing NPCs to differentiate into neural cells, wherein the spheres have a size of 40-60 pm and wherein the subsequently added spheres are layered above the initial monolayer; treating the oriented 3D neural network with a drug candidate, optionally before or after creation of the in vitro model; treating the oriented 3D neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder, or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder; analyzing the effect of the drug candidate on the onset, the reduction, or the abolition of the disease in the neural cells of the in vitro model.

[0062] In one other aspect, the invention relates to a method for the preparation of a two- dimensional in vitro model for human neurological disorders, the method comprising providing human iPSCs (HiPSCs), developing neural progenitor cells (NPCs) from the HiPSCs, preparing a two-dimensional neural network based on said NPCs, comprising growing the NPCs on a growth surface having cone-shaped Matrigel® or collagen protrusions, and differentiating and expanding the NPCs into neural cells, treating the 2D neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder, or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0063] In another aspect, the invention relates to a method for the preparation of a two-dimensional in vitro model for human neurological disorders, the method comprising providing human iPSCs (HiPSCs), developing neural progenitor cells (NPCs) from the HiPSCs, preparing a two-dimensional neural network based on said NPCs, comprising growing the NPCs on a growth surface comprising Matrigel®, and differentiating and expanding the NPCs into neural cells, treating the 2D neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder, or treating the neural network with recombinant peptides characteristic for said neurological disorder, or treating the neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0064] Brief description of the figures

[0065] Figures 1 and 2 present reconstructions of Z-stack fluorescent microscopy images from two distinct areas of a 3D in vitro model according to the invention, under control (Fig. 1) and Tau- treated (Fig. 2) conditions. The field of view corresponds to a volume of 340 pm x 340 pm x 22.5 pm. Neurons were stained with an anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was detected using the AT8 antibody (green), and cell nuclei were counterstained with DAPI (blue). The bottom row shows a magnified view of the region highlighted by the yellow dashed box. In the control condition (Fig. 1), the arrow indicates an area displaying a specific AT8 signal located outside neurons, indicative of the absence of Tau pathology, whereas in the Tau-treated condition (Fig. 2), the arrows indicate regions with strong AT8 signal within neurons, indicative of Tau pathology.

[0066] Figure 3A shows fluorescent microscopy images of a 3D in vitro model in different conditions. Neurons were stained with anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was stained with AT8 antibody (green), and DAPI was used to stain cell nuclei (blue).

[0067] Figure 3B shows the mean AT8 antibody signal intensity in the culture of Figure 3A. Figure 4A shows fluorescent microscopy images of a 2D in vitro model in different conditions. Neurons were stained with anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was stained with AT8 antibody (green), and DAPI was used to stain cell nuclei (blue).

[0068] Figure 4B shows the mean AT8 antibody signal intensity in the culture of Figure 4A.

[0069] Figure 5 shows a 2D in vitro model of a human neurological disorder for drug testing. Human neural cells were immunostained with Tujl (neuronal marker), AT8 (Tau phosphorylated at Ser202 / Thr205), and DAPI (nuclear counterstain), (a) Control (untreated), (b) Control + methylene blue (6 pM). (c) FloCells™. (d) FloCells™ + methylene blue.

[0070] Detailed description of the invention

[0071] The present invention relates to a method for the preparation of an in vitro model fora human neurological disorder. Moreover, the invention relates to the in vitro model, produced according to the inventive method disclosed herein. In addition, the invention relates to the use of the in vitro model of the invention in drug testing. In addition, the invention relates to methods for testing the efficacy of drugs in the prevention, delayand amelioration of a human neurological disorder.

[0072] Advantages of the invention

[0073] Working with the present inventions based on HiPSCs allows to understand and analyze pathophysiological mechanisms underlying the induction and propagation of human proteinopathies, especially tauopathies. The invention thus provides for the first time an in vitro model for proteinopathies, such as AD and tauopathies, giving precious insights on characteristic filaments such as tau strain properties. Thus, beyond providing precious information on the diversity of proteinopathies, the present invention will allow to screen specific biomarkers and therapeutic molecules for their effect on neural cells opening the possibility to specifically diagnose and treat human proteinopathies.

[0074] The present invention is a mature three-dimensional network of human neural cells that can survive for up to 13 months under healthy conditions (healthy in vitro model resembling human brain tissue). This environment is ideally suited for replicating sporadic neurodegenerative diseases that occur with age.

[0075] The present invention's three-dimensional network is maintained in a non-organized architecture and non-tissue structure. Thus, the model is kept simple and enables to analyze and control cellular communication, disease induction and propagation, and to precisely characterize mechanistic changes. The model of the invention uses human induced pluripotent cells coming from healthy controls contra rily to the prior art using cells coming from (non-healthy) patients or artificially mutated cells. By working with healthy cells, also ethical issues associated with sourcing cells from patients with dementia is avoided.

[0076] The present invention provides models for any neurodegenerative disorder including familial as well as sporadic forms of a respective neurodegenerative disorder.

[0077] Prior art often focuses on familial forms of neurodegenerative diseases. For instance, numerous prior art documents relate to familial AD, which accounts, however, for only 1% of real life cases. In only 1% of cases, genetic mutation in several genes lead to early-onset familial AD (FAD), typically manifesting in young individuals around age of 45. FAD is caused by genetic mutations that run-in families and is inherited in an autosomal dominant manner. This means that if one parent carries the mutated gene, each child has a 50% chance of inheriting it and potentially developing the disease.

[0078] Contrarily, the present invention accurately replicates the pathology seen in 99% of AD cases, namely sporadic AD cases. The majority of neurodegenerative diseases are sporadic, occurring randomly and not following hereditary transmission patterns.

[0079] The present invention specifically provides models for sporadic neurodegenerative diseases. For instance, the present invention provides a model for sporadic AD. The present invention models the pathophysiological changes seen in sporadic AD or other tauopathies, which lead to dementia. The term sporadic refers to a disease that occurs randomly and unpredictably without a clear family history. This is in contrast to a familial condition that is caused by abnormalities or mutations in a person's DNA. These abnormalities can be inherited from one or both parents, or they can occur spontaneously.

[0080] The invention provides robust results by reliably inducing sporadic A|3 and tau pathologies, compared to other models based on culturing HiPSCs from sporadic AD patients, which only exhibit signs of neurodegeneration and do not reproduce amyloid plaques or tangles (Foveau et al., Journal of Alzheimer's Disease, vol. 67(3):893-910).

[0081] The cells comprised in the model mimic human brain pathology, providing direct access to brain pathological biomarkers of A|3 and tau pathologies, which is crucial for developing diagnostic tools.

[0082] The present invention can precisely mimic specific sporadic neurodegenerative processes in vitro, offering a highly reliable and reproducible platform for research and drug development. With unprecedented precision in testing, the invention can accelerate the early identification and optimization of both therapeutic and diagnostic candidates. Definitions

[0083] The term "oriented" in "oriented three-dimensional neural network" means that it has a direction, one cell can communicate with another.

[0084] The expression "three-dimensional neural network" is interchangeable with the expression "neural 3D network", "3D neural network", "3D-oriented cellular network" or "in vitro model", wherein the "in vitro model" is either a healthy in vitro model or a diseased in vitro model. Thus, the three-dimensional neural network / the model is present at two different states, a "healthy" state and a "diseased state". The diseased state is provided after treatment of the healthy model with human brain homogenate, rodent brain homogenate, or recombinant proteins. The in vitro model for human neurological disorder is an in vitro model in a diseased state, i.e. an in vitro model which has been treated with postmortem human brain tissue.

[0085] The term "neural cells" or "neural" refers collectively to neurons, astrocytes, oligodendrocytes, microglia.

[0086] The term "neuronal" refers to neurons.

[0087] "Neural stem cells (NSCs)" refer to neural stem cells derived from human induced pluripotent stem cells. The terms NPC and NSC may be used interchangeably.

[0088] "Neural progenitor cells (NPCs)" as used in the present invention refer to neural stem cells derived from human induced pluripotent stem cells that have an ability to undergo cellular proliferation, to regenerate exact copies of themselves (self-renew), and to generate cellular progeny of uniquely differentiated cells. The progeny of NPCs can be either neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes). The terms NPC and NSC may be used interchangeably.

[0089] The term "monolayer" as used herein refers to a layer of spheres, wherein the spheres are in contact with each other only at their sides. There is no other sphere below or on top of the spheres of the monolayer.

[0090] The term "filaments" as used herein refers to proteins linked to each other via beta sheet structures. An example are tau filaments.

[0091] The term "tau" as used herein is the abbreviation for "tubulin associated unit" and refers to the microtubule associated protein Tau; the Tau protein.

[0092] The term "disorder" is used interchangeably with the term "disease" and describes a non- healthy status or condition. The term "differentiation cues" refers to instructions or triggers that guide how cells behave or develop in response to their environment. Such cues would in this case lead to differentiation of the cells, here of NPCs.

[0093] The term "Matrigel" as used herein is a composition derived from Engelbreth-Holm-Swarm mouse sarcomas (Kleinman et al., Biochemistry, 1982). Matrigel is a mixture that is not precisely defined chemically, but generally comprises laminin, collagen IV, heparin sulfate proteoglycans, entactin, and growth factors. Matrigel is commonly used as a matrix for cell cultivation. Matrigel is a trade name. Therefore, it is referred to herein as Matrigel®.

[0094] The term "day" when referring to the method of preparing the model of the invention means "culture day" or "day of cell culture" and refers to a 24 hours time span on the indicated day. The terms may be used interchangeably.

