New method for detecting neuronal antibodies

Abstract

The present invention provides methods for obtaining human induced pluripotent stem cell (hiPSC)-derived neuronal cells, which can be used in the diagnosis of neurological disorders associated with the presence of neuronal antibodies. In particular, the present invention provides methods for obtaining hiPSC-derived neuronal cells, wherein the method comprises culturing neural progenitor cells in a neurotrophic medium for at least three weeks under optimized conditions. The present invention also covers the use of neuronal cells obtained by the methods of the present invention as diagnostic tests in neurological disorders associated with the presence of neuronal antibodies and kits comprising the same.

Description

Technical Field

[0001] This invention belongs to the field of stem cells. In particular, this invention provides a method for obtaining neurons derived from human-induced pluripotent stem cells (hiPSCs), which can be used to detect autoantibodies against the surface of neurons in patients with autoimmune neurological disorders. Background Technology

[0002] Neurological disorders associated with neuronal antibodies, such as autoimmune encephalitis (AE), are a new class of diseases caused by an immune attack on proteins present in the patient's brain cells. Clinical symptoms include neurological and psychiatric manifestations, which vary between patients and throughout the disease course. The prevalence of AE in the United States (1995–2015) was 0.8 per 100,000, and a previous study showed that nearly one-third of patients with encephalitis were diagnosed with AE (Deng Q. et al., “The antibody assay in suspected autoimmune encephalitis from positive rate to test strategies”, Front Immunol., 2022;13:803854). Rapid diagnosis and the use of immunotherapy are crucial for the prevention of symptom exacerbation, admission to the intensive care unit, and prolonged hospital stays. Clinical guidelines allow for the identification of suspected AE cases, but a definitive diagnosis of AE requires detection of specific associated antibodies in the patient's serum or cerebrospinal fluid (CSF). Despite recent advances in the discovery of AEs and the increasing number of AE-associated neuronal antigens described over the past few years (see, for example, Varley, JA, et al., “Autoimmune encephalitis: recent clinical and biological advances”, JNeurol 2023, 270, 4118–4131), a significant proportion of patients remain undiagnosed. This is the case for patients who genuinely have AEs but whose samples yield negative antibody results when measured with currently available diagnostic procedures (McCracken et al., 2017). Commercially available tests designed to detect AE antibodies are based on cell-based assays (CBAs), in which heterologous non-neuronal cells are transfected with specific plasmids to express neuronal proteins (known antigenic targets of AE antibodies). For each specific antibody (approximately 15 described in AEs and related diseases), a separate CBA must be performed to detect or rule out its presence. Ideally, a single patient sample (serum or CSF) can be tested with a commercial kit that provides a combination of six predefined antigens. When the target antigen is not included in commercial tests, the assay provides a negative result, even though “undetectable” AE antibodies are actually present in the subject sample. Furthermore, some patients may have antibodies against novel, previously undescribed neuronal antigens that are not constitutively expressed by the cell lines used for CBA.Currently, there is no available diagnostic test that serves as the first-line screening step for detecting the presence of neuronal surface antibodies in patients with suspected acute exacerbations (AEs).

[0003] Table 1 shows examples of diagnostic methods for acute exacerbations (AEs).

[0004] Table 1.

[0005]

[0006] ( ) Deng Q. et al., "The antibody assay in suspected autoimmuneencephalitis from positive rate to test strategies", Front Immunol, 2022;13:803854;

[0007] ( For example, Athea Diagnostics test code 4722 (NeoEncephalitis Paraneoplastic Evaluation with Recombx®).

[0008] ( EUROIMMUN Medizinische Labordiagnostika AG, an autoimmune encephalitis biochip.

[0009] Therefore, there is a need to improve the detection of neuronal antibodies and the diagnosis of associated neurological disorders such as acute exacerbations (AEs) and related diseases. Summary of the Invention

[0010] This invention addresses the aforementioned need and provides a method for obtaining hiPSC-derived neuronal cells, wherein the method comprises culturing neural progenitor cells (NPCs) in a proneural medium for at least three weeks (e.g., 21 days). This invention therefore provides a population of hiPSC-derived neuronal cells obtained by the method of this invention.

[0011] The present invention also provides the use of the methods and / or cells of the present invention for the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related diseases.

[0012] The present invention also provides the use of the hiPSC-derived neurons of the present invention for identifying the presence of neuronal antibodies. Furthermore, the present invention provides the use of the hiPSC-derived neurons of the present invention for identifying target antigens of: autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE.

[0013] In another aspect, the present invention provides a method for identifying neuronal antibodies associated with autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, more preferably AE and / or related diseases, wherein the method comprises:

[0014] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from the object, preferably, the object has suspected autoimmune neurological disorders, more preferably has suspected autoimmune neurological disorders associated with the presence of neuronal antibodies, more preferably has suspected AE-related diseases, and even more preferably has suspected AE;

[0015] b. Detect and identify human antibodies present in biological fluids of subjects suffering from the diseases mentioned in a., wherein the human antibodies specifically bind to hiPSC-derived neuronal cells.

[0016] Furthermore, the present invention provides a method for identifying target antigens for autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as autoimmune ataxia (AE) and / or related disorders, the method comprising:

[0017] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from a subject having suspected neurological autoimmune disorders, preferably having suspected neurological disorders associated with the presence of neuronal antibodies, more preferably having suspected autoimmune encephalitis (AE)-related diseases, and even more preferably having suspected AE.

[0018] b. Identify antigens bound to human antibodies present in the biological fluids of the subjects mentioned in a. in hiPSC-derived neuronal cells.

[0019] The present invention also provides an in vitro method for diagnosing autoimmune neurological disorders in subjects, wherein the autoimmune neurological disorders are preferably autoimmune neurological disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE, the method comprising:

[0020] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from the object;

[0021] b. Determine whether the antibodies present in the biofluid obtained from the subject bind to the antigens present in the hiPSC-derived neuronal cells of the present invention, wherein a positive immunoreactivity detected by immunocytochemistry using the biofluid exposed to the hiPSC-derived neuronal cells indicates the presence of neuronal antibodies in the subject, and thus indicates the presence of an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE.

[0022] Finally, the present invention provides a kit suitable for in vitro diagnosis of neurological autoimmune disorders in subjects, wherein the neurological autoimmune disorders are preferably neurological disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE, wherein the kit contains hiPSC-derived neuronal cells of the present invention. Attached Figure Description

[0023] Figure 1 Characterization of neural progenitor cells (NPCs) and neurons derived from human iPSCs. (A) Schematic representation of the protocol used to derive NPCs and neurons from iPSCs. (B) NPCs express neural progenitor cell markers such as Nestin, Sox2, and Pax6, as well as the proliferation marker Ki67. Nuclei are labeled with DAPI. (C) Four weeks after differentiation, most human neurons derived from NPCs express the neuronal maturation marker (MAP2), and approximately 70% of them express GABA, indicating the presence of GABAergic neurons in the culture. (D) Percentage of MAP2-positive neurons to DAPI, and the percentage of GABA-positive cells to MAP2-positive cells. (E) Human neuron cultures also express vGlut and CTIP2, confirming the presence of glutamatergic and cortical neurons. (F) Four weeks after differentiation, NPC-derived neurons express the synaptic markers Synapsin and Gephyrin, as well as ion channel receptors such as GlyR or AMPAR(G). (H) Human neurons are functional, as shown by the calcium wave representation of a single active neuron. Cell nuclei are stained with DAPI.

[0024] Figure 2Analytical performance of NeurAntigen neurons. (A) Schematic representation of the experimental procedure used to incubate patient samples with live NeurAntigen neurons. (B) Human neurons exhibit a complex morphology in culture without forming aggregates. Positive dotted patterns can be observed after incubation with serum containing neuronal surface antibodies from patients with autoimmune encephalitis, followed by incubation with secondary anti-human IgG antibodies. Note the difference between the negative control (NHS: normal human serum, from healthy donors) and the positive detection of 14 different neuronal surface antibodies on NeurAntigen neurons.

[0025] Figure 3 NeurAntigen neurons can be used for both serum and cerebrospinal fluid samples. After incubation with patient CSF or serum, positive immunostaining with a characteristic dotted pattern can be detected on human neurons. Note the lack of reactivity compared to the corresponding negative control (NHCSF: normal human CSF, from a healthy donor).

[0026] Figure 4 The NeurAntigen protocol stabilizes and improves neuronal attachment and allows visualization of complex dendrites for proper detection of neuronal antibodies. (A) A schematic representation of non-optimal conditions for human neuronal differentiation, where neuronal cultures show areas of neuronal aggregates and poorly attached cells. (B) Optimal conditions for human neuronal differentiation, including lower seeding numbers, O2 levels, and shorter culture times. Using the NeurAntigen protocol, neurons maintain good attachment and uniform distribution, thus allowing detection of neuronal antigens via patient antibodies.

[0027] Figure 5 NeurAntigen improves neuronal differentiation and maturation. (A) Representative images of mature neuron (MAP2, green) and GABAergic marker (GABA, red) expression in cell cultures after 2 and 3 weeks of neuronal differentiation using the protocol published by Yan et al. or the NeurAntigen protocol. (B) The percentage of mature neurons (MAP2+) in the culture was significantly increased after 3 weeks of treatment with the NeurAntigen protocol compared to the culture obtained according to the protocol obtained by Yan et al. (C) Positive signals with a characteristic speckled pattern of neuronal surface antibodies were detected only on NeurAntigen neurons differentiated at 3 weeks.

[0028] Figure 6Human iPSC-derived astrocytes can be used to detect glial antibodies associated with other types of autoimmune neurological disorders. (A) Representative images of aquaporin-4 (AQP4) and fibrillary acidic protein (GFAP) expressed on astrocytes using commercial antibodies. (B) Positive immunostaining detected on human astrocytes after incubation with serum from patients with neuromyelitis optica spectrum disorder containing AQP4 antibodies (top panel) or CSF from patients with meningoencephalomyelitis containing GFAP antibodies (bottom panel), followed by incubation with secondary anti-human IgG antibodies. Note the lack of reactivity compared to the corresponding negative controls (NHS: normal human serum, NHCSF: normal human CSF, both from healthy donors). Detailed Implementation

[0029] definition

[0030] All terms used herein, unless otherwise stated, shall be understood to have their conventional meaning as known in the art. For certain terms used in this application, other more specific definitions are provided below and are intended to apply uniformly throughout the specification and claims, unless otherwise expressly provided for a broader definition.

