Method for hindbrain neuron generation

Abstract

The present invention refers an in vitro method for inducing differentiation of human pluripotent stem cells (hPSCs) generating hindbrain neurons and compositions thereof. The invention also refers to the hindbrain neurons and compositions thereof for use in diagnostic screening and for use in the prevention and / or treatment of neurological disorders.

Description

[0001] METHOD FOR HINDBRAIN NEURON GENERATION

[0002] FIELD OF THE INVENTION

[0003] The present invention refers to a method for generating hindbrain neurons and compositions thereof. The invention also refers to the hindbrain neurons and compositions thereof for use in diagnostic screening and for use in the prevention and / or treatment of neurological disorders.

[0004] BACKGROUND OF THE INVENTION

[0005] Human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), and their derivatives have been used as disease models and cell replacement therapy in the context of neurodevelopmental, neuroimmunological, neuropsychiatric, and / or neurodegenerative disorders. Directed differentiation methods starting from hPSCs have been used to generate central nervous system (CNS) cells of interest to model brain diseases such as autism, depression, schizophrenia, encephalitis, Parkinson’s disease, and Alzheimer’s disease. However, the results of these studies show that the recapitulation of in vivo phenotypes is dependent on generating disease-relevant, and thus region-specific, neuronal subtypes in vitro. For instance, regarding human hindbrain neurons, there are no methods at present that disclose how to obtain them using hPSCs. Thus far, the only method for generating a very specific subtype of the ventral hindbrain, namely serotonergic neurons, has been described in Lu, J., Zhong, X, Liu, H. et al., 20161and US2020087620A1. Nevertheless, serotonergic neuron cells differ from most hindbrain neuron subtypes and are therefore not broadly applicable for modelling, screening, and treating of the disorders affecting most parts of the human hindbrain.

[0006] Therefore, there is still a need for improved methods for hindbrain neuron generation, suitable for modelling, screening, and treating neurological disorders, such as neurodevelopmental, neuroimmunological, neuropsychiatric, and / or neurodegenerative disorders, in particular those affecting the hindbrain.

[0007] SUMMARY OF THE INVENTION

[0008] With the present invention, it has now been found how to overcome the critical gap of the lack of disease-relevant, region-specific, neuronal cell types that recapitulate in vivo phenotypes of hindbrain neurons. The present invention provides a method for generating hindbrain neurons and compositions comprising such cells. The invention also provides the hindbrain neurons and compositions thereof for use in diagnostic screening, and / or for preventing and / or treating neurodevelopmental, neuroimmunological, neuropsychiatric, and / or neurodegenerative disorders.

[0009] It is therefore an object of the invention an in vitro method for inducing differentiation of human pluripotent stem cells (hPSCs), comprising a step of a) culturing hPSCs in a culture medium comprising at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signalling, and at least one activator of wingless (Wnt) signalling from DIV [days in vitro 0 to DIV 2, and in a culture medium comprising at least one inhibitor of SMAD signalling from DIV 2 to DIV 11, to obtain a cell population of cells, wherein at least about 75% of the cells express at least one hindbrain neural stem cell (NSC) marker; and further comprising a step of b) culturing hindbrain NSCs obtained in (a) in a culture medium comprising at least one inhibitor of Notch signalling for about at least 10 days, preferably for about at least 12 days from DIV 15, to obtain a cell population of differentiated cells, wherein at least about 75% of the differentiated cells express at least one hindbrain neuron marker.

[0010] In an embodiment, for culturing in step (b) “for about at least 10 days, preferably for about at least 12 days from DIV 15”, it is also intended culturing in a culture medium comprising at least one inhibitor of Notch signalling from DIV 15 onwards.

[0011] According to the invention, in the step a) hPSCs are seeded at density of 250,000-300,000 cells / cm2, said hPSCs being 100% confluent.

[0012] In an embodiment, at least one inhibitor of SMAD signalling is selected from inhibitors of TGFp / Activin-Nodal signalling, inhibitors of bone morphogenetic protein (BMP) signalling, and combinations thereof.

[0013] In an embodiment, at least one inhibitor of TGFp / Activin- Nodal signalling comprises an inhibitor of ALK5. Preferably, it comprises SB431542, or a derivative, or a mixture thereof. In more preferred embodiment it comprises SB431542.

[0014] In certain embodiments, at least one inhibitor of BMP signalling comprises LDN193189, Noggin, dorsomorphin, a derivative thereof, or a mixture thereof. In a preferred embodiment it comprises LDN193189. In an embodiment, at least one activator of Wnt signalling comprises a GSK3 inhibitor. Preferably, it comprises CHIR99021, derivatives thereof, and combinations thereof. In more preferred embodiment it comprises CHIR99021.

[0015] In an embodiment, at least one inhibitor of Notch signalling comprises a y-secretase inhibitor. Preferably, it comprises DAPT, derivatives thereof, or mixtures thereof. In more preferred embodiment it comprises DAPT.

[0016] In an embodiment, in a) the culture medium from DIV 0 to DIV 2 comprises SB431542, LDN193189 and CHIR99021, and from DIV 2 to DIV 11 the culture medium comprises SB431542 and LDN193189; and in b) culture medium comprises DAPT. Preferably, SB431542 is comprised at a concentration of about 10 pM; LDN193189 is comprised at a concentration of about lOOnM; CHIR99021 is comprised at a concentration of about 1.5 pM to 3.5 pM, preferably of 3 pM; and in culture medium in b) DAPT is comprised at a concentration of about 10 pM.

[0017] In an embodiment, in a) at least one hindbrain NSC marker is selected from Homeobox A2 (HOXA2), Homeobox B 1 (HOXB 1), Homeobox B2 (HOXB2), Homeobox B4 (HOXB4), MKI67, IRX3 and combinations thereof; and / or wherein in b) the at least one hindbrain neuron marker is selected from Microtubule-Associated Protein 2 (MAP2), and / or Homeobox A2 (HOXA2), HOXA2, HOXB2, HOXB4, ASCL1, LMX1 A, LMX1B, TLX1, TLX3, DRG11, and combinations thereof.

[0018] In an embodiment, in a) hindbrain NSCs do not express FOXG1; and in b) the differentiated cells do not express any of markers selected from the group consisting of: MKI67, SRY-box transcription factor 10 (SOXIO), BRACHYURY (T), SRY-box transcription factor 17 (SOX17), FOXG1, EMX1, OTX2, and combinations thereof.

[0019] In an embodiment, the hindbrain NSCs obtained in a) are incubated in neural differentiation medium for about four days, preferably from DIV 12 to DIV 15, then replated and / or optionally cryopreserved.

[0020] In an embodiment, hPSCs are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

[0021] It is also an object of the invention, an isolated cell population of in vitro differentiated hindbrain neuron cells, wherein the in vitro differentiated hindbrain neuron cells are obtained according to the method of the invention for use in preventing and / or treating and / or diagnosis of neurological disorder. Said neurological disorder is selected from the group of neurodevelopmental, neuroimmunological, neuropsychiatric and / or neurodegenerative disorder, such as Alzheimer’s disease, Parkinson’s disease, Olivopontocerebellar Atrophy, and IgLON5 encephalitis.

[0022] A composition comprising the cell population for use as indicated above is also an object of the invention.

[0023] In an embodiment, the above indicated composition further comprises a pharmaceutically acceptable carrier.

[0024] It is also an object of the invention a kit for performing the method as herein disclosed, comprising: i. at least one inhibitor of SMAD signalling; ii. at least one activator of Wnt signalling; iii. at least one inhibitor of Notch signalling; and optionally further comprising: iv. instructions for inducing differentiation of human pluripotent stem cells into a population of differentiated cells expressing at least one hindbrain neuron marker.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be described by means of non-limiting examples, referring to the following figures:

[0027] Figure 1. (a) Schematic representation depicting a method of hindbrain neuron generation as disclosed in Materials and Methods section; differentiation protocol, (b) Bright-field images of cells after 5, 10, and 15 days from the beginning of the method, showing progressive and robust differentiation of hPSCs in hindbrain neurons. Abbreviations: VTN, vitronectin; PO, Poly-L- Omithine; L, Laminin; F, Fibronectin; CHIR, CHIR99021; LDN, LDN-193189; Y27, Y-27632 (ROCK inhibitor) DIV, days in vitro.

