WO2025078508A1

WO2025078508A1 - New methods for the detection of neuronal antibodies

Info

Publication number: WO2025078508A1
Application number: PCT/EP2024/078537
Authority: WO
Inventors: Antonella CONSIGLIO; Ángel Raya Chamorro; Meritxell PONS ESPINAL; Irene FERNÁNDEZ CARASA; Carles CALATAYUD ARISTOY; Josep DALMAU OBRADOR; Eugenia MARTÍNEZ HERNÁNDEZ
Original assignee: Universitat de Barcelona UB; Fundacio Privada Institut dInvestigacio Biomedica de Bellvitge IDIBELL; Institucio Catalana de Recerca i Estudis Avancats ICREA; Hospital Clinic de Barcelona; Institut d'Investigacions Biomèdiques August Pi i Sunyer
Current assignee: Universitat de Barcelona UB; Fundacio Privada Institut dInvestigacio Biomedica de Bellvitge IDIBELL; Institucio Catalana de Recerca i Estudis Avancats ICREA; Hospital Clinic de Barcelona; Institut d'Investigacions Biomèdiques August Pi i Sunyer
Priority date: 2023-10-10
Filing date: 2024-10-10
Publication date: 2025-04-17
Anticipated expiration: 2026-04-10

Abstract

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

Description

NEW METHODS FOR THE DETECTION OF NEURONAL ANTIBODIES

FIELD OF THE TECHNOLOGY

The present invention belongs to the field of stem cells. In particular, the present invention provides methods for obtaining human-induced pluripotent stem cells (hiPSCs)-derived neuronal cells which are useful for the detection of autoantibodies directed against the neuronal surface, present in patients with autoimmune neurological disorders.

BACKGROUND OF THE TECHNOLOGY

Neurological disorders associated with the presence of neuronal antibodies, such as autoimmune encephalitis (AE), are a new category of diseases that result from an immune attack against proteins present in the patient's brain cells. Clinical symptoms include neurological and psychiatric manifestations that differ between patients and during disease course. The prevalence (1995-2015) of AE was 0.8/100,000 in the United States, and a previous study demonstrated 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). Quick diagnosis and treatment with immunotherapy are essential to prevent worsening of the symptoms, admission to intensive care units, and prolonged hospital stays. Clinical guidelines allow the identification of probable AE cases, but definite AE diagnosis requires detection of the specific associated antibody in the serum or cerebrospinal fluid (CSF) of the patient. Despite recent advances in the discovery of AE, and the increasing number of AE-associated neuronal antigens described in the last years (see, e.g., Varley, J. A. et al. "Autoimmune encephalitis: recent clinical and biological advances", J Neurol 2023, 270, 4118-4131), a substantial proportion of patients remain nowadays without an established diagnosis. This is the case of patients that truly have AE but, when their samples are assayed with currently available diagnostic procedures, give antibody-negative results (McCracken et al., 2017). Commercial tests designed for the detection of AE-antibodies are based on a cell-based assay (CBA), in which heterologous non-neural cells are transfected with a specific plasmid to express a neuronal protein (known antigenic target of AE-antibodies). For each specific antibody (about 15 described in AE and related diseases) one different CBA has to be performed, to detect or rule out its presence. In the best scenario, one patient sample (serum or CSF) can be tested with a commercial kit that provides a combination of 6 pre-determined antigens. When the antigen of interest is not included in the commercial test the assay provides a negative result, although "undetected" AE-antibodies are in fact present in the patient sample. Moreover, some patients may have antibodies directed against novel, non-previously described, neuronal antigens, which are not constitutively expressed by cell lines used for CBAs. Currently, there is no available diagnostic test as first screening step to detect the presence of neuronal surface antibodies in patients with suspected AE.

Table 1 shows examples of AE diagnostic means.

Table 1.

(*) Deng Q. et al., "The antibody assay in suspected autoimmune encephalitis from positive rate to test strategies", Front Immunol, 2022;13:803854;

(**) E.g., Athea Diagnostics Test code 4722 (NeoEncephalitis Paraneoplastic Evaluation with Recombx®);

(***) EURO I MM UN Medizinische Labordiagnostika AG, Autoimmune Encephalitis biochip.

Hence, there is a need for improvement of the detection of neuronal antibodies, and the diagnosis of the associated neurological disorders such as AE and related diseases.

SUMMARY OF THE INVENTION

The present invention addresses the above needs and provide methods for obtaining hiPSCs- derived neuronal cells, wherein the method comprises culturing neural progenitor cells (NPC) for at least three weeks (e.g., 21 days) in proneural medium. The invention thus provides a population of hiPSC-derived neuronal cells obtained by the method of the present invention.

The present invention further provides the use of the method 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.

The invention further provides the use of the hiPSC-derived neuronal cells of the present invention for the identification of the presence of neuronal antibodies. In addition, the invention provides the use of the hiPSC-derived neuronal cells of the present invention for the identification of target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE.

In a further aspect, the invention provides a method for the identification of neuronal antibodies associated with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, more preferably AE and/or related diseases, wherein the method comprises: a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from a subject, preferably a subject with a suspected autoimmune neurological disorder, preferably with a suspected autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably with a suspected AE- related disease, even more preferably with suspected AE; b. Detecting human antibodies present in the biological fluid of a subject suffering a disease as mentioned in a. and identifying them, wherein the human antibodies specifically bind to the hiPSC-derived neuronal cells.

Further, the invention provides a method for the identification of target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorder, the method comprising: a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of neuronal antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Identifying antigens in the hiPSC-derived neuronal cells to which human antibodies present in the biological fluid of a subject as mentioned in a. bind.

The invention further provides an in vitro method for the diagnosis of an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably with an AE-related disease, even more preferably with AE in a subject, the method comprising a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from a subject; b. Determining whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived neuronal cells of the present invention, wherein a positive immunoreactivity detected by immunocytochemistry, using the biological fluid exposed to the hiPSC-derived neuronal cells, is indicative of the presence of neuronal antibodies and thus of an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably an AE-related disease, even more preferably AE, in the subject.

Finally, the invention provides a kit suitable for the in vitro diagnosis of neurological autoimmune disorders, preferably neurological disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, even more preferably AE in a subject, wherein the kit comprises the hiPSC-derived neuronal cells of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Characterization of human iPSC-derived Neural progenitor cells (NPC) and neurons. (A) Schematic representation of the protocol used for NPC and neuronal derivation from iPSCs. (B) NPCs express neural progenitor markers such as Nestin, Sox2 and Pax6, as well as the proliferation marker Ki67. Nuclei are labeled with DAPI. (C) After 4 weeks of differentiation, the majority of NPC-derived human neurons express markers of neuronal maturity (MAP2), and around 70% of them express GABA, indicating the presence of GABAergic neurons in the culture. (D) Proportion of MAP2 positive neurons over DAPI, and fraction of GABA positive cells over MAP2 positive cells. (E) Human neuronal cultures also express vGlut and CTIP2, confirming the presence of glutamatergic and cortical neurons. (F) Following 4 weeks of differentiation NPC-derived neurons express the synaptic markers Synapsin and Gephyrin, as well as ion channels receptors such as GlyR or AMPAR (G). (H) Human neurons are functional as shown by the representation of calcium waves of single active neurons. Nuclei are stained with DAPI.

Figure 2. Analytical performance of NeurAntigen neurons. (A) Schematic representation of the experimental procedure used to incubate patients' samples with live NeurAntigen neurons. (B) Human neurons display complex morphologies in culture, without forming aggregates. After incubation with sera from patients with autoimmune encephalitis containing neuronal surface antibodies, and later with a secondary anti-human IgG antibody, a positive punctate pattern is appreciated. Note the difference of a negative control (NHS: normal human serum, from a healthy donor), compared with the positive detection of 14 different neuronal surface antibodies on NeurAntigen neurons.

Figure 3. NeurAntigen neurons can be used both with serum and cerebrospinal fluid samples. After incubation with patients' CSF or serum, a positive immunostaining with the characteristic punctate pattern can be detected on human neurons. Note the difference with the absence of reactivity of the corresponding negative controls (NHCSF: normal human CSF, from a healthy donor). Figure 4. NeurAntigen protocol stabilizes and improves neuronal attachment and allows the visualization of complex dendrites for the appropriate detection of neuronal antibodies. (A) Schematic representation of non-optimized conditions for human neuronal differentiation, in which neuronal cultures show some regions of neuronal aggregates and poorly attached cells. (B) Optimized conditions for human neuronal differentiation, including lower number of cells seeded, levels of 02 and shorter time of culture. Using NeurAntigen protocol the neurons remain properly attached and are uniformly distributed, allowing the detection of neuronal antigen by patients' antibodies.