[0031] Throughout the specification and claims, the word "comprising" and variations thereof (e.g., "comprising," "having," "including," "containing") are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" covers situations where it means "consisting of." Additional objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the specification, or may be learned by practicing the invention.

[0032] In this specification and claims, in the context of describing the invention (especially in the context of the appended claims), nouns and similar references without quantifiers shall be interpreted to cover both singular and plural forms, unless otherwise indicated herein or clearly contradicted by the context.

[0033] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or can determine, many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by this invention.

[0034] The use of any and all examples or exemplary language (e.g., "such") provided herein is intended only to better illustrate the invention and, unless otherwise stated, does not constitute a limitation on the scope of the invention. No language in the specification should be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0035] When the term "about" is used with a numerical value throughout the specification and claims, it indicates an accuracy range familiar and acceptable to those skilled in the art. For example, the term "about" means an indicated value ± 1% of its value, or the term "about" means an indicated value ± 2% of its value, or the term "about" means an indicated value ± 5% of its value, the term "about" means an indicated value ± 10% of its value, or the term "about" means an indicated value ± 20% of its value, or the term "about" means an indicated value ± 30% of its value; preferably, the term "about" means an exact indicated value (±0%).

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the many terms used in this disclosure. Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form.

[0037] The range of values ​​includes the numbers that define that range.

[0038] The headings provided herein are not intended to limit the various aspects or embodiments of the disclosure. Furthermore, the invention encompasses all possible combinations of the specific aspects and embodiments described herein.

[0039] The accompanying drawings and experimental sections / exemplary embodiments are for further illustration of the invention only and should not be construed or understood as limiting the scope of the invention and / or the appended claims in any way, unless otherwise expressly indicated herein.

[0040] The embodiments illustrated and discussed in this specification are intended only to teach inventors of the best known methods of making and using the invention without departing from the invention, as will be understood by those skilled in the art in light of the foregoing teachings. Therefore, it should be understood that the invention can be practiced in ways other than those specifically described within the scope of the claims and their equivalents.

[0041] The method of the present invention

[0042] In a first aspect, the present invention provides a method for obtaining neuronal cells derived from human induced pluripotent stem cells. In the context of this invention, “human induced pluripotent stem cells” refers to a class of pluripotent stem cells that can be directly generated from somatic cells. Stem cells are cells capable of self-renewal and differentiation into specialized cell types. As described in the review by Romito A. and Cobellis G. (Romito A, Cobellis G. “Pluripotent Stem Cells: Current Understanding and Future Directions”, Stem Cells Int. 2016; 2016:9451492), the definition of “pluripotent stem cells” is based on two characteristics: self-renewal and potency. Self-renewal is the ability of stem cells to divide indefinitely, producing unaltered daughter cells that retain the same characteristics as the progenitor cells. Under specific conditions or specific signals, stem cells can cease self-renewal and initiate a process leading to differentiation into specialized cell types derived from the three germ layers (ectoderm, endoderm, and mesoderm).

[0043] The method of this invention includes culturing neural progenitor cells (NPCs) in a neuropromoting medium for at least three weeks / at least 21 days. "Neural progenitor cells" are progenitor cells of the central nervous system (CNS) that generate many (if not all) glial cell and neuronal cell types that constitute the CNS. NPCs do not generate non-neuronal cells, such as immune system cells, that are also present in the CNS. NPCs are present in the brains of newborns and mature adults and are therefore not embryonic stem cells. For characterization of NPCs based on their location, morphology, gene expression profiles, temporal distribution, and function in the brain, see, for example, Martínez-Cerdeño V. and Noctor SC., "Neural progenitor cellterminology", Front Neuroanat, 2018, 12:104.

[0044] In one embodiment, the NPC is cultured under static conditions (i.e., without stirring, shaking, etc.). Furthermore, the temperature of the culture medium is typically maintained at approximately 35 to 39°C, preferably approximately 36 to 38°C, for example, approximately 37°C.

[0045] The NPCs are then cultured under suitable conditions in a neurotrophic medium for at least 21 days (3 weeks). The inventors unexpectedly discovered that when NPCs are cultured in a neurotrophic medium for at least 21 days (3 weeks), they differentiate into mature neurons, including the expression of mature neuronal components (such as MAP2, GABA, vGlut, CITP2) and synaptic components (such as synaptic proteins and pontocetin). Therefore, in the method of the present invention, NPCs are cultured for at least 21 days, for example, three weeks, or 21 days, or longer, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or longer, for example, up to 35 days. Preferably, NPCs are cultured in a neurotrophic medium for at least 21 days. Therefore, in the method of the present invention, NPCs are cultured under suitable conditions in a neurotrophic medium for at least 3 weeks, for example, 3 weeks, 4 weeks, or 5 weeks.

[0046] In the context of this invention, "neurotropic culture medium" refers to a cell culture medium comprising at least the following:

[0047] Dulbecco's Modified Eagle Medium F12 (DMEM / F12) (e.g., Gibco 21331020) is a widely used basal medium for supporting the growth of many different mammalian cells. This medium comprises a 1:1 mixture of DMEM and Ham's F12 medium, combining the high concentrations of glucose, amino acids, and vitamins in DMEM with a wide range of components present in Ham's F12 medium. In addition to the glucose, amino acids, and vitamins found in DMEM, DMEM F12 also contains other components present in Ham's F12 medium, including zinc, putrescine, hypoxanthine, and thymidine. DMEM F12 may also contain other factors such as L-glutamine and phenol red indicator. Examples of DMEM / F12 medium components are shown in Table 2;

[0048] Neurobasal medium (e.g., Gibco 21103049) contains 25 mM D-glucose, 0.22 mM sodium pyruvate, amino acids, vitamins, inorganic salts, and other components. Examples of the components of neurobasal medium are shown in Table 3.

[0049] Vitamin A-free B27 supplements (e.g., Gibco 12587010, 50X). For example, vitamin A-free B27 supplements may contain the following components: biotin, DL-α-tocopheryl acetate, DL-α-tocopherol, BSA, fatty acid-free fraction V, catalase, recombinant human insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and T3 (triiodo-L-thyroxine). Preferably, the vitamin A-free B27 supplement is present in an amount of 0.5X.

[0050] N2 supplements (e.g., Gibco 17502048, 100X) are chemically-defined, serum-free supplements based on Bottenstein's N-1 formulation. Preferably, the amount of N2 supplement is 0.5X. Examples of the components contained in N2 supplements are shown in Table 4; and

[0051] Ultraglutamine (e.g., Lonza H3BE17-605E / U1 or Lonza™ BE17605E / U1, e.g., 200 mM) is an extremely stable dipeptide form of L-glutamine (alanyl-L-glutamine). Preferably, ultraglutamine is present in a neurotrophic medium at a concentration of about 1%.

[0052] In addition to the components mentioned above, the neurotrophic medium may contain epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2 or FGF-basic). When used for culturing NPCs (to obtain neurons derived from human induced pluripotent stem cells (hiPSCs), the neurotrophic medium... No It contains EGF and FGF2. However, when used to culture cells for the production of NPCs from NEP-rosette, the neurotrophic medium also contains FGF2 and EGF, see, for example, Figure 1A. For example, these two proteins are available from Peprotech, catalog numbers AF-100-15 and 100-18B, respectively. Recombinant human EGF is a 6.2 kDa globular protein containing 53 amino acid residues, including 3 intramolecular disulfide bonds (see SEQ ID NO: 1: NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR). FGF-2 is one of the 23 known members of the FGF family. Recombinant human FGF-basic is a 17.2 kDa protein composed of 154 amino acid residues (see SEQ ID NO: 2: AAGSITTLPAPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS). If present, EGF is present at a concentration of approximately 10 ng / ml. If present, FGF2 is present at a concentration of approximately 10 ng / ml.

[0053] The neurotropic culture medium may also contain a reducing agent, such as β-mercaptoethanol (e.g., Life Technologies 31350010, e.g., 55 mM) and / or an antibiotic, such as penicillin / streptomycin (e.g., Lonza 17-602E, e.g., 10,000 U / ml). Preferably, β-mercaptoethanol is present at a concentration of about 50 μM.

[0054] Table 2. Components of DMEM / F12 medium Gibco 21331020

[0055]

[0056]

[0057]

[0058]

[0059] Table 3. Components of the neural basal culture medium Gibco 21103049

[0060]

[0061]

[0062]

[0063] N2 supplement Gibco 17502048 components

[0064]

[0065] In a preferred embodiment, the neurotropic culture medium comprises or is composed of the following components:

[0066] - DMEM / F-12 medium (e.g., Gibco 21331020)

[0067] - Neural basal culture medium (e.g., Gibco 21103049)

[0068] - Vitamin B27 supplements that do not contain Vitamin A (e.g., Gibco 12587010)

[0069] - N2 supplements (e.g., Gibco 17502048)

[0070] - UltraGlutamine (e.g., Lonza H3BE17-605E / U1)

[0071] - β-mercaptoethanol (e.g., Life Technologies 31350010).

[0072] As mentioned above, when used for culturing NPCs, the neurotrophic medium does not contain EGF and FGF2. However, when used for culturing cells to produce NPCs from NEP-flowering, the neurotrophic medium preferably also contains:

[0073] - EGF (e.g., Peprotech AF-100-15); and

[0074] - FGF2 (e.g., Peprotech 100-18B).

[0075] See, for example Figure 1 A.

[0076] The culture medium may also contain penicillin / streptomycin (e.g., Lonza 17-602E).

[0077] More preferably, the neurotrophic culture medium comprises or is composed of the following:

[0078] - DMEM / F-12 medium (e.g., Gibco 21331020);

[0079] - Neural basal culture medium (e.g., Gibco 21103049);

[0080] - 0.5x Vitamin B27 supplement without Vitamin A (e.g., Gibco 12587010);

[0081] - 0.5x N2 supplement (e.g., Gibco 17502048);

[0082] - 1% UltraGlutamine (e.g., Lonza H3BE17-605E / U1);

[0083] - 50 μM β-mercaptoethanol (e.g., Life Technologies 31350010); and

[0084] - 1% penicillin / streptomycin (e.g., Lonza 17-602E).