[0028] Figure 2. (a) Schematic representation of the hindbrain and HOX gene expression, (b) qRT-PCR data of CHIR99021 titration and anterior to posterior patterning of hindbrain progenitors. Abbreviations: HOX, Homeobox; Rh, Rhombomere; OTX2, Orthodenticle homeobox 2; FOXG1, Forkhead box protein Gl; EN1, Homeobox protein engrailed-1; GBX2, Gastrulation Brain Homeobox 2.

[0029] Figure 3. (a) Schematic representation depicting the H0XA2 hPSC tdTomato reporter cell line, (b, c) Generation of a highly pure population of hindbrain progenitors of a H0XA2 identity ()80% H0XA2+) after 15 days upon treatment. Data represent two independent differentiations, (d) qRT- PCR data demonstrate the requirement of CHIR99021 to obtain a specific and robust induction of the hindbrain progenitor specific markers H0XA2, H0XB2, H0XB1, and H0XB4 by gene expression. Abbreviations: P2A, 2A peptide derived from the porcine teschovirus-1; CHIR, CFUR99021; H0XA2, Homeobox A2; tdT, tandem dimer Tomato; XAV, XAV939.

[0030] Figure 4. Hindbrain neurons maintain robust tdTomato (H0XA2) expression throughout culture and neuronal maturation (Days in vitro [DIV]17, 23, and 38).

[0031] Figure 5. (a) qRT-PCR data show the absence of forebrain (PAX6) and non-CNS specific markers (SOXIO, T, and SOX17) at 40 days of culture, (b) qRT-PCR data show the stable and robust expression of hindbrain neuron specific markers at 40 days of differentiation and neuronal culture, (c) Immunofluorescence images show that Ki67+ progenitor cells are depleted and a pure population of MAP2+ neurons, enriched for tdTomato+ (HOXA2) cells, are generated and maintained by day 40 of differentiation. Abbreviations: PAX6, Paired box protein; SOXIO, SRY- box transcription factor 10; T, BRACHYURY; SOX17, SRY-box transcription factor 17; MAP2, Microtubule-associated protein 2: DIV, Days in vitro'. XAV, XAV939; CHIR, CHIR99021; HOX, Homeobox.

[0032] For inhibitor of Small Mothers Against Decapentaplegic protein signalling (SMAD inhibitor), it is intended at least one inhibitor of TGFp / Activin-Nodal signalling (such as SB431542) and / or at least one inhibitor of Bone Morphogenic Proteins (BMP) signalling (such as LDN-193189). The dual-SMAD inhibition is required to prevent endodermal and mesodermal patterning, which leads to ectodermal differentiation.

[0033] Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor beta (TGFP)ZActivin-Nodal signalling (referred to as “TGFp / Activin-Nodal inhibitor”), and inhibitors of bone morphogenetic proteins (BMP) signalling. In certain embodiments, the TGFp / Activin- Nodal inhibitor can neutralize the ligands including TGFPs, BMPs, Nodal, and activins, and / or block their signal pathways through blocking the receptors and downstream effectors. Nonlimiting examples of TGFp / Activin-Nodal inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul l;30(7):715-20 (2012), all of which are incorporated by reference in their entireties herein for all purposes. In certain embodiments, the at least one TGFp / Activin-Nodal inhibitor is selected from inhibitors of ALK5, inhibitors of ALK4, inhibitors of ALK7, and combinations thereof. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGFp / Activin-Nodal inhibitor is a small molecule selected from SB431542, derivatives thereof, and mixtures thereof. “SB431542” refers to a molecule with a number CAS 301836-41-9, a molecular formula of C22H18N4O3, and a name of 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamide, for example, see structure below:

[0034] In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01 (CAS No. 909910-43-6). In a preferred embodiment it comprises SB431542. SB and SB431542 are herein used interchangeably.

[0035] In certain embodiments, the at least one SMAD inhibitor comprises an inhibitor of BMP signalling (referred to as BMP inhibitor). Non-limiting examples of BMP inhibitors include those disclosed in WO2011 / 149762, Chambers et al, Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks etal, Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul l;30(7):715-20, all of which are incorporated by reference in their entireties. In certain embodiments, the BMP inhibitor is a small molecule selected from LDN193189, dorsomorphin (CAS No 866405-64-3), derivatives thereof, and mixtures thereof. LDN193189 (CAS No, 1062368-24-4) refers to a small molecule DM-3189, IUPAC name 4- (6-(4-(piperazin-l-yl)phenyl)pyrazolo[l,5-a]pyrimidin-3- yl)quinoline, with a chemical formula of C25H22N6 with the following formula:

[0036] LDN193189 is capable of functioning as a SMAD signalling inhibitor. LDN193189 is also highly potent small-molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinases (PTK), inhibiting signalling of members of the ALK1 and ALK3 families of type I TGF0 receptors, resulting in the inhibition of the transmission of multiple biological signals, including the bone morphogenetic proteins (BMP) BMP2, BMP4, BMP6, BMP7, and Activin cytokine signals and subsequently SMAD phosphorylation of Smadl, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14: 1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18: 4388-4392, herein incorporated by reference).

[0037] Noggin is a 32 kDa glycoprotein secreted by Spemann organizer of Xenopus embryos, and is found to rescue dorsal development in the ultraviolet-induced ventralized embryos. Noggin binds to BMP-2 and BMP -4 with high affinity and to BMP-7 with low affinity, and prevent BMPs from binding to its receptors. Human noggin is a 205 amino acid glycoprotein and is secreted as a covalently linked homodimer of approximately 32kDa with a similar structure to BMP-7. As used herein, the expression “Noggin” refers to the isolated protein and derivatives thereof, including chimeric noggin molecules comprising at least a portion of noggin, such as those described in W02007 / 028212.

[0038] In certain embodiments, the BMP inhibitor comprises LDN193189. In certain embodiments, the BMP inhibitor comprises Noggin. In a preferred embodiment it comprises LDN193189.

[0039] In certain embodiments, the at least one SMAD inhibitor comprises a TGFp / Activin-Nodal inhibitor. In certain embodiments, the one TGFp / Activin- Nodal inhibitor is SB431542 or A83- 01. In certain embodiments, the stem cells are exposed to two SMAD inhibitors (dual SMAD inhibitors). In certain embodiments, the two SMAD inhibitors are a TGFp / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In a preferred embodiment, the hPSCs are exposed to SB431542 and LDN193189. Preferably, SB431542 is used in an amount of about 10 pM; and LDN193189 is used in an amount of about 100 nM. In an embodiment, hPSCs are exposed to SB431542 and LDN193189 for at least about 8 days from DIV 3 to DIV 11, or for at least about 9 days from DIV 2 to DIV 11. Preferably, hPSCs are exposed to SB431542 and LDN193189 for about 9 days from DIV 2 to DIV 11.

[0040] For inhibitor of Notch signalling it is intended at least one y-secretase inhibitor that inhibits Notch. Non-limiting examples of Notch inhibitors include DAPT (Dovey et al., Journal of neurochemistry 76, 173-181 (2001)), Begacestat (5-Chloro-N-[(lS)-3,3,3-trifhroro-l- (hydroxymethyl)-2- (trifluoromethyl)propyl]-2-thiophenesulfonamide) (Mayer et al., J.Med.Chem. 51 :7348 (2008)), DBZ (N-[(lS)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H- dibenz[b,d]azepin-7-yl]amino]-l- methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide) (van Es et al., Nature 435:959 (2005)), BMS 299897 (2-[(lR)-l-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5- fluorobenzenebutanoic acid] (Goldstein et al., J.Pharmacol.Exp.Ther. 323: 102 (2007)), Compound W (3,5-Bis(4- nitrophenoxy)benzoic acid) (Okochi et al., J.Biol.Chem. 281 :7890 (2006)), Flurizan ((R)-2-Fluoro-a-methyl[l,r-biphenyl]-4-acetic acid) (Eriksen et al., J. Clin. Invest. 112:440 (2003)), L-685,458 ((5S)-(tert-Butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)- benzylhexanoyl)-L-leucy-L-phenylalaninamide) (Shearman et al., Biochemistry 39:8698 (2000)), JLK 6 (7 -Amino-4-chl oro-3 -methoxy-lH-2 -benzopyran) (Petit et al., Nat.Cell.Biol. 3:507 (2001)), MRK 560 (N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5- difluorophenyl)cyclohexyl]-l,l,l- trifluoromethanesulfonamide) (Best et al., J.Pharm.Exp.Ther. 317:786 (2006)), PF 3084014 hydrobromide ((2S)-2-[[(2S)-6,8- Difluoro-l,2,3,4-tetrahydro-2-naphthalenyl]amino]-N-[l-[2- [(2,2- dimethylpropyl)amino]-l,l-dimethylethyl]-lH-imidazol-4-yl]pentanamide dihydrobromide) (Lanz et al., J.Pharmacol.Exp.Ther. 334:269 (2010)), or derivatives thereof.