Figure 5. NeurAntigen improves neuronal differentiation and maturation. (A) Representative images showing the expression of mature neuronal (MAP2, green) and GABAergic markers (GABA, red) in the cell cultures, either using the protocol published by Yan et al., or NeurAntigen protocol for 2 and 3 weeks of neuronal differentiation. (B) Following NeurAntigen protocol for 3 weeks significantly increases the percentage of mature neurons (MAP2+) in the culture as compared to the ones obtained following Yan et al. protocol. (C) A positive signal with the characteristic punctate pattern of neuronal surface antibodies can be detected only on NeurAntigen neurons differentiated for 3 weeks.

Figure 6. Human iPSC-derived astrocytes can be used for the detection of glial antibodies, associated with other types of autoimmune neurological disorders. (A) Representative images of aquaporin-4 (AQP4) and glial fibrillary acidic protein (GFAP) expression on astrocytes, using commercial antibodies. (B) After incubation with serum from a patient with neuromyelitis optica spectrum disorder containing AQP4 antibodies (upper panel) or CSF from a patient with meningoencephalomyelitis containing GFAP antibodies (lower panel), and later with a secondary anti-human IgG antibody, a positive immunostaining is detected on human astrocytes. Note the difference with the absence of reactivity of the corresponding negative controls (NHS: normal human serum, NHCSF: normal human CSF, both from healthy donors). DESCRIPTION

Definitions

All terms as used herein, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the description and claims unless an otherwise expressly set out definition provides a broader definition.

Throughout the description and claims the word "comprise" and variations of the word (e.g., "comprising", "having", "including", "containing"), are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of". Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

In this specification and claims, the use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. If the term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term "about" means the indicated value ± 1% of its value, or the term "about" means the indicated value ± 2% of its value, or the term "about" means the indicated value ± 5% of its value, the term "about" means the indicated value ± 10% of its value, or the term "about" means the indicated value ± 20% of its value, or the term "about" means the indicated value ± 30% of its value; preferably the term "about" means exactly the indicated value (± 0%).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. 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 one of skill with a general dictionary of many of the terms used in this disclosure. Units, prefixes, and symbols are denoted in their "Systeme International de Unites" (SI) accepted form.

Numeric ranges are inclusive of the numbers defining the range.

The headings provided herein are not limitations of the various aspects or embodiments of the disclosure. Furthermore, the present invention covers all possible combinations of particular aspects and embodiments described herein.

The figures and the experimental part/examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicitly indicated otherwise herein.

The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

The method of the present invention

In a first aspect, the present invention provides a method for obtaining human-induced pluripotent stem cells-derived neuronal cells. In the context of the present invention, a "human-induced pluripotent stem cell" refers to a type of pluripotent stem cell that can be generated directly from a somatic cell. Stem cells are cells which are able to self-renew and to differentiate into specialized cell types. As reviewed 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 cell" is based on two properties: self-renewal and potency. The self-renewal is the capacity of the stem cells to divide indefinitely, producing unaltered cell daughters maintaining the same properties of the progenitor cell. In particular conditions or under specific signals, a stem cell is able to exit from self-renewal and engage a program leading to differentiate into specialized cell types deriving from the three germ layers (ectoderm, endoderm, and mesoderm).

The method of the present invention comprises the step of culturing neural progenitor cells (NPC) for at least three weeks / for at least 21 days in proneural medium. "Neural progenitor cells" are the progenitor cells of the central nervous system (CNS) that give rise to many, if not all, of the glial and neuronal cell types that populate the CNS. NPCs do not generate the non- neural cells that are also present in the CNS, such as immune system cells. NPCs are present in the neonatal and mature adult brain, and therefore are not embryonic stem cells. NPCs are characterized based on their location in the brain, morphology, gene expression profile, temporal distribution and function, see, e.g., Martinez-Cerdeno V. and Noctor SC., "Neural progenitor cell terminology", Front Neuroanat, 2018, 12:104.

In one embodiment, the NPCs are cultured under static conditions, i.e., without agitation, shaking and the like. Further, the temperature of the culture medium is generally maintained at from about 35 to 39°C, preferably from about 36 to 38°C, such as about 37°C. Then NPCs are cultured under suitable conditions and in proneural medium for at least 21 days (3 weeks). The inventors have surprisingly found that, when the NPCs are cultured in proneural medium for at least 21 days (3 weeks), NPCs differentiate into mature neurons, which includes the expression of mature neuron-components such as MAP2, GABA, vGlut, CITP2, and synaptic components such as Synapsin and Gephyrin. Hence, in the method of the present invention, the NPCs are cultured for at least 21 days, such as for three weeks, or for 21 days, or more, such as for 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, or more, such as up to 35 days. Preferably, the NPCs are cultured for at least 21 days in proneural medium. Hence, in the method of the present invention, the NPCs are cultured under suitable conditions and in proneural medium for at least 3 weeks, such as for 3 weeks, or for 4 weeks or for 5 weeks.

In the context of the present invention "proneural medium" refers to a cell medium comprising at least:

Dulbecco's Modified Eagle Medium F12 (DMEM/F12) medium (e.g., Gibco 21331020), which 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 media, and combines DMEM's high concentrations of glucose, amino acids, and vitamins with the wide variety of components found in Ham's F12 media. Along with the glucose, amino acids, and vitamins in DMEM, DMEM/F12 contains additional components found in Ham's F12 media, including zinc, putrescine, hypoxanthine, and thymidine. DMEM F12 may also include other factors such as L-glutamine and phenol red indicator. An example of DMEM/F12 medium components can be seen in Table 2;

Neurobasal medium (e.g., Gibco 21103049), which contains 25 mM D-glucose, 0.22 mM sodium pyruvate, amino acids, vitamins, inorganic salts, and other components. An example of neurobasal medium components can be seen in Table 3;

B27 supplement without vitamin A (e.g., Gibco 12587010, 50X). For instance, B27 supplement without vitamin A may comprise the following components: Biotin, DL Alpha Tocopherol Acetate, DL Alpha-Tocopherol, BSA, fatty acid free Fraction V, Catalase, Human Recombinant Insulin, Human Transferrin, Superoxide Dismutase, Corticosterone, D-Galactose, Ethanolamine HCI, Glutathione (reduced), L-Carnitine HCI, Linoleic Acid, Linolenic Acid, Progesterone, Putrescine 2HCI, Sodium Selenite and T3 (triodo-l-thyronine). Preferably, B27 supplement without vitamin A is present in an amount of 0,5X; N2 supplement (e.g., Gibco 17502048, 100X), which is a chemically-defined, serum- free supplement based on Bottenstein's N-l formulation. Preferably, the amount of N2 supplement is 0,5X. An example of components comprised in N2 supplement can be found in Table 4; and

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

Proneural medium may comprise, in addition to the above components, epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2 or FGF-basic). When used for culturing NPCs (to obtain human-induced pluripotent stem cells (hiPSC)-derived neuronal cells), proneural medium does not comprise EGF and FGF2. However, when used for culturing cells for the generation of NPCs from NEP-rosettes, proneural medium further comprises FGF2 and EGF, see, e.g., Figure 1A. For instance, these two proteins can be obtained from Peprotech, with catalogue 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 23 known members of the FGF family. Recombinant Human FGF-basic is a 17.2 kDa protein consisting of 154 amino acid residues (see SEQ ID NO.: 2: AAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVV SIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPG QKAILFLPMSAKS). If present, EGF is present at a concentration of about 10 ng/ml. If present, FGF2 is present at a concentration of about 10 ng/ml.

Proneural medium may further comprise a reducing agent such as p-mercaptoethanol (e.g., Life Technologies 31350010, e.g., 55 mM) and/or antibiotics such as Penicillin/Streptomycin (e.g., Lonza 17-602E, e.g., 10.000 U/ml). Preferably, |3-mercaptoethanol is present at a concentration of about 50 pM.

Table 2. DMEM/F12 medium Gibco 21331020 components

Table 3. Neurobasal medium Gibco 21103049 components

Table 4. N2 supplement Gibco 17502048 components

In a preferred embodiment, proneural medium comprises or consists of the following components:

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

Neurobasal medium (e.g., Gibco 21103049)

B27 w/o Vit. A supplement (e.g., Gibco 12587010) - N2 supplement (e.g., Gibco 17502048)

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

P-mercaptoethanol (e.g., Life Technologies 31350010). When used for culturing NPCs, proneural medium does not comprise EGF and FGF2, as described above. However, when used for culturing cells for the generation of NPCs from NEP-rosettes, proneural medium preferably further comprises:

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

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

See, e.g., Figure 1A.

The medium may further comprise Penicillin/Streptomycin (e.g., Lonza 17-602E).