[0085] As mentioned above, when used for culturing NPCs, the neurotrophic medium does not contain EGF and FGF2. However, when used for culturing cells to produce NPCs from NEP-flowering, the neurotrophic medium preferably also contains:

[0086] - 10 ng / ml FGF2 (e.g., Peprotech 100-18B); and

[0087] - 10 ng / ml EGF (e.g., Peprotech AF-100-15).

[0088] In a preferred embodiment, the neurotrophic culture medium does not contain glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and / or platelet-derived growth factor-AA (PDGF-AA, which has two A subunits).

[0089] In a preferred embodiment, NPCs are prepared at a concentration of approximately 35,000 to approximately 40,000 cells / cm². 2 The NPCs are seeded at a density and then cultured (incubated) in a neurotrophic medium for at least three weeks (at least 21 days), as described above. Preferably, the NPCs are seeded on polyornithine / lamin (PO / Lam) coated plates. Thus, in a preferred embodiment, the method of the present invention comprises culturing about 30,000 to about 40,000 NPCs / cm² in a neurotrophic medium. 2(Optimal training of approximately 37,000 NPCs / cm) 2 At least three weeks (at least 21 days) are required to obtain hiPSC-derived neurons.

[0090] Therefore, in a preferred embodiment, the method of the present invention further includes the following steps:

[0091] - At approximately 30,000 to approximately 40,000 cells / cm 2 NPCs were inoculated at a high density.

[0092] - Preferred inoculation is performed on PO / Lam coated plates; and

[0093] - Incubate the NPC for at least 21 days (at least 3 weeks), as described above.

[0094] Therefore, in a preferred embodiment, the NPC has a concentration of at least about 30,000 cells / cm². 2 For example, at least approximately 35,000 cells / cm² 2 Or higher, for example, about 37,000 cells / cm³ 2 For example, approximately 40,000 cells / cm³ 2 The preferred seeding density is approximately 30,000 to approximately 40,000 cells / cm². 2 The preferred seeding density is approximately 35,000 to approximately 40,000 cells / cm³. 2 Crop density, or even better, at approximately 37,000 cells / cm². 2 Cells are seeded at a density of at least about 30,000 to about 40,000 cells / cm². Cells are preferably seeded on plates pre-coated with polyornithine and laminin. In a preferred embodiment, the plate is first coated with polyornithine. Subsequently, the plate is washed, for example with PBS, and laminin is added. In this way, the plate is simultaneously coated with both polyornithine and laminin. The polyornithine / laminin coating promotes the attachment, spread, and proliferation of NPCs on the coated plate. The inventors unexpectedly discovered that, as described above, when NPCs are seeded at a density of at least about 30,000 to about 40,000 cells / cm², the cell density is optimal. 2 When seeded at high cell density, the differentiation of NPCs into hiPSC-derived neurons was improved, see, for example... Figure 4 And item 3 in the "Results" section - "Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons".

[0095] Furthermore, NPCs are preferably cultured under hypoxic conditions (e.g., about 5% O2). Physiological in vivo oxygen concentrations can range from 1% to 15%. However, most cell cultures are maintained under normal atmospheric oxygen conditions (about 21% O2, “normative”). The value of O2 control, in particular, has been shown, for example, influencing gene expression profiles and phenotypic changes. The authors have observed better results when NPCs are cultured under hypoxic conditions (e.g., about 5% O2) compared to normoxic conditions (e.g., about 21% O2), for example, optimizing hiPSC-derived neurons, especially for their use in detecting neuronal antibodies, see, for example... Figure 4 And item 3 in the "Results" section - "Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons".

[0096] Those skilled in the art are familiar with the means used to obtain the NPCs used in the methods of this invention. For example, Chambers, S. et al. (“Highly efficient neural conversion of human ES and iPS cells by dualinhibition of SMAD signaling”, Nat Biotechnol, 2009, 27, 275–280) described the differentiation of human pluripotent stem cells into neural derivatives. As described by Yan Y. et al. (“Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells”, Stem Cells Transl Med, 2013, 2(11):862-70), the standard protocol requires suspension culture to generate embryoid bodies, which can then be plated as adherent cultures to generate neural progenitor cells that can be mechanically or enzymatically separated.

[0097] In a preferred embodiment of the present invention, the NPC used in the method of the present invention is obtained by the following:

[0098] a. Culture hiPSC cells in an extracellular matrix-based hydrogel, preferably until they reach approximately 70% to 80% confluence;

[0099] b. Production of embryoid bodies (EB);

[0100] c differentiates EB into neuroepithelial (NEP) flower clusters; and

[0101] d differentiates NEP flower knots into NPCs.

[0102] Preferably, the extracellular matrix-based hydrogel is "Matrigel". Matrigel is a basement membrane dissolving formulation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is a tumor rich in ECM proteins such as laminin (major component), collagen IV, heparan sulfate proteoglycan, entactin / nidogen, and many growth factors. Its use improves cell adhesion and differentiation. It can be obtained, for example, from Corning (e.g., catalog number 354234). Extracellular matrix-based hydrogels (preferably Matrigel) may contain approximately 60% laminin, approximately 30% collagen IV, approximately 8% nestin, heparan sulfate proteoglycan (perlecan), transforming growth factor (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF-1), fibroblast growth factor (bFGF), tissue plasminogen activator, and other growth factors naturally present in EHS tumors. They may also contain residual matrix metalloproteinases derived from tumor cells.

[0103] For example, an extracellular matrix-based hydrogel (preferably Matrigel) may contain the following growth factors in the amounts listed in Table 5.

[0104] Table 5. Growth factor components of extracellular matrix-based hydrogels (preferably Matrigel)

[0105]

[0106] "Confluence" is the percentage of the culture dish area covered by adherent cells (e.g., 50% confluence means that 50 out of 100 growth surfaces are occupied by cells). Those skilled in the art are familiar with methods for estimating cell confluence. For example, cell confluence can be estimated visually, by using image analysis software (image processing methods), or with the aid of chemical dyes (e.g., thymidine, alamar blue, XTT). As described above, hiPSC cells are cultured in an extracellular matrix-based hydrogel, preferably until they reach approximately 70% to 80% confluence. Preferably, hiPSC cells are cultured in a cell-free medium (e.g., mTeSR1 medium), as described below.

[0107] In the context of this invention, an "embryomorph" (EB) is a three-dimensional aggregate of pluripotent stem cells (hiPSCs herein). EBs facilitate the initiation of lineage-specific differentiation toward many lineages, such as the neural lineage. Those skilled in the art are aware of methods for generating EBs from hiPSCs. Embryomorphs (EBs) can be generated during normally arranged passage by seeding hiPSC plates into non-tissue culture-treated culture dishes to prevent attachment.

[0108] Preferably, EB is generated by dissociating cultured hiPSCs as clumps in a feeder-free cell culture medium (e.g., mTeSR1 medium, a cGMP feeder-free maintenance medium for human ES and iPS cells available from StemCell (e.g., catalog number #85850),) and centrifuging the dissociated cells to obtain EB. The feeder-free cell culture medium (e.g., mTeSR1 medium) used to generate EB is a complete, serum-free, defined formulation designed for feeder-free maintenance and expansion of human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells in an undifferentiated state.

[0109] In one implementation, EB is generated by the following:

[0110] - Seed iPSCs onto an extracellular matrix-based hydrogel (e.g., "Matrigel"), as described above;

[0111] - When the cells reach 70% to 80% confluence, the cells are physically separated from the plate as clumps;

[0112] - Incubate cells in a cone-shaped plate in the presence of a feeder-free medium (such as the aforementioned mTeSR™1, Stemcell Technologies, #85850);

[0113] - Inoculate EB into uncoated plates and incubate with feeder-free medium (e.g., mTeSR1 medium as defined above, or mTeSR™ Plus, Stemcell Technologies, #100-0276) for approximately 24 hours;

[0114] Replace the culture medium with a neurotrophic medium and update it every 24 hours until EB forms well (round and dense).

[0115] In a further step, EB can be differentiated into neuroepithelial (NEP) rosettes. NEP rosettes are microscopic flower-like structures that appear transiently during the development of the central nervous system. Technicians are also familiar with methods for differentiating EB into NEP rosettes. Preferably, EB can be seeded onto PO / Lam-coated plates and incubated with a neuropromoting medium for several days (e.g., at least 8 days, such as at least 10 days, or at least 12 days), preferably supplemented with noggin and also preferably in the presence of a TGF-β inhibitor, see, for example... Figure 1 A. For example, a TGF-β inhibitor could be SB431542. SB431542 is a selective and potent inhibitor of the TGF-β / activin / NODAL pathway, inhibiting ALK5 (IC50) by competing for ATP binding sites. 50 = 94 nM), ALK4 (IC 50 = 140 nM) and ALK7. It does not inhibit BMP type I receptors ALK2, ALK3, and ALK6. It is available from Stemcell Technologies (e.g., #100-1051).

[0116] Finally, NEP flower clusters can be differentiated into NPCs used in the method of this invention. Those skilled in the art also know of methods for differentiating NEP flower clusters into NPCs. This is described, for example, in Chambers, S. et al. (see full reference above) or Topol A. et al., “A guide to generating and using hiPSC derived NPCs for the study of neurological diseases”, J Vis Exp, 2015;(96):e52495. For example, NEP flower clusters can be dissociated and deagglomerated from the plate, resuspended in a neurotrophic medium supplemented with FGF2 and EGF, and seeded onto PO / Lam-coated plates. NPCs can be expanded for about 3 to about 16 generations, preferably about 5 to 12 generations, until the cell population is homogeneous and the phenotype is stable, see [reference missing]. Figure 1 A.

[0117] The cells of the present invention

[0118] The method of this invention yielded novel hiPSC-derived neurons. These hiPSC-derived neurons were optimized for the detection of human antibodies on the neuronal surface and were functional, see, for example, item 3 in the "Results" section – "Optimized expression of mature biomarkers and detection of neuronal surface antibodies by NeurAntigen neurons".