[0041] In certain embodiments, the term DAPT refers to one example of a y-secretase inhibitor that inhibits NOTCH, which is described as a dipeptidic y-secretase-specific inhibitor otherwise known as N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine- 1, 1-dimethyl ethyl ester; LY- 374973, N — [N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester; with a chemical formula of C 23H26F2N2O4. One example of a DAPT derivative is DAP-BpB (N — [N- (3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8-bioti-namido)octylamino)benzoyl) benzyl)methylamide), a photoactivable DAPT derivative. In certain embodiments, DAPT has the following structure:

[0042] In certain embodiments, the at least one Notch inhibitor is selected from DAPT, BMS 299897, Compound E (y-Secretase Inhibitor XXI, CAS 209986-17-4), Compound W (3,5-Bis(4- nitrophenoxy)benzoic acid), DBZ (N-[(lS)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H- dibenz[b,d]azepin-7-yl]amino]-l-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide), L-685,458 (292632-98-5), PF 3084014 hydrobromide (CAS No. 1290543-63-3), derivatives thereof, and combinations thereof. Preferably, the Notch inhibitor is DAPT. In an exemplary embodiment, DAPT is used from DIV15 onwards, for at least about 10 days, preferably for at least about 12 days; to ensure that hindbrain neural stem cells (NSCs) exit cell cycle and become post-mitotic hindbrain neurons. In an embodiment it is used at a concentration of about 10 pM.

[0043] According to the invention, the initial contact or exposure of the cells with or to the Notch inhibitor is at least about 10 days, at least about 15 days, or at least about 20 days from the initial contact or exposure of the cells with or to the at least one SMAD inhibitor. In certain embodiments, the initial contact or exposure of the cells with or to the at least one Notch inhibitor is no later than about 15 days, no later than about 20 days, or no later than about 25 days from initial exposure of the stem cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or exposure of the cells with or to the at least one Notch inhibitor is between about 10 days and about 25 days, between about 10 days and about 20 days, between about 10 days and about 15 days, or between about 15 days and about 20 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells to the at least one Notch inhibitor is about 15 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0044] For a WNT or wingless signalling pathway it is intended a signalling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRP Derailed / RYK receptors, mediated with or without P-catenin. In certain embodiments, the WNT signalling pathway include mediation by P-catenin, e.g., WNT / P-catenin.

[0045] For activator of wingless (Wnt) signalling it is intended molecules that bind Wnt proteins and activate the transduction of cellular signals that facilitate the expression of genes involved in cell proliferation, survival, differentiation, and migration. As used herein, a Wnt signalling pathway activator can be interchanged with a “Wnt signalling pathway agonist,” “Wnt agonist,” “Wnt pathway activator,” or “Wnt activator”. In some embodiment the Wnt signalling activator is selected from BIO-Acetoxime (6Bromoindirubin3’ acetoxime, Meijer L et al. Chem Biol 10(12): 1255-66.); TWS119 (3[[6(3aminophenyl)7Hpyrrolo[2,3d]pyrimidin4yl]oxy]phenol, Ding S et al. Proc Natl Acad Sci USA 100(13): 7632-7; SB216763 (3-(2,4-Dichlorophenyl)-4-(l-methyl-lH- indol-3-yl)-lH-pyrrole-2, 5-dione, Coghlan MP et al. Chem Biol 7(10): 793-803.); CHIR98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(lH-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6- pyridinediamine, Guerrero F et al. PLoS One 9(2): e89179); IQ-1 (2-[2-(4-acetylphenyl)diazenyl]- 2-(3,4-dihydro-3,3-dimethyl-l(2H)-isoquinolinylidene)-acetamide, Miyabayashi T et al. Proc Natl Acad Sci USA 104(13): 5668-73.); BIO, (2'Z,3'E)-6-Bromoindirubin-3 '-oxime; XAV939 (2-[4- (trifluoromethyl)phenyl]-l,5,7,8-tetrahydrothiopyrano[4,3-d]pyrimidin-4-one, Huang S-MA et al. Nature 461(7264): 614-20); IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo- 3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide, Berge D et al. Nat Cell Biol 13(9): 1070- 5), and CHIR99021 6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2- yl)amino)ethyl) amino)nicotinonitrile (CAS No. : 252917-06-9).

[0046] In certain embodiments, the at least one Wnt activator is selected from BIO-Acetoxime, TWS119, SB216763, CHIR98014, IQ-1, BIO, XAV939, IWP-2, CHIR99021, derivatives thereof, and combinations thereof. Preferably, the WNT activator is CHIR99021. In an embodiment CHIR99021 is used at a concentration of about 1.5 pM to 3.5 pM, preferably at a concentration of 3pM, and preferably for at least about 2 days from DIV 0 to DIV 2, to ensure correct patterning along the anterior to posterior axis of the developing neurectoderm.

[0047] For an inhibitor of the ROCK (Rho Kinase) signalling it is intended a molecule that targets the Rho kinase (ROCK) and inhibits the ROCK pathway.

[0048] For Rho kinase (or Rho-associated kinase) it is intended a kinase belonging to the AGC (PKA / PKG / PKC) family of serine-threonine specific protein kinases. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton.

[0049] In some embodiment the ROCK signalling inhibitor is selected from Thiazovivin (N- (phenylmethyl)-2-(4-pyrimidinylamino)-4-thiazolecarboxamide, Li D et al Stem Cell Reports. 2016 May 10;6(5):717-28.); HA-1077 (Fasudil and Hydroxyfasudil) (5-((l,4-diazepan-l- yl)sulfonyl)isoquinoline hydrochloride, Shi J et al J Cardiovasc Pharmacol. 2013 Oct;62(4):341- 54.; AT-13148 ((S)-l-(4-(lH-pyrazol-4-yl)phenyl)-2-amino-l-(4-chlorophenyl)ethanol, Yap TA et al, Clin Cancer Res. 2012 Jul 15; 18(14):3912-23.; P-Elemene (P-Elemene, (lS,2S,4R)-(-)-2,4- Diisopropenyl- 1 -methyl- 1 -vinylcyclohexane, ( 1 S,2S,4R)- 1 -Ethenyl- 1 -methyl-2,4-bis( 1 - methylethenyl)cyclohexane, Zhai et al Int J Nanomedicine. 2018; 13: 6279-6296); Belumosudil (2-[3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(l-methylethyl)-acetamide, Lee JH et al Ann Clin Transl Neurol. 2014 Jan 1; 1(1):2-14); Chroman 1 ((3S)-N-[2-[2- (dimethylamino)ethoxy]-4-(lH-pyrazol-4-yl)phenyl]-6-methoxy-3,4-dihydro-2H-chromene-3- carboxamide, (Chen et al Nat Methods. 2021 May; 18(5): 528-541); DJ4 ((2E,5Z)-5-((3a,7a- dihydro-lH-pyrrolo[2,3-b]pyridin-3-yl)methylene)-2-(phenethylimino)thiazolidin-4-one, Kale VP Cancer Lett. 2014 Nov 28; 354(2): 299-310.); GSK-576371 (Phrommintikul A, et al Am. J. Physiol. Heart Circ. Physiol. 2008;294(4):H1804-H1814); GSK429286A (N-(6-fluoro-lH- indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-l,4,5,6-tetrahydropyridine-3- carboxamide, Nichols RJ et al Biochem J. 2009 Oct 23;424(l):47-60.); H-1152 (4-Methyl-5- [[(2S)-2-methyl-l,4-diazepan-l-yl]sulfonyl]isoquinoline, Tamura et al Biochim Biophys Acta. 2005 Dec 30;1754(l-2):245-52); LX-7101 (3-(4-(aminomethyl)-l-(5-methyl-7H-pyrrolo[2,3- d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate, Harrison et al ACS Med Chem Lett. 2014 Nov 24;6(l):84-8); Netarsudil (AR-11324, Benzoic acid, 2,4-dimethyl-, (4-((lS)- l-(aminomethyl)-2-(6-isoquinolinylamino)-2-oxoethyl)phenyl)methyl ester, Sturdivant et al Bioorg Med Chem Lett. 2016 May 15;26(10):2475-2480); RKI-1447 (l-(3-Hydroxybenzyl)-3-(4- (pyridin-4-yl)thiazol-2-yl)urea, Patel et al Cancer Res. 2012 Oct l;72(19):5025-34); TC-S 7001 (6-Chloro-N4-[3,5-difluoro-4-[(3-methyl-lH-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl]-2,4- pyrimidinediamine, Kast R et al British Journal of Pharmacology. 152 (7): 1070-80); Verosudil (AR- 122862-(dimethylamino)-N-(l -oxo- 1 ,2-dihydroisoquinolin-6-yl)-2-(thi ophen-3 - yl)acetamide, Skaat A et al J Glaucoma. 2016 Sep;25(9):e807-14); Y-30141 ((lR,4r)-4-((R)-l- aminoethyl)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)cyclohexane-l-carboxamide, Ishizaki T et al Mol Pharmacol. 2000 May;57(5):976-83); Y-33075 ((R)-4-(l-aminoethyl)-N-(lH-pyrrolo[2,3- b]pyridin-4-yl)benzamide, Gao C et al Neuroimmunomodulation. 2013;20(6):334-40); Y-39983 ((R)-4-(l-aminoethyl)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)benzamide Yang Z et al Oncol Rep. 2013 Mar;29(3): 1140-6). Preferably, Y-27632 ((lR,4R)-4-((R)-l-aminoethyl)-N-(pyridin-4- yl)cyclohexanecarboxamide, Qian et al Methods Mol Biol. 2018 Feb 25) is used to prevent cell death when cells are at a single-cell stage (for seeding or replating).