More preferably, proneural medium comprises or consists of:

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

Neurobasal medium (e.g., Gibco 21103049);

0,5x B27 w/o Vit. A supplement (e.g., Gibco 12587010);

0,5x N2 supplement (e.g., Gibco 17502048) ;

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

50 pM p-mercaptoethanol (e.g., Life Technologies 31350010); and

1% Penicillin/Streptomycin (e.g., Lonza 17-602E).

When used for culturing NPCs, proneural medium does not comprise EGF and FGF2, as described above. However, when used for culturing cells for the generation of NPCs from NEP-rosettes, proneural medium preferably further comprises:

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

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

In a preferred embodiment, the proneural medium does not comprise glial cell line-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and/or platelet-derived growth factor-AA (PDGF-AA, with two A subunits). In a preferred embodiment, the NPCs are seeded with a density of about 35.000 to about 40.000 cells/cm² and then cultured (incubated) for at least three weeks (for at least 21 days) in proneural medium, as described above. Preferably, the NPCs are seeded on Polyornithine/Laminin (PO/Lam) coated plates. Hence, in a preferred embodiment, the method of the present invention comprises culturing about 30.000 to about 40.000 NPCs/cm², preferably culturing about 37.000 NPCs/cm² for at least three weeks (for at least 21 days) in proneural medium, to obtain the hiPSC-derived neuronal cells.

Thus, in a preferred embodiment, the method of the present invention further comprises the steps of:

Seeding the NPCs with a density of about 30.000 to about 40.000 cells/cm²;

Preferably on PO/Lam coated plates; and

Incubating the NPCs for at least 21 days (at least 3 weeks), as described above.

Hence, in a preferred embodiment, the NPCs are seeded at a density of at least about 30.000 cells/cm², such as for instance at least about 35.000 cells/cm², or more, such as about 37.000 cells/cm², e.g., about 40.000 cells/cm², preferably at a density of about 30.000 to about 40.000 cells/cm², more preferably at a density of about 35.000 to about 40.000 cells/cm², even more preferably at a cell density of about 37.000 cells/cm². The cells are preferably seeded on plates which have been previously coated with polyornithine and laminin. In a preferred embodiment, the plates are first coated with polyornithine. Afterwards, the plates are washed, e.g., with PBS, and the laminin is added. This way, the plates are coated with both polyornithine and laminin. The polyornithine/laminin coating promotes the attachment, spreading and proliferation of NPCs on the coated plates. The present inventors have surprisingly found that, when the NPCs are seeded at a cell density of at least about 30.000 to about 40.000 cells/cm², as described above, the differentiation of NPCs into hiPSC-derived neuronal cells is improved, see, e.g., Figure 4 and "Results" section, item 3- "Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons".

In addition, preferably, the NPCs are cultured in hypoxia conditions (e.g., about 5% O2).

Physiological in vivo oxygen concentrations can range from 1% to 15%. However, most cell cultures are maintained in normal atmosphere oxygen conditions (~ 21% O2, "normoxia"). The value of inter alia O2 control has been shown to, e.g., influence gene expression profiles and phenotypic changes. The present authors have observed that, when the NPCs are cultured in hypoxia conditions (e.g., about 5% O2), better results are observed as compared with culture in normoxia conditions (e.g., about 21% O2), e.g., the hiPSC-derived neuronal cells, are optimized, in particular for their use for the detection of neuronal antibodies, see, e.g., Figure 4 and "Results" section, item 3- "Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons".

The skilled person is aware of means for obtaining the NPCs used in the method of the present invention. For instance, Chambers, S. et al. ("Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling", Nat Biotechnol, 2009, 27, 275-280) describes 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(ll):862-70), the standard protocol requires a suspension culture to generate embryoid bodies, which can then be plated as an adherent culture to generate neural precursors that can be mechanically or enzymatically isolated.

In a preferred embodiment of the present invention, the NPCs used in the method of the present invention are obtained by: a. Culturing hiPSC cells in an extracellular matrix-based hydrogel, preferably until they reach between about 70-80% confluence; b. Generating embryoid bodies (EBs); c. Differentiating the EBs to neuroepithelial (NEP)-rosettes; and d. Differentiating the NEP-rosettes to NPCs.

Preferably, the extracellular matrix-based hydrogel is "Matrigel". Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in such ECM proteins as laminin (a major component), collagen IV, heparan sulfate proteoglycans, entactin/nidogen, and a number of growth factors. Its use improves the attachment and differentiation of cells. It can be obtained, e.g., from Corning (e.g.. Cat No. 354234). The extracellular matrix-based hydrogel (preferably Matrigel) may comprise about 60% laminin, about 30% collagen IV, about 8% entactin, heparan sulfate proteoglycan (perlecan), transforming growth factor (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor (IGF-1), fibroblast growth factor (bFGF), tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor. There are also residual matrix metalloproteinases derived from the tumor cells.

For instance, the extracellular matrix-based hydrogel (preferably Matrigel) may comprise the following growth factors in the following amounts, see Table 5.

Table 5. Extracellular matrix-based hydrogel (preferably Matrigel) growth factor components

"Confluency" is the percentage of the culture dish area covered by adherent cells (e.g., 50% confluency indicates that 50 out of 100 parts of the growth surface are occupied by cells). The skilled person is aware of means of estimating cell confluence. For instance, cell confluence can be estimated by visual estimation, by using an image analysis software (image processing methods) or with the help of chemical dyes (e.g., Thymidine, Alamar blue, XTT). As stated above, the hiPSC cells are cultured in an extracellular matrix-based hydrogel preferably until they reach between about 70-80% confluence. Preferably, the hiPSC cells are cultured in free cell culture medium, such as mTeSRl medium, as described below.

In the context of the present invention, "embryoid bodies" (EBs) are three-dimensional aggregates of pluripotent stem cells, in this case of hiPSCs. EBs are beneficiary in the initiation of lineage-specific differentiation towards many lineages such as neural. The skilled person is aware of means of generating EBs from hiPSCs. Embryoid Bodies (EBs) can be generated at a normally scheduled passage by plating hiPSCs into non-tissue culture-treated dishes to prevent attachment.

Preferably, the EBs are generated by detaching the cultured hiPSC as clumps in feeder-free cell culture medium, such as mTeSRl medium, a cGMP, feeder-free maintenance medium for human ES and iPS cells available from StemCell, e.g., Catalog #85850) and centrifuging the detached cells to obtain EBs. The feeder-free cell culture medium used in the generation of EBs, such as mTeSRl medium, is a complete, serum-free, defined formulation designed for the feeder-free maintenance and expansion of human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells in the undifferentiated state.

In one embodiment, the EBs are generated as follows: iPSCs are seeded on an extracellular matrix-based hydrogel, such as "Matrigel", as described above;

When cells reach 70-80% confluence, cells are physically detached as clumps from the plate;

Cells are incubated in conical bottom plates in the presence of a feeder-free culture medium (such as mTeSR™l, Stemcell Technologies, #85850, as described above); The EBs are seeded in non-coated plates and incubated for about 24 h with a feeder-free culture medium (e.g., mTeSRl medium as defined above, or mTeSR™ Plus, Stemcell Technologies, #100-0276);

The medium is changed to proneural medium and refreshed every 24 h until the EBs are well formed (round and compact).

In a further step, the EBs may be differentiated to neuroepithelial (NEP)-rosettes. NEP- rosettes are microscopic flower-like structures that occur transiently during the development of the central nervous system. The skilled person is also aware of means for differentiating EBs to NEP-rosettes. Preferably, the EBs can be seeded on PO/Lam coated plates and incubated for several days (e.g., for at least 8 days, such as for at least 10 days, or for at least 12 days) with proneural medium, preferably supplemented with Noggin and also preferably in the presence of a TGF- ? inhibitor, see, e.g., Figure 1A. For instance, the TGF-/? inhibitor may be SB431542. SB431542 is a selective and potent inhibitor of the TGF-P/Activin/NODAL pathway that inhibits ALK5 (I C₅₀ = 94 nM), ALK4 (IC₅₀ = 140 nM), and ALK7 by competing for the ATP binding site. It does not inhibit the BMP type I receptors ALK2, ALK3, and ALK6. It can be purchased from Stemcell Technologies (e.g., #100-1051).

Finally, the NEP-rosettes may be differentiated into the NPCs used in the method of the present invention. The skilled person is also aware of means of differentiating NEP-rosettes into NPCs. This is described, e.g., in Chambers, S. et al. (see complete reference above) or in Topol A. et al., “ guide to generating and using hiPSC derived NPCs for the study of neurological diseases", J Vis Exp, 2015;(96):e52495. For instance, the NEP-rosettes may be detached from the plate and disaggregated, resuspended in proneural medium supplemented with FGF2 and with EGF and seeded on PO/Lam coated plates. NPCs may be expanded for about 3 to about 16 passages, preferably from about 5 to 12 passages, until the cell population is homogeneous and phenotypically stable, see Figure 1A.

