Cell surface markers of arcuate nucleus cell types

Abstract

The present invention provides in-vitro cell populations of arcuate nucleus cell types, such as tuberal progenitor cells, neurons and / or tanycytes, characterized by the expression of cell surface markers. The present invention also provides methods for 5 identifying populations of arcuate nucleus cell types expressing said markers. The invention also discloses positive and negative sorting selection steps based on expression of said markers. The present invention finally provides kits for performing the methods described herein.

Description

[0001] Cell surface markers of arcuate nucleus cell types

[0002] Technical field

[0003] The present invention relates to arcuate nucleus cell populations, such as tuberal progenitors, tanycytes, and neuron populations, defined by novel cell-surface markers. Said markers can also find useful applications for instance in methods for identifying, sorting or isolating said cell populations, for example during methods of cell differentiation into said arcuate nucleus progenitor cells populations.

[0004] Background

[0005] Stem cells can differentiate into various cell types through a process known as differentiation. In the context of neurons, stem cells can differentiate into neural progenitor cells, which are specialized intermediate cell types with the potential to become neurons. We have hundreds of different subtypes of neurons in the adult brain, all with specific structures and functions. Having accessible and easy-to-use models of these neurons is paramount for understanding brain development, for disease modelling, and for developing regenerative therapies for neurodegenerative diseases. Human embryonic stem cells (hESCs) are a useful tool for generating neuronal cell types of human origin thus bypassing issues arising from species-to-species differences and enhancing biological relevance. Neuroscientists have previously generated human-specific neuronal cell types in vitro for uncovering important molecular mechanisms underlying diseases such as Huntington’s Disease (Faedo et al., 2017) and Parkinson’s Disease (Nolbrant et al., 2017). An understudied area of the human brain in the context of human in vitro models is the hypothalamus. The hypothalamus houses neuronal cell populations that are critical to many metabolic processes such as thermoregulation, sleep, and appetite (Xie & Dorsky, 2017).

[0006] Human neurons of the arcuate nucleus (ARC) of the hypothalamus include for instance the Pro-opiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons, which have been known for decades to have opposing effects on appetite regulation (Morton et al., 2014), as well as growth hormone-releasing hormone (GHRH), tyrosine hydroxylase (TH) and thyrotropin-releasing-hormone (TRH) neurons and Prepronocictin neurons (PNOC) (Huisman et al., 2019). These subtypes are some of the first responders to many circulating peripheral hormones in the body that are sensed and integrated in the brain to control metabolism. Additionally, tanycytes (Beta 1 and 2), a specialized type of glial cells, play a very important role in regulating energy homeostasis (Yoo et al., 2021). They lay adjacent to the arcuate nucleus and play a pivotal role in facilitating the transport of these circulating hormones (Miranda-Angulo et al., 2014).

[0007] However, the in-vitro characterization of said human arcuate nucleus cell types, such as during cell differentiation protocols to generate said cells, remains a challenge. Despite their importance for appetite regulation and energy consumption, surface markers to characterize and purify arcuate nucleus cells are not well described. There is a need for surface markers enabling identification and sorting of the various cell populations of the arcuate nucleus.

[0008] Summary

[0009] Recently, the inventors have been successful at developing and optimising an advanced protocol for generating both tanycytes and mature neuronal subtypes of the arcuate nucleus, from hESC’s (exemplified in Figure 1A). As access to human brain tissue is limited, this protocol provides neuroscientists, both in academia and industry, an easy-to-use biological model that is human-specific. This model can be upscaled and shipped as a product to users and with limited neuronal culturing techniques required, can be grown to provide an in vitro model of the arcuate nucleus of the hypothalamus. Accompanying this initial invention, the inventors have surprisingly identified a selection of novel cell surface markers via 10X RNA-single cell / nucleus sequencing of the hESC derived ARC cells, at different developmental timepoints of the neuronal differentiation process. These cell surface markers can find helpful applications for instance to purify desired ARC cell types of interest, especially at the early developmental timepoints, when the neuronal progenitors are fate-committed but still proliferative and able to be replated. The novel markers therefore also allow a method to obtain enhanced purity of the desired cell types, thus increasing specificity for research and potential therapeutic applications. This is particularly important when working with differentiated cultures of ARC cells from several different hPSC lines since each line may differentiate with varying efficacy and purity. The populations thus identified herein may also be useful in enhancing reproducibility and reliability of experiments making use of said population, and also enable a more detailed study of a disease mechanism for conditions that affect these specific cell types.