[0095] Method for the preparation of an in vitro model for human neurological disorders

[0096] In one aspect, the invention relates to a method for the preparation of an in vitro model for human neurological disorders. The method comprises providing human induced pluripotent stem cells (HiPSCs), developing neural progenitor cells (NPCs) from the HiPSCs, expanding the NPCs, and preparing an oriented three-dimensional neural network comprising NPC seeding and growth on a monolayer of spheres until attachment of the cells to the spheres, adding another layer of spheres onto the first monolayer of spheres and attached cells and seeding more NPCs to the subsequently added spheres, adding at least one more time another layer of spheres and more NPCs when the previously added cells are attached to the previously added spheres in order to form a three-dimensional neural network. The attached NPCs are differentiated into neural cells. With this, a healthy in vitro model is generated. This is followed by treating the oriented neural network (healthy in vitro model) with brain homogenates of postmortem brain tissue derived from humans who suffered from said neurological disorder, or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0097] In a preferred embodiment, the human neurological disorder is a sporadic neurological disorder. In another embodiment, the human neurological disorder is a familial neurological disorder.

[0098] In an embodiment, the human neurological disorder is a proteinopathy.

[0099] The proteinopathy may be a sporadic or familial proteinopathy. Proteinopathies are neurodegenerative disorders that are characterized by the accumulation of specific filamentous proteins within neural cells or in the brain parenchyma. Most prevalent examples for typical proteinopathies are Alzheimer's disease (AD) and Parkinson's disease (PD). In healthy brain, these proteins are unstructured as a monomer, serving most likely as the physiological form. In a disease conditions, the unstructured proteins experience a conformational change leading to small oligomers that eventually will aggregate into higher ordered structures. Thus, the general principle of proteinopathies is that the proteins change their conformation thereby gaining toxic activity or losing the normal function. The most prominent type of a typical proteinopathy is AD although many more exist such as PD, Lewy body disease, prion diseases, and tauopathies. In nearly all instances, the disease-causing molecular configuration involves an increase in beta-sheet secondary structure of the protein. In an embodiment, the human neurological disorder comprises the presence of an p-amyloid peptide (A|3). In an embodiment, the human neurological disorder comprises the presence of Tau protein. In an embodiment, the human neurological disorder comprises the presence of a-synuclein. In an embodiment, the human neurological disorder comprises the presence of filaments forming a p-sheet. In another embodiment, the human neurological disorder comprises the presence of PrPsc.

[0100] In an embodiment, the proteinopathy is selected from the group comprising AD, cerebral |3- amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, prion diseases, PD and other synucleinopathies, tauopathies, frontotemporal lobar degeneration (FTLD), FTLD-FUS (fused in sarcoma), Huntington's disease and other trinucleotide repeat disorders, familial British dementia, familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHWA-I), CADASIL, Alexander disease, Pelizaeus-Merzbacher disease, familial amyloidotic neuropathy, or senile systemic amyloidosis.

[0101] In a preferred embodiment, the proteinopathy is AD. In another preferred embodiment, the proteinopathy is PD. In a preferred embodiment, the proteinopathy is sporadic AD. In another preferred embodiment, the proteinopathy is sporadic PD. In another embodiment, the proteinopathy is familial AD or familial tauopathies. In an embodiment, the familial AD comprises a mutation on the APP or PS gene. Such mutations lead to A|3 deposition and subsequent tau pathology (i.e. tau phosphorylation and / or tau aggregation). In an embodiment, the familial tauopathies comprise one or more mutations in the tau gene. Tau mutations may be associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17T), a condition that is clinically characterized by a range of symptoms, many of which resemble Parkinson's disease. Neuropathologically, these diseases are marked by the deposition of abnormal tau protein in the brain, without the presence of amyloid-beta (AP) pathology, which distinguishes them from other neurodegenerative conditions like Alzheimer's disease.

[0102] In a preferred embodiment, the human neurological disorder is a tauopathy.

[0103] Tauopathies are a class of neurodegenerative diseases characterized by the aggregation of abnormal tau protein. Hyperphosphorylation of tau proteins causes them to dissociate from microtubules and form insoluble aggregates with different morphologies including neurofibrillary tangles. Tauopathies comprise primary and secondary tauopathies. Primary tauopathies comprise disorders where the predominant feature is the deposition of tau protein. Secondary tauopathies comprise diseases exhibiting tau pathologies attributed to different and varied underlying causes. In an embodiment, the tauopathy is selected from the group comprising AD, Pick's disease (PiD), progressive supranuclear palsy (PSP), tangle-only dementia (TD), chronic traumatic encephalopathy (CTE), argyrophilic grain disease (AGD), and corticobasal degeneration (CBD). In a preferred embodiment, the tauopathy is Alzheimer's disease.

[0104] In a preferred embodiment, the in vitro model for a human neurological disorder is an in vitro model for sporadic Alzheimer's Disease.

[0105] Neural cell culture generation from human pluripotent stem cell (HiPSCs)

[0106] Providing human induced pluripotent stem cells

[0107] HiPSC culture is preferably conducted on day -21 to day 0 of culture.

[0108] Induced pluripotent stem cells (iPSCs) are a type of stem cells generated by reprogramming a variety of mature, specialized somatic cells into an embryonic-like pluripotent state. The HiPSCs used in the present invention are derived from dermal fibroblast or peripheral blood mononuclear cells of a healthy human being, established from single clones and expanded in feeder-free conditions. Such HiPSCs can be purchased from companies such as the Coriell Institute. These expanded HiPSCs are ready to use preferably within 2-3 weeks of culture for differentiation into neural progenitor cells (NPCs). Optionally, the cultures HiPSCs may be cryopreserved at this stage in a controlled rate freezing container. At this point, cells can be stored in liquid nitrogen for a prolonged time thus allowing to thaw cryotubes and start differentiation into neurons at any desired time.

[0109] During this initial culture time, HiPSCs should be maintained in an undifferentiated state and cultured in appropriate conditions before initiating differentiation. Accordingly, HiPSCs may preferably be cultured in a stem cell medium on a coated surface, preferably a surface coated with a polymer like Matrigel® (e.g. Corning, 354277). In an embodiment, the HiPSCs are cultured on a poly-L-ornithone and / or laminin coated surface. Suitable media are mTeSRl™, StemFlex™, or E8. The HiPSCs of the present invention are not produced in a process that involves modifying the germ line genetic identity of a human being or which involves the use of a human embryo for industrial or commercial purposes. In an embodiment, the stem cell medium may be mTeSRl™ (STEM CELL TECHNOLOGIES) that supports the maintenance of pluripotency. The cells are preferably maintained in a standard incubator at 37°C, 5% CO2. For splitting, the HiPSCs are treated with Accutase™ (e.g., Innovative Cell Technologies) and plated as dissociated cells in well of a 24-well plate at a cell density of l-5xio4cells / cm2, preferably around 2.6xl04cells / cm2. Preferably, the medium is changed daily, and cells are passaged every 4-5 days when they reach 70-80% confluence, which ensures that they are in an undifferentiated state. Human pluripotent stem cells are typically identified by the expression of key markers such as OCT4, NANOG, and SOX2, which are transcription factors essential for maintaining pluripotency. Surface markers like SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 are also commonly used. Additionally, alkaline phosphatase (AP) activity and the ability to form teratomas in vivo are functional indicators of pluripotency. These markers help confirm that the cells are undifferentiated and pluripotent.

[0110] In an embodiment, the HiPSC culture is conducted on day -30 to day 0. Preferably, the HiPSC culture is conducted on day -21 to day 0. In an embodiment, HiPSC culture is conducted on day -18 to day 0. In an embodiment, HiPSC culture is conducted on day -15 to day 0.

[0111] In an embodiment, the HiPSC culture is conducted on day -20 to day 0. In an embodiment, the HiPSC culture is conducted on day -19 to day 0. In an embodiment, the HiPSC culture is conducted on day -17 to day 0. In an embodiment, the HiPSC culture is conducted on day -16 to day 0. In an embodiment, the HiPSC culture is conducted on day -14 to day 0. In an embodiment, the HiPSC culture is conducted on day -14 to day 0. In an embodiment, the HiPSC culture is conducted on day -13 to day 0. In an embodiment, the HiPSC culture is conducted on day -12 to day 0. In an embodiment, the HiPSC culture is conducted on day -11 to day 0. In an embodiment, the HiPSC culture is conducted on day -10 to day 0. In an embodiment, the HiPSC culture is conducted on day -9 to day 0. In an embodiment, the HiPSC culture is conducted on day -8 to day 0. In an embodiment, the HiPSC culture is conducted on day -7 to day 0. In an embodiment, the HiPSC culture is conducted on day -6 to day 0. In an embodiment, the HiPSC culture is conducted on day -5 to day 0. In an embodiment, the HiPSC culture is conducted on day -4 to day 0. In an embodiment, the HiPSC culture is conducted on day -3 to day 0. In an embodiment, the HiPSC culture is conducted on day -2 to day 0. In an embodiment, the HiPSC culture is conducted on day -1 to day 0.

[0112] The cultured HiPSCs are collected on day 0 of culture.

[0113] Neural induction

[0114] Developing neural progenitor cells (NPCs) from the HiPSCs

[0115] Developing of NPCs, i.e., neural induction, is preferably conducted from day 0 to day 7 of cell culture.

[0116] In an embodiment, developing of NPCs conducted from day 0 to day 4 of cell culture. In an embodiment, developing of NPCs conducted from day 0 to day 5 of cell culture. In an embodiment, developing of NPCs conducted from day 0 to day 6 of cell culture. In an embodiment, developing of NPCs conducted from day 0 to day 8 of cell culture. In an embodiment, developing of NPCs conducted from day 0 to day 9 of cell culture. In an embodiment, developing of NPCs conducted from day 0 to day 10 of cell culture. For the development of HiPSCs to NPCs, the HiPSCs are cultured in neural induction medium (NIM).