The cells of the present invention

With the method of the present invention, new hiPSC-derived neuronal cells are obtained. These hiPSC-derived neuronal cells are optimized for the detection of neuronal surface human antibodies, and are functional see, e.g., "Results" section, item 3- "Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons".

Hence, the present invention provides a population of hiPSC-derived neuronal cells directly obtained by the method of the present invention ("the cells of the present invention" from now on). The cells of the present invention preferably express at least one, preferably all of the following proteins:

GABA (y-Aminobutyric acid);

MAP2 (Microtubule-associated protein 2);

TUJ1 (class III beta-tubulin);

CTIP2 (transcription factor Cti p2, also known as Bclllb); vGlut (vesicular glutamate transporter); Synapsin;

Gephyrin;

AM PAR (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor)

NMDAR (/V-methyl-D-aspartate receptor);

LGI1 (leucine-rich glioma inactivated 1)

GABAbR (GABAB receptor);

Caspr2 (contactin-associated protein-like 2);

- lgLON5;

GABAaR (GABA receptor);

DPPX (Dipeptidyl-peptidase-like protein-6)

GlyR (glycine receptor); mGluR5 (Metabotropic glutamate receptor 5); mGluRl (Metabotropic glutamate receptor 1)

Neurexin (NRXN);

SEZ6L2 (Seizure Related 6 Homolog Like 2); and/or

GluK2 (glutamate receptor, ionotropic, kainate 2).

The above list is not exhaustive, and the cells of the present invention may express other proteins.

Hence, the hiPSC-derived neuronal cells of the present invention express neuronal surface antigens associated with the autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably an AE-related disease, even more preferably AE, wherein the neuronal surface antigens are selected from the list comprising or consisting of AMPAR, NMDAR, LGI1, GABAbR, Caspr2, lgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluRl, Neurexin, SEZ6L2, and GluK2.

In the context of the present invention, a "target antigen" for autoimmune neurological disorders associated with the presence of autoantibodies may refer to any antigen present in the surface neurons which are specifically recognized by autoantibodies from a subject. For instance, a target neuronal antigen may be selected from AMPAR, NMDAR, LGI1, GABAbR, Caspr2, lgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluRl, Neurexin, SEZ6L2, and GluK2. The skilled person is aware of means for ascertaining whether a certain cell or cell population expresses one or more of the above proteins. For instance, the skilled person may make use of antibodies specific to the proteins antigens to detect their expression in a certain cell or cell population. In addition, markers of neuronal maturation and synaptic proteins, receptors and ion channels are commercially available, e.g., MAP2 (Synaptic Systems (188 004); 1:1000) and TUJ1 (Biolegend (801202); 1:500), cortical layers CTIP2 (Abeam (abl8465); 1:500), neurotransmitters GABA (Sigma (A2052); 1:1000), synaptic vesicles vGlut (Synaptic Systems (135 302); 1:1000), synaptic markers such as Synapsin (Calbiochem (574777); 1:1000), and Gephyrin (SYSY 147011; 1:200), and receptors such as AMPAR (Millipore 07-598; 1:100), GlyR (Sigma HPA016502; 1:50).

In a preferred embodiment, the hiPSC-derived neuronal cells of the present invention are forebrain cortical neurons which contain neurotransmitters and synaptic vesicles (see, e.g., Figure 1 D and E). The hiPSC-derived neuronal cells of the present invention are able to form synapses, as indicated by the positive immunostaining of Synapsin and Gephyrin (Figure IF), and express ion channel receptors, such as AMPAR or GlyR (see, e.g., Figure 1G). The cells of the present invention are functionally active for at least two weeks of differentiation, preferably for at least three weeks of differentiation, more preferably for at least four weeks of differentiation, and up to five weeks of differentiation, see, e.g., Figure 1H and "Results" section, item 1- "Characterization of human iPSC- derived neural progenitor cells and NeurAntigen neurons".

A wide variety of human antibodies associated with autoimmune encephalitis and related diseases can be detected using the hiPSC-derived neuronal cells of the present invention, as shown in the Examples. In particular, the hiPSC-derived neuronal cells of the present invention express all the antigens detected by antibodies of patients with AE and related diseases described until now (e.g., NMDAR, LGI1, AMPAR, GABAbR, Caspr2, lgLON5, etc.). In addition, the hiPSC-derived neuronal cells of the present invention are suitable to detect human antibodies directed against other neuronal surface antigens associated with AE and related diseases, such as GABAaR, DPPX, GlyR, mGluR5, mGluRl, Neurexin, SEZ6L2 and/or GluK2. Hence, the hiPSC-derived neuronal cells of the present invention are able to express at least one, preferably all of these antigens. Hence, the cells of the present invention are able to express at least one, preferably two or more, even more preferably at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, preferably all of the following antigens: NMDAR, LGI1, AMPAR, GABAbR, Caspr2, lgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluRl, Neurexin, SEZ6L2 and/or GluK2.

Hence, the cells of the present invention are particularly suited for their use in the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, even more preferably AE. For this the hiPSC-derived neuronal cells of the present invention may be put in contact (incubated) with a biological fluid obtained from a subject, preferably from a subject with a suspected neurological disorder associated with the presence of neuronal antibodies, such as cerebrospinal fluid, or serum, or any other biological fluid, 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 the case (positive immunoreactivity), and the biological fluid comprises antibodies which bind to antigens present in the hiPSC-derived neuronal cells, the subject may have a neurological disorder associated with the presence of neuronal antibodies, such as autoimmune encephalitis, like AE-related disease.

The cells of the present invention may also be able to express additional antigens associated with autoimmune neurological disorders, preferably autoimmune neurological disorders, such as AE and related diseases, such as neuronal surface antigens which have not yet been described as target of patients' antibodies. That is, the cells of the present invention may be used for the identification of new antibody reactivities associated with autoimmune neurological disorders, preferably autoimmune neurological disorders, such as AE and related diseases, as described in detail below. For this, a biological fluid (e.g., CSF or serum) from a subject with suspected AE, or a neurological autoimmune disorder, preferably autoimmune neurological disorders, such as AE and related diseases may be put in contact with the cells of the present invention, and a positive reactivity against antigens of the hiPSC-derived neuronal cells can be identified, indicating that neuronal antibodies are present. New neuronal antibodies and the antigens to which they bind may thus be identified.

The present disclosure also relates to astrocytes (or astrocyte population) which have been generated by 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. In particular, the generation of the astrocytes is described in detail in the Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17. Briefly, iPSCs were differentiated into spherical neurospheres or spherical neural masses (SNMs) containing neuroectodermal progenitors and then differentiated toward an astrocytic lineage following a previously published protocol (Serio et al., 2013). First, the SNMs were grown in suspension for 28 days with Induction Medium (DMEM/F12, 1% N2 supplement, 0.1% B27 supplement (Life, 17504-044), 1% nonessential amino acids (NEAA), 1% penicillin/streptomycin (PenStrep), 1% Glutamax) supplemented with 20 ng/mL LIF (Sigma) and 20 ng/mL EGF (R&D Systems), and then for further 21 days with 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, SNMs were incubated with accutase (LabClinics) for 15 minutes at 37^C, mechanically dissaggregated and plated on matrigel-coated plates as a monolayer. The monolayer of neural progenitors was cultured for 14 more days in Propagation Medium and then for 14 more days in CNTF medium (Neurobasal, 1% Glutamax, 1% PenStrep, 1% NEAA, 0.2% B27 supplement, 10 ng/mL CNTF (Prospec Cyt-272), a stage in which they were considered astrocyte progenitors and therefore characterized. These astrocyte progenitors were successfully frozen in Astrocyte Freezing Medium (90% FBS and 10% DMSO) and stored in liquid nitrogen for future use. When needed for an experiment, vials were thawed in medium containing FBS, resuspended in CNTF medium and plated on matrigel-coated plates. Cells were passaged four times before considered mature and then further characterized. Experiments were performed with astrocytes growing on Thermanox™ plastic coverslips (Thermofisher) coated with matrigel in 24-well plates. Hence, the hiPSC-derived astrocytes of the present disclosure can be obtained by differentiating iPSCs into spherical neurospheres containing neuroectodermal progenitors and then differentiated toward an astrocytic lineage. The hiPSC-derived astrocytes express markers selected from the list comprising or consisting of CD44, glial fibrillary acidic protein (GFAP), and S100 calcium-binding protein R (S100R), as well as of the excitatory amino acid transporter 2 (EAAT2, also known as GLT1).