[0010] A first aspect of the present invention therefore relates to an in-vitro cell population of arcuate nucleus cell types, wherein said population expresses one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

[0011] A second aspect of the present invention relates to a method for identifying a population of arcuate nucleus cell types from an in-vitro mixed cell population, said method comprising the step of: a) detecting the expression of one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3 on single cells of the in-vitro mixed cell population, thereby identifying a population of arcuate nucleus cell types from the in- vitro mixed population.

[0012] A third aspect of the present invention relates to a kit for isolating the in-vitro population of arcuate nucleus cell types as described herein, said kit comprising : a) labelled binding members arranged to bind one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably wherein said binding members are antibody(ies), nanobodies or microbodies specific for said cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for said cell-surface markers; b) Instructions performing the method for identifying a population of arcuate nucleus cell types from an in-vitro mixed cell population described herein. Description of Drawings

[0013] Figure 1.

[0014] A- Schematic of in-vitro protocol for deriving ARC progenitor cells from human embryonic stem cells. SB = SB413542, NOG = Noggin, SHH = Sonic Hedgehog protein, CHIR = CHIR99021 , BMP4 = Bone Morphogenic Protein 4, IGFBP3 = Insulin-like growth factor binding protein-3, BDNF = brain-derived neurotrophic factor, AA = ascorbic acid.

[0015] B- ScRNAseq data from day 16 dataset containing 4 batches of ARC progenitor cells. Cluster annotations based on Leiden clustering and gene expression of each cluster in the. Clusters of the LIMAP corresponding to neurons and progenitors, respectively, are circled.

[0016] C- Feature plots in LIMAP space of important reference markers that distinguish cell types of interest. SOX2=neuronal progenitor marker, STMN2=mature neuronal marker, RAX+ / TBX3+ / NKX2.1+ / FGF10+ cells are indicative of posterior tuberal hypothalamus. RAX+ / SHH+ cells are indicative of anterior tuberal hypothalamus.

[0017] D- Feature plots of cell surface candidates for the purification of posterior tuberal cells E- Feature plots of cell surface candidates for the purification of both anterior and poster tuberal cells.

[0018] For all feature plots, intensity of expression is provided as log normalised, scaled mRNA expression values.

[0019] Figure 2.

[0020] A- LIMAP projections of the scRNAseq data of the same 4 ARC batches as used in Figure 1 , at day 25. Clusters of the LIMAP corresponding to neurons clusters, and tanycytes are circled.

[0021] B- Feature plots of important reference markers that distinguish cell types of interest, DIO2= tanycytes specific marker, SOX2=neuronal progenitor marker, STMN2=mature neuronal marker.

[0022] C- mRNA expression of cell surface candidate DSCAM for the purification of ARC neurons and / or depletion of tanycytes populations.

[0023] D- Feature plots of cell surface candidates for the purification of tanycytes at day 25. E- Feature plot of important reference markers for neurons-STMN2 or tanycytes- DIO2 at day 50 and 70 of differentiation to support the specification of day 25 cell surface candidates for the purification of tanycytes.

[0024] F- mRNA expression of cell surface candidates for the purification of tanycytes that are also specific at later stages (day 50 and day 70) of differentiation.

[0025] For all feature plots, intensity of expression is provided as log normalised, scaled mRNA expression values.

[0026] Figure 3: LIMAP highlighting the ARC specific neuronal clusters used to calculate the percentage of cells expressing DSCAM.

[0027] Figure 4: Feature plots in LIMAP space of the three selected cell surface candidates at day 25 and their FACS plots, showing the gating used for sorting the positive and negative cell populations.

[0028] A- CD99- Early tanycyte marker.

[0029] B- GPC3- Early tanycyte marker.

[0030] C- DSCAM- early neuronal marker.