[0117] In an embodiment, the neural induction medium comprises Neurobasal™ Medium with L- glutamine (e.g. Capricorn Scientific), supplemented with N-2 (e.g. Thermo Fisher Scientific) and B-27 without vitamin A (e.g. Thermo Fisher Scientific) for neuroectodermal differentiation, and two SMAD inhibitors. In a preferred embodiment, the NIM comprises a TGF-P receptor inhibitor and a BMP inhibitor. In a more preferred embodiment, the NIM comprises SMAD inhibitors SB431542 and LDN193189.

[0118] For neural induction, HiPSCs are plated in a 6-well or 12-well plate preferably at a density of 6.3xl04-2.6xl05cells / cm2. This will allow the cells to reach confluence by day 3 of culture. The surface of the wells / culture dishes is preferably coated with a polymer, preferably Matrigel® for enhanced cell attachment. In an embodiment, the cells are cultured in wells coated with poly-L-ornithine and laminin. Cells may be cultured in a neural induction medium (NIM) supplemented with SB431542 and LDN193189 to promote neural lineage commitment.

[0119] The medium is preferably changed daily to fresh NIM containing inhibitors to promote the differentiation of HiPSCs into neural progenitor cells (NPCs). In an embodiment, the NIM comprises SMAD inhibitors. In a preferred embodiment, the NIM comprises SMAD inhibitors SB431542 and LDN193189. The inhibitors drive neural differentiation by blocking non-neural signaling pathways, which are crucial for driving the differentiation towards neural lineages.

[0120] After about 3-4 days (days 3-4 of culture), the cells will begin to flatten and start to form clusters of columnar-shaped cells (neural rosettes) demonstrative of neural induction.

[0121] After about 5-7 days (days 5-7 of culture), cell culture is continued in NIM. Neural rosettes continue to form and grow, and more defined NPCs should emerge by Day 7. Neural rosettes can be easily identified visually because they resemble a flower-like structure, typically formed by tight clusters of neural progenitor cells that organize into a central lumen surrounded by columnar-like cells.

[0122] NPCs are typically stained for the expression of Pax6, SOX1, and Nestin. It is recommended to stain for at least two of these three neural markers (e.g., Pax6 and SOX1, Pax6 and Nestin, or SOX1 and Nestin) to confirm NPC identity. Nestin should not be used alone to assess induction efficiency, as it can also be expressed by neural crest cells, which are the most common non- NPC cell type present in neural induction cultures. Additional markers such as OCT4 (pluripotency) and SOXIO (neural crest) may be used as negative controls to assess induction specificity. Neural progenitor cell expansion

[0123] Expanding NPCs

[0124] The expansion of NPCs is preferably conducted on day 7 to day 14 of culture.

[0125] At this point, neural rosettes comprising the NPCs can be manually dissected or dissociated. Once rosettes have formed, the rosettes are gently dissociated using Accutase™. The dissociated cells are plated in neural progenitor medium (NPM) and fed with fresh medium every 2-3 days to expand the NPC population.

[0126] In an embodiment, the neural progenitor medium comprises Neurobasal™ Medium (with L- glutamine) (Thermo Fisher Scientific) supplemented with N-2 Supplement and B-27 Serum- Free Supplement with vitamin A (Thermo Fisher Scientific), EGF and bFGF. EGF and bFGF have preferably a final concentration of 20-50 ng / ml and 10-20 ng / mL respectively.

[0127] The NPCs should maintain their undifferentiated state, which is identified by a monolayer of proliferating NPCs. Differentiation cues should be avoided during this phase.

[0128] In an embodiment, the expansion of NPCs is preferably conducted on day 5 to day 14. In an embodiment, the expansion of NPCs is preferably conducted on day 6 to day 14. In an embodiment, the expansion of NPCs is preferably conducted on day 8 to day 14. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 15. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 16. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 13. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 12. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 11. In an embodiment, the expansion of NPCs is preferably conducted on day 7 to day 10. In an embodiment, the expansion of NPCs is preferably conducted on culture day 5-7 to day 13-16.

[0129] Neuronal Differentiation and onset of 3D culture

[0130] Preparing an oriented three-dimensional neural network using the cultured NPCs

[0131] Neuronal differentiation is conducted while a 3D neural network is formed. This is performed preferably from day 14-16 to day 28-42 of culture.

[0132] In an embodiment, NPC development and expansion is performed for 2-3 weeks.

[0133] In an embodiment, the growth and differentiation of NPCs comprise the transfer of NPCs to spheres, wherein the spheres form a monolayer and the attachment of the cells to the spheres.

[0134] On day 14 to 16 of culture, when the neural progenitor cells reach 70-80% confluence, the NPCs are collected and transferred into neuronal differentiation medium (NDM). In an embodiment, the neuronal differentiation medium comprises Neurobasal™ Medium supplemented with B-27 (with vitamin A) and Glutamine, further supplemented with SMAD inhibitors to promote neural lineage commitment, neurotrophic factors, preferably BDNF, GDNF, NT-3, cAMP to promote synaptic function and neuronal maturation, and retinoic acid to drive terminal differentiation and enhance synaptic plasticity.

[0135] In an embodiment, SMAD inhibitors SB431542 and LDN193189 are used to promote neural lineage commitment.

[0136] Sterilized spheres are provided as a monolayer. The cells are transferred onto the monolayer of spheres.

[0137] For this step, three to five single layers of spheres are arranged in three to five wells of a 12- or 24- or 48-well plate to create monolayers. For the 3D assembly of the neural network, the previously cultured NPCs are plated on each of the three to five monolayers of spheres in NDM medium. In an embodiment, the NPCs may be plated on the monolayers of spheres at a concentration of 1X104-1X106cells / cm2. In an embodiment, the NPCs may be plated on the monolayers of spheres at a concentration of 1X105-2.1X105cells / cm2. In a preferred embodiment, the NPCs are plated on the monolayers of spheres at a density of 1.25xl05cells / cm2. The NPCs will immediately start to attach to the spheres. These monolayers with NPCs are cultured for 1-7 days of culture. Preferably, the monolayers with NPCs are cultured for 3-7 days of culture. The spheres do not attach to the bottom of the well in the well-plate.

[0138] Once monolayers of spheres are completely covered with attached cells, preferably after 3-7 days of culture, the 3D in vitro model can be stacked / layered. For the establishment of the 3D in vitro model, a first monolayer with attached NPCs is chosen as the basic monolayer. Another monolayer with attached NPCs is used for the first transfer of spheres. Therefore, the spheres of the monolayer to which the NPCs are attached are transferred with a pipet to the basic monolayer, wherein the basic monolayer is covered with the transferred spheres in order to form a two-layered structure. The two-layered structure is incubated for 1-3 days of culture, preferably for 1 day. After incubation, another monolayer of the initially prepared three to five monolayers with attached NPCs is used for the second transfer of spheres. Therefore, the spheres of the monolayer to which the NPCs are attached are transferred with a pipet to the two-layered structure, wherein the second layer is covered with the transferred spheres in order to form a three-layered structure.

[0139] This layering is performed another one to two times resulting in a four- to five-layered structure.

[0140] This repetitive process will result in the formation of a tightly packed 3D neural network. The formation of these three-dimensional structures provide favorable conditions to create cell interactions and form networks in about three weeks. During the whole process of attaching to spheres and layering, the NPCs are cultured in neuronal differentiation medium. Neuronal cell differentiation will be initiated once the NPCs are cultured in NDM. Thus, neuronal cell differentiation takes place as soon as the seeding on NPCs on the monolayers of spheres is initiated.

[0141] The NDM drives neuronal differentiation but does at the same time not block glial differentiation. This is favorable, as this keeps the cellular environment as close as possible to the one in the living brain. In addition, in many tauopathies there is glial deposition of tau protein, meaning that tau protein is in a pathological form in astrocytes and oligodendrocytes.

[0142] The spheres used for the preparation of the oriented three-dimensional neural network have preferably a size of about 50 pm. In an embodiment, the spheres have a size of between BO- GO pm. In an embodiment, the spheres have a size of between 40-50 pm. In an embodiment, the spheres have a size of between 45-50 pm. In an embodiment, the spheres used for the oriented three-dimensional neural network are culture beads. In an embodiment, the spheres are transparent spheres. Transparent spheres allow for optical imaging.

[0143] In an embodiment, the spheres are Matrigel® coated glass spheres. In an embodiment, the spheres are Geltrex® coated glass spheres. In an embodiment, the spheres are Matrigel® coated glass spheres with a size of 40-50 pm, more preferably 45-50 pm.

[0144] In a preferred embodiment, the spheres are soda-lime glass spheres (e.g. mo-sci, GL0191B5 / 45-53). These spheres are preferably coated, preferably with Matrigel® or Geltrex® (ThermoFischer Scientific, A1569601). Such coated spheres improve cell adhesion and support neuronal maturation. While coating the spheres they should be kept on ice. Prior to the addition of the NPCs, the coated spheres are pre-warmed in an incubator at 37°C and 5% CO2, to equilibrate at 37°C for at least 60 minutes.

[0145] In an embodiment, the spheres are poly(L-lysine) coated glass spheres. In an embodiment, the spheres are laminin coated glass spheres. In an embodiment, the spheres are Matrigel® coated glass spheres with a size of 40-50 pm, more preferably 45-50 pm. In another embodiment, the spheres are coated silica spheres. In an embodiment, the spheres are poly(L-lysine) coated silica spheres. In an embodiment, the spheres are laminin coated silica spheres. In an embodiment, the spheres are Matrigel® coated silica spheres. In an embodiment, the spheres are Geltrex® coated silica spheres. In an embodiment, the spheres are poly(L-lysine) coated silica spheres with a size of 40-50 pm, more preferably 45-50 pm. The spheres may preferably have a diameter of around 45-50 pm.

[0146] The term "sphere" is interchangeable with the term "bead". The spheres of the invention have a substantially round shape. The spheres of the invention have a round shape.