As shown in Figure 6, the astrocytes as described herein (i.e., generated following the method described in di Domenico A., etal., "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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17) are optimized for the detection of glial surface antibodies, preferably astrocyte human antibodies of patients with an autoimmune neurological disorder.

The uses of the present invention

In a further 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, the hiPSC-derived neuronal cells obtained by the method of the present invention are optimized for the detection of neuronal surface antibodies, preferably neuronal surface human antibodies. Hence, the method and cells of the present invention can be used to detect neuronal surface antibodies, which is useful in the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related diseases.

Further, the invention provides the use of the astrocytes or astrocyte population as described herein (generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17) for the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of glial antibodies, such as NMOSD with AQP4 antibodies, meningoencephalomyelitis with GFAP antibodies or related diseases. Hence, these astrocytes can be used to detect astrocyte antibodies, which are useful in the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of glial antibodies, such as NMOSD, meningoencephalomyelitis or related diseases.

The invention further relates to the use of the hiPSC-derived neuronal cells obtained by the method of the present invention and/or of the astrocytes as described herein, in combination, to detect autoantibodies, which is useful in the diagnosis of autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related diseases.

In the context of the present invention, an "autoantibody" is an antibody produced by the immune system that is directed against one or more of the individual's own proteins. For instance, a subject suffering from an autoimmune neurological disorder may produce antibodies against his or her own cells (i.e., autoantibodies), such as antibodies against his or her own neurons (i.e., "neuronal antibodies" or "anti-neuronal antibodies", both terms being used indistinctively along the present description) or antibodies against one type of glial cells, his or her own astrocytes ("astrocyte antibodies" or "anti-astrocyte antibodies", both terms being used indistinctively along the present description), such as a neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related diseases, or with the presence of astrocyte antibodies, such as NMOSD, meningoencephalomyelitis or related diseases.

In the context of the present invention, a "neurological disorder associated with the presence of neuronal antibodies" refers to an expanding group of diseases or disorders which are mediated by antibodies against neuronal surface proteins. For instance, the neurological disorder associated with the presence of neuronal antibodies may be an autoimmune neurological disorder. In the context of the present invention, a "autoimmune neurological disorder" refers to an immune-mediated disorder in which the patients' own immune system recognizes the nervous system and attacks it, causing inflammation and damage. In a preferred embodiment, the autoimmune neurological disorder is an AE-related disease or disorder. "Autoimmune encephalitis (AE)" refers to a class of severe brain inflammatory diseases that are mediated, at least in part, by neuronal antibodies, mostly causing memory and behavioral problems, seizures, movement disorders, language dysfunction, dysautonomia or sleep disorders as main clinical manifestations. The disease is associated with antibodies against neuronal cell surface and synaptic proteins. AE is an immune-mediated condition characterized by the presence of autoantibodies, resulting from an inflammatory response directed to neuronal antigens. AE are associated with antibodies to different neuronal cellsurface proteins such as /V-methyl-D-aspartate receptor (NMDAR), leucine-rich glioma- inactivated 1 (LGI1), contactin-associated protein-like 2 (CASPR2), gamma-aminobutyric-acid B receptor (GABABR), etc. When bound to target protein, the antibodies cause neuronal dysfunction. AE are also known as antibody-mediated encephalitis. See, e.g., Gole S, Anand A., "Autoimmune Encephalitis". [Updated 2023 Jan 2], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: htt s://www.ncbi.nlm.nih.gov/books/NBK578203/. The AE may also be associated with the presence of tumors.

Hence, the autoimmune neurological disorder may be a neurological disorder associated with the presence of neuronal antibodies, such as AE-related disorder or AE. In a preferred embodiment, the neurological disorder is AE.

Further, the method and cells of the present invention may be used for the identification of neuronal antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorders, as explained in detail above.

The astrocytes as defined herein (generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17) may be used for the identification of glial antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of astrocyte autoantibodies, such as NMOSD, meningoencephalitis or related disorders, as explained in detail above.

In addition, the method and cells of the present invention may be used for the identification of target antigens of autoimmune neurological disorders, preferably neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorders. As explained above, with the method of the present invention, the obtained hiPSC-derived neuronal cells are optimized for the expression of neuronal maturation markers and AE- antigens. Hence, the cells of the present invention are optimized for their use in the identification of target antigens s associated with AE or related disorders (as explained above).

Hence, in a further aspect, the present invention provides a method for 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 related disorders. The method comprises the steps of: a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid (preferably CSF or serum) obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of neuronal antibodies, more preferably with AE and/or related diseases; b. Detecting human antibodies present in the biological fluid (preferably CSF or serum) of a patient suspect of suffering a disease as mentioned in a. and identifying them, wherein the human antibodies specifically bind to surface antigens present in the hiPSC-derived neuronal cells.

In a further aspect, the present invention provides a method for the detection of autoantibodies such as 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 comprises the steps of: a. Incubating the astrocytes as described herein (generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC- derived astrocyte generation and culture", p. 17) with a biological fluid (preferably CSF or serum) obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of glial autoantibodies, more preferably with NMOSD, meningoencephalomyelitis or related diseases; b. Detecting human antibodies present in the biological fluid (preferably CSF or serum) of a patient suspect of suffering a disease as mentioned in a. and identifying them, wherein the human antibodies specifically bind to antigens present in the astrocytes.

In a further embodiment, the present invention provides a method for the identification of target antigens associated with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorder, the method comprising: a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid (preferably CSF or serum) obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of neuronal antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Identifying antigens in the hiPSC-derived neuronal cells to which human antibodies present in the biological fluid of a patient suspect of suffering a disease as mentioned in a. specifically bind. Hence, the neuronal antibodies present in the biological fluid of the patient will specifically bind to the antigens present in the hiPSC-derived neuronal cells of the present invention. The skilled person understands the term "specifically bind" in the context of antigen-antibody of the present invention. The specific binding of an antibody to its antigen(s) excludes nonspecific binding, such as for instance non-specific antibody (Ab) binding to endogenous Fc receptors (FcRs) or non-specific binding due to ionic and/or hydrophobic interactions. The skilled person is able to detect and identify neuronal antibodies specifically binding to the hiPSC-derived neuronal cells of the present invention, as well as the antigen to which the antibodies specifically bind. Therefore, by using the methods and cells of the present invention, new antibodies and antigens associated with autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of anti-neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE, can be detected and identified.

The present invention further provides methods for treating a subject suffering from an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably with an AE-related disease, even more preferably with AE, NMOSD or meningoencephalitis, wherein the method comprises:

1. The detection of autoantibodies associated with autoimmune neurological disorders, preferably neurological disorders with neuronal antibodies and/or astrocyte antibodies, such as AE and/or related disorders or NMOSD, meningoencephalitis or related disorders, by

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

(ii). Identifying human antibodies present in the biological fluid of the subject which bind to the hiPSC-derived neuronal cells or astrocytes; and

2. Administering the subject a treatment to effectively treat the autoimmune neurological disorder, such as corticoids, immunoglobulins, Rituximab, Tocilizumab, etc. Hence, the present invention further provides a method of treating an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably an autoimmune encephalitis (AE)-related disease, even more preferably AE, comprising: administering a treatment to effectively treat the autoimmune neurological disorder in a subject who has been diagnosed as having an autoimmune neurological disorder based on the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies detected in a biological fluid obtained from a subject as described herein.

Suitable treatments to effectively treat the autoimmune neurological disorder are known to the skilled in the art. For instance, Table 4 of Bhagavati, S., "Autoimmune disorders of the nervous system: pathophysiology, clinical features, and therapy", Front. Neurol., 14 April 2021, Sec. Multiple Sclerosis and Neuroimmunology. Volume 12 - 2021, provides example of therapies of autoimmune diseases of the nervous system. In one embodiment, the therapy comprises corticoids, immunoglobulins, Rituximab, Tocilizumab, Bortezomib, etc.

The diagnostic method of the present invention

The present invention further provides an in vitro method for the diagnosis of autoimmune neurological disorders, preferably disorders associated with the presence of neuronal antibodies, more preferably AE-related diseases, even more preferably AE, in a subject, the method comprising: a. Incubating the hiPSC-derived neuronal cells of the present invention with a biological fluid obtained from a subject (preferably CSF or serum); b. Determining whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived neuronal cells of the present invention, wherein a positive immunoreactivity, for instance detected by immunocytochemistry, using the biological fluid (preferably CSF or serum) exposed to the hiPSC-derived neuronal cells, is indicative of the presence of neuronal antibodies and thus of an autoimmune neurological disorder, preferably a disorder associated with the presence of neuronal antibodies, more preferably an AE- related disease, even more preferably AE.