[0031] Figure 5: Immunocytochemistry assay at day 30 of the three selected cell surface candidates.

[0032] A- Tanycyte cell surface candidates GPC3 and CD99 antibodies were incubated with the tanycyte marker S100b and DNA stain DAPI.

[0033] B- Early neuron cell surface candidate DSCAM antibody was incubated together with DNA stain DAPI.

[0034] Figure 6: mRNA expression levels of reference markers that distinguish cell types of interest in the FACS-sorted positive and negative populations. Cells were sorted after three different cell surface candidates for the purification of ARC neurons and / or depletion of tanycytic populations: CD99, GPC3 and DSCAM. The data shows that CD99 and GPC3 enriches cells of tanycyte fate and depletes cells of neuronal fate, whereas DSCAM enriches cells of neuronal fate and depletes cells of tanycyte fate. A- DIO2, CRYM, NFIA = tanycyte specific markers.

[0035] B- PRDM12, OTP = early neuron specific markers. Detailed description

[0036] Definitions

[0037] As used herein “progenitor cells” refers to cells capable of differentiating into a certain type of cells.

[0038] As used herein, "arcuate nucleus cell type” refer to cells with molecular characteristics of the arcuate nucleus of the hypothalamus, including stem cell-derived in vitro populations of neural progenitor cells, tanycytes, and mature neurons expressing markers specific to the ARC. Arcuate nucleus cell types therefore comprise, but are not limited to, tuberal progenitor cells, such as posterior and anterior tuberal cells, arcuate nucleus neurons, and subtypes thereof, and tanycytes.

[0039] Human protein and gene nomenclature used herein are according to the nomenclature guidelines established by the National Center for Biotechnology Information (NCBI), unless otherwise specified. As used herein, “PTPRM” refers to the Protein Tyrosine Phosphatase Receptor Type M, “ERBB4” refers to the Erb-B2 Receptor Tyrosine Kinase 4, “PTPRP”, also known as PTPRN2 refers to the Protein Tyrosine Phosphatase Receptor Type N2, “SEMA5A” refers to Semaphorin 5A,”PCDH9” refers to Protocadherin 9, “BRINP3” refers to BMP / Retinoic Acid Inducible Neural Specific 3, “CNTN6” refers to Contactin 6, “EDNRB” refers to Endothelin Receptor Type B, “DSCAM” refers to DS Cell Adhesion Molecule, “GPC3” refers to Glypican 3, “SDC2” refers to Syndecan 2, “SLCO1C1” refers to the Solute Carrier Organic Anion Transporter Family Member 1C1 , “HTR2C” refers to the 5-Hydroxytryptamine Receptor 2C, “CD99” refers to Cluster of differentiation 99 also known as CD99 molecule (Xg blood group) or MIC2 , “FAT1” refers to the FAT atypical cadherin 1 , “NR3C2” relates to the Nuclear Receptor Subfamily 3 Group C Member 2, and “TRPM3” refers to the Transient Receptor Potential Cation Channel Subfamily M Member 3, “SOX2” refers to SRY-Box Transcription Factor 2, “RAX” refers to Retina And Anterior Neural Fold Homeobox, “NKX2-1” refers to NK2 Homeobox 1 , “TBX3” refers to T-Box Transcription Factor 3, “SHH” relates to Sonic Hedgehog Signaling Molecule, “FGF10” relates to Fibroblast Growth Factor 10, “DIO2” refers to lodothyronine Deiodinase 2, “DLX6-AS1” relates to DLX6 Antisense RNA 1, “FLI1” refers to Fli-1 proto-oncogene / ETS transcription factor, “PRDM12” refers to PR / SET Domain 12, “NR5A2” refers to Nuclear Receptor Subfamily 5 Group A Member 2, “ONECUT3” refers to One Cut Homeobox 3, “OTP” relates to Orthopedia Homeobox, “POMC” relates to Proopiomelanocortin, “CRYM” refers to crystallin mu or NADP-regulated thyroid-hormone-binding protein (THBP), “NFIA” refers to nuclear factor I A, and cover genes, transcripts of genes, and encoded proteins.