[0147] During formation of a 3D in vitro model, the cells in the model are cultured in neuronal differentiation medium (NDM). Between days 17-21 of culture, it can be observed that further neuronal processes extend between the spheres. Highly interconnected networks are being formed, all without the need for astrocyte-derived conditioned medium or a glial feeder layer. As states above, although the NPCs are differentiated in neuronal differentiation medium, it could be observed that the 3D neural networks also comprise glial cells.

[0148] Astrocytes may be present in the model with a ration of up to 40%. 60% of the mature model may comprise neurons and oligodendrocytes.

[0149] Between days 21-28 of culture, is has been observed that neurons mature further, and dendritic structures and synaptic connections start to form. At day 28 of culture, most of the neurons are mature. From day 28 onwards, further maturation and maintenance of the neuronal cells is observed.

[0150] Day 28 onward the cells in the model are continued to be cultured in NDM. By this point, most neurons are mature. Homeostasis is reached at about 5-6 weeks from initiation of the 3D model; this means from days 35-42 of culture.

[0151] The model can be kept in culture for several weeks to months to achieve further maturation. To maintain health and viability of the cells, the medium should be replaced twice a week. During this phase, the neurons can be analyzed for electrophysiological activity (e.g., spontaneous action potentials, synaptic activity). Neuronal differentiation may be assessed by immunocytochemistry using Anti-Beta-Tubulin III Antibody, Clone TUJ1 (StemCell, Catalog #60052). The presence of synapses can be assessed by evaluating the expression and localization of synapsin.

[0152] At neuronal differentiation days 28-42, preferably days 35-42, more preferably on about day 42 of culture, cells are mature and form an in vitro model resembling a healthy cell state. These in vitro models may be used as control models. At this stage, the 3D neural network is thus referred to as "healthy" in vitro model. In other words, without the treatment of the 3D neural network with brain homogenate, the network can be maintained in culture for up to 13 months, and probably even longer, and is from day 28 of culture referred to as a healthy in vitro model.

[0153] After differentiation and maturation of NPCs to neural cells the healthy in vitro model can be treated with human brain homogenate, in order to produce diseased cells, and thus an in vitro model specific for a certain neurological disorder, preferably neurodegenerative disorder is provided.

[0154] Brain homogenates

[0155] In an embodiment the brain homogenate comprises disease specific filaments, wherein a filament is a protein filament or a peptide characteristic for the respective neurodegenerative disease. In an embodiment, the filament is one or more selected from the group comprising tau filament, p-amyloid peptide (AP), a-synuclein, the PrPscisoform of the prion protein (scrapie). The brain homogenate is added to the oriented three-dimensional model in order to initiate a specific neurological disorder.

[0156] In another embodiment, the brain homogenate from a transgenic rodent model for said neurological disorders comprises a disease specific protein filament or a peptide characteristic for the respective neurodegenerative disorder. In an embodiment, the recombinant protein is one or more selected from the group comprising tau filament, amyloid- peptide (AP), a- synuclein, the PrPscisoform of the prion protein (scrapie). The recombinant protein is added to the oriented three-dimensional model in order to initiate a specific neurological disorder.

[0157] In another embodiment, the recombinant protein characteristic for a neurodegenerative disorder comprises a disease specific protein filament or a peptide characteristic for the respective neurodegenerative disorder. In an embodiment, the recombinant protein is one or more selected from the group comprising tau filament, amyloid-p peptide (AP), a-synuclein, the PrPscisoform of the prion protein (scrapie). The recombinant protein is added to the oriented three-dimensional model in order to initiate a specific neurological disorder.

[0158] Preparation of the brain homogenates

[0159] Total brain homogenates were prepared as follows, resulting in a 1:10 (w / v) homogenate.

[0160] Postmortem rodent brain material was derived from transgenic animal models for proteinopathies.

[0161] Postmortem human brain material was derived from neuropathologically confirmed cases of sporadic and familial proteinopathies. Brain regions were chosen due to their high burden of protein inclusions, e.g. tau inclusions, AP, a-synuclein, orthe PrPscisoform of the prion protein (scrapie). Brain tissue from age-matched healthy controls is prepared likewise and may be used as a control treatment of the healthy in vitro model.

[0162] Brain tissue was homogenized at a concentration of 10% (w / v) in sterile phosphate-buffered saline (PBS), briefly sonicated (Branson 450, output 2, 5 x 0.9 s) and centrifuged at 3,000g at 4 °C for 5 min. The 3,000g supernatant contains soluble and insoluble proteins (solution of first supernatant).

[0163] In an embodiment, the first supernatant may be used for the treatment of the in vitro model to induce the respective neurological disorder.

[0164] The supernatant was then centrifuged at 100,000 x g at 4 °C for 20 minutes. The resulting supernatant, containing soluble protein, was aliquoted and stored at -80 °C until use (solution of second supernatant). The pellet contains insoluble proteins, e.g. tau filaments. The pellets containing insoluble proteins are resuspended in the original volume of PBS (solution of first dissolved pellet).

[0165] Supernatant and pellet fractions may be analyzed by Western blotting to detect protein bands, e.g. human tau protein bands, with the use of respective antibodies, e.g. anti-tau antibodies.

[0166] Preferably, a second round of ultracentrifugation of the first dissolved pellet is performed at 100,000 x g at 4°C for 20 minutes to concentrate the insoluble aggregates. The pellet is resuspended in the original volume of PBS (solution of second dissolved pellet). The dissolved pellet was aliquoted and stored at -80°C until use. It could be observed that the respective pathologies could be transferred more efficiently (faster and in higher quantity) to the model by using this second supernatant.

[0167] Thus, in a preferred embodiment, the brain homogenate is a solution of the second dissolved pellet.

[0168] In alternative embodiments, the brain homogenate is a solution of first supernatant.

[0169] In alternative embodiments, the brain homogenate is a solution of second supernatant.

[0170] In yet another embodiment, the brain homogenate is a solution of the first dissolved pellet.

[0171] In a preferred embodiment, the brain homogenate (solution of a first supernatant, second supernatant, first dissolved pellet or second dissolved pellet) is further diluted 1:10 in PBS before use.

[0172] The presently disclosed way of producing brain homogenate concentrated in disease specific protein / filaments is performed in order to avoid the use of chemical products, which can be toxic to the model. The advantage is to have only human brain material with no artificial chemicals.

[0173] Synthetic filaments

[0174] In some embodiments, recombinant human preformed filaments may be used to apply on the 2D or 3D oriented cellular networks.

[0175] Commercially available purified proteins specific for certain neurodegenerative diseases may be used for the treatment of the healthy in vitro model, in order to generate the model of the invention.

[0176] Recombinant tau may be used. Commercially available purified tau (15 pM) produced by bacteria such as E. coli was incubated with 40 pg / mL HP sodium (#411210010, ACROS Organics) to induce tau aggregation. The incubation was carried out at 37°C in buffer B, which consisted of 30 mM Tris-HCI (pH 7.5), 5 mM DTT, and 0.1% sodium azide to prevent microbial contamination and stabilize protein integrity. The reaction was maintained with gentle shaking at 200 rpm to promote uniform mixing and ensure that the tau protein remained in solution throughout the incubation period. The incubation lasted for 7 days, allowing sufficient time for potential aggregation or conformational changes in tau in the presence of the selected salts under these specific conditions.

[0177] Treatment of the oriented neural network with brain homogenate

[0178] By the treatment of the oriented neural network with the respective brain homogenate a certain disease is "transferred" to the so far healthy in vitro model. The resulting in vitro model of the invention thus corresponds to the clinical picture of the patient or of the rodent model from whom the brain homogenate was obtained. In an embodiment, where recombinant protein is used to transfer the disease, the resulting in vitro model of a human neurological disorder corresponds to the clinical picture related to this specific protein.

[0179] Thus, in an embodiment the brain homogenate is derived from human brain tissue derived from a biobank, wherein the human was postmortem diagnosed for a neurodegenerative disorder. An in vitro model for a certain neurodegenerative disorder is prepared by treating the oriented three-dimensional network with a brain homogenate derived from patients with such neurodegenerative disorder. The present in vitro model may thus be established for any human neurological disorder by the treatment of the oriented three-dimensional neural network with brain homogenate obtained from patients who suffered from the desired disorder. Any brain homogenate disclosed herein is obtained from human tissue of dead human bodies who were ante or postmortem diagnosed with a specific neurodegenerative disorder. The brain homogenates are based on brain tissue areas with recordable disease.

[0180] In an embodiment, an in vitro model for AD is prepared by treating the oriented three- dimensional network with a brain homogenate derived from AD patients. In an embodiment, an in vitro model for PD is prepared by treating the oriented three-dimensional network with a brain homogenate derived from PD patients.

[0181] Leveraging its long-term culture capabilities, the three-dimensional neural network may be treated with brain homogenate at different stages of maturation and homeostasis. The healthy three-dimensional neural network can be sustained for up to 12 months.

[0182] The neural 3D network comprising mature neural cells, preferably at culture days 28-42, were treated once with brain homogenate. The brain homogenate solution is preferably at a 1:1000 dilution. In other embodiments, 1:10.000, 1:100.000 or 1:1.000.000 dilutions may be used. Since the 3D in vitro model can be kept in culture over at least 13 months, treatment with brain homogenate can be performed also after day 42 of culture.

[0183] The brain homogenate is incubated on the three-dimensional neural network for 6 to 72 hours. Preferably, the brain homogenate is incubated on the three-dimensional neural network for 18 to 26 hours. More preferably, the brain homogenate is incubated on the three- dimensional neural network for 20 to 24 hours. Most preferably, the brain homogenate is incubated on the three-dimensional neural network for about 24 hours.

[0184] About twenty-four hours after the addition of the brain extract to the three-dimensional network, the medium was completely replaced with fresh conditioned medium to remove any residual filaments from the extracellular space.

[0185] Use of the in vitro model for analyzing drug candidates for their efficacy in treating neurodegenerative disorders

[0186] The in vitro model for human neurological disorders of the invention may be used for analyzing drug candidates or for the neuropathological analysis and characterization of specific diseased neural cells.