In one embodiment, the biological fluid (also referred to as "body fluid" in the present description) obtained from the subject is CSF. In another embodiment, the biological fluid is serum. In 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.

In another embodiment, the present invention provides an in vitro method for the diagnosis of autoimmune neurological disorders, preferably disorders associated with the presence of astrocytes antibodies, more preferably AE-related diseases, such as NMOSD or meningoencephalomyelitis, in a subject, the method comprising: a. Incubating the hiPSC-derived astrocytes as described herein (generated following the method described in di Domenico A., et al., "Patientspecific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease", Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC- derived astrocyte generation and culture", p. 17) with a biological fluid obtained from a subject (preferably CSF or serum); b. Determining whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived astrocytes, wherein a positive immunoreactivity, for instance detected by immunocytochemistry, using the biological fluid (preferably CSF or serum) exposed to the hiPSC-derived astrocytes, is indicative of the presence of astrocyte antibodies and thus of an autoimmune neurological disorder, preferably a disorder associated with the presence of autoantibodies, more preferably an AE-related disease such as NMOSD or meningoencephalomyelitis. In the context of the method of the present invention, the term "incubating the hiPSC-derived neuronal cells with a biological fluid obtained from a subject" or "incubating the astrocytes as defined herein with a biological fluid obtained from a subject" means contacting the cells with the biological fluid, under the conditions and for a period of time which is sufficient for the determination of the presence of auto-antibodies in the fluid. As used herein, the term "contact" or "contacting" means bringing together, either directly or indirectly, the hiPSC- derived neuronal cells and/or astrocytes with the biological fluid. Contacting may occur, for example, in any number of buffers, salts, solutions, or in cell culture medium. In a preferred embodiment, the biological fluid obtained from a subject (e.g., CSF or serum) is incubated for about 1 hour at about 37^C with the hiPSC-derived neuronal cells of the present invention (preferably live neuronal cells, where the cells have not been previously fixated or permeabilized) and/or with the astrocytes as defined herein.

The determination of whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived neuronal cells of the present invention and/orto the astrocytes as defined herein or not can be performed by routine methods known to the skilled person. For instance, the determination may be performed by immunocytochemistry. In one embodiment, the determination is performed as follows. After the about one hour of incubation of the biological fluid with the hiPSC-derived neuronal cells of the present invention and/or with the astrocytes as defined herein, the cells are washed, e.g., with PBS, fixed, e.g., with 4% paraformaldehyde for about 5 minutes, and incubated with a labelled secondary anti-human immunoglobulin antibody, e.g., with a green fluorescent secondary anti-human immunoglobulin antibody, for about 1 hour at room temperature (RT). The cell nuclei may also be labelled, e.g., with DAPL The labelled secondary antibody allows for the visualization of the primary antibody, originally present in the biological fluid and bound to the antigen expressed in the hiPSC-derived neuronal cells of the present invention and/or astrocytes.

Hence, a positive immunoreactivity, for instance detected by immunocytochemistry, as explained above, in the biological fluid exposed to the hiPSC-derived neuronal cells and/or astrocytes may be indicative of the presence of anti-neuronal and/or anti-astrocyte antibodies. The presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies in the biological fluid obtained from the subject may be indicative of an autoimmune neurological disorder, such as a neurological disorder associated with autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, for instance AE or related diseases, such as such as NMOSD or meningoencephalomyelitis.

The present invention thus allows for a rapid, sensible and efficient identification of the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies in the biological fluid, which may be indicative of the presence of an autoimmune neurological disorder, such a neurological disorder associated with autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, such as AE or related diseases, e.g., such as NMOSD or meningoencephalomyelitis (diagnostic method).

The kit of the present invention

In a further aspect, the present invention provides a kit suitable for the in vitro diagnosis of an autoimmune neurological disorder, such as a neurological disorder associated with autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, for instance AE or related diseases. The kit comprises the hiPSC-derived neuronal cells of the invention, or a composition comprising the same, and instructions for use. The kit may also comprise a composition comprising the hiPSC-dervied neuronal cells of the invention and the control cells as described herein, and/or the astrocytes as described herein. In certain embodiments, the kit may also comprise a sterile container such as boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, dishes or other suitable container forms known in the art. Such containers can be made of plastic, glass or other materials suitable for holding cells. The kit may also comprise suitable cell culture medium, shipping medium, buffers and any other reactive that may become necessary for detecting neuronal antibodies and/or astrocyte antibodies. The kit may also comprise anti-human immunoglobulin antibodies, preferably labelled, such as fluorescently labelled, and/or any other reagent used in immunocytochemistry.

The kit may also further comprise the iPSC-derived astrocytes as defined herein (generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17).

In certain embodiments, the kit may further comprise a cell population non-related to autoimmune neurological disorders. Such a cell population may preferably be non-relevant for neurological diseases , preferably a cell population of an origin different from the nervous system, such as fibroblasts, to be used as negative control in the kit of the present invention.

The kit of the present invention may also be suitable for the identification of autoantibodies such as neuronal antibodies and/or astrocyte antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of autoantibodies such as neuronal antibodies and/or astrocyte antibodies, such as AE and/or related disorders, NMOSD, meningoencephalomyelitis or related disorders. This composition of the kit, including separately iPSC-derived neurons and iPSC-derived astrocytes could be relevant in those cases of patients presenting with neurological symptoms common to several autoimmune neurological diseases, such as memory and behavioral problems, seizures, movement disorders, etc., which can be present for example in both AE and meningoencephalomyelitis, and associate with either neuronal or glial antibodies.

In certain embodiments, binding of antibodies present in a biological fluid obtained from the subject to antigens present in the hiPSC-derived neuronal and/or iPSC-derived astrocytes as defined herein, and absence of binding to the control cells as defined herein, is indicative of the presence of anti-neuronal or anti-astrocyte antibodies and thus of an autoimmune neurological disorder in the subject. In contrast, binding of antibodies present in the biological fluid obtained from the subject to antigens present in the hiPSC-derived neuronal cells or iPSC- derived astrocytes as defined herein and also to the cell population unrelated to an autoimmune neurological disorder (negative control) may not be indicative of the presence of specific anti-neuronal or anti-astrocyte antibodies and thus is not indicative of an autoimmune neurological disorder. This composition of the kit, including iPSC-derived neurons or iPSC-derived astrocytes and control cells could be relevant to interpret the test results. It serves as an internal control of the diagnostic method, for example, if a non-specific immunoreactivity is found using the biological fluid obtained from the subject (positive signal detected with neurons and with fibroblast), one can easily detect that it does not correspond with specific neuronal antibodies.

Hence, the present invention provides the use of the kit of the present invention for the in vitro diagnosis of an autoimmune neurological disorder, such as a neurological disorder associated with neuronal antibodies, for instance AE or related diseases and for the identification of neuronal antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or for the identification of target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE.

The kit of the invention can also be used for the in vitro diagnosis of an autoimmune neurological disorder, such as a neurological disorder associated with astrocyte antibodies, for instance AE-related diseases, such as NMOSD or meningoencephalomyelitis, and for the identification of astrocyte antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of astrocyte antibodies, such as AE-related diseases and/or for the identification of target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of astrocyte antibodies, more preferably AE-related diseases, e.g., NMOSD or meningoencephalomyelitis.

Items

The present invention further provides the following items:

1. A method for obtaining human-induced pluripotent stem cells (hiPSC)-derived neuronal cells, wherein the method comprises culturing about 30.000 to about 40.000 cells/cm² neuronal progenitor cells (NPCs) for at least three weeks in proneural medium, to obtain hiPSC-derived neuronal cells. 2. The method according to item 1, wherein the NPCs are cultured in hypoxia conditions (5% O₂).

3. The method according to any one of items 1 or 2, wherein the NPCs are obtained by: a. Culturing hiPSC cells in an extracellular matrix-based hydrogel until they reach between about 70-80% confluence; b. Generating embryoid bodies (EBs); c. Differentiating the EBs to neuroepithelial (NEP)-rosettes; and d. Differentiating the NEP-rosettes to neuronal progenitor cells.

4. The method according to item 3, wherein step b. is performed by detaching the cultured hiPSC of step a. as clumps in serum-free cell culture medium and centrifuging the detached cells to obtain EBs.

5. The method according to any one of items 1 to 4, wherein step c. is performed by seeding the EBs of step b. on Polyornithine/Laminin (Po/Lam)-coated plates in the presence of Noggin and at least one TGF- inhibitor, and incubating them in proneuronal medium for at least 8 days, preferably wherein the TGF- inhibitor is SB431542.