[0119] Therefore, the present invention provides a population of hiPSC-derived neurons obtained directly by the method of the present invention (hereinafter referred to as "the cells of the present invention"). The cells of the present invention preferably express at least one, preferably all, of the following proteins:

[0120] - GABA (γ-aminobutyric acid);

[0121] - MAP2 (microtubule-associated protein 2);

[0122] - TUJ1 (Class III β-tubulin);

[0123] - CTIP2 (transcription factor Ctip2, also known as Bcl11b);

[0124] - vGlut (vesicle glutamate transporter);

[0125] - Synaptic proteins;

[0126] - Bridge tail protein;

[0127] - AMPAR (α-amino-3-hydroxy-5-methyl-4-isocyanate) (Zyroxadione receptor);

[0128] - NMDAR (N-methyl-D-aspartate receptor);

[0129] - LGI1 (Leucine-rich glioma inactivation 1);

[0130] - GABAbR (GABA B receptor);

[0131] - Caspr2 (contactin-associated protein-like 2);

[0132] - IgLON5;

[0133] - GABAaR (GABA A receptor);

[0134] - DPPX (dipeptidyl peptidase-like protein-6).

[0135] - GlyR (glycine receptor);

[0136] - mGluR5 (metabolic glutamate receptor 5);

[0137] - mGluR1 (metaboloid glutamate receptor 1);

[0138] - Neurexin (NRXN);

[0139] - SEZ6L2 (Seizure-associated 6 homolog-like 2); and / or

[0140] - GluK2 (glutamate receptor, ionotropic, phycocyanine 2).

[0141] The above list is not exhaustive, and the cells of this invention may express other proteins.

[0142] Therefore, the hiPSC-derived neurons of the present invention express neuronal surface antigens associated with autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE, wherein the neuronal surface antigens are selected from a list comprising or consisting of the following: AMPAR, NMDAR, LGI1, GABAbR, Caspr2, IgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluR1, neuroconnectin, SEZ6L2, and GluK2.

[0143] In the context of this invention, the term "target antigen" for an autoimmune neurological disorder associated with the presence of autoantibodies can refer to any antigen present in surface neurons that is specifically recognized by autoantibodies from the subject. For example, target neuronal antigens may be selected from AMPAR, NMDAR, LGI1, GABAbR, Caspr2, IgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluR1, neuroconnectin, SEZ6L2, and GluK2.

[0144] Those skilled in the art are familiar with the means used to determine whether a particular cell or cell population expresses one or more of the aforementioned proteins. For example, those skilled in the art may use antibodies specific to protein antigens to detect their expression in a particular cell or cell population. In addition, neuronal maturation markers, as well as synaptic proteins, receptors, and ion channels, are commercially available, such as MAP2 (Synaptic Systems (188 004); 1:1000) and TUJ1 (Biolegend (801202); 1:500), cortical CTIP2 (Abcam (ab18465); 1:500), neurotransmitter GABA (Sigma (A2052); 1:1000), synaptic vesicle vGlut (Synaptic Systems (135 302); 1:1000), synaptic markers (such as synaptic proteins (Calbiochem (574777); 1:1000) and pontocin (SYSY 147011; 1:200)) and receptors (such as AMPAR (Millipore 07-598; 1:100) and GlyR (Sigma HPA016502; 1:50)).

[0145] In a preferred embodiment, the hiPSC-derived neurons of the present invention are precortical neurons containing neurotransmitters and synaptic vesicles (see, for example...). Figure 1 (D and E). The hiPSC-derived neurons of this invention are capable of forming synapses (as indicated by positive immunostaining of synaptic proteins and pontocetin). Figure 1 F), and expresses ion channel receptors such as AMPAR or GlyR (see, for example) Figure 1 G). The cells of the present invention are functionally active within a period of differentiation of at least two weeks, preferably at least three weeks, more preferably at least four weeks and at most five weeks, see, for example Figure 1 H and item 1 of the "Results" section - "Characteristics of neural progenitor cells and NeurAntigen neurons derived from human iPSCs".

[0146] As illustrated in the examples, the hiPSC-derived neurons of the present invention can be used to detect a variety of human antibodies associated with autoimmune encephalitis and related diseases. Specifically, the hiPSC-derived neurons of the present invention express all antigens (e.g., NMDAR, LGI1, AMPAR, GABAbR, Caspr2, IgLON5, etc.) detected by antibodies against AE and related diseases described to date. Furthermore, the hiPSC-derived neurons of the present invention are suitable for detecting human antibodies against other neuronal surface antigens associated with AE and related diseases, such as GABAaR, DPPX, GlyR, mGluR5, mGluR1, neuroconnectin, SEZ6L2, and / or GluK2. Therefore, the hiPSC-derived neurons of the present invention are capable of expressing at least one, preferably all, of these antigens. Therefore, the cells of the present invention are capable of expressing at least one of the following antigens, preferably two or more, even more preferably at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least eleven, or at least twelve, or at least thirteen, or at least fourteen, preferably all of them: NMDAR, LGI1, AMPAR, GABAbR, Caspr2, IgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluR1, neural connectin, SEZ6L2 and / or GluK2.

[0147] Therefore, the cells of the present invention are particularly suitable for diagnosing autoimmune neurological disorders, preferably those associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE. For this purpose, the hiPSC-derived neuronal cells of the present invention can be contacted (incubated) with a biological fluid (e.g., cerebrospinal fluid, serum, or any other biological fluid) obtained from a subject (preferably from a subject with a suspected neurological disorder associated with the presence of neuronal antibodies), see below. In a next step, immunocytochemistry can be used to determine whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived neuronal cells. If this is indeed the case (positive immunoreactivity), and the biological fluid contains antibodies that bind to antigens present in the hiPSC-derived neuronal cells, then the subject may have a neurological disorder associated with the presence of neuronal antibodies, such as autoimmune encephalitis, as in AE-related diseases.

[0148] The cells of the present invention are also capable of expressing additional antigens associated with autoimmune neurological disorders (preferably autoimmune neurological disorders such as AE and related diseases), such as neuronal surface antigens not yet described as targets of patient antibodies. That is, the cells of the present invention can be used to identify novel antibody reactivity associated with autoimmune neurological disorders (preferably autoimmune neurological disorders such as AE and related diseases), as detailed below. For this purpose, a biological fluid (e.g., CSF or serum) from a subject with suspected AE or an autoimmune neurological disorder (preferably autoimmune neurological disorders such as AE and related diseases) can be contacted with the cells of the present invention, and a positive reactivity against hiPSC-derived neuronal cell antigens can be identified, indicating the presence of neuronal antibodies. New neuronal antibodies and the antigens they bind to can thus be identified.

[0149] This disclosure also relates to astrocytes (or astrocyte populations) generated using the method described in di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019. Specifically, the generation of astrocytes is described in detail in the “iPSC-derived astrocyte generation and culture” section (page 17) of the Supplemental Information, “Supplemental experimental procedures” part of that article. In short, iPSCs are differentiated into spherical neural masses (SNMs) containing neuroectodermal progenitor cells, and subsequently differentiated into astrocyte lineages according to a previously published protocol (Serio et al., 2013). First, SNM was cultured in suspension for 28 days in induction medium (DMEM / F12, 1% N2 supplement, 0.1% B27 supplement (Life, 17504-044), 1% nonessential amino acid (NEAA), 1% penicillin / strepmycin (PenStrep), 1% Glutamax) supplemented with 20 ng / mL LIF (Sigma) and 20 ng / mL EGF (R&D Systems). Then, it was further cultured for 21 days in propagation medium (DMEM / F12, 1% N2 supplement, 0.1% B27 supplement, 1% NEAA, 1% PenStrep, 1% Glutamax) containing 20 ng / mL FGF-2 (PeproTech) and 20 ng / mL EGF (R&D Systems). Finally, SNM was incubated with acutase (LabClinics) at 37°C for 15 minutes, mechanically depolymerized, and then inoculated onto a matrigel-coated plate as a monolayer.Neural progenitor cell monolayers were cultured for 14 days in proliferation medium, followed by another 14 days in CNTF medium (neural basis, 1% Glutamax, 1% PenStrep, 1% NEAA, 0.2% B27 supplement, 10 ng / mL CNTF (Prospec Cyt-272)). At this stage, they were considered astrocyte progenitors and were therefore characterized. These astrocyte progenitors were successfully frozen in astrocyte cryopreservation medium (90% FBS and 10% DMSO) and stored in liquid nitrogen for future use. When needed, vials were thawed in FBS-containing medium, resuspended in CNTF medium, and plated on matrigel-coated plates. Cells were considered mature after four passages and then further characterized. Experiments were performed using astrocytes grown on matrigel-coated Thermanox™ plastic coverslips (Thermofisher) in 24-well plates.

[0150] Therefore, the hiPSC-derived astrocytes of this disclosure can be obtained by differentiating iPSCs into spherical neurospheres containing neuroectodermal progenitor cells and subsequently differentiating them into astrocyte lineages. The hiPSC-derived astrocytes express markers selected from the list of those included or composed of: CD44, glial fibrillary acidic protein (GFAP), and S100 calcium-binding protein β (S100β), as well as excitatory aminoacid transporter 2 (EAAT2, also known as GLT1).

[0151] like Figure 6 As shown, the astrocytes described in this article (i.e., generated according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, page 17)) were optimized for detecting glial surface antibodies, preferably human antibodies from astrocytes of patients with autoimmune neurological disorders.

[0152] Uses of the present invention

[0153] In another embodiment, the present invention provides the use of the method and cells of the present invention for the diagnosis of autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related diseases). As described above, hiPSC-derived neuronal cells obtained by the method of the present invention are optimized for the detection of neuronal surface antibodies, preferably human antibodies on the neuronal surface. Therefore, the method and cells of the present invention can be used to detect neuronal surface antibodies, which can be used to diagnose autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related diseases.

[0154] Furthermore, this invention provides the use of the astrocytes or astrocyte populations described herein (generated according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-Derived Astrocytes”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, page 17) for the diagnosis of autoimmune neurological disorders (preferably neurological disorders associated with the presence of glial antibodies, such as NMOSD with AQP4 antibody, meningoencephalomyelitis with GFAP antibody, or related diseases). Therefore, these astrocytes can be used to detect astrocyte antibodies, which can be used to diagnose autoimmune neurological disorders, preferably neurological disorders associated with the presence of glial antibodies, such as NMOSD, meningoencephalitis, or related diseases.