[0050] In some embodiments the cells are contacted with medium supplemented with Y-27632 at a concentration of 10 pM. In an embodiment, cells are exposed to Y-27632 at the seeding and / or replating.

[0051] For derivative it is intended a chemical compound with a similar core structure, a salt, solvate, isomer, tautomer thereof.

[0052] For combination it is intended that the respective described substances are paired or present in a specific physical form. The term mixture is alternatively used throughout the invention with the same meaning of combination.

[0053] For a cell population it is intended a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population may be a pure population comprising one cell type, such as a population of hindbrain neurons, or a population of undifferentiated stem cells. Alternatively, the population may comprise more than one cell type, for example a mixed cell population.

[0054] For human pluripotent stem cell or hPSC it is intended cells that possess the capacity to self-renew and to differentiate into all cell types of the adult body.

[0055] For human embryonic stem cell or hESC it is intended a type of pluripotent stem cells derived from early stage human embryos, up to and including the blastocyst stage, that is capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

[0056] For induced pluripotent stem cell or iPSC it is intended a type of pluripotent stem cell formed by the introduction of certain embryonic genes (such as but not limited to OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference) into a somatic cell.

[0057] For differentiation it is intended the process during which pluripotent or multipotent cells take on individual characteristics and reach their mature (specialized) form and function.

[0058] For cell culture it is intended to a growth of cells in vitro in an artificial medium for research or medical treatment.

[0059] For culture medium it is intended a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.

[0060] In some embodiments cells are contacted with various solutions and material to promote cell dissociation and / or cell adherence to the plastic of the plates and / or to assure cell growth. A non- exhaustive list of materials and solutions includes: Accutase (Innovative Cell Technologies #AT104), Geltrex-coated plastic plates (Thermo Fisher #A1413202), E6 medium, Stem Cell banker (Amsbio #11924), DMEM / F12, N2 supplement (Thermo Fisher Scientific, #17502-048), B27 without vitamin A (Thermo Fisher Scientific #12587010), penicillin / streptomycin (Thermo Fisher Scientific #15-140-122), L-glutamine (Thermo Fisher Scientific, #25030081), dibutyryl cAMP (Sigma-Aldrich, #D0627), DAPT (Tocris, #2634), ascorbic acid (Sigma-Aldrich, #A4034), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF). For in vitro it is intended an artificial environment and / or processes and / or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

[0061] For marker or cell marker it is intended a gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker, markers may refer to a “pattern” of markers such that a designated group of markers may identity a cell or cell type from another cell or cell type.

[0062] For hindbrain it is intended one of the three major regions of our brains, located at the lower back part of the brain. It connects the brain to the spinal cord and coordinates many vital functions, such as breathing and heartbeat, motor control, and sensory processing.

[0063] For hindbrain neuron it is intended the functional and structural unit of the hindbrain.

[0064] For hindbrain neural stem cell, it is intended a type of neural stem cell derived from the hindbrain region of the human fetal brain. In the context of the present invention, neural stem cell progenitor is the synonym.

[0065] For neural stem cell it is intended a unique cell with the capacity to self-renew and differentiate into various cell types found in the nervous system, such as neurons, including hindbrain neurons, astrocytes, and oligodendrocytes.

[0066] The non-limiting examples of hindbrain markers include: GBX2, KROX20, H0XA1, H0XA2, H0XA3, H0XA4, H0XB1, H0XB2, H0XB3 and H0XB4. Single-cell RNA sequencing (scRNA-seq) performed on hindbrain neurons throughout the first 20 days of differentiation (compared to cortical neurons) demonstrated that all these markers are expressed, although at varying levels.

[0067] In an embodiment the hindbrain NSCs are marked by the presence of the following markers: H0XA2, HOXB2, HOXB1, HOXB4, MKI67, and IRX3.

[0068] In an embodiment the hindbrain NSCs are marked by the absence of FOXG1.

[0069] In an embodiment the hindbrain neurons are marked by the presence of the following markers: MAP2, H0XA2, HOXB2, HOXB4, ASCL1, LMX1A, LMX1B, TLX1, TLX3, DRG11, GBX2 and KROX20.

[0070] In an embodiment the hindbrain neurons are marked by the absence of any of forebrain and midbrain markers: FOXG1, EMX1, OTX2, NKX2.1, SIX3, EN1, SIM1 and LIM1; non-CNS specific markers: SOXIO, T, and SOX17; and MKI67. Preferably, by the absence of any of markers selected from the group consisting of MKI67, SRY-box transcription factor 10 (SOXIO), BRACHYURY (T), SRY-box transcription factor 17 (SOX17), FOXG1, EMX1, OTX2, and combinations thereof.

[0071] For Gastrulation Brain Homeobox 2 (GBX2) it is intended a transcription factor for cell pluripotency and differentiation in the embryo. GBX2, accession no: Uniprot P52951 For Early growth response protein 2 (EGR2; also known as KROX20) it is intended a sequencespecific DNA-binding transcription factor. It plays a role in hindbrain segmentation by regulating the expression of a subset of homeobox containing genes. It binds to two Early Growth Response 2 (EGR2)-consensus sites EGR2A (5'-CTGTAGGAG-3') and EGR2B (5'-ATGTAGGTG-3') in the HOXB3 enhancer and promotes HOXB3 transcriptional activation. It also binds to specific DNA sites located in the promoter region of H0XA4, HOXB2 and ERBB2. It regulates hindbrain segmentation by controlling the expression of Hox genes, such as H0XA4, HOXB3 and HOXB2, and promotes the expression of HOXB3 in the hindbrain. KROX20 accession no: Uniprot Pl 1161. For Homeobox Al (HOXA1) it is intended a sequence-specific transcription factor. It regulates multiple developmental processes including brainstem, inner and outer ear, abducens nerve and cardiovascular development and morphogenesis as well as cognition and behavior. Also, it is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. It acts on the anterior body structures. H0XA1, accession no: Uniprot P49639.

[0072] For Homeobox A2 (HOXA2) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. The protein encoded by H0XA2 may be involved in the placement of hindbrain segments in the proper location along the anterior-posterior axis during development. H0XA2 accession no: Uniprot 043364.

[0073] For Homeobox A3 (H0XA3) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. H0XA3 accession no: Uniprot 043365.

[0074] For Homeobox A4 (H0XA4) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. H0XA4, accession no: Uniprot Q00056. For Homeobox Bl (HOXB1) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis, and acts on the anterior body structures. H0XB1 accession number: Uniprot P14653.