6 The method according to item 5, wherein the EBs are incubated for a period between 8 to 12 days.

7. The method according to any one of items 1-6, wherein step d. is performed by disaggregating the NEP-rosettes, seeding the cells in PO/Lam-coated plates, incubating them in proneural medium supplemented with FGF2 and EGF and passaging the cells for about 5-12 passages.

8. Use of the method as defined in any of the preceding items for the diagnosis of an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably an autoimmune encephalitis (AE)-related disease, even more preferably AE. 9. A population of hiPSC-derived neuronal cells obtained by the method as defined in any one of items 1-7.

10. Use of the cells as defined in item 9 and/or a population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17, for the diagnosis of autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE.

11. Use of the cells as defined in item 9 and/or a population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17, for the identification of: neuronal and/or astrocyte antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, such as AE and/or related disorders, and/or target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE. 12. A method for the identification of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, the method comprising: a. Incubating the hiPSC-derived neuronal cells as defined in item 9 and/or a population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17, with a biological fluid obtained from a subject, preferably a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Detecting human antibodies present in the biological fluid of the subject as mentioned in a. and identifying them, wherein the human antibodies specifically bind to surface antigens present in the hiPSC-derived neuronal cells and/or to surface antigens present in the astrocytes.

13. A method for the identification of target antigens associated with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, such as AE and/or related disorder, the method comprising: a. Incubating the hiPSC-derived neuronal cells as defined in item 9 and/or a population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC- derived astrocyte generation and culture", p. 17, with a biological fluid obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Identifying antigens in the hiPSC-derived neuronal cells to which human autoantibodies, such as neuronal antibodies and/or astrocyte antibodies present in the biological fluid of a patient suspect of suffering a disease as mentioned in a. specifically bind. An in vitro method for the diagnosis of autoimmune neurological disorders, preferably autoimmune neurological disorders associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE, in a subject, the method comprising a. Incubating the hiPSC-derived neuronal cells as defined in item 9 and/or a population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17 with a biological fluid obtained from a subject; b. Determining whether antibodies present in the biological fluid obtained from the subject bind to antigens present in the hiPSC-derived neuronal cells as defined in item 9 and/or to the population of astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17, wherein a positive immunoreactivity, preferably detected by immunocytochemistry, using the biological fluid exposed to the hiPSC- derived neuronal cells and/or to the astrocytes, is indicative of the presence of neuronal and/or astrocyte antibodies and thus of an autoimmune neurological disorder, such as a neurological disorder associated with the presence of autoantibodies, such as neuronal antibodies and/or astrocyte antibodies, preferably an autoimmune encephalitis (AE)-related disease, even more preferably AE, in the subject.

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

16. The kit according to item 15, wherein the kit further comprises iPSC-derived astrocytes generated following 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, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17.

17. The kit according to item 15 or 16, wherein the kit further comprises a cell population unrelated to an autoimmune neurological disorder, preferably a non-neural cell population, more preferably fibroblasts. EXAMPLES

Materials and experimental methods

1- Obtention of neuronal cells derived from human-induced pluripotent stem cells: the

NeurAntigen protocol a. Culturing human-induced pluripotent stem cells:

Human-induced pluripotent stem cells (hiPSC) are cultured in an extracellular matrix-based hydrogel until they reach 70 to 80% confluency. Embryoid bodies (EBs) are generated by detaching iPSC as clumps in serum-free cell culture medium and centrifuging the detached cells. EBs are then seeded on poly-L-ornithine-laminin (PO/Lam) coated plates, incubated for 8 to 12 days with proneural cell culture medium containing Noggin and TGF-b inhibitor (SB431542), and differentiated into neuroepithelial (NEP)-rosettes. Later, NEP-rosettes are disaggregated, seeded on PO/Lam coated plates, incubated with proneural medium containing FGF2 and EGF, and differentiated into neural progenitor cells (NPC). b. Culturing neural progenitor cells:

NPC are incubated on PO/Lam coated plates with proneural medium containing FGF2 and EGF, and can be passaged approximately for 5 to 12 times. c. Differentiation of neuronal cells:

NPC are seeded on PO/Lam coated plates, with a density of 35.000 to 40.000 cells/cm², incubated for at least 21 days with proneural medium without growing factors, preferably in hypoxia conditions (5% 02), and differentiated into neurons.

2- Characterization of neural progenitor cells

Immunostaining of NPC using markers of neural linage such as Nestin (Santa Cruz (sc-23927); 1:250), SOX2 (R&D System (MAB2018); 1:50), and Pax6 (Covance (PRB-278P); 1:100), and a marker of proliferation Ki67 (Abeam (Abl5580); 1:2000). In brief, the above-mentioned primary antibodies are incubated for 48 hours at 4^C, and revealed with the corresponding secondary fluorescent antibodies (2 hours at room temperature); cells nuclei are labeled with DAPI. 3- Characterization of neuronal cells derived from human-induced pluripotent stem cells a. Immunocytochemistry:

Immunostaining of hiPSC-derived neurons using markers of neuronal maturation such as MAP2 (Synaptic Systems (188 004); 1:1000) and TUJ1 (Biolegend (801202); 1:500), cortical layers CTIP2 (Abeam (abl8465); 1:500), neurotransmitters GABA (Sigma (A2052); 1:1000), synaptic vesicles vGlut (Synaptic Systems (135 302); 1:1000), synaptic markers such as Synapsin (Calbiochem (574777); 1:1000), and Gephyrin (SYSY 147011; 1:200), and receptors such as AMPAR (Millipore 07-598; 1:100), GlyR (Sigma HPA016502; 1:50). Similarly, primary antibodies are incubated for 48 hours at 4^C, and revealed with the corresponding secondary fluorescent antibodies (2 hours at room temperature); cells nuclei labeled with DAPL b. Calcium i

We used calcium fluorescent imaging to determine the spontaneous activity of hiPSCderived neurons, after 4 weeks of differentiation, as previously reported (e.g., Carola, G., Malagarriga, D., Calatayud, C. et al., "Parkinson's disease patient-specific neuronal networks carrying the LRRK2 G2019S mutation unveil early functional alterations that predate neurodegeneration", npj Parkinsons Dis. 7, 55 (2021)).

4- Immunostaining of biological samples from patients with autoimmune encephalitis on NeurAntigen neurons (hiPSC-derived neurons obtained by the NeurAntigen protocol)

Immunocytochemistry on live hiPSC-derived neurons obtained by the NeurAntigen protocol, using serum or cerebrospinal fluid (CSF) from patients or healthy donors. To demonstrate the wide expression of human autoantigens by these mature neurons, and their recognition by patients' autoantibodies, samples of different individuals with autoimmune encephalitis containing specific antibodies against known neuronal surface antigens, or samples of healthy controls without detectable antibodies, are exposed to NeurAntigen neurons, without prior fixation or permeabilization. Serum (1:100 dilution) or CSF (1:5) samples are incubated for 1 hour at 37^C, later the cultures of neurons are washed and fixed with 4% paraformaldehyde for 5 minutes, and incubated with a green fluorescent secondary anti-human immunoglobulin antibody (1:1000 dilution) for 1 hour at room temperature; cells nuclei labeled with DAPL 5- Comparison of NeurAntigen neurons and neuronal cells obtained by a previously reported method

To demonstrate that NeurAntigen neurons are optimized forthe detection of neuronal surface human antibodies, we compared two maturation steps of our protocol (at 2 and 3 weeks) and a different method of obtaining neurons from hiPSC (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(ll):862-70). Expression of mature neuronal markers (MAP2, and GABA), and immunostaining of serum samples (1:100 dilution) from patients with autoimmune encephalitis or healthy controls.

6- Obtention of astrocytes derived from human-induced pluripotent stem cells

The obtention of astrocytes derived from iPSC is described in di Domenico A., et al., "Patientspecific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease", Stem Cell Reports, Vol. 12, 213-229, February 12, 2019, Supplemental Information of this article, section "Supplemental experimental procedures", subsection "iPSC-derived astrocyte generation and culture", p. 17. Briefly, iPSCs were differentiated into spherical neurospheres containing neuroectodermal progenitors and then differentiated toward an astrocytic lineage following a previously published protocol (Serio et al., 2013). First, the SNMs were grown in suspension for 28 days with Induction Medium (DMEM/F12, 1% N2 supplement, 0.1% B27 supplement (Life, 17504-044), 1% nonessential amino acids (NEAA), 1% penicillin/streptomycin (PenStrep), 1% Glutamax) supplemented with 20 ng/mL LIF (Sigma) and 20 ng/mL EGF (R&D Systems), and then for further 21 days with 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, SNMs were incubated with accutase (LabClinics) for 15 minutes at 37^C, mechanically dissaggregated and plated on matrigel-coated plates as a monolayer. The monolayer of neural progenitors was cultured for 14 more days in Propagation Medium and then for 14 more days in CNTF medium (Neurobasal, 1% Glutamax, 1% PenStrep, 1% NEAA, 0.2% B27 supplement, 10 ng/mL CNTF (Prospec Cyt-272), a stage in which they were considered astrocyte progenitors and therefore characterized. These astrocyte progenitors were successfully frozen in Astrocyte Freezing Medium (90% FBS and 10% DMSO) and stored in liquid nitrogen for future use. When needed for an experiment, vials were thawed in medium containing FBS, resuspended in CNTF medium and plated on matrigel-coated plates. Cells were passaged four times before considered mature and then further characterized. Experiments were performed with astrocytes growing on Thermanox™ plastic coverslips (Thermofisher) coated with matrigel in 24-well plates.