[0155] The present invention also relates to the use of hiPSC-derived neuronal cells obtained by the method of the present invention and / or astrocytes described herein in combination for the detection of autoantibodies, which can be used to diagnose autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related diseases.

[0156] In the context of this invention, an "autoantibody" is an antibody produced by the immune system against one or more proteins of an individual. For example, an individual with an autoimmune neurological disorder may produce antibodies against their own cells (i.e., autoantibodies), such as antibodies against their own neurons (i.e., "neuronal antibodies" or "anti-neuronal antibodies," both terms used interchangeably in this specification) or antibodies against a type of glial cell (their own astrocytes) ("astrocytocyte antibodies" or "anti-astrocytocyte antibodies," both terms used interchangeably in this specification), for example, in the presence of neurological disorders (e.g., AE and / or related diseases) associated with neuronal antibodies, or in the presence of neurological disorders (e.g., NMOSD, meningoencephalitis, or related diseases) associated with astrocyte antibodies.

[0157] In the context of this invention, "neurological disorders associated with the presence of neuronal antibodies" refers to a growing group of diseases or disorders mediated by antibodies targeting neuronal surface proteins. For example, neurological disorders associated with the presence of neuronal antibodies can be autoimmune neurological disorders. In the context of this invention, "autoimmune neurological disorders" refers to immune-mediated disorders in which the patient's own immune system recognizes and attacks the nervous system, causing inflammation and damage. In a preferred embodiment, an autoimmune neurological disorder is an AE-related disease or disorder. "Autoimmune encephalitis (AE)" refers to a class of severe inflammatory diseases of the brain that are at least partially mediated by neuronal antibodies, primarily causing memory and behavioral problems, seizures, motor disorders, language dysfunction, autonomic dysfunction, or sleep disorders as the main clinical manifestations. The disease is associated with antibodies targeting neuronal cell surface and synaptic proteins. AE is an immune-mediated condition characterized by the presence of autoantibodies resulting from an inflammatory response to neuronal antigens. Antibody-aspartate (AE) is associated with antibodies targeting various neuronal cell surface proteins, such as N-methyl-D-aspartate receptor (NMDAR), leucine-rich glioma inactivation 1 (LGI1), contactin-associated protein-like 2 (CASPR2), and gamma-aminobutyric acid B receptor (GABABR). When bound to the target protein, the antibody induces neuronal dysfunction. AE is also known as antibody-mediated encephalitis. See, for example, Gole S, Anand A., “Autoimmune Encephalitis”. [Updated 2023 Jan 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK578203 / . AE may also be associated with the presence of tumors.

[0158] Therefore, autoimmune neurological disorders can be neurological disorders associated with the presence of neuronal antibodies, such as AE-related disorders or AE. In a preferred embodiment, the neurological disorder is an AE.

[0159] Furthermore, the methods and cells of the present invention can be used to identify neuronal antibodies in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related disorders), as detailed above.

[0160] The astrocytes defined herein (generated according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-Derived Astrocytes” in di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, p. 17) can be used to identify glial antibodies in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of astrocyte autoantibodies, such as NMOSD, meningoencephalitis, or related disorders), as detailed above.

[0161] Furthermore, the methods and cells of the present invention can be used to identify target antigens for autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related disorders). As described above, the hiPSC-derived neurons obtained by the methods of the present invention are optimized for the expression of neuronal maturation markers and AE antigens. Therefore, the cells of the present invention are optimized for their use in identifying target antigens associated with AE or related disorders (as described above).

[0162] Therefore, in another aspect, the present invention provides a method for identifying neuronal antibodies in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related disorders). The method includes the following steps:

[0163] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid (preferably CSF or serum) obtained from a subject having a suspected neurological autoimmune disorder, preferably a neurological disorder suspected to be associated with the presence of neuronal antibodies, and more preferably having AE and / or related diseases;

[0164] b. Detect and identify human antibodies present in biological fluids (preferably CSF or serum) of patients suspected of having the disease mentioned in a., wherein the human antibodies specifically bind to surface antigens present in hiPSC-derived neuronal cells.

[0165] In another aspect, the present invention provides a method for detecting autoantibodies (e.g., astrocyte antibodies) in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of glial autoantibodies, such as NMOSD, meningoencephalomyelitis, or related disorders). The method includes the following steps:

[0166] a. The astrocytes described herein (generated according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, p. 17) are incubated with a biological fluid (preferably CSF or serum) obtained from a subject with a suspected neurological autoimmune disorder, preferably a neurological disorder suspected of being associated with the presence of glial autoantibodies, more preferably NMOSD, meningoencephalomyelitis or related diseases;

[0167] b. Detect and identify human antibodies present in the biological fluid (preferably CSF or serum) of patients suspected of having the disease mentioned in a., wherein the human antibodies specifically bind to antigens present in astrocytes.

[0168] In another embodiment, the present invention provides a method for identifying target antigens associated with autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and / or related disorders), the method comprising:

[0169] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid (preferably CSF or serum) obtained from a subject having suspected neurological autoimmune disorders, preferably having suspected neurological disorders associated with the presence of neuronal antibodies, more preferably having suspected autoimmune encephalitis (AE)-related diseases, and even more preferably having suspected AE.

[0170] b. Identify antigens in hiPSC-derived neuronal cells, and specifically bind to said antigens in the biofluids of patients suspected of having the disease mentioned in a.

[0171] Therefore, neuronal antibodies present in the patient's biofluid will specifically bind to antigens present in the hiPSC-derived neuronal cells of the present invention. Those skilled in the art will understand the term "specific binding" in the antigen-antibody context of the present invention. Specific binding of an antibody to its antigen excludes non-specific binding, such as non-specific binding of an antibody (Ab) to an endogenous Fc receptor (FcR), or non-specific binding due to ionic and / or hydrophobic interactions. Those skilled in the art can detect and identify neuronal antibodies that specifically bind to the hiPSC-derived neuronal cells of the present invention, as well as antigens that are specifically bound to the antibodies. Therefore, by using the methods and cells of the present invention, novel antibodies and antigens related to autoimmune neurological disorders (preferably autoimmune neurological disorders associated with the presence of anti-neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE) can be detected and identified.

[0172] The present invention also provides a method for treating a subject suffering from an autoimmune neurological disorder (preferably an autoimmune neurological disorder associated with the presence of autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), more preferably an AE-related disease, and even more preferably an AE, NMOSD, or meningoencephalitis), wherein the method comprises:

[0173] 1. Detect autoantibodies associated with autoimmune neurological disorders (preferably those with neuronal antibodies and / or astrocyte antibodies, such as AE and / or related disorders or NMOSD, meningoencephalitis, or related disorders) by the following methods:

[0174] (i) Incubating the hiPSC-derived neuronal cells of the present invention and / or the astrocytes defined herein with a biological fluid obtained from the object; and

[0175] (ii) Identify human antibodies present in the target biological fluid that bind to hiPSC-derived neurons or astrocytes; and

[0176] 2. Administer treatments to the subject to effectively treat autoimmune neurological disorders, such as corticosteroids, immunoglobulins, rituximab, tocilizumab, etc.

[0177] Therefore, the present invention also provides a method for treating autoimmune neurological disorders (preferably autoimmune neurological disorders associated with the presence of autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE), comprising: administering a treatment to effectively treat an autoimmune neurological disorder in a subject, the subject being diagnosed with an autoimmune neurological disorder based on the presence of autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies) detected in a biological fluid obtained from the subject, as described herein.

[0178] Appropriate treatments for effectively treating autoimmune neurological disorders are known to those skilled in the art. For example, Table 4 of Bhagavati, S., “Autoimmune disorders of the nervous system: pathophysiology, clinical features, and therapy”, Front. Neurol., 14 April 2021, Sec. MultipleSclerosis and Neuroimmunology. Volume 12-2021, provides examples of treatments for autoimmune diseases of the nervous system. In one implementation, the treatment includes corticosteroids, immunoglobulins, rituximab, tocilizumab, bortezomib, etc.

[0179] The diagnostic method of the present invention

[0180] The present invention also provides an in vitro method for diagnosing autoimmune neurological disorders (preferably disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE) in subjects, the method comprising:

[0181] a. Incubate the hiPSC-derived neuronal cells of the present invention with a biological fluid (preferably CSF or serum) obtained from the subject;

[0182] b. Determine whether the antibodies present in the biofluid obtained from the object bind to the antigens present in the hiPSC-derived neuronal cells of the present invention, wherein, for example by immunocytochemistry, a positive immunoreactivity detected using a biofluid (preferably CSF or serum) exposed to hiPSC-derived neuronal cells indicates the presence of neuronal antibodies and thus indicates the presence of autoimmune neurological disorders, preferably disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, and even more preferably AE.

[0183] In one embodiment, the biological fluid obtained from the subject (also referred to herein as "body fluid") is CSF. In another embodiment, the biological fluid is serum. In yet another embodiment, the biological fluid is any other type of fluid known in the art, such as blood, plasma, amniotic fluid, or saliva. Preferably, the biological fluid obtained from the subject is CSF or serum.

[0184] In another embodiment, the present invention provides an in vitro method for diagnosing autoimmune neurological disorders (preferably disorders associated with the presence of astrocyte antibodies, more preferably AE-related diseases such as NMOSD or meningoencephalomyelitis) in subjects, the method comprising:

[0185] a. The hiPSC-derived astrocytes described herein (produced according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes”, “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson’s disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, page 17)) were incubated with a biological fluid (preferably CSF or serum) obtained from the subject.

[0186] b. Determine whether antibodies present in a biological fluid obtained from the subject bind to antigens present in astrocytes derived from hiPSCs, wherein, for example by immunocytochemistry, a positive immunoreactivity detected using a biological fluid (preferably CSF or serum) exposed to astrocytes derived from hiPSCs indicates the presence of astrocyte antibodies and thus indicates the presence of an autoimmune neurological disorder, preferably a disorder associated with the presence of autoantibodies, more preferably AE-related diseases such as NMOSD or meningoencephalitis.