[0075] For Homeobox B2 (H0XB2) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. H0XB2, accession number: UniProt P0C1T1.

[0076] For Homeobox B3 (H0XB3) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. H0XB3, accession no: UniProt P14651.

[0077] For Homeobox B4 (H0XB4) it is intended a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. H0XB4: accession number Uniprot P17483.

[0078] For Marker of proliferation Kiel 67 (MKI67) it is intended a protein that plays a role in genome maintenance by maintaining individual mitotic chromosomes dispersed in the cytoplasm following nuclear envelope disassembly. It prevents chromosomes from collapsing into a single chromatin mass by forming a steric and electrostatic charge barrier: the protein has a high net electrical charge and acts as a surfactant, dispersing chromosomes and enabling independent chromosome motility. In the context of the present invention, Antigen Kiel 67 and Ki-67 are the synonyms. MKI67, accession no: Uniprot P46013.

[0079] For Iroquois homeobox 3 (IRX3) it is intended a transcription factor involved in Sonic hedgehog (SHH)-dependent neural patterning. Together with NKX2-2 and NKX6-1 acts to restrict the generation of, among others, motor neurons to the appropriate region of the neural tube. Belongs to the class I proteins of neuronal progenitor factors, which are repressed by SHH signals. Involved in the transcriptional repression of MNX1 in non-motor neuron cells. Acts as a regulator of energy metabolism. IRX3, accession no: Uniprot P78415.

[0080] For Microtubule-associated protein 2 (MAP2) it is intended a marker of neural growth, axonal regeneration and synaptic plasticity. MAP2, accession number: Uniprot Pl 1137.

[0081] For Achaete-scute homolog 1 (ASCL1) it is intended a transcription factor that plays a key role in neuronal differentiation: acts as a pioneer transcription factor, accessing closed chromatin to allow other factors to bind and activate neural pathways. It plays a role at early stages of development of specific neural lineages in most regions of the Central Nervous System (CNS) , and of several lineages in the Peripheral Nervous System (PNS). It is essential for the generation of olfactory and autonomic neurons. ASCL1, accession no: P50553.

[0082] For Homeobox transcription factor 1, alpha (LMX1A) is intended a transcriptional activator for specification of dorsal cell fates in the Central Nervous System and developing vertebrae. LMX1 A accession no: Uniprot Q8TE12.

[0083] For Homeobox transcription factor 1, beta LMX1B is intended a transcription factor essential for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. LMX1B accession no: Uniprot 060663

[0084] For T-cell leukemia homeobox 1 (TLX1) it is intended a member of the divergent T-cell leukemia translocation (H0X1 l / Tlx) homeobox gene family. It is expressed, together with TLX3, in differentiating neurons of both the peripheral and central nervous systems. TLX1, accession no: Uniprot 31314.

[0085] For T-cell leukemia homeobox 3 (TLX3) it is intended a member of the divergent T-cell leukemia translocation (H0X1 l / Tlx) homeobox gene family, and it is expressed together with TLX1, in differentiating neurons of both the peripheral and central nervous systems. TLX3, accession no: 043711.

[0086] For Dorsal Root Ganglion Homeobox (DRGX or DRG11) it is intended a paired homeodomain transcription factor expressed in both the developing dorsal horn and in sensory neurons, but not in the ventral spinal cord. DRG11, accession no: Q6NW40.

[0087] For Forkhead box G1 (F0XG1) is intended a ttranscription repression factor which plays an important role in the establishment of the regional subdivision of the developing brain. F0XG1, accession no: P55316.

[0088] For Empty spiracles homeobox 1 (EMX1) is intended a transcription factor, which in cooperation with EMX2, acts to generate the boundary between the roof and archipallium in the developing brain. May function in combinations with 0TX1 / 2 to specify cell fates in the developing central nervous system. EMX1, accession no: Uniprot Q04741.

[0089] For Orthodenticle homeobox 2 (0TX2) is intended a transcription factor involved in the development of the brain and the sense organs. 0TX2, accession no: Uniprot P32243. For NK2 homeobox 1 (NKX2.1) is intended a transcription factor that binds and activates the thyroglobulin promoter and regulates the expression of thyroid-specific genes but has also been shown to regulate the expression of genes involved in morphogenesis. It may play a role in lung development and surfactant homeostasis. It forms a regulatory loop with Grainyhead Like Transcription Factor 2 (GRHL2) that coordinates lung epithelial cell morphogenesis and differentiation. It activates the transcription of Gonadotropin-releasing hormone receptor (GNRHR) and plays a role in enhancing the circadian oscillation of its gene expression. It represses the transcription of the circadian transcriptional repressor Nuclear receptor subfamily 1, group D, member 1 (NR1D1). NKX2.1 accession no: Uniprot P43699.

[0090] For SIX homeobox 3 (SIX3) is intended a transcriptional regulator which can act as both a transcriptional repressor and activator by binding an ATTA homeodomain core recognition sequence on these target genes. It plays a role in eye development by suppressing WNT1 expression and in dorsal -ventral patterning by repressing BMP signalling pathway. SIX3 accession no: Uniprot 095343.

[0091] For Homeobox protein engrailed-1 (EN1) is intended a transcription factor required for proper formation of the apical ectodermal ridge and correct dorsal -ventral patterning in the limb. EN1 accession no: Uniprot Q05925.

[0092] For single-minded family bHLH transcription factor 1 (SIM1) is intended a transcription factor expressed in the developing kidney and central nervous system and is essential for formation of the hypothalamus. SIM1 accession no: P81133.

[0093] For LIM homeobox 1 (LIM1 or LHX1) is intended a transcription factor that plays a role in early mesoderm formation and later in lateral mesoderm differentiation and neurogenesis. LIM1 accession no: P48742.

[0094] For SRY-box transcription factor 10 (SOXIO) is intended a transcription factor that plays a central role in developing and mature glia. Specifically, it activates expression of myelin genes, during oligodendrocyte (OL) maturation. SOXIO accession no: P56693.

[0095] For BRACHYURY (T or TBXT), it is intended a transcription factor involved in the transcriptional regulation of genes required for mesoderm formation and differentiation. T accession no: Uniprot 015168.

[0096] For SRY-box transcription factor 17 (SOX17) is intended a transcription regulator that binds target promoter DNA and bends the DNA. It inhibits Wnt signalling, and plays a key role in the regulation of embryonic development. It is also required for normal development of the definitive gut endoderm, and for normal looping of the embryonic heart tube. In addition, it plays an important role in embryonic and postnatal vascular development, including development of arteries, and also in postnatal angiogenesis, where it is functionally redundant with SOX18. It is required for the generation and maintenance of fetal hematopoietic stem cells, and for fetal hematopoiesis. SOX17 accession no: Q9H6I2.

[0097] In an embodiment, according to the method of the invention, an obtained cell population of in vitro differentiated hindbrain neuron cells or a composition comprising thereof, is for use in preventing and / or treating and / or diagnosis of neurological disorder, said neurological disorder is being selected in the group of neurodevelopmental, neuroimmunological, neuropsychiatric and / or neurodegenerative disorder, such as Alzheimer’s disease, Parkinson’s disease, Olivopontocerebellar Atrophy, and IgLON5 encephalitis.

[0098] In an embodiment of the invention, the hindbrain neuronsobtained by the method of the invention or a composition comprising thereof can be administered to a subject suffering from a neurodegenerative disorder, a neurodevelopmental disorder, and / or a neuropsychiatric disorder. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0099] For composition it is also intended the cell culture comprising cell population of hindbrain neurons, or graft of hPSC-derived hindbrain neurons. In an exemplary embodiment, a cell population of hindbrain neurons and compositions thereof are used in diagnostic screening and in the prevention and / or treatment of neurological disorders; i.e.

[0100] - Diagnostic screening: used as a cell-based assay to screen for auto antigens that target the hindbrain in the context of autoimmune encephalitis and / or other neurological disorders and therefore to identify affected patients.

[0101] Treatment / prevention: cell replacement therapy for neurological disorders in which there is a degeneration on the hindbrain.