7- Immunostaining of biological samples from patients with autoimmune neurological disorders on the astrocytes derived from human iPSC.

Inmunocytochemistry on hiPSC-derived astrocytes obtained as described in di Domenico A., et al., using serum or CSF from patients or healthy donors. To demonstrate the expression of human autoantigens by these astrocytes, and their recognition by patients' autoantibodies, samples of different individuals with NMOSD containing antibodies against AQP4, or individuals with meningoencephalomyelitis or related disorders containing antibodies against GFAP, or samples of healthy controls without detectable glial antibodies, are exposed to astrocytes. For AQP4 detection, serum samples are incubated (1:50 dilution) for 1 hour at 37^C, later the cultures of astrocytes are washed and fixed with 4% PFA for 10 minutes, and incubated with a green fluorescent secondary anti-human immunoglobulin G antibody (1:1000 dilution) for 1 hour at room temperature; cells nuclei labeled with DAPL For GFAP detection, CSF samples are incubated (1:10 dilution) overnight at 4^C, after the astrocytes are fixed and permeabilized with PFA and Triton, and then incubated with a red fluorescent secondary anti-human IgG antibody (1:1000). Figure 6 A.

Results

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

The scheme of cell culture steps to obtain NPC from hiPSC (after 45 days) and differentiated neurons (21 days later) is shown on Figure 1A. NPC generated from hiPSC express Nestin, SOX2 and PAX6, markers of neural linage, as well as the proliferation marker Ki67 (Figure IB). After differentiation, mature neurons stained with MAP2 show long neuronal processes and positive immunostaining for the neurotransmitter GABA (Figure 1C). Quantification of these cultured cells shows that 80% are mature neurons (MAP2 and DAPI positive), and 70% are GABAergic neurons (MAP2 and GABA positive; Figure ID). Differentiated neurons stained with TUJ1 or MAP2 were also positive for CTIP2 and vGlut, showing that NeurAntigen cells are cortical neurons and contain excitatory synaptic vesicles (Figure IE). These neurons form synapses as indicated by the positive immunostaining of Synapsin and Gephyrin (Figure IF), and express ion channel receptors, such as AMPAR or GlyR (Figure 1G). Moreover, at 4 weeks of differentiation, NeurAntigen neurons are functionally active, as shown by the calcium waves detected on single cells (Figure 1H).

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

The scheme for using NeurAntigen neurons to diagnose autoimmune encephalitis is shown on Figure 2A: in 2 hours, a sample from a patient with suspected autoimmune encephalitis (or a related neurological disorder) is screened for the detection of neuronal surface autoantibodies. Figure 2B shows examples of the positive immunoreactivity of patients' serum, seen with a thin bright green punctate pattern on the neuronal processes, in contrast to a negative result shown with a control serum (NHS). NeurAntigen live neurons are able to detect not only human antibodies directed against the common and uncommon neuronal surface antigens associated with autoimmune encephalitis (including but not limited to NMDAR, LGI1, AMPAR, GABAbR, Caspr2, lgLON5, GABAaR, DPPX, GlyR, mGluR5, mGluRl) or have been more recently described (Neurexin, SEZ6L2, GluK2). In addition, patients' CSF incubated with NeurAntigen live neurons shows a similar immunoreactivity pattern than the patient serum, as opposed to the negative result seen with a control CSF (NHCSF), indicating that both serum and CSF antibodies can be detected with our test (Figure 3).

3- Optimized expression of maturation markers and detection of neuronal surface antibodies by NeurAntigen neurons

A comparative scheme of cell culture conditions to differentiate neurons from hiPSC-derived NPC is shown on Figure 4. Under optimized conditions, that include a lower number of NPC seeded, incubation in hypoxia, and fora limited period of time, NeurAntigen neurons are more stable during the cell culture, better attached to the plate surface, with a more uniform distribution, and show better the morphology of the neuronal processes (Figure 4B, insets 1 and 2). The specific differences between the NeurAntigen protocol and another method of obtaining neurons from hiPSC are detailed in Table 6. Figure 5 shows how extending the differentiation period from NPC to neurons (2 vs 3 weeks) and using the NeurAntigen protocol instead of previously reported methods, the presence of MAP2 and GABA double-positive mature neurons increases significantly (Fig 5 A-B). Moreover, the detection of patients' neuronal surface antibodies is only possible with NeurAntigen neurons after 3 weeks (at least 21 days) of differentiation, as shown in Figure 5C.

6. Protocol specifications for NeurAntigen kit in comparison with Yan et al.

Claims

1. A method for obtaining human-induced pluripotent stem cells (hiPSC)-derived neuronal cells, wherein the method comprises culturing about 30.000 to about 40.000 cells/cm² neuronal progenitor cells (NPCs) for at least three weeks in proneural medium, to obtain hiPSC-derived neuronal cells.

2. The method according to claim 1, wherein the NPCs are cultured in hypoxia conditions (5% O₂).

3. The method according to any one of claims 1 or 2, wherein the NPCs are obtained by: a. Culturing hiPSC cells in an extracellular matrix-based hydrogel until they reach between about 70-80% confluence; b. Generating embryoid bodies (EBs); c. Differentiating the EBs to neuroepithelial (NEP)-rosettes; and d. Differentiating the NEP-rosettes to neuronal progenitor cells.

4. The method according to claim 3, wherein step b. is performed by detaching the cultured hiPSC of step a. as clumps in serum-free cell culture medium and centrifuging the detached cells to obtain EBs.

5. The method according to any one of claims 1 to 4, wherein step c. is performed by seeding the EBs of step b. on Polyornithine/Laminin (Po/Lam)-coated plates in the presence of Noggin and at least one TGF- inhibitor, and incubating them in proneuronal medium for at least 8 days, preferably wherein the TGF- inhibitor is SB431542.

6 The method according to claim 5, wherein the EBs are incubated for a period between 8 to 12 days.

7. The method according to any one of claims 1-6, wherein step d. is performed by disaggregating the NEP-rosettes, seeding the cells in PO/Lam-coated plates, incubating them in proneural medium supplemented with FGF2 and EGF and passaging the cells for about 5-12 passages.

8. Use of the method as defined in any of the preceding claims for the diagnosis of an autoimmune neurological disorder, preferably an autoimmune neurological disorder associated with the presence of neuronal antibodies, more preferably an autoimmune encephalitis (AE)-related disease, even more preferably AE.

9. A population of hiPSC-derived neuronal cells obtained by the method as defined in any one of claims 1-7.

10. 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, even more preferably AE.

11. Use of the cells as defined in claim 9 for the identification of: neuronal antibodies in patients with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorders, and/or target antigens for autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, more preferably autoimmune encephalitis (AE)-related diseases, even more preferably AE.

12. A method for the identification of neuronal antibodies, the method comprising: a. Incubating the hiPSC-derived neuronal cells as defined in claim 9 with a biological fluid obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of neuronal antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Detecting human antibodies present in the biological fluid of a patient suspect of suffering a disease as mentioned in a. and identifying them, wherein the human antibodies specifically bind to surface antigens present in the hiPSC-derived neuronal cells.

13. A method for the identification of target antigens associated with autoimmune neurological disorders, preferably with neurological disorders associated with the presence of neuronal antibodies, such as AE and/or related disorder, the method comprising: a. Incubating the hiPSC-derived neuronal cells as defined in claim 9 with a biological fluid obtained from a subject with a suspected neurological autoimmune disorder, preferably with a suspected neurological disorder associated with the presence of neuronal antibodies, more preferably with a suspected autoimmune encephalitis (AE)-related disease, even more preferably with suspected AE; b. Identifying antigens in the hiPSC-derived neuronal cells to which human neuronal antibodies present in the biological fluid of a patient suspect of suffering a disease as mentioned in a. specifically bind.

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

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