[0187] In the context of the methods of this invention, the terms "incubating hiPSC-derived neurons with a biofluid obtained from the object" or "incubating astrocytes as defined herein with a biofluid obtained from the object" mean contacting the cells with the biofluid for a period of time and under conditions sufficient to determine the presence of autoantibodies in the fluid. As used herein, the term "contact" or variations thereof means contacting hiPSC-derived neurons and / or astrocytes directly or indirectly with the biofluid. Contact can occur, for example, in any quantity of buffer, salt, solution, or cell culture medium. In a preferred embodiment, a biofluid (e.g., CSF or serum) obtained from the object is incubated with hiPSC-derived neurons of the present invention (preferably live neurons, wherein the cells have not been previously fixed or permeabilized) and / or with astrocytes as defined herein at about 37°C for about 1 hour.

[0188] Determining whether antibodies present in the biofluid obtained from the subject bind to antigens present in the hiPSC-derived neurons of the present invention and / or astrocytes as defined herein can be performed by conventional methods known to those skilled in the art. For example, it can be determined by immunocytochemistry. In one embodiment, the determination is performed as follows: After incubating the biofluid with the hiPSC-derived neurons of the present invention and / or astrocytes as defined herein for about one hour, the cells are washed, for example, with PBS, fixed, for example, with 4% paraformaldehyde for about 5 minutes, and incubated with a labeled secondary anti-human immunoglobulin antibody (e.g., green fluorescent secondary anti-human immunoglobulin antibody) at room temperature (RT) for about 1 hour. The cell nuclei may also be labeled, for example, with DAPI. The labeled secondary antibody allows the manifestation of the primary antibody, which is initially present in the biofluid and binds to antigens expressed in the hiPSC-derived neurons of the present invention and / or astrocytes.

[0189] Therefore, as described above, a positive immunoreactivity, detected by immunocytochemistry, in biofluids exposed to hiPSC-derived neurons and / or astrocytes can indicate the presence of anti-neuronal and / or anti-astrocytic antibodies. The presence of autoantibodies (e.g., neuronal and / or astrocyte antibodies) in biofluids obtained from the subject can indicate autoimmune neurological disorders, such as neurological disorders associated with autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), such as acute exacerbations (AEs) or related diseases, such as NMOSD or meningoencephalomyelitis.

[0190] Therefore, this invention allows for the rapid, sensitive, and efficient identification of the presence of autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies) in biological fluids, which can indicate the presence of autoimmune neurological disorders, such as neurological disorders associated with autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), such as AE or related diseases, such as NMOSD or meningoencephalomyelitis (diagnostic methods).

[0191] The reagent kit of the present invention

[0192] In another aspect, the present invention provides a kit suitable for the in vitro diagnosis of autoimmune neurological disorders, such as neurological disorders associated with autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), such as acute exacerbations (AEs) or related diseases. The kit comprises hiPSC-derived neuronal cells of the present invention or compositions comprising them, along with instructions for use. The kit may also comprise a composition containing hiPSC-derived neuronal cells of the present invention and control cells and / or astrocytes as described herein. In some embodiments, the kit may also comprise a sterile container, such as a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, dish, or other suitable container form known in the art. Such a container may be made of plastic, glass, or other materials suitable for containing cells. The kit may also comprise suitable cell culture media, transport media, buffers, and any other reagents that may become necessary for the detection of neuronal antibodies and / or astrocyte antibodies. The kit may also comprise anti-human immunoglobulin antibodies (preferably labeled, e.g., fluorescently labeled) and / or any other reagents for immunocytochemistry.

[0193] The kit may also contain additional iPSC-derived astrocytes as defined herein (produced according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes” in diDomenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, p. 17).

[0194] In some embodiments, the kit may also contain a cell population unrelated to autoimmune neurological disorders. Such a cell population is preferably unrelated to neurological diseases, and more preferably a cell population of a different origin from the nervous system (e.g., fibroblasts), to serve as a negative control in the kit of the present invention.

[0195] The kit of this invention can also be applied to identify autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies) in patients with autoimmune neurological disorders (preferably those with neurological disorders associated with autoantibodies (e.g., neuronal antibodies and / or astrocyte antibodies), such as acute exacerbations (AEs) and / or related disorders, NMOSD, meningoencephalitis, or related disorders). The composition in the kit (containing iPSC-derived neurons and iPSC-derived astrocytes, respectively) may be relevant in cases where patients exhibit neurological symptoms common to multiple autoimmune neurological diseases (e.g., memory and behavioral problems, seizures, motor disorders, etc., which may coexist in, for example, both AEs and meningoencephalitis, and are associated with neuronal or glial antibodies).

[0196] In some embodiments, antibodies present in the biofluid obtained from the subject bind to antigens present in hiPSC-derived neurons and / or iPSC-derived astrocytes as defined herein, but not to control cells as defined herein, indicating the presence of anti-neuronal or anti-astrocytic antibodies in the subject, and thus indicating the presence of an autoimmune neurological disorder. In contrast, antibodies present in the biofluid obtained from the subject bind to antigens present in hiPSC-derived neurons or iPSC-derived astrocytes as defined herein. and It also binds to cell populations unrelated to autoimmune neurological disorders (negative control), and therefore does not indicate the presence of specific anti-neuronal or anti-astrocytic antibodies, and thus cannot indicate autoimmune neurological disorders. The composition in the kit (containing iPSC-derived neurons or iPSC-derived astrocytes and control cells) may be relevant to the interpretation of test results. It serves as an internal control for diagnostic methods; for example, if nonspecific immunoreactivity is detected using biological fluids obtained from the subject (positive signals detected in neurons and fibroblasts), it can be easily detected that it does not correspond to specific neuronal antibodies.

[0197] Therefore, the present invention provides the use of the kit of the present invention in the in vitro diagnosis of autoimmune neurological disorders (e.g., neurological disorders associated with neuronal antibodies, such as AE or related diseases), and in the identification of neuronal antibodies in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, such as AE), and / or in the identification of target antigens of autoimmune neurological disorders (preferably neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE) related diseases, and even more preferably AE).

[0198] The kit of this invention can also be used for the in vitro diagnosis of autoimmune neurological disorders (e.g., neurological disorders associated with astrocyte antibodies, such as AE-related diseases like NMOSD or meningoencephalomyelitis), and for the identification of astrocyte antibodies in patients with autoimmune neurological disorders (preferably neurological disorders associated with the presence of astrocyte antibodies, such as AE-related diseases), and / or for the identification of target antigens of autoimmune neurological disorders (preferably neurological disorders associated with the presence of astrocyte antibodies, more preferably AE-related diseases, such as NMOSD or meningoencephalomyelitis).

[0199] item

[0200] The present invention also provides the following:

[0201] 1. A method for obtaining neurons derived from human induced pluripotent stem cells (hiPSCs), wherein the method comprises culturing approximately 30,000 to approximately 40,000 cells / cm² in a neurotrophic medium. 2 Neural progenitor cells (NPCs) are cultured for at least three weeks to obtain hiPSC-derived neurons.

[0202] 2. The method according to item 1, wherein the NPC is cultured under hypoxic conditions (5% O2).

[0203] 3. The method according to any one of items 1 or 2, wherein the NPC is obtained by:

[0204] a. Culture hiPSC cells in an extracellular matrix-based hydrogel until they reach approximately 70% to 80% confluence;

[0205] b. Production of embryoid bodies (EB);

[0206] c. Differentiate the EB into a neuroepithelial (NEP) rosette; and

[0207] d. Differentiate the NEP flower clusters into neural progenitor cells.

[0208] 4. The method according to item 3, wherein step b. is performed by dissociating the hiPSCs cultured in step a. as clumps in serum-free cell culture medium and centrifuging the dissociated cells to obtain EB.

[0209] 5. The method according to any one of items 1 to 4, wherein step c. is carried out by: seeding the EB from step b. onto a polyornithine / laminusoidal (Po / Lam) coated plate in the presence of head protein and at least one TGF-β inhibitor, and incubating it in a neurotrophic medium for at least 8 days, preferably wherein the TGF-β inhibitor is SB431542.

[0210] 6. The method according to item 5, wherein the EB is incubated for a period of 8 to 12 days.

[0211] 7. The method according to any one of items 1 to 6, wherein step d. is performed by: depolymerizing the NEP flower clusters, seeding the cells on a PO / Lam-coated plate, incubating them in a neurotrophic medium supplemented with FGF2 and EGF, and passaged the cells for about 5 to 12 generations.

[0212] 8. The use of the method as defined in any of the preceding items for the diagnosis of autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE.

[0213] 9. hiPSC-derived neuronal cell populations obtained by the methods defined in any of items 1 to 7.

[0214] 10. The use of the cells defined in item 9 and / or the astrocyte population produced according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-Derived Astrocytes” (page 17) of di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, for the diagnosis of autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of autoantibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AEs, such as neuronal antibodies and / or astrocyte antibodies.

[0215] 11. The use of the astrocyte populations generated by the cells defined in item 9 and / or according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-Derived Astrocytes” (page 17) of di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, for the identification of:

[0216] - Neuronal and / or astrocyte antibodies in a patient with an autoimmune neurological disorder, preferably with a neurological disorder associated with the presence of autoantibodies, such as adverse events (AEs) and / or related disorders, such as neuronal antibodies and / or astrocyte antibodies, and / or

[0217] - Target antigens of autoimmune neurological disorders, preferably neurological disorders associated with the presence of autoantibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE, wherein the autoantibodies are, for example, neuronal antibodies and / or astrocyte antibodies.

[0218] 12. A method for identifying autoantibodies, such as neuronal antibodies and / or astrocyte antibodies, the method comprising:

[0219] a. Incubate hiPSC-derived neurons as defined in item 9 and / or a population of astrocytes produced according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes” (page 17) of di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, with a biological fluid obtained from a subject, preferably a subject with suspected neurological autoimmune disorders, more preferably with suspected neurological disorders associated with the presence of autoantibodies, more preferably with suspected autoimmune encephalitis (AE)-related disease, and even more preferably with suspected AE, the autoantibodies being, for example, neuronal antibodies and / or astrocyte antibodies;

[0220] b. Detect and identify human antibodies present in the biological fluid of the object mentioned in a., wherein the human antibodies specifically bind to surface antigens present in the hiPSC-derived neuronal cells and / or surface antigens present in the astrocytes.