[0187] With the use of the in vitro models, effective treatments may be found which reverses the changes induced in the diseased cells of the respective model. Neuropathological characterization of different disease models will help to understand the behavior of such diseased cells and their functional changes over time.

[0188] In one aspect, the present invention relates to the use of the herein disclosed in vitro model for human neurological disorders for the analysis of drug candidates. In an embodiment, the disclosed in vitro model is used for the analysis of drug candidates for their efficacy in treating neurodegenerative disorders. In an embodiment, the analysis of drug candidates is the analysis of the efficacy of certain drug candidates in delaying the progression of the specific neurodegenerative disorder displayed by the respective in vitro model. In an embodiment, the analysis of drug candidates is the analysis of the impact of certain drug candidates in the onset of the specific neurodegenerative disorder of the respective in vitro model. In an embodiment, the disclosed in vitro model is used for the analysis of effective treatments which reverse the changes induced in the diseased cells of the in vitro model. In yet another embodiment, the disclosed in vitro model is used for the neuropathological analysis and characterization of specific diseased neural cells.

[0189] In an embodiment, the in vitro model is used to analyze the behavior of diseased cells.

[0190] In an embodiment, the in vitro model is used to analyze functional changes of diseased cells over time.

[0191] The "diseased cells" relate to the respectively treated two- or three-dimensional neural network. Thus, in an embodiment, the diseased cells are cells of an in vitro model for human AD. In another embodiment, the diseases cells are cells of an in vitro model for human PD. In yet another embodiment, the diseases cells are cells of an in vitro model for a human tauopathy. In order to analyze drug efficacy, an in vitro model of a certain disease comprising neurons and glial cells.

[0192] Drugs of interest may be added at various time points prior to and following brain treatment with brain homogenate ranging from day 1 to day 14 after treatment or immediately after filament removal. The drugs may be applied daily, every other day, or as a single dose. The state of the pathology may be compared between control and treated conditions by confocal imaging. The therapeutic efficacy may be determined by a reduction in staining with specific antibodies detecting pathological proteins, e.g. tau, indicating decreased disease formation.

[0193] This approach enables the evaluation of therapeutic efficacy at different stages of disease prevention and progression, from early development to later stages, providing an opportunity to prevent and / or treat tauopathies.

[0194] Method for analyzing drug candidates

[0195] In another aspect, the present invention is directed to a method for analyzing drug candidates for their efficacy in treating or preventing neurological disorders or for their efficacy in postponing the onset of the disease. The method comprises providing the in vitro model for human neurological disorders as disclosed herein, treating the model with a drug candidate, and analyzing the effect of the drug candidate on the diseased neural cells of the in vitro model.

[0196] In an embodiment, the method comprises determining the suitability of the tested drug candidate to treat the neurological disorder.

[0197] Alternative 3D in vitro model

[0198] An alternative 3D in vitro model is prepared in line with the previously described in vitro model. However, instead of layers of spheres, a 3D structure is prepared prior to cell seeding. Therefore, cone-like 3D structures made of Matrigel® or Geltrex® may be formed on a culture dish. The formed cones are then used as a matrix on which the cells are allowed to grow. While creating these structures, the culture dish should be kept on ice. Prior to adding the cells, the culture dish is pre-warmed in an incubator at 37°C and 5% CO2 to equilibrate the dish at 37°C for at least 60 minutes.

[0199] NPCs are added in NDM as outlined above once. In an embodiment, the NPCs may be plated on the cone-like structures at a concentration of 1X104-1X106cells / cm2. In an embodiment, the NPCs may be plated on the cone-like structures at a concentration of 1X105-2X105cells / cm2. In a preferred embodiment, the NPCs are plated on the cone-like structures at a concentration of 1.25xl05cells / cm2.

[0200] The cells are cultured in NDM and are then treated with brain homogenate as described for the 3D in vitro model above. In an embodiment, the NPCs are developed from HiPSCs within about 7 days of culture.

[0201] In an embodiment, the NPCs are cultured in neuronal differentiation medium.

[0202] The 3D neural network is fully generated by day 28 of culture.

[0203] The 3D model is then treated with brain homogenate in order to prepare an in vitro model of a human neurological disorder, or left untreated, in order to use the cells as a healthy 3D in vitro model for control measurements.

[0204] Mature neural cells at about culture day 28-42 are treated with the desired concentration of human brain homogenate, i.e. brain homogenate is diluted to 1:1000, 1:10.000, 1:100.000 or 1:1.000.000. The brain homogenate is incubated on the cells for 24 hours. In a preferred embodiment, the final brain homogenate dilution in the well is 1:10000. For the treatment, the brain homogenate is added once to the respective cell cultures. The day after, the total cell media was replaced in order to remove filaments from the extracellular space.

[0205] The brain homogenate is prepared as disclosed for the 3D in vitro model above.

[0206] 24 hours after addition of the brain homogenate, the medium is exchanged to fresh NDM. The model is kept in NDM for the further days of culture.

[0207] The human 3D model is ready to use as a 3D in vitro model for a human neurological disorder after around 7 days post treatment with brain homogenate.

[0208] Thus, in one aspect, the invention relates to a method for the preparation of a three- dimensional in vitro model for human neurological disorders, the method comprising providing human iPSCs, developing neural progenitor cells (NPCs) from the HiPSCs, preparing a three-dimensional neural network comprising, comprising NPC cell growth on cone-shaped Matrigel structures and cell differentiation and expansion of the NPCs into neural cells, treating the neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0209] 2D in vitro model

[0210] The inventors found that the two-dimensional culture can also be used for the preparation of an in vitro model of a human neurodegenerative disease and the analysis of drug candidates for delaying the onset or the progression of human neurological, preferably neurodegenerative disorders.

[0211] Thus, the invention also relates to a method for the preparation of a two-dimensional in vitro model for human neurological disorders.

[0212] In one aspect, the invention relates to a method for the preparation of a two-dimensional in vitro model for human neurological disorders, the method comprising providing human iPSCs, developing neural progenitor cells (NPCs) from the HiPSCs, preparing a two-dimensional neural network based on said NPCs, comprising NPC cell growth on a monolayer of spheres and cell differentiation and expansion of the NPCs into neural cells, treating the neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder.

[0213] Induced pluripotent stem cells (iPSCs) are a type of stem cells generated by reprogramming a variety of mature, specialized somatic cells into an embryonic-like pluripotent state. The HiPSCs used in the present invention are derived from dermal fibroblast or peripheral blood mononuclear cells of a healthy human being, established from single clones and expanded in feeder-free conditions. Such HiPSCs can be purchased from companies. The HiPSCs are expanded as described for the 3D in vitro model herein.

[0214] These expanded HiPSCs are differentiated into NPCs as described for the 3D in vitro model above, before optional cryopreservation in a controlled rate freezing container. At this point, cells can be stored in liquid nitrogen for a prolonged time thus allowing to thaw cryotubes and start differentiation into neurons at any desired time.

[0215] Subsequently, NPCs are expanded as described for the 3D in vitro model above.

[0216] A monolayer of spheres is provided in a 6-28-well plate. The same spheres are used as described for the 3D in vitro model of the invention above.

[0217] NPCs are added in NDM as outlined above once. In an embodiment, the NPCs may be plated on the monolayer of spheres at a concentration of 1X104-1X106cells / cm2. In an embodiment, the NPCs may be plated on the monolayer at a concentration of 1X105-2X105cells / cm2. In a preferred embodiment, the NPCs are plated on the monolayer at a concentration of 1.25xl05cells / cm2.

[0218] During formation of a 2D in vitro model, the cells in the model are cultured in neuronal differentiation medium (NDM) as described for the 3D in vitro model. In an embodiment, the NPCs are developed from HiPSCs within about 7 days of culture.

[0219] In an embodiment, the NPCs are cultured in neural differentiation medium. Preferably, 50% of the medium is replaced every day.

[0220] The 2D neural network is fully generated by day 28 of culture.

[0221] The 2D model is then treated with brain homogenate in order to prepare an in vitro model of a human neurological disorder, or left untreated, in order to use the cells as a healthy 2D in vitro model for control measurements.

[0222] Mature neural cells at about culture day 28-42 are treated with the desired concentration of brain homogenate, such as human brain homogenate, i.e. brain homogenate is diluted to 1:1000, 1:10.000, 1:100.000 or 1:1.000.000. The brain homogenate is incubated on the cells for 24 hours. In a preferred embodiment, the final brain homogenate dilution in the well is 1:10000. For the treatment, the brain homogenate is added once to the respective cell cultures. The day after, the total cell media was replaced in order to remove filaments from the extracellular space.

[0223] The brain homogenate is prepared as disclosed for the 3D in vitro model above.

[0224] 24 hours after addition of the brain homogenate, the medium is exchanged to fresh NDM. The model is kept in NDM for the further days of culture.

[0225] The human 2D model is ready to use as a 2D in vitro model for a human neurological disorder after around 7 days post treatment with brain homogenate.

[0226] In another aspect, the invention relates to a method for the preparation of a two-dimensional in vitro model for human neurological disorders, the method comprising providing human iPSCs, developing neural progenitor cells (NPCs) from the HiPSCs, preparing a two-dimensional neural network based on said NPCs, comprising NPC cell growth as a monolayer and cell differentiation and expansion of the NPCs into neural cells, treating the neural network with brain homogenate of postmortem brain tissue derived from humans who suffered from said neurological disorder or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder, or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

[0227] Induced pluripotent stem cells (iPSCs) are a type of stem cells generated by reprogramming a variety of mature, specialized somatic cells into an embryonic-like pluripotent state. The HiPSCs used in the present invention are derived from dermal fibroblast or peripheral blood mononuclear cells of a healthy human being, established from single clones and expanded in feeder-free conditions. Such HiPSCs can be purchased from companies. The HiPSCs are expanded as described for the 3D in vitro model herein.