[0040] In vitro cell populations of arcuate nucleus cell types and markers

[0041] A first aspect of the invention relates to an in-vitro cell population of arcuate nucleus cell types, wherein said population expresses one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3.

[0042] In some embodiments, said population is a posterior tuberal progenitor cells population and said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3, PTPRP, EDNRB and ERBB4.

[0043] In other embodiments, said population is a posterior tuberal progenitor cells population and said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4.

[0044] In further embodiments, said population is a posterior tuberal progenitor cells population and said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

[0045] In other embodiments, said population is an arcuate nucleus neurons population and said one or more cell-surface markers is DSCAM.

[0046] In yet other embodiments, said population is a tanycyte population and said one or more cell surface markers are selected from the group consisting of: GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably said one or more cell-surface markers are selected from the group consisting of: GPC3 and SDC2, even more preferably, wherein said one or more cell-surface markers are GPC3, and / or SDC2, and SLCO1C1. For some applications, it may be beneficial to characterize the in-vitro cell populations of arcuate nucleus cell types of the present invention by both positive markers (markers expressed in said population) and negative markers (markers not expressed in said population).

[0047] The skilled person will appreciate that positive identification based on markers expressed by a population of cells of interest allows for characterization, and in some applications also isolation, of said cell populations, and may provide advantages, for example in downstream applications, such as in studies based on said population, drug screening or diagnostic assays.

[0048] The skilled person will also know that negative identification, alone, but preferably in combination with positive selection, helps to exclude unwanted cell types, such as cell types not belonging to the cell population of interest, to enhance the purity of the isolated population.

[0049] Both types of identification, preferably in combination, enhance the specificity and reliability of the identification of the population of cells of interest, and may be advantageous in some applications making use of said cell population of interest, to obtain more robust and accurate results, such as in biomedical research, such as drug screening, for example for appetite-disorders or metabolic disorder drug screening, diagnostics, and therapeutic applications.

[0050] In some applications, it is for instance beneficial that the arcuate nucleus cell type population expresses markers of a tanycyte population, such as the tanycyte markers of the present invention, while not expressing markers of the tuberal progenitors population.

[0051] Therefore in some embodiments, said population does not express any of the cellsurface markers PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB.

[0052] In some applications, it is for instance beneficial that the arcuate nucleus cell type population expresses markers of the tuberal progenitor and / or neuron population, such as the tuberal progenitor and / or neuron population markers of the present invention, while not expressing markers of the tanycyte population. Thus in other embodiments, said population does not express any of the cell-surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

[0053] In some applications, it is for instance beneficial that the arcuate nucleus cell type population expresses markers of the neuron population, such as the neuron population markers of the present invention, while not expressing markers of the tanycyte and / or tuberal progenitor population.

[0054] Thus in other embodiments, the in-vitro cell population of arcuate nucleus cell types does not express the cell-surface marker DSCAM.

[0055] The skilled person will appreciate that the expression of markers within a cell population may depend for instance on the origin of the cell population, such as the protocol by which said cells are obtained, and / or culture conditions, the heterogeneity of cell types within the cell population, which may be in turn dependent on the degree of purification or isolation of specific cell types within said population.

[0056] The skilled person will thus know that homogenous populations of specific cell types expressing specific markers, for example populations obtained after a step of sorting or isolation against said specific markers (negative and / or positive markers), or for example populations obtained from a differentiation protocol allowing the generation of specific cell types, will typically have a higher percentage of expression of the markers specific to the cells of said population, compared to more heterogenous cell populations, for example compared to unsorted populations, or compared to populations sorted to a lesser degree, or for example compared to cells obtained from differentiation protocols leading to the generation of less specific cell types.

[0057] Therefore, in preferred embodiments of the in-vitro cell population of arcuate nucleus cell types as described herein: a) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; b) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; c) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; d) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; e) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; f) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; g) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; h) said arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; i) said arcuate nucleus cell type is arcuate nucleus neurons and the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 14%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; j) said arcuate nucleus cell type is tanycytes and the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; k) said arcuate nucleus cell type is tanycytes and the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of the population; l) said arcuate nucleus cell type is tanycytes and the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; m) said arcuate nucleus cell type is tanycytes and the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; n) said arcuate nucleus cell type is tanycytes and the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; o) said arcuate nucleus cell type is tanycytes and the cell surface marker FAT 1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; p) said arcuate nucleus cell type is tanycytes and the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population; and / or q) said arcuate nucleus cell type is tanycytes and the cell surface marker TRPM3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population.