[0102] The stem-cell derived hindbrain neurons obtained by the method of the invention or compositions can be administered or provided systemically or directly to a subject for treating or preventing a neurodegenerative disorder, a neurodevelopmental disorder, and / or a neuropsychiatric disorder. In certain embodiments, the cells or compositions are directly injected into an organ of interest (e.g., the central nervous system (CNS)). In certain embodiments, the cells or compositions are directly injected into the hindbrain.

[0103] The stem-cell derived hindbrain neurons obtained by the method of the invention or compositions can be administered in any physiologically acceptable vehicle. The cells or compositions can be administered via localized intracranial injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the cells or compositions are administered to a subject suffering from a neurodegenerative disorder via intracranial injection, e.g., localized intracranial injection into the CNS.

[0104] The stem-cell derived hindbrain neurons obtained by the method of the invention or compositions can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising the presently disclosed stem-cell -derived hindbrain neurons, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g, methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0105] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum inurn monostearate and gelatin. Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g ., liquid dosage form (e.g, whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid- filled form).

[0106] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the presently disclosed stemcell-derived precursors. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0107] One consideration concerning the therapeutic use of the cells is the quantity of cells necessary to achieve an optimal effect. An optimal effect includes, but is not limited to, repopulation of CNS regions, specifically the hindbrain, of a subject suffering from a neurodegenerative disorder, and / or improved function of the subject’s CNS.

[0108] In certain embodiments, the composition comprises an effective amount of the stem-cell derived hindbrain neurons obtained by the method of the invention. As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in at least one doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the neurodegenerative disorder or neuroimmunological disorder, or otherwise reduce the pathological consequences of the neurodegenerative disorder. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

[0109] In certain embodiments, an effective amount of the cells is an amount that is sufficient to repopulate CNS regions of a subject suffering from a neurodegenerative disorder, a neurodevelopmental disorder, and / or a neuropsychiatric disorder. In certain embodiments, an effective amount of the cells is an amount that is sufficient to improve the function of the CNS of a subject suffering from a neurodegenerative disorder, a neurodevelopmental disorder, and / or a neuropsychiatric disorder, e.g., the improved function can be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or about 100% of the function of a normal person’s CNS.

[0110] The quantity of cells to be administered will vary for the subject being treated. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0111] The invention also provides a culture medium that is capable of promoting differentiation of hPSCs into hindbrain neurons, said culture medium comprising:

[0112] (a) a neural basal medium;

[0113] (b) at least one SMAD inhibitor;

[0114] (c) at least one WNT activator;

[0115] (d) at least one inhibitor of Notch signalling.

[0116] The invention also provides a kit comprising the above culture medium and optionally further comprising instructions for inducing differentiation of human pluripotent stem cells into a population of differentiated cells expressing at least one hindbrain neuron marker.

[0117] EXAMPLES

[0118] Materials and methods

[0119] Differentiation protocol The differentiation of hESCs or iPSCs (referred to jointly as hPSCs) into hindbrain neurons was induced by dual-SMAD inhibition (Chambers et al., 20092), with correct anterior-to-posterior patterning achieved by activation of the Wnt pathway. Briefly, hPSCs were dissociated into a single-cell suspension with Accutase (Innovative Cell Technologies #AT104) and seeded on Geltrex (Thermo Fisher #A1413202)-coated plastic plates at a density of 300,000 cells / cm2 in Essential 8 medium (E8 medium) supplemented with 10 M ROCK inhibitor (Y-27632; 10 pM; R&D Systems #1254 / 10). The cells were then transferred to neural induction medium for 11 days. This medium consisted of Essential 6 medium (E6 medium), (Stem Cell banker; Amsbio #11924), supplemented with LDN193189 (100 nM; Reprocell #04-0074) and SB431542 (10 pM; R&D Systems #1614 / 50), with the addition of CHIR99021 (3 pM; Tocris Bioscience #4423) for the first two days. After 11 days, this medium was replaced by neural differentiation medium consisting of 1 : 1 DMEM / F12 and Neurobasal, 1 x N2 supplement (Thermo Fisher Scientific, #17502-048), 1 x B27 without vitamin A (Thermo Fisher Scientific #12587010), and 1xpenicillin / streptomycin (Thermo Fisher Scientific #15-140-122). The cells were incubated in this neural differentiation medium for four days, and hindbrain neural progenitors were then either cryopreserved in Stem Cellbanker (Amsbio #11924) or replated onto poly-omithine / laminin / fibronectin plates at a density of 2 x 10A5 cells / cm2 in maturation medium consisting of Neurobasal medium, 1 x B27 without vitamin A, 1 x penicillin / streptomycin, 2 mM L-glutamine (Thermo Fisher Scientific, #25030081), 200 pM dibutyryl cAMP (Sigma-Aldrich, #D0627), 10 pM DAPT (Tocris, #2634), 200 pM ascorbic acid (Sigma-Aldrich, #A4034), 20 ng / mL glial cell line-derived neurotrophic factor (GDNF), and 20 ng / mL brain-derived neurotrophic factor (BDNF). We added 10 pM Y- 27632 at replating. The medium was replaced entirely on day 16, followed by half medium changes every five days until day 40, when the hindbrain neurons were used for experiments. Hindbrain neurons however can be kept in the culture for prolonged periods of time if required. The brief representation of differentiation protocol is shown in Fig. la.

[0120] Cell culture hPSCs were maintained on vitronectin (Thermo Fisher Scientific) at 37°C in 5% CO2 with Essential 8 medium (E8) and passaged once a week with EDTA. The detailed hPSCs culture protocol is herein reported as follows (from day -3 to day 26 and onwards):

[0121] Day -3

[0122] Thawing of Geltrex (Thermo Fisher #A1413202) over night on ice at 4°C . Day -2

[0123] 1. Coating of 6 well plates with Geltrex (1 :40 in DMEM F-12, over night at 4°C), by using 1 mL / well for 6 well.

[0124] Day -1

[0125] 1. Placing Geltrex-coated 6 well plates for at least 1 hour in incubator at 37°C.

[0126] 2. Detaching the cells using Accutase (Innovative Cell Technologies #AT104) for 15 min at 37°C (5 mL / 10 cm dish).

[0127] 3. In the meantime, Geltrex is removed from the plate and 4 mL of E8 media + 10 uM Y- 27632 is added.

[0128] 4. Addition of the media to dilute Accutase (5 mL media in 10 cm dish).

[0129] 5. Counting the cells and centrifuging for 5 minutes at 200g.

[0130] 6. Plating 300,000 cells / cm2in E8 + 10 pM Y-27632.

[0131] Day 0

[0132] 1. The cells should look like a healthy monolayer (no holes to be observed); 100% confluent cells.

[0133] 2. Washing the cells with 2 mL of E6 media.

[0134] 3. Changing the media: 5 mL / well of E6 + 100 nM LDN, 10 uM SB, 3 pM CHIR.

[0135] Day 1

[0136] 1. Changing the media: 5 mL / well of E6 + 100 nM LDN, 10 pM SB, 3 pM CHIR.

[0137] Day 2 to 10

[0138] 1. Changing the media: 5 mL / well of E6 + 100 nM LDN, 10 pM SB.

[0139] Day 11

[0140] 1. Medium Switch by changing the media to: 6 mL / well of Neurobasal:DMEM / F12 1 : 1 supplemented with, 1 x penicillin / streptomycin (Thermo Fisher Scientific #15-140-122), 1 x N2 supplement (Thermo Fisher Scientific, #17502-048), 1 x B27 without vitamin A (Thermo Fisher Scientific #12587010).

[0141] Day 12 to 14

[0142] 1. Changing the media: 6mL / well Neurobasal:DMEM / F12 1 : 1 supplemented with, 1 x penicillin / streptomycin, 1 x N2 supplement, 1 x B27 without vitamin A.

[0143] 2. Preparation for replating at DAY 15. - On day 13: Poly-Ornithine (Sigma-Aldrich, P3655-100MG) (1 : 1000) is diluted in PBS and 96-well plate is coated (150 pL / well), 6-well plate (1 mL / well) at 37°C over night.

[0144] - On day 14: washing ornithine-coated plates 3 times with PBS, then coating the plates on top with Laminin (Sigma-Aldrich, L2020-1MG) (1 :500) and Fibronectin (Enzo Life Sciences, NOV-PT_74124-lmg)(l : 100) in PBS, over night at 37°C.

[0145] Day 15

[0146] 1. W ashing the cell s with PB S

[0147] 2. Adding 2 mL of Accutase and incubating at 37°C for 40 minutes.