[0228] These expanded HiPSCs are differentiated into NPCs as described for the 3D in vitro model above, before optional cryopreservation in a controlled rate freezing container. At this point, cells can be stored in liquid nitrogen for a prolonged time thus allowing to thaw cryotubes and start differentiation into neurons at any desired time.

[0229] Subsequently, NPCs are expanded as described for the 3D in vitro model above.

[0230] A monolayer of cells is provided in a 6-28-well plate.

[0231] NPCs are added in NDM as outlined above once. In an embodiment, the NPCs may be plated at a concentration of 1X104-1X106cells / cm2. In an embodiment, the NPCs may be plated at a concentration of 1X105-2X105cells / cm2. In a preferred embodiment, the NPCs are plated at a concentration of 1.25xl05cells / cm2.

[0232] During formation of a 2D in vitro model, the cells in the model are cultured in neuronal differentiation medium (NDM) as described for the 3D in vitro model.

[0233] In an embodiment, the NPCs are developed from HiPSCs within about 7 days of culture.

[0234] In an embodiment, the NPCs are cultured in neural differentiation medium. Preferably, 50% of the medium is replaced every day.

[0235] The 2D neural network is fully generated by day 28 of culture.

[0236] The 2D model is then treated with brain homogenate in order to prepare an in vitro model of a human neurological disorder, or left untreated, in order to use the cells as a healthy 2D in vitro model for control measurements.

[0237] Mature neural cells at about culture day 28-42 are treated with the desired concentration of brain homogenate, such as human brain homogenate, i.e. brain homogenate is diluted to 1:1000, 1:10.000, 1:100.000 or 1:1.000.000. The brain homogenate is incubated on the cells for 24 hours. In a preferred embodiment, the final brain homogenate dilution in the well is 1:10000. For the treatment, the brain homogenate is added once to the respective cell cultures. The day after, the total cell media was replaced in order to remove filaments from the extracellular space.

[0238] The brain homogenate is prepared as disclosed for the 3D in vitro model above.

[0239] 24 hours after addition of the brain homogenate, the medium is exchanged to fresh NDM. The model is kept in NDM for the further days of culture. 1 The human 2D model is ready to use as a 2D in vitro model for a human neurological disorder after around 7 days post treatment with brain homogenate.

[0240] Cell fixation for immunofluorescence staining

[0241] All cells and in vitro models described herein may be fixated and stained for imaging.

[0242] Cells can be fixated at any time point of the protocol. The in vitro model as such can be fixated. For fixation, cells are fixated with 4% PFA. Cells may be stained using different antibodies.

[0243] Fixation may be with 4% PFA for 20 minutes at room temperature. The models can then be stored at 4°C until staining with the desired antibodies.

[0244] For fixation and staining, the in vitro model is not disrupted but used in one piece.

[0245] Imaging

[0246] Confocal microscopy Z series images are preferably acquired on a Zeiss LSM510 confocal microscope with 63x dipping objective (0.9 N.A.), and a Zeiss LSM 510 Axiovert 200 using a 20x air objective and 40x oil objective (1.3 N.A.). Laser power, photomultiplier gain, and filter sets are preferably selected to minimize bleaching and bleed-through between channels (In nm, Alexa488 and GFP. ex 488, em 500-550; Cy3: ex543, em. 565-615; Alexa 647: ex 633, em 650-700).

[0247] Examples

[0248] Example 1 - preparation of an in vitro model for Alzheimer's Disease

[0249] Providing human induced pluripotent stem cells on culture days -21 to 0

[0250] HiPSCs were purchased from Coriell Institute and cultured in mTeSRl™ basal medium supplemented with mTeSRl supplement, Rock inhibitor at a concentration of 10 pM and P / S (PSC medium) in matrigel-coated wells of a 6-well plate. Cells were cultured for 21 days. The medium was changed daily and the cells were passages every 4-6 days when they reach 70- 80% confluence using Accutase.

[0251] The HiPSCs are detached and collected on day 0 of culture, washed and diluted in PSC medium in a concentration of 2.6xl04cells / cm2.

[0252] Developing neural progenitor cells on days O to 7 of culture

[0253] For neural induction, HiPSCs were plated in a 6-well or 12-well plate preferably at a density of 6.3xl04-2.1xl05cells / cm2. Cells were cultured in neural induction medium (N I M) comprising Neurobasal Medium with L-glutamine (e.g. Capricorn Scientific), supplemented with N-2 (e.g. Thermo Fisher Scientific) and B-27 without vitamin A (e.g. Thermo Fisher Scientific) for neuroectodermal differentiation, and two SMAD inhibitors SB431542 and LDN193189.

[0254] Medium was changed on day 1 and then daily with fresh NIM supplemented with SB431542 and LDN193189 to promote neural lineage commitment.

[0255] Cells were maintained in this medium until day 7 of culture.

[0256] Culturing NPCs in neural progenitor medium from day 7 to day 14 of culture

[0257] Around day 7, cellular rosettes have formed. The rosettes were gently dissociated using Accutase, and plated in neural progenitor medium (NPM). Medium was changed every 2-3 days. Neural progenitor medium comprised Neurobasal™ Medium (Thermo Fisher Scientific) supplemented N-2 supplement and with B-27 Serum-Free Supplement with vitamin A (Thermo Fisher Scientific), EGF (at a final concentration of 20 ng / mL) and bFGF (at a final concentration of 20 ng / mL).

[0258] Preparing an oriented three-dimensional neural network on days 14-28 of culture

[0259] NPCs were confluent on day 14. NPCs are detached with Accutase and transferred into neuronal differentiation medium. The neuronal differentiation medium (NDM) comprised Neurobasal™ Medium supplemented with B-27 (with vitamin A) and Glutamine, and was further supplemented with SMAD inhibitors, neurotrophic factors BDNF, GDNF, NT-3, cAMP, and retinoic acid. The NPCs were plated on the first layer of spheres at a concentration of 1.25xl05cells / cm2.

[0260] Four monolayers of Matrigel-coated soda lime glass spheres were prepared in a well on a 12- well plate, respectively. The spheres had a diameter of 50 pm.

[0261] The NPCs were seeded on the monolayers of spheres. Cells were allowed to attach to the spheres for 5 days. After cell attachment to the spheres, one of these monolayers with attached cells was chosen to be the basic layer. The spheres comprising attached NPCs of another of these monolayers were gently isolated by use of a pipet and transferred onto the basic monolayer in order to create a two-layered structure. After 24 hours, this transfer was repeated using the spheres comprising the attached NPCs in order to create a three-layered structure. This step was repeated once more after 24 hours with the spheres with attached NPCs of the last remaining prepared monolayer. With this, a four-layered structure of spheres was formed.

[0262] During layering and until day 28 of culture, cells were kept in NDM. The 3D model was formed within two weeks. On day 28 of culture, the 3D model was complete. Preparation of brain homogenate - before day 28 of culture

[0263] Postmortem brain tissue from an individual diagnosed with AD was homogenized at a concentration of 10% (w / v) in sterile phosphate-buffered saline (PBS), briefly sonicated (Branson 450, output 2, 5 x 0.9 s) and centrifuged at 3,000g at 4 °C for 5 min. The supernatant contains soluble proteins (solution of first supernatant).

[0264] The supernatant was then centrifuged at 100,000 x g at 4 °C for 20 minutes. The resulting supernatant, containing soluble tau, was aliquoted and stored at -80 °C until use. The supernatant contained the soluble protein (solution of second supernatant), whereas the pellet contained insoluble proteins, e.g. tau filaments. The pellets containing insoluble tau were resuspended in the original volume of PBS (solution of first dissolved pellet).

[0265] Supernatant and pellet fractions were analyzed by Western blotting to detect human tau protein bands with the use of anti-tau antibodies. The slowest migrating tau species, which represent the insoluble proteins, are immunoreactive with antibody AT100. Soluble pathological tau proteins are immunoreactive with other phosphorylation-dependent anti-tau antibodies such as AT8.

[0266] A second round of ultracentrifugation of the first dissolved pellet at 100,000 x g at 4°C for 20 minutes was performed to concentrate the insoluble aggregates. The pellet is resuspended in the original volume of PBS (solution of second dissolved pellet). The dissolved pellet was aliquoted and stored at -80°C until use.

[0267] The solution of the second dissolved pellet was used for treatment of the 3D neural network.

[0268] Treating the 3D neural network with brain homogenate on day 28 of culture

[0269] The 3D models were treated with the brain homogenate of human AD brain on day 28 of culture. Therefore, the brain homogenate solution of the second dissolved pellet (solution of the second dissolved pellet) was used at a 1:1000 dilution.

[0270] Thus 1 pl of the brain homogenate was pipetted per ml culture volume into the wells with the 3D models and was incubated for 24 hours. After 24 hours, the medium was completely replaced with fresh NDM to remove any residual filaments from the extracellular space.

[0271] After another 7 days of culture, the 3D in vitro model for Alzheimer's Disease was completed.

[0272] It was subsequently fixed and analyzed using confocal imaging. Cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature and stained with different anti- tau antibodies (Tau5, AT8, AT100) in order to assess tau pathology with a confocal microscope.

[0273] Healthy control models

[0274] Some of the 3D models were not treated with the brain homogenate. These models were kept for the same duration in NDM and were used as the healthy control. Confocal imaging visualized diverse proteins after immunostaining. Localization of pathology within the cells could be analyzed by double staining with neural or neuronal specific proteins and pathological tau epitopes. The cells could be analyzed in a 3D manner and the pathology could be quantified.