[0221] 13. A method for identifying target antigens associated with an autoimmune neurological disorder, preferably a neurological disorder associated with the presence of autoantibodies, such as acute exacerbations (AEs) and / or related disorders, the autoantibodies being such as neuronal antibodies and / or astrocyte antibodies, the method comprising:

[0222] a. Incubate hiPSC-derived neurons as defined in item 9 and / or a population of astrocytes produced according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes” (page 17) of di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, with a biological fluid obtained from subjects having suspected neurological autoimmune disorders, preferably neurological disorders suspected of being associated with the presence of autoantibodies, more preferably suspected autoimmune encephalitis (AE)-related disease, or even more preferably suspected AE, the autoantibodies being, for example, neuronal antibodies and / or astrocyte antibodies;

[0223] b. Identify antigens in the hiPSC-derived neuronal cells, and specifically bind to the antigens in the biofluids of patients suspected of having the diseases mentioned in a., such as neuronal antibodies and / or astrocyte antibodies.

[0224] 14. An in vitro method for diagnosing an autoimmune neurological disorder in a subject, wherein the autoimmune neurological disorder is preferably an autoimmune neurological disorder associated with the presence of autoantibodies, more preferably an autoimmune encephalitis (AE)-related disease, and even more preferably an AE, wherein the autoantibodies are, for example, neuronal antibodies and / or astrocyte antibodies, the method comprising:

[0225] a. Incubate hiPSC-derived neurons as defined in item 9 and / or a population of astrocytes generated according to the methods described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-derived Astrocytes” (page 17) of the article published on February 12, 2019, with a biological fluid obtained from the subject.

[0226] b. Determine whether the antibodies present in the biological fluid obtained from said object bind to antigens present in hiPSC-derived neuronal cells as defined in item 9, and / or bind to antigens present in the hiPSC-derived neuronal cells according to the description of Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229. On February 12, 2019, the supplementary information of this article, in the section "Supplementary Experimental Procedures" under the subsection "Generation and Culture of iPSC-Derived Astrocytes" (page 17), describes whether the astrocyte population produced is bound, wherein a positive immunoreactivity indicates the presence of neuronal and / or astrocyte antibodies in the subject, and thus indicates the presence of an autoimmune neurological disorder, such as a neurological disorder associated with the presence of autoantibodies, preferably autoimmune encephalitis (AE)-related disease, or even more preferably AE, said positive immunoreactivity preferably detected by immunocytochemistry using a biofluid exposed to said hiPSC-derived neuronal cells and / or said astrocytes, said autoantibodies such as neuronal antibodies and / or astrocyte antibodies.

[0227] 15. A kit for in vitro diagnosis of autoimmune neurological disorders in subjects, such as autoimmune neurological disorders associated with the presence of autoantibodies, preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE, the autoantibodies being such as neuronal antibodies and / or astrocyte antibodies, wherein the kit comprises hiPSC-derived neurons as defined in item 9 and instructions for use.

[0228] 16. The kit according to item 15, wherein the kit further comprises iPSC-derived astrocytes produced according to the method described in the supplementary information “Supplementary Experimental Procedures” section “Generation and Culture of iPSC-Derived Astrocytes” of the article by di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019 (page 17).

[0229] 17. The kit according to item 15 or 16, wherein the kit further comprises a cell population unrelated to autoimmune neurological disorders, preferably a non-neural cell population, more preferably fibroblasts.

[0230] Example

[0231] Materials and Experimental Methods

[0232] 1- Obtaining neurons derived from human induced pluripotent stem cells: The NeurAntigen protocol

[0233] a. Culturing human induced pluripotent stem cells:

[0234] Human induced pluripotent stem cells (hiPSCs) were cultured in an extracellular matrix-based hydrogel until they reached 70% to 80% confluence. The iPSCs were dissociated as clumps in serum-free cell culture medium, and the dissociated cells were centrifuged to generate embryoid bodies (EBs). The EBs were then seeded onto poly-L-ornithine-laminus (PO / Lam)-coated plates and incubated for 8 to 12 days in a neurotrophic medium containing head protein and a TGF-β inhibitor (SB431542) to differentiate into neural epithelial (NEP) rosettes. Subsequently, the NEP rosettes were depolymerized, seeded onto PO / Lam-coated plates, and incubated in a neurotrophic medium containing FGF2 and EGF to differentiate into neural progenitor cells (NPCs).

[0235] b. Culture neural progenitor cells:

[0236] NPCs were incubated on PO / Lam-coated plates using a neurotrophic medium containing FGF2 and EGF, and could be passaged approximately 5 to 12 times.

[0237] c. Differentiation of neuronal cells:

[0238] NPCs were calculated at 35,000 to 40,000 cells / cm². 2 The cells were seeded at a density on PO / Lam-coated plates and incubated for at least 21 days in a neurotrophic medium without growth factors, preferably under hypoxic conditions (5% O2), and differentiated into neurons.

[0239] 2-Characteristics of neural progenitor cells

[0240] NPCs were immunostained using neural lineage markers, such as Nestin (Santa Cruz (sc-23927); 1:250), SOX2 (R&D System (MAB2018); 1:50), and Pax6 (Covance (PRB-278P); 1:100), as well as the proliferation marker Ki67 (Abcam (Ab15580); 1:2000). Briefly, the primary antibodies were incubated at 4°C for 48 hours and then imaged with the corresponding secondary fluorescent antibodies (2 hours at room temperature); cell nuclei were labeled with DAPI.

[0241] 3-Characteristics of neurons derived from human induced pluripotent stem cells

[0242] a. Immunocytochemistry

[0243] Immunostaining was performed on hiPSC-derived neurons using neuronal maturation markers, such as MAP2 (SynapticSystems (188 004); 1:1000) and TUJ1 (Biolegend (801202); 1:500), cortical CTIP2 (Abcam (ab18465); 1:500), neurotransmitter GABA (Sigma (A2052); 1:1000), synaptic vesicle vGlut (SynapticSystems (135 302); 1:1000), synaptic markers (e.g., synaptic proteins (Calbiochem (574777); 1:1000) and pontocin (SYSY 147011; 1:200)) and receptors (e.g., AMPAR (Millipore 07-598; 1:100), GlyR (Sigma HPA016502; 1:50)). Similarly, the primary antibody was incubated at 4°C for 48 hours and then imaged with the corresponding secondary fluorescent antibody (2 hours at room temperature); the cell nuclei were labeled with DAPI.

[0244] b. Calcium imaging measurement:

[0245] We used calcium fluorescence imaging to determine the spontaneous activity of hiPSC-derived neurons 4 weeks after differentiation, as previously reported (e.g., Carola, G., Malagarriga, D., Calatayud, C. et al., “Parkinson's diseasepatient-specific neuronal networks carrying the LRRK2 G2019S mutation unveil early functional alterations that predate neurodegeneration”, npj ParkinsonsDis. 7, 55 (2021)).

[0246] 4- Immunostaining of biological samples from patients with autoimmune encephalitis on NeurAntigen neurons (hiPSC-derived neurons obtained via the NeurAntigen protocol).

[0247] Immunocytochemistry was performed on live hiPSC-derived neurons obtained via the NeurAntigen protocol using serum or cerebrospinal fluid (CSF) from patients or healthy donors. To confirm the extensive expression of human autoantigens in these mature neurons and the recognition of them by patient autoantibodies, samples from different individuals with autoimmune encephalitis containing specific antibodies against known neuronal surface antigens, or healthy control samples without detectable antibodies, were exposed to NeurAntigen neurons without pre-fixation or permeabilization. Serum (1:100 dilution) or CSF (1:5) samples were incubated at 37°C for 1 hour, followed by washing of neuronal cultures and fixation with 4% paraformaldehyde for 5 minutes, and incubation at room temperature for 1 hour with a green fluorescent secondary anti-human immunoglobulin antibody (1:1000 dilution); cell nuclei were labeled with DAPI.

[0248] Comparison of 5-NeurAntigen neurons with neurons obtained by previously reported methods

[0249] To confirm that NeurAntigen neurons were optimized for detecting human antibodies on the neuronal surface, we compared two maturation steps of our protocol (at 2 and 3 weeks) with different methods of obtaining neurons from hiPSCs (Yan Y. et al., "Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells. Stem Cells Transl Med. 2013 Nov;2(11):862-70). The expression of mature neuronal markers (MAP2 and GABA) and immunostaining of serum samples (1:100 dilution) from subjects with autoimmune encephalitis or healthy controls were also compared.

[0250] 6- Obtaining astrocytes derived from human induced pluripotent stem cells

[0251] Obtaining astrocytes derived from iPSCs is described in the supplemental information of di Domenico A. et al., “Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease”, Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, in the section “Generation and Culture of iPSC-derived Astrocytes” (page 17). In short, iPSCs are differentiated into spherical neurospheres containing neuroectodermal progenitor cells, and subsequently differentiated into astrocyte lineages according to a previously published protocol (Serio et al., 2013). First, SNM was cultured in suspension for 28 days in induction medium (DMEM / F12, 1% N2 supplement, 0.1% B27 supplement (Life, 17504-044), 1% non-essential amino acids (NEAA), 1% penicillin / strepmycin (PenStrep), 1% Glutamax) supplemented with 20 ng / mL LIF (Sigma) and 20 ng / mL EGF (R&D Systems), followed by a further 21 days in proliferation medium (DMEM / F12, 1% N2 supplement, 0.1% B27 supplement, 1% NEAA, 1% PenStrep, 1% Glutamax) containing 20 ng / mL FGF-2 (PeproTech) and 20 ng / mL EGF (R&D Systems). Finally, SNM was incubated with acutase (LabClinics) at 37°C for 15 minutes, mechanically depolymerized, and inoculated onto Matrigel-coated plates as a monolayer. Neural progenitor cell monolayers were cultured for 14 days in proliferation medium, followed by another 14 days in CNTF medium (Neurobasal, 1% Glutamax, 1% PenStrep, 1% NEAA, 0.2% B27 supplement, 10 ng / mL CNTF (Prospec Cyt-272)). At this stage, they were considered astrocyte progenitors and were therefore characterized. These astrocyte progenitors were successfully frozen in astrocyte cryopreservation medium (90% FBS and 10% DMSO) and stored in liquid nitrogen for future use. When needed, the vials were thawed in FBS-containing medium, resuspended in CNTF medium, and plated on Matrigel-coated plates. Cells were considered mature after four passages and then further characterized.The experiment was conducted using astrocytes grown on matrigel-coated Thermanox™ plastic coverslips (Thermofisher) in 24-well plates.