[0058] The skilled person will appreciate that several methods exist in the field for measuring the expression of genes, transcripts of genes, and encoded proteins thereof. The skilled person will thus know that gene expression analysis may be performed by, but not limited to, quantitative PCR (qPCR), reverse Transcription PCR (RT-PCR), or quantitative reverse Transcription PCR (qRT-OCR), RNA Sequencing (RNA-seq) such as single cell or single nucleus RNA sequencing, In situ Hybridization (ISH). The skilled person will know that protein expression analysis may be performed by, but not limited to immunocytochemistry (ICC) approaches, flow cytometry, Western Blotting, Enzyme- Linked Immunosorbent Assay (ELISA) or mass spectrometry. In preferred embodiments of the present invention, the expression of the markers in the in-vitro cell population of arcuate nucleus cell types is measured by single-cell RNA sequencing (scRNA-seq) or single-cell nucleus sequencing (snRNA-seq).

[0059] In some embodiments, the expression of the markers is provided as a percentage of cells of the in-vitro cell population expressing said markers within a subgroup of arcuate nucleus types. For each subgroup, certain markers are known to be associated with the cell type.

[0060] In some embodiments, said subgroup is anterior or posterior tuberal progenitors, defined by the expression of S0X2, RAX and NKX2-1.

[0061] In other embodiments, said subgroup is anterior tuberal progenitors, defined by the expression of S0X2, RAX, NKX2-1 and SHH.

[0062] In further embodiments, said subgroup is posterior tuberal progenitors, defined by the expression of S0X2, RAX, NKX2-1 , TBX3 and FGF10 and the absence of SHH expression.

[0063] In yet other embodiments, said subgroup is tanycytes, defined by S0X2 and DIO2 expression. In some embodiments, tanycytes are also defined by CRYM and NFIA expression.

[0064] In other embodiments, said subgroup is neurons, such as the neuron population defined by the expression of STMN2, preferably ARC neurons defined by expression of DLX6-AS1 , FLI1 / PRDM12, NR5A2 / ONECUT3, OTP, POMC / PRDM12.

[0065] Table 1 hereafter summarizes the cell surface markers, the cluster they relate to (Name of cluster), and the percentage of cells in the population that express cell surface candidate (% in cluster). The percentage was calculated based on positive cells, and a positive cell was defined as any cell with a number of detected transcripts above 0.

[0066] [Table 1]

[0067] * ARC neurons at day 25 was defined by cells expressing ARC specific markers DLX6- AS1 , FLI1 / PRDM12, NR5A2 / ONECUT3, OTP, POMC / PRDM12 and the percentage of DSCAM positive cells was calculated from these clusters (see Fig. 3). “Day 25” refers to cell obtained at day 25 of the protocol exemplified in Fig 1A, i.e. 25 days after initiation of the differentiation by addition of the dual SMAD inhibitors SB413542 and Noggin (SB + NOG), and wherein cells are further grown from day 16 to 25 in a medium supporting growth of neuronal stem cells comprising ascorbic acid (AA) used at 0.2 mM, Brain-derived neurotrophic factor (BDNF) used at 20 pg / mL dibutyryl-cAMP used at 500pM, y-secretase inhibitor (DAPT) used at 1 M, and Glial cell line-derived neurotrophic factor (GDNF) used at 10ng / mL .

[0068] Differentiation methods

[0069] Directed differentiation is a process used to guide the development of stem cells into specific mature cell types, such as neurons, in a controlled and predictable manner. It involves mimicking the natural signalling cues and environmental conditions that cells experience during normal organismal development. The skilled person will appreciate that cell differentiation methods may comprise sequential phases from initiating the differentiation of cells, such as pluripotent stem cells, to obtaining partially- or fully-differentiated cells.

[0070] Methods for differentiation of cells that can be used in the present invention are described in co-pending application EP24160215.0, filed on 28 February 2024, hereby incorporated by reference.