[0275] Example 2: human cerebral model of tauopathies

[0276] A two- and three-dimensional neural network comprising glial cells was prepared as described herein. Tau filaments were either extracted from brain tissue homogenate of patients who had died from well-defined AD, progressive supranuclear palsy (PSP), Pick's disease (PiD) and chronic traumatic encephalopathy (CTE), respectively or were prepared from recombinant Tau protein as described herein. Once the cells have reached maturity in the healthy 3D model, here maturation was proceeded to between days 28 and 56 of culture, filaments can be added to the culture medium. Tau filament incubation was performed for a duration of 6 hours, extending up to 72 hours. After the incubation period, the culture medium is replaced with fresh medium, and the cells were maintained in culture for a minimum of 24 hours and up to 14 days, allowing sufficient time for the filaments to enter the cells and induce the desired pathological effects. Cells were then fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature and stained with different anti-tau antibodies (Tau5, AT8, AT100) in order to assess tau pathology with a confocal microscope.

[0277] With confocal imaging diverse proteins could be visualized after immunostaining. Localization of pathology within the cells could be analyzed by double staining with neural or neuronal specific proteins and pathological tau epitopes. The cells could be analyzed in a 3D manner, and the pathology could be quantified.

[0278] Example 3: 2D drug testing on human cerebral model of tauopathies

[0279] 3D AD-diseased and healthy control human 3D models were prepared as described in Example 1. Drugs of interest were added at various time points prior to and following brain treatment ranging from day 1 to day 14 after treatment. The drugs were applied daily, every other day, or as a single dose immediately after filament removal. After the therapeutic treatment, cells were fixed with 4% PFA and stained with different anti-tau antibodies to quantify and compare pathology between control and treated conditions by confocal imaging. The therapeutic efficacy was determined by a reduction in staining with specific antibodies detecting pathological tau proteins, indicating decreased disease formation.

[0280] This approach enables the evaluation of therapeutic efficacy at different stages of disease prevention and progression, from early development to later stages, providing an opportunity to prevent and / or treat tauopathies. Example 4: Preparation of an in vitro model for Frontotemporal dementia with parkinsonism (FTDP17T, P301S Tau mutation)

[0281] 3D human models were prepared as described in Example 1.

[0282] Preparation of brain homogenate

[0283] Postmortem brain tissue of mice transgenic for human P3O1S tau and age-matched non- transgenic control mice (C57BI / 6 mice) stored at -80 °C were processed on ice under sterile conditions. Tissue was first weighed in a sterile cup and transferred to a 2 mL KIMBLE Dounce tissue grinder (D8938, KIMBLE). PBS (ThermoFisher, 11580456) was added at a 1:10 (w / v) ratio, and the tissue was allowed to thaw on ice. Homogenization was performed using the Dounce grinder by first applying approximately 10 gentle strokes with pestle A until the tissue passed smoothly under the pestle with minimal resistance, followed by approximately 10 additional strokes with pestle B until only small fragments remained. The homogenizer and pestles were rinsed sequentially with distilled water, 70% ethanol, and distilled water. The homogenate was then transferred to a pre-chilled microtube and subjected to sonication using a ThermoFisher FB120EUK-220 sonicator. Before and after sonication, the probe was rinsed with distilled water, 70% ethanol, and distilled water. Samples were sonicated in a beaker containing an ice-water mixture to prevent warming, using the following settings: 1 s ON / 1 s OFF pulses for a total of 5 pulses (10 s), at 20% power (amplitude settings may require optimization). Following sonication, samples were centrifuged at 3,000 g for 5 min at 4 °C (Eppendorf CR22N), and the resulting supernatant was collected, aliquoted, and stored at -80 °C until use.

[0284] Treatment of the neural cells

[0285] The 3D models were treated with the brain homogenate of P301S mouse brainstem on day 28 of culture. Therefore, the brain homogenate solution of the first supernatant was used at a 1:1000 dilution.

[0286] Thus 1 pl of the brain homogenate was pipetted per ml culture volume into the wells with the 3D models and was incubated for 24 hours. After 24 hours, the medium was completely replaced with fresh NDM to remove any residual filaments from the extracellular space.

[0287] After another 7 days of culture, the 3D in vitro model for FTDP17T was completed.

[0288] It was subsequently fixed and analyzed using confocal imaging. Cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature and stained with different anti- tau antibodies (AT8, AT100) in order to assess tau pathology with a confocal microscope. Healthy control models

[0289] Some of the 3D models were not treated with the brain homogenate. These models were kept for the same duration in NDM and were used as the healthy control.

[0290] Immunocytochemistry

[0291] The in vitro models were fixed by gently removing the culture medium, rinsing with DPBS, and incubating cells for 5 min at room temperature. After removing the buffer, cells were fixed with cold 4% paraformaldehyde in PBS for 15 min at 4 °C, washed three times with DPBS, and kept in DPBS until further processing. For permeabilization, cells were incubated in DPBS containing 0.2% Triton X-100 for 15 min at 4 °C, followed by blocking for 1 h at room temperature in a solution containing 3% goat serum, 3% horse serum, and 0.1% Tween-20. Cells were then incubated overnight at 4 °C with primary antibodies diluted in blocking solution: mouse anti-AT8 (1:200, Invitrogen), mouse anti-ATlOO (1:200, Invitrogen), and rabbit anti-Tujl (1:500, Invitrogen). After three DPBS washes, cells were incubated for 1 h at room temperature with secondary antibodies diluted in blocking solution (Alexa Fluor 488 antirabbit IgG, 1:1000, Invitrogen; Alexa Fluor 555 anti-mouse IgG, 1:800, Invitrogen), washed three times with DPBS and once with water, and mounted using VECTASHIELD Antifade Mounting Medium with DAPL Slides were dried, stored at 4 °C, and sealed with nail polish prior to imaging.

[0292] Confocal imaging visualized diverse proteins after immunostaining. Localization of pathology within the cells could be analyzed by double staining with neural specific proteins and pathological tau epitopes. The cells could be analyzed in a 3D manner and the pathology could be quantified.

[0293] Image Analysis

[0294] The in vitro models were stained with anti-AT8 to detect hyperphosphorylated tau, Tujl to visualize neuronal morphology, and DAPI for nuclei. Images were acquired on an Olympus CKX53 confocal microscope equipped with a Hamamatsu ORCA-spark camera at 20x magnification, using identical settings for all conditions (NT, B6, and P301S). Image analysis was performed in Fiji. The Tujl channel was first adjusted for brightness / contrast and smoothed with a Gaussian blur to enhance cell borders. Segmentation was carried out by thresholding and, when needed, applying the Watershed function or manual correction. A binary mask was then generated to extract cell area and AT8-related measures (Integrated Density and Mean Intensity). Regions of interest were saved in the ROI Manager and applied to the corresponding raw images for final quantification.

[0295] Figures 1 and 2 present reconstructions of Z-stack fluorescent microscopy images from two distinct areas of a 3D in vitro model according to the invention, under control (Fig. 1) and Tau- treated (Fig. 2) conditions. The field of view corresponds to a volume of 340 pm x 340 pm x 22.5 pm. Neurons were stained with an anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was detected using the AT8 antibody (green), and cell nuclei were counterstained with DAPI (blue). The bottom row shows a magnified view of the region highlighted by the yellow dashed box. In the control condition (Fig. 1), the arrow indicates an area displaying a specific AT8 signal located outside neurons, indicative of the absence of Tau pathology, whereas in the Tau-treated condition (Fig. 2), the arrows indicate regions with strong AT8 signal within neurons, indicative of Tau pathology.

[0296] Figure 3A shows fluorescent microscopy images of a 3D in vitro model in different conditions. Neurons were stained with anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was stained with AT8 antibody (green), and DAPI was used to stain cell nuclei (blue). In control conditions, AT8 staining remains negative in cell bodies whereas, in Tau conditions, AT8 signal is detected within neurons indicative of Tau pathology.

[0297] In Figure 3B, the quantification of AT8 mean signal intensity shows a statistically significant increase of more than 2.5-fold in Tau conditions compared with the control.

[0298] Example 5: 2D human in vitro model of tauopathy and drug testing

[0299] HiPSC-derived glutamatergic neurons (ioGlutamatergic Neurons™; bit. bio) were used in this study. Cells were diluted in BrainPhys™ Neuronal Growth Medium (b:GN medium; STEMCELL Technologies), centrifuged at 200g for 3 min at room temperature, and resuspended in Complete BrainPhys™ Neuronal Growth Medium supplemented with differentiation factors (comp:GN + D medium; STEMCELL Technologies). The cell suspension was diluted to a final concentration of 1.14 x 105cells / mL, corresponding to 30,000 cells / cm2, and plated at 100 pL per well onto 96-well Screenstar plates sequentially coated with poly-D-lysine (PDL) and Geltrex. Plates were transferred to a humidified incubator (37°C, 5% CO2) and left undisturbed for 48 h. Medium was first changed at Day 2 and replaced with fresh, pre-warmed Complete BrainPhys™ Neuronal Growth Medium supplemented with differentiation factors and DAPT (comp:GN + D + DAPT medium; STEMCELL Technologies). At Day 4, 90% of the medium was gently replaced with pre-warmed comp:GN medium. From Day 6 onwards, 50% of the medium was exchanged every 48 h with fresh, pre-warmed comp:GN medium until neuronal maturation (day 11-12). Mature neurons were then exposed for 24 h to mouse brain extracts derived from P301S or C57BI / 6J mice (1:1000 dilution) or left untreated, followed by a 7-day recovery period in fresh medium. Cells were fixed on day 18-19 using 4% paraformaldehyde and processed for immunofluorescence staining. Imaging was performed by confocal microscopy on day 20, and quantitative image analysis was carried out using ImageJ software.