[0148] 3. Adding 3 mL of Neurobasal medium to inhibit Accutase.

[0149] 4. Counting the cells and spinning them at 180g for 5 minutes at RT.

[0150] 5. Discarding the supernatant and resuspending the cells in Neurobasal, medium 1 x B27 without vitamin A, 1 x penicillin / streptomycin, 2 mM L-glutamine (Thermo Fisher Scientific, #25030081), 200 pM dibutyryl cAMP (Sigma-Aldrich, #D0627), 10 pM DAPT (Tocris, #2634), 200 pM ascorbic acid (Sigma-Aldrich, #A4034), 20 ng / mL glial cell line-derived neurotrophic factor (GDNF), and 20 ng / mL brain-derived neurotrophic factor (BDNF) and 10 pM Y-27632.

[0151] 6. Plating 200,000 cells / cm2on Poly-Ornithine / Laminin / Fibronectin coated plates.

[0152] Day 16

[0153] Changing entire medium with Neurobasal medium 1 x B27 without vitamin A, 1 x penicillin / streptomycin, 2 mM L-glutamine, 200 pM dibutyryl cAMP, 10 pM DAPT, 200 pM ascorbic acid, 20 ng / mL GDNF, and 20 ng / mL BDNF, to fully remove Y-27632.

[0154] Day 21

[0155] Changing one half of the media with Neurobasal medium 1 x B27 without vitamin A, 1 x penicillin / streptomycin, 2 mM L-glutamine, 200 pM dibutyryl cAMP, 10 pM DAPT, 200 pM ascorbic acid, 20 ng / mL GDNF, and 20 ng / mL BDNF.

[0156] Day 26 - onwards

[0157] Changing one half of the media with Neurobasal medium 1 x B27 without vitamin A, 1 x penicillin / streptomycin, 2 mM L-glutamine (Thermo Fisher Scientific, #25030081), 200 pM dibutyryl cAMP, 200 pM ascorbic acid, 20 ng / mL GDNF, and 20 ng / mL BDNF, every 5 days (from day 26 onwards DAPT is optional).

[0158] Immunofluorescence staining, imaging, and data analysis Cells were fixed with 4% paraformaldehyde (PF A) for 15 minutes at room temperature. Permeabilization and blocking step were done using 1% BSA (Sigma Aldrich), 5% normal donkey serum (Jackson Immunoresearch) and 0,3% Triton-X (Sigma Aldrich) in PBS for one hour at room temperature. Primary antibody incubation was performed overnight at 4°C in 1% BSA, 5% normal donkey serum and 0,1% Triton-X in PBS. Cells were washed three times in PBS and secondary antibody incubation was performed in antibody solution for 1 hour at room temperature. To visualize cell nuclei, cells were incubated with DAPI (ThermoFisher Scientific, 62248) for 10 minutes at room temperature. Samples were washed three times in PBS and mounted with Mowiol 4-88 + DABCO mountant (Sigma Aldrich). Acquisitions were done using spinning disk confocal microscope, that comprised a Nikon Ti2-E inverted microscope equipped with a CrestOptics V3 Light, which was coupled to Photometries Prime 95B 25 mm camera, configured with a 50-micron pinhole, and to a Celesta Lumencor as light source. The imaging process was conducted using a PlanApo LambdaS 40X / 1.25 Sil objective, capturing Z-stacks to encompass the complete volume of the sample. Subsequent processing and analysis of the acquired datasets were performed using ImageJ software.

[0159] Table 1. Primary and secondary antibodies used for immunofluorescence staining. qRT-PCR

[0160] Cells from three independent wells for each timepoint (DIVIO, DIV40) were lysed with 350pL RLT Buffer (Qiagen, Cat. No. 79216). RNA extraction was performed through RNAeasy Minikit (Qiagen, Cat. No. 74104) following manufacturer’s instructions, then RNA samples were treated with DNAse (DNA-free™ DNA Removal Kit by Invitrogen, Cat. No. AM1906). For each sample, 1 pg of RNA was retrotranscribed to cDNA with RevertAid RT Reverse Transcription Kit (Thermo Scientific, Cat. No. K1691) with the following thermic protocol: 5 minutes 25°C, 1 minute 42°C, 5 minutes 70°C. The obtained cDNA was diluted 1 : 10 in ddH2O for qPCR with the Sybr Green method (SYBR Green PCR Master Mix, Applied Biosystems, Cat. No. 4309155). qPCR primers sequences, listed below (in Table 2), were designed through Origene website or selected from Lu, J., Zhong, X, Liu, H. et al., 20161. The expression of the genes was normalized to the PSMB2 housekeeping gene and displayed as 2A(-ACt).

[0161] Table 2. Primers list. Flow cytometry

[0162] Cells were washed in Dulbecco’s Phosphate Buffer Saline w / o calcium and magnesium (DPBS; Euroclone, ECB4004L), and incubated in let Accutase Cell Detachment Solution (Innovative Cell Technologies, AT104) for 1 hour at 37° 5% CO2. After blocking Accutase with DPBS 10% Fetal Bovine Serum (FBS; Fisher Scientific, 26-140-079), cells were spun for 5 minutes at 400 ref, resuspended in DPBS 2% FBS and strained with 5 ml Polystyrene Round-Bottom Tube with Cell- Strainer Cap (Coming, 352235). Propidium iodide (Merck, 25535-16-4) was added to the cell suspension to stain dead cells, at a final concentration of 1 pg / ml. Cells were analysed through flow cytometry using Cytoflex LX (Beckman Coulter) and data were analysed with FlowJo 10.8.2. For each sample, 20.000 live cells were acquired.

[0163] Results

[0164] With an aim to generate in vitro human hindbrain neurons to study the development and disease of the human hindbrain, the Inventors herein described the method to differentiate hPSCs (both embryonic stem cells [hESCs] and induced pluripotent stem cells [iPSCs] to hindbrain neurons by activating Wnt signalling (Fig. la). First, the Inventors differentiated hPSCs to hindbrain neural stem cells (NSCs) in a chemically defined medium containing transforming growth factor (TGF)P inhibitor: SB431542, BMP inhibitor: LDN and GSK3-P inhibitor: CHIR99021 (to activate Wnt) (Fig. la, b). Additionally, the Inventors, performed qRT-PCR to evaluate anterior to posterior patterning of hindbrain progenitors upon CHIR99021 titration, demonstrating a loss of forebrain markers such as FOXG1 and expression of hindbrain markers, such as H0XA2 and H0XB1 (Fig. 2b). To evaluate H0XA2, a key hindbrain marker expressed in rhombomere 2-3 of the hindbrain, Inventors generated an hPSC_HOXA2::tdTomato reporter line through CRISPR / Cas9 geneediting (Zhong A, Li M, Zhou T, 2020s), as shown in Fig.3a. Differentiation of hPSCs into hindbrain neurons, but not into cortical neurons, led to expression of tdTomato using the hPSC_HOXA2::tdTomato reporter line (Fig 3.b). The hindbrain NSCs expressed key hindbrain markers, including H0XA2, H0XB1, HOXB2, and HOXB4 without the expression of forebrain marker FOXG1 (Fig. 3b, c, d). This line allowed for quantification of H0XA2+ cells throughout differentiation (Fig. 3c), and continued to express it at the postmitotic neuron stage up to 38 days in vitro (DIV) (Fig. 4).

[0165] To assess the differentiation and maturation potential of hindbrain NSCs, the Inventors seeded them as single cells at DIV15 in neural differentiation medium including DAPT. After 25 days of neuronal maturation, hindbrain neurons continued to express hindbrain-specific markers such as H0XA2, HOXB2, and HOXB4, without the expression of off-target markers SOX10 (neural crest marker), T (mesoderm marker), and SOX17 (endoderm marker) (Fig. 5a, b). Hindbrain neuron cultures matured up to DIV40 and developing highly pure MAP2+ neuronal cultures which were enriched for H0XA2+ cells (Fig. 5c). Discussion

[0166] Dual-SMAD inhibition is a common step in differentiation of central nervous system cells, as it patterns cells into neurectoderm, from which the central nervous system develops. Such examples are cortical neurons (Ciceri et al., 202-14) and serotonergic neurons (method described in: Lu, J., Zhong, X, Liu, H. et al., 20161'). However, differentiation of serotonergic neurons, and differentiation of hindbrain neurons of the present invention consists in the different starting material that influences the differentiation method, in particular on Mouse Embryonic Fibroblasts (MEF) feeder cells (for serotonergic neurons) versus feeder free cultured hPSCs (for hindbrain neurons), and the differential activation of Hedgehog signalling. The activation of Wnt is then needed to generate hindbrain neurons specifically (in contrast to cortical neurons for example). Furthermore, the protocol to generate serotonergic neurons (Lu, J., Zhong, X, Liu, H. etal., 20161), relies on the addition of purmorphamine to ventralize the hindbrain progenitors. This is an evident difference compared to the method of the invention.