[0071] In some embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention may be obtained by methods comprising, or consisting in, one or more of the three following general phases of differentiation:

[0072] Phase I, preferably spanning between day 0 and day 11 of differentiation, and referred to herein as a cell Patterning phase.

[0073] Phase II, preferably spanning between day 11 day 16 of differentiation, and referred to herein as a cell Expansion phase.

[0074] Phase III, preferably spanning from day 16 of differentiation, and referred to herein as a cell Maturation phase.

[0075] In some preferred embodiments of the present invention, day 0 of differentiation, corresponding to the day of initiation of the Patterning phase described in more detail herein below, is the day on which the step of culturing pluripotent stem cells in the presence of one or more inhibitors of the SMAD signalling pathway is initiated.

[0076] The skilled person will appreciate that once the differentiation is initiated, following initiation of the Patterning phase, pluripotent stem cells start differentiating and can be generally referred to as pluripotent stem cell-derived cells, or pluripotent stem cell- derived cultures, obtained from said pluripotent stem cells.

[0077] Methods for differentiating pluripotent stem cells into arcuate nucleus progenitor cells

[0078] The in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention may be obtained in some embodiments by methods enabling the generation of region-specific neural progenitor cells of the arcuate nucleus of the hypothalamus. Said methods combine timing and serial addition of compounds enabling the differentiation of pluripotent stem cells into arcuate nucleus progenitor cells.

[0079] In some embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by a method for differentiating pluripotent stem cells into arcuate nucleus progenitor cells, said method comprising a cell patterning and expansion phase together comprising the steps of: a. Culturing said pluripotent stem cells in the presence of one or more sonic hedgehog pathway activator(s); b. Culturing the cells from step a. in the presence of bone morphogenetic protein 4 (BMP4) and / or bone morphogenetic protein 7 (BMP7), and Insulin-like growth factor-binding protein 3 (IGFBP3), thereby obtaining arcuate nucleus progenitor cells.

[0080] In the Patterning phase of said methods, Sonic Hedgehog pathway activators supports the induction of ventral (ventral region of the hypothalamus) identity, and ventral forebrain orientation, in the differentiating cells (e.g. stem cells).

[0081] The inventors further found that BMP signalling activation, such as mediated by BMP4 and / or BMP7 at specific timepoints is crucial for correct Arcuate nucleus (ARC) patterning, and in generating bona fide ARC progenitor cells {RAX, TBX3 and NKX2-1 positive) during patterning and expansion phase, as well as for subsequent fates of the anterior and posterior tuberal hypothalamus regions during the subsequent maturation phase. In particular, the timepoints of addition and duration of exposure to BMP4 (and / or BMP7), such as the ones of the ARC V2 protocol (Figure 1A), were also found by the inventors to be important for maintaining SHH down-regulation, and avoid the loss of important markers of posterior tuberal fate, i.e. TBX3 and DIO2 as well as an increased contamination of the cultures with F0XG1+ telencephalic fates, thereby improving accurate ARC patterning in vitro.

[0082] In addition, the inventors identified that IGFBP3 addition during the patterning and expansion phase was important in increasing ARC cell types markers expression (e.g. AGRP and POMC) at later stages of differentiation, such as during the maturation phase. Optionally, said methods may further include a step of culturing the pluripotent stem cells in the presence of a Rho kinase (ROCK) inhibitor, preferably wherein the ROCK inhibitor is Y-27632. The presence of the ROCK inhibitor supports cell viability, and said step is preferably performed after plating (subculturing) cells, such as after plating the cells for the step of initiating the Patterning phase, and / or after plating cells for the step of initiating the cell expansion phase, and / or after plating cells for the initiation of the maturation phase.

[0083] Initiation of the differentiation of the pluripotent stem cells to neuroectodermal lineages is typically triggered by contacting said cells with one or more inhibitor(s) of the SMAD signalling pathway.