[0300] Preparation of brain homogenate

[0301] Postmortem brain tissue of mice transgenic for human P301S tau and age-matched non- transgenic control mice (C57BI / 6 mice) stored at -80 °C were processed on ice under sterile conditions. Tissue was first weighed in a sterile cup and transferred to a 2 mL KIMBLE Dounce tissue grinder (D8938, KIMBLE). PBS (ThermoFisher, 11580456) was added at a 1:10 (w / v) ratio, and the tissue was allowed to thaw on ice. Homogenization was performed using the Dounce grinder. The homogenate was subjected to sonication using a ThermoFisher FB120EUK-220 sonicator using the following settings: 1 s ON / 1 s OFF pulses for a total of 5 pulses (10 s), at 20% power. Following sonication, samples were centrifuged at 3,000 g for 5 min at 4 °C (Eppendorf CR22N), and the resulting supernatant was collected, aliquoted, and stored at -80 °C until use. This fraction was used to treat human cells in order to prepare an in vitro model for human neurological disease.

[0302] Treatment of the neural cells

[0303] The 2D models were treated with the brain homogenate of P301S mouse brainstem on day 12 of culture. Therefore, the brain homogenate solution of the first supernatant was used at a 1:1000 dilution.

[0304] Thus 1 pl of the brain homogenate was pipetted per ml culture volume into the wells with the 2D models and was incubated for 24 hours. After 24 hours, the medium was completely replaced with fresh medium to remove any residual filaments from the extracellular space.

[0305] After another 7 days of culture, the 2D in vitro model for FTDP17T was completed.

[0306] It was subsequently fixed and analyzed using confocal imaging. Cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature and stained with different anti- tau antibodies (AT8, AT100) in order to assess tau pathology with a confocal microscope.

[0307] Healthy control models

[0308] Some of the 2D models were not treated with the brain homogenate. These models were kept for the same duration in NDM and were used as the healthy control.

[0309] Drug testing in the in vitro models

[0310] Methylene Blue (MB), a phenothiazine-derived small molecule that modulates tau pathology by inhibiting tau aggregation and enhancing the clearance of toxic tau species, was applied on day 4 following treatment with brain homogenates (P301S or B6). MB was administered once at a final concentration of 6.25 pM. After 24 h, the medium was fully replaced with fresh culture medium to remove any residual compound. Three days after MB application (i.e., seven days after brain homogenate exposure), cells were fixed with 4% PFA and stained with anti-tau antibodies (AT8, AT100) to quantify and compare tau pathology by confocal imaging.

[0311] Therapeutic efficacy was assessed as a reduction in pathological tau signal relative to untreated conditions. Immunocytochemistry on human neural cell cultures

[0312] The 2D in vitro models were fixed by gently removing the culture medium, rinsing with DPBS, and incubating cells for 5 min at room temperature. After removing the buffer, cells were fixed with cold 4% paraformaldehyde in PBS for 15 min at 4 °C, washed three times with DPBS, and kept in DPBS until further processing. For permeabilization, cells were incubated in DPBS containing 0.2% Triton X-100 for 15 min at 4 °C, followed by blocking for 1 h at room temperature in a solution containing 3% goat serum, 3% horse serum, and 0.1% Tween-20. Cells were then incubated overnight at 4 °C with primary antibodies diluted in blocking solution: mouse anti-AT8 (1:200 , Invitrogen), mouse anti-ATlOO (1:200, Invitrogen), and rabbit anti-Tujl (1:500, Invitrogen). After three DPBS washes, cells were incubated for 1 h at room temperature with secondary antibodies diluted in blocking solution (Alexa Fluor 488 anti-rabbit IgG, 1:1000, Invitrogen; Alexa Fluor 555 anti-mouse IgG, 1:800, Invitrogen), washed three times with DPBS and once with water, and mounted using VECTASHIELD Antifade Mounting Medium with DAPI. Slides were dried, stored at 4 °C, and sealed with nail polish prior to imaging.

[0313] Image Analysis

[0314] The tauopathy in vitro models were stained with anti-AT8 to detect hyperphosphorylated tau, Tujl to visualize neuronal morphology, and DAPI for nuclei. Images were acquired on an Olympus CKX53 confocal microscope equipped with a Hamamatsu ORCA-spark camera at 20x magnification, using identical settings for all conditions (NT, B6, and P301S). Image analysis was performed in Fiji. The Tujl channel was first adjusted for brightness / contrast and smoothed with a Gaussian blur to enhance cell borders. Segmentation was carried out by thresholding and, when needed, applying the Watershed function or manual correction. A binary mask was then generated to extract cell area and AT8-related measures (Integrated Density and Mean Intensity). Regions of interest were saved in the ROI Manager and applied to the corresponding raw images for final quantification.

[0315] Figure 4A shows fluorescent microscopy images of a 2D in vitro model in different conditions. Neurons were stained with anti-Tuj-1 antibody (red), abnormal phosphorylated Tau was stained with AT8 antibody (green), and DAPI was used to stain cell nuclei (blue). In control conditions, AT8 staining remains negative in cell bodies whereas, in Tau conditions, AT8 signal is detected within neurons indicative of Tau pathology.

[0316] In Figure 4B, the quantification of AT8 mean signal intensity shows a statistically significant increase of more than 2.5-fold in Tau conditions compared with the control.

[0317] Figure 5 shows an in vitro model of a human neurological disorder for drug testing. Human neural cells were immunostained with Tujl (neuronal marker), AT8 (Tau phosphorylated at Ser202 / Thr205), and DAPI (nuclear counterstain), (a) Control (untreated). Neurons exhibit a well-organized Tujl-positive network consistent with normal hiPSC-derived neuronal development. AT8 immunoreactivity is sparse and faint, (b) Control + methylene blue (6 pM). In the absence of Tau filaments, treatment with methylene blue, a phenothiazine derivative that has shown significant interest as a modulator of tau pathology, preserves the typical Tujl- positive neurite network. AT8 staining remains minimal and is further reduced compared with the untreated control, (c) FloCells™. Exposure to Tau filaments derived from P3O1S Tau transgenic mouse brain homogenate results in a robust AT8-positive signal in neuronal cell bodies and processes, indicating pronounced Tau pathology, while the Tujl network remains intact, (d) FloCells™ + methylene blue. In P301S-treated neurons, methylene blue markedly reduces AT8 immunoreactivity to levels comparable to those observed in control conditions (a-b), without affecting the integrity of the Tujl-positive network.

Claims

CLAIMS1. A method for the preparation of an in vitro model for a human neurological disorder, the method comprising(a) providing human induced pluripotent stem cells (HiPSCs),(b) developing neural progenitor cells (NPCs) from the HiPSCs,(c) expanding NPCs(d) preparing an oriented three-dimensional neural network based on the NPCs of step (c), comprising the steps of(i) preparing monolayers of spheres(ii) seeding the NPCs of step (c) on each of the monolayers of spheres,(ii) transferring to one of these monolayers the spheres of another monolayer, wherein the transferred spheres comprise attached NPCs,(iii) repeating step (ii) for at least one more time in order to form a three- dimensional neural network,(iv) allowing NPCs to differentiate into neural cells, wherein the spheres have a size of 40-60 pm and wherein the subsequently added spheres are layered above the initial monolayer;(e) treating the oriented neural network with brain homogenate of post mortem brain tissue derived from humans who suffered from said neurological disorder or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder.

2. The method according to claim 1, wherein the disorder is a neurodegenerative disorder, preferably Alzheimer's disease.

3. The method according to claim 1 or 2, wherein the spheres are lime-soda glass spheres, preferably coated lime-soda glass spheres.

4. The method according to claims 1-3, wherein step (a) is performed on day 0 of cell culture, step (b) is performed on days 0-7 of cell culture, step (c) is performed on days 7-14 of cell culture, step (d) is performed on days 14-28 of cell culture, and step (e) may be performed on days 28-42 of culture.

5. The method according to claims 1-4, wherein the brain homogenate is incubated on the three-dimensional neural network for around 6 to 72 hours, preferably for around 24 hours.

6. An in vitro model for a human neurological disorder, wherein the in vitro model has been produced according to the method of claims 1-5, preferably wherein the in vitro modelfor a human neurological disorder is an in vitro model of a sporadic form of Alzheimer's Disease.

7. Use of the in vitro model of claim 6 for drug candidate analysis.

8. A method for analyzing drug candidates for their efficacy in treating a human neurological disorder, the method comprising providing an in vitro model for a human neurological disorder according to claim 6 or preparing an in vitro model for a human neurological disorder according to claims 1-5, treating the in vitro model with a drug candidate, and analyzing the effect of the drug candidate on the diseased neural cells of the in vitro model.

9. The method for analyzing drug candidate according to claim 8, further comprising determining the suitability of the tested drug candidate to treat the neurological disorder of the cells in the in vitro model.

10. A method for analyzing drug candidates for their efficacy in preventing or delaying the onset of human neurological disorders, the method comprising providing a healthy oriented 3D neural network by performing following consecutive steps:(a) providing human induced pluripotent stem cells (HiPSCs),(b) developing neural progenitor cells (NPCs) from the HiPSCs,(c) expanding NPCs(d) preparing an oriented three-dimensional neural network based on the NPCs of step (c), comprising the steps of(i) preparing monolayers of spheres(ii) seeding the NPCs of step (c) on each of the monolayers of spheres,(ii) transferring to one of these monolayers the spheres of another monolayer, wherein the transferred spheres comprise attached NPCs,(iii) repeating step (ii) for at least one more time in order to form a three- dimensional neural network,(iv) allowing NPCs to differentiate into neural cells, wherein the spheres have a size of 40-60 pm and wherein the subsequently added spheres are layered above the initial monolayer; treating the oriented 3D neural network with a drug candidate;treating the oriented 3D neural network with brain homogenate of post mortem brain tissue derived from humans who suffered from said neurological disorder or treating the oriented neural network with recombinant peptides characteristic for said neurological disorder or treating the oriented neural network with brain homogenate from a transgenic rodent model for said neurological disorder. analyzing the effect of the drug candidate on the onset of the disease in the neural cells of the in vitro model.

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