[0167] In order to achieve the differentiation of hPSCs into hindbrain neurons, further to SMAD inhibition, there are additional important steps to be considered, such as:

[0168] The seeding density of preferably 250,000-300,000 cells / cm2(100% confluent hPSCs): the initial number of hPSCs seeded at DIV0 ensures both correct patterning of the hindbrain neural stem cells (NSCs), as well as maintaining a healthy culture during expansion of NSCs.

[0169] The exposure to Wnt agonists: activation of Wnt signalling, for example by using CHIR99021, is the most critical component of the herein described method, as the titration of Wnt activation is what defines the regional identity of hindbrain NSCs and neurons.

[0170] The timing of replating: considering the different pace of development of hindbrain NSCs compared to forebrain NSCs (as shown by Baumann et al., 20233), it is important to replate NSCs at between about 10 days and about 15 days, or between about 15 days and about 20 days from the initial contact of the cells with the at least one SMAD inhibitor. For example, optimal results have been obtained by replating at DIV 15 (rather than at later timepoints as in other protocols for generating forebrain cortical neurons). The exposure to y-secretase inhibitors: the exposure to DAPT (y-secretase inhibitor) ensures that, upon replating, NSCs are expelled from cell cycle and thus become postmitotic hindbrain neurons. BIBLIOGRAPHY

[0171] 1. Lu, J., Zhong, X., Liu, H. et al. Generation of serotonin neurons from human pluripotent stem cells. Nat Biotechnol 34, 89-94 (2016). https: / / doi.org / 10.1038 / nbt.3435

[0172] 2. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling [published correction appears in Nat Biotechnol. 2009 May;27(5):485], Nat Biotechnol.

[0173] 2009;27(3):275-280. doi:10.1038 / nbt,1529

[0174] 3. Natalia Baumann, Robin Wagener, Awais Javed, Philipp Abe, Andrea Lopes, Adrien Lavalley, Daniel Fuciec, Elia Magrinelli, Sabine Fievre, Denis Jabaudon. Regional Differences in Progenitor Consumption Dynamics Shape Brain Growth during Development. bioRxiv 2023.08.21.553891; doi: https: / / doi.org / 10.1101 / 2023.08.21.553891

[0175] 4. Ciceri, G., Baggiolini, A., Cho, H.S. et al. An epigenetic barrier sets the timing of human neuronal maturation. Nature 626, 881-890 (2024). https: / / doi.org / 10.1038 / s41586-023-06984-8

[0176] 5. Zhong A, Li M, Zhou T. Protocol for the Generation of Human Pluripotent Reporter Cell Lines Using CRISPR / Cas9. STAR Protoc. 2020; 1(2): 100052. doi: 10.1016 / j.xpro.2020.100052

Claims

CLAIMS1. An in vitro method for inducing differentiation of human pluripotent stem cells (hPSCs), comprising a step of a) culturing hPSCs in a culture medium comprising at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signalling, and at least one activator of wingless (Wnt) signalling from DIV [days in vitro} 0 to DIV 2, and in a culture medium comprising at least one inhibitor of SMAD signalling from DIV 2 to DIV 11, to obtain a cell population of cells, wherein at least about 75% of the cells express at least one hindbrain neural stem cell (NSC) marker; and further comprising a step of b) culturing hindbrain NSCs obtained in (a) in a culture medium comprising at least one inhibitor of Notch signalling for about at least 10 days, preferably for about at least 12 days from DIV 15, to obtain a cell population of differentiated cells, wherein at least about 75% of the differentiated cells express at least one hindbrain neuron marker.

2. The method according to claim 1, wherein in the step (a) hPSCs are seeded at density of 250,000-300,000 cells / cm2, said hPSCs being 100% confluent.

3. The method according to any one of claims 1 -2, wherein the at least one inhibitor of SMAD signalling is selected from inhibitors of TGFp / Activin-Nodal signalling, inhibitors of bone morphogenetic protein (BMP) signalling, and combinations thereof.

4. The method according to claim 3, wherein the inhibitor of TGFp / Activin- Nodal signalling comprises an inhibitor of ALK5, preferably comprises SB431542, or a derivative, or a mixture thereof.

5. The method according to claim 3, wherein the inhibitor of BMP signalling comprises LDN193189, Noggin, dorsomorphin, a derivative thereof, or a mixture thereof, preferably comprises LDN193189.

6. The method according to any one of claims 1 to 5, wherein the at least one activator of Wnt signalling comprises a GSK3 inhibitor, preferably comprises CHIR99021, derivatives thereof, and combinations thereof, more preferably comprises CHIR99021.

7. The method according to any one of claims 1 to 6, wherein the at least one inhibitor of Notch signalling comprises a y-secretase inhibitor, preferably comprises DAPT, derivatives thereof, or mixtures thereof, more preferably comprises DAPT.

8. The method according to any one of claims 1 to 7, wherein in a) the culture medium from DIV 0 to DIV 2 comprises SB431542, LDN193189 and CHIR99021, and from DIV 2 toDIV 11 the culture medium comprises SB431542 and LDN193189; and in b) culture medium comprises DAPT.

9. The method according to any one of claims 1 to 8, wherein in culture medium in a) SB431542 is comprised at a concentration of about 10 pM; LDN193189 is comprised at a concentration of about lOOnM; CHIR99021 is comprised at a concentration of about 1.5 pM to 3.5 pM, preferably of 3 pM; and in culture medium in b) DAPT is comprised at a concentration of about 10 pM.

10. The method according to any one of claims 1 to 9, wherein in a) at least one hindbrain NSC marker is selected from Homeobox A2 (H0XA2), Homeobox Bl (HOXB1), Homeobox B2 (HOXB2), Homeobox B4 (HOXB4), MKI67, IRX3 and combinations thereof; and / or wherein in b) the at least one hindbrain neuron marker is selected from Microtubule- Associated Protein 2 (MAP2), and / or Homeobox A2 (HOXA2), HOXA2, HOXB2, HOXB4, ASCL1, LMX1A, LMX1B, TLX1, TLX3, DRG11, and combinations thereof.

11. The method according to any one of claims 1 to 10, wherein in a) hindbrain NSCs do not express FOXG1; and in b) the differentiated cells do not express any of markers selected from the group consisting of: MKI67, SRY-box transcription factor 10 (SOX10), BRACHYURY (T), SRY-box transcription factor 17 (SOX17), FOXG1, EMX1, OTX2 and combinations thereof.

12. The method according to any one of claims 1 to 11, wherein the hindbrain NSCs obtained in a) are incubated in neural differentiation medium for about four days, preferably from DIV 12 to DIV 15, then replated and optionally cryopreserved.

13. The method according to any one of claims 1 to 12, wherein hPSCs are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

14. An isolated cell population of in vitro differentiated hindbrain neuron cells, wherein the in vitro differentiated hindbrain neuron cells are obtained according to the method of any one of previous claims and composition thereof, for use in preventing and / or treating and / or diagnosis of neurological disorder, said neurological disorder is selected from the group of neurodevelopmental, neuroimmunological, neuropsychiatric and / or neurodegenerative disorder, such as Alzheimer’s disease, Parkinson’s disease, Olivopontocerebellar Atrophy, and IgLON5 encephalitis.

15. The composition for use according to claim 14, which further comprises a pharmaceutically acceptable carrier.

16. A kit for performing the method according to any one of claims 1 to 13, comprising: i. at least one inhibitor of SMAD signalling; ii. at least one activator of Wnt signalling; iii. at least one inhibitor of Notch signalling; and optionally further comprising: iv. instructions for inducing differentiation of human pluripotent stem cells into a population of differentiated cells expressing at least one hindbrain neuron marker.

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