[0252] 7- Immunostaining of biological samples from patients with autoimmune neurological disorders on astrocytes derived from human iPSCs.

[0253] Using hiPSC-derived astrocytes obtained as described by di Domenico A. et al., immunocytochemistry was performed using serum or CSF from patients or healthy donors. To confirm the expression of human autoantigens by these astrocytes and the recognition of them by patient autoantibodies, samples from individuals with NMOSD containing antibodies against AQP4, or from individuals with meningoencephalomyelitis or related disorders containing antibodies against GFAP, or from healthy control samples without detectable glial antibodies, were exposed to astrocytes. For AQP4 detection, serum samples (1:50 dilution) were incubated at 37°C for 1 hour, followed by washing of astrocyte cultures and fixation with 4% PFA for 10 minutes, and incubation with green fluorescent secondary anti-human immunoglobulin G antibody (1:1000 dilution) at room temperature for 1 hour; cell nuclei were labeled with DAPI. For GFAP detection, CSF samples (1:10 dilution) were incubated overnight at 4°C, then astrocytes were fixed and permeabilized with PFA and Triton, and subsequently incubated with red fluorescent secondary anti-human IgG antibody (1:1000). Figure 6 A.

[0254] result

[0255] Characterization of 1-human iPSC-derived neural progenitor cells and NeurAntigen neurons

[0256] The cell culture steps for obtaining NPCs (after 45 days) and differentiated neurons (after 21 days) from hiPSCs are illustrated in the diagram. Figure 1 As shown in Figure A, NPCs generated by hiPSC express nestin, SOX2, and PAX6 (neural lineage markers) as well as the proliferation marker Ki67. Figure 1 B). After differentiation, mature neurons, when stained with MAP2, show long neuronal processes and are immunopositive for the neurotransmitter GABA. Figure 1 C). Quantitative analysis of these cultured cells showed that 80% were mature neurons (MAP2 and DAPI positive), and 70% were GABAergic neurons (MAP2 and GABA positive); Figure 1D). Differentiated neurons stained with TUJ1 or MAP2 were also positive for CTIP2 and vGlut, indicating that NeurAntigen cells are cortical neurons and contain excitatory synaptic vesicles (D). Figure 1 E). As shown by positive immunostaining of synaptic proteins and pontocin, these neurons form synapses ( Figure 1 F), and expresses ion channel receptors, such as AMPAR or GlyR (F ...). Figure 1 G). Furthermore, at 4 weeks of differentiation, NeurAntigen neurons exhibit functional activity, as indicated by calcium waves detected on single cells (G). Figure 1 H).

[0257] 2- Detection of human antibodies associated with autoimmune encephalitis using NeurAntigen neurons

[0258] An illustration of using NeurAntigen neurons to diagnose autoimmune encephalitis is shown in [the image / image / etc.]. Figure 2 A shows that samples from patients with suspected autoimmune encephalitis (or related neurological disorders) are screened within 2 hours to detect autoantibodies on the surface of neurons. Figure 2 B illustrates an example of positive immunoreactivity in patient serum, showing a fine, bright green speckled pattern on neuronal processes, contrasting with the negative result shown in control serum (NHS). NeurAntigen live neurons can detect human antibodies against not only common and uncommon neuronal surface antigens associated with autoimmune encephalitis (including, but not limited to, NMDAR, LGI1, AMPAR, GABAbR, Caspr2, IgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluR1), but also those recently described (neuronexins, SEZ6L2, GluK2). Furthermore, patient CSF incubated with NeurAntigen live neurons showed an immunoreactive pattern similar to patient serum, unlike the negative result observed in control CSF (NHCSF), indicating that both serum and CSF antibodies can be detected by our test. Figure 3 ).

[0259] 3-Optimized expression of maturation markers and detection of neuronal surface antibodies by Neuron Antigen

[0260] A comparison of cell culture conditions for differentiating hiPSC-derived NPCs into neurons is illustrated in [the diagram]. Figure 4As shown in the figure. Under optimized conditions (including seeding with a lower number of NPCs, incubation under hypoxia, and a limited duration), NeurAntigen neurons were more stable during cell culture, attached better to the plate surface, had a more uniform distribution, and exhibited better morphology of neuronal processes. Figure 4 B, Insert Figure 1 and 2 ).

[0261] The specific differences between the NeurAntigen approach and another method for obtaining neurons from hiPSCs are detailed in Table 6. Figure 5 This demonstrates how extending the differentiation time from NPC to neuron (2 weeks vs. 3 weeks) and using the NeurAntigen protocol instead of previously reported methods significantly increased the presence of mature neurons with both MAP2 and GABA positivity. Figure 5 (A to B). Furthermore, neuronal surface antibodies from patients can only be detected when using NeurAntigen neurons differentiated at least 3 weeks (at least 21 days) prior, such as... Figure 5 As shown in C.

[0262] Table 6. Description of the NeurAntigen kit compared to Yanet al.

[0263]

Claims

1. A method for obtaining neurons derived from human induced pluripotent stem cells (hiPSCs), wherein the method comprises culturing approximately 30,000 to approximately 40,000 cells / cm² in a neurotrophic medium. 2 Neural progenitor cells (NPCs) are cultured for at least three weeks to obtain hiPSC-derived neurons.

2. The method according to claim 1, wherein the NPC is cultured under hypoxic conditions (5% O2).

3. The method according to any one of claims 1 or 2, wherein the NPC is obtained by: a. Culture hiPSC cells in an extracellular matrix-based hydrogel until they reach approximately 70 to 80% confluence; b. Production of embryoid bodies (EB); c. Differentiate the EB into a neuroepithelial (NEP) rosette; and d. Differentiate the NEP flower clusters into neural progenitor cells.

4. The method of claim 3, wherein step b. is performed by dissociating the hiPSCs cultured in step a. as clumps in serum-free cell culture medium and centrifuging the dissociated cells to obtain EB.

5. The method according to any one of claims 1 to 4, wherein step c. is carried out by: seeding the EB from step b. onto a polyornithine / laminusoidal (Po / Lam) coated plate in the presence of head protein and at least one TGF-β inhibitor, and incubating it in a neurotrophic medium for at least 8 days, preferably wherein the TGF-β inhibitor is SB431542.

6. The method of claim 5, wherein the EB incubation period is 8 to 12 days.

7. The method according to any one of claims 1 to 6, wherein step d. is performed by: depolymerizing the NEP flower clusters, seeding the cells on a PO / Lam-coated plate, incubating them in a neurotrophic medium supplemented with FGF2 and EGF, and passaged the cells for about 5 to 12 generations.

8. The use of the method as defined in any of the preceding claims for diagnosing autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE.

9. hiPSC-derived neuronal cell populations, obtained by the method defined in any one of claims 1 to 7.

10. The use of the cells as defined in claim 9, for the diagnosis of autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of anti-neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE.

11. The use of the cells as defined in claim 9, for the identification of: - Neuronal antibodies in a patient with an autoimmune neurological disorder, preferably with a neurological disorder associated with the presence of neuronal antibodies, such as autoimmune ataxia (AE) and / or related disorders, and / or - The following target antigens: autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE.

12. A method for identifying neuronal antibodies, the method comprising: a. Incubating hiPSC-derived neuronal cells as defined in claim 9 with a biological fluid obtained from an object having suspected neurological autoimmune disorders, preferably having suspected neurological disorders associated with the presence of neuronal antibodies, more preferably having suspected autoimmune encephalitis (AE)-related diseases, and even more preferably having suspected AE; b. Detect and identify human antibodies present in the biological fluid of patients suspected of having the disease mentioned in a., wherein the human antibodies specifically bind to surface antigens present in the hiPSC-derived neuronal cells.

13. A method for identifying a target antigen, said target antigen being associated with an autoimmune neurological disorder, preferably a neurological disorder associated with the presence of neuronal antibodies, such as acute exacerbations (AEs) and / or related disorders, said method comprising: a. Incubating hiPSC-derived neuronal cells as defined in claim 9 with a biological fluid obtained from an object having suspected neurological autoimmune disorders, preferably having suspected neurological disorders associated with the presence of neuronal antibodies, more preferably having suspected autoimmune encephalitis (AE)-related diseases, and even more preferably having suspected AE; b. Identify antigens in the hiPSC-derived neuronal cells, and specifically bind to the antigens in the biofluids of patients suspected of having the disease mentioned in a.

14. An in vitro method for diagnosing an autoimmune neurological disorder in a subject, wherein the autoimmune neurological disorder is preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably an autoimmune encephalitis (AE)-related disease, and even more preferably AE, the method comprising: a. Incubate hiPSC-derived neuronal cells as defined in claim 9 with a biological fluid obtained from the object; b. Determine whether the antibodies present in the biofluid obtained from the object bind to the antigens present in the hiPSC-derived neuronal cells as defined in claim 9, wherein a positive immunoreactivity indicates the presence of neuronal antibodies in the object and thus indicates the presence of an autoimmune neurological disorder, such as a neurological disorder associated with the presence of anti-neuronal antibodies, preferably autoimmune encephalitis (AE)-related disease, or even more preferably AE, said positive immunoreactivity preferably detected by immunocytochemistry using a biofluid exposed to the hiPSC-derived neuronal cells.

15. A kit for in vitro diagnosis of autoimmune neurological disorders in subjects, such as autoimmune neurological disorders associated with the presence of neuronal antibodies, preferably autoimmune encephalitis (AE)-related diseases, and even more preferably AE, wherein the kit comprises hiPSC-derived neuronal cells as defined in claim 9 and instructions for use.

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