[0084] Therefore, in preferred embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by methods for differentiating pluripotent stem cells into arcuate nucleus progenitor cells as described herein, wherein step a. further comprises a step of culturing the pluripotent stem cells in the presence of one or more inhibitor(s) of the SMAD signalling pathway, said step initiating the Patterning phase, preferably initiated simultaneously with step a., and preferably wherein the inhibitors of the SMAD signalling pathway are SB413542 and Noggin.

[0085] In some embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by methods for differentiating pluripotent stem cells into arcuate nucleus progenitor cells of the s described herein, wherein step a. further comprises a step of culturing the pluripotent stem cells in the presence of an activator of the WNT signalling pathway, preferably said step is initiated 2 days after initiating the Patterning phase, and for 7 days, and preferably the activator of the WNT signalling pathway is CHIR99021.

[0086] Said step aims to mimic the control of anterior to posterior orientation controlled by a gradient of WNT signalling activation in the developing neural tube.

[0087] In some embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by methods for differentiating pluripotent stem cells into arcuate nucleus progenitor cells as described herein wherein step a. further comprises a step of culturing the pluripotent stem cells in the presence of BMP4 wherein said step is initiated 5 days after initiating the cell patterning phase, and continues for 2 days. Said step is therefore initiated prior to the step of culturing cells in the presence of bone morphogenetic protein 4 (BMP4) and / or bone morphogenetic protein 7 (BMP7), and Insulin-like growth factor-binding protein 3 (IGFBP3), i.e. prior to IGFBP3 addition to the cell medium.

[0088] Following the Patterning phase, it may be beneficial to support the expansion phase of neural progenitor cells, such as ARC progenitor cells, by providing compounds supporting their survival, proliferation and / or differentiation. Thus, in some embodiments the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by methods for differentiating pluripotent stem cells into arcuate nucleus progenitor cells as described herein, wherein step b. further comprises a step of culturing the pluripotent stem cells in the presence of one or more components selected from the group consisting of : Brain-derived neurotrophic factor (BDNF) and ascorbic acid (AA). Said step is preferably initiated 11 days after initiating the Patterning phase, thereby initiating the cell expansion phase, and continues for 5 days.

[0089] Therefore, in preferred embodiments, the in vitro cell populations of arcuate nucleus cell types or the in vitro mixed cell populations of the present invention are obtained by methods comprising the ARC V2 protocol (or v2 ARC protocol or ARC differentiation protocol version 2 (V2)) found by the inventors, wherein the method for differentiating pluripotent stem cells into arcuate nucleus progenitor cells as described herein comprises a cell Patterning and Expansion phase together comprising the steps of: a. Culturing said pluripotent stem cells in the presence of the sonic hedgehog pathway activator SHH, at 300ng / mL, for 9 days; b. Optionally, culturing said pluripotent stem cells in the presence of a ROCK inhibitor, preferably wherein said ROCK inhibitor is Y-27632, simultaneously with the initiation of step a., for 1 day; c. Culturing said pluripotent stem cells in the presence of the inhibitors of the SMAD signalling pathway SB413542 and Noggin at 10pM and 100ng / mL, respectively, thereby initiating the Patterning phase, simultaneously with step a., for 9 days; d. Culturing the cells of step c. in the presence of the activator of the WNT signalling pathway CHIR99021, at 0.3pM, 2 days after initiating the Patterning phase, for 7 days; e. Culturing the cells of step d., in the presence of BMP4, at 50ng / mL, 5 days after initiating the Patterning phase, for 2 days; f. Culturing the cells from step e. in the presence of BMP4 and IGFBP3, at 50ng / mL and 400ng / mL, respectively, 7 days after initiating the Patterning phase, for 7 days; g. Culturing the cells of step f. in the presence of Brain-derived neurotrophic factor (BDNF) and ascorbic acid, at 20pg / mL and 0.2mM, respectively, thereby initiating the cell expansion phase, 11 days after initiating the Patterning phase, for 5 days; and h. Optionally culturing the cells of step g. in the presence of a ROCK inhibitor, preferably wherein said ROCK inhibitor is Y-27632, simultaneously with the initiation of step g., for 1 day, thereby obtaining arcuate nucleus progenitor cells.

[0090] The person of skill in the art is aware that the precise concentration of compounds and the precise duration of exposure to these as well as the relative timing of the steps can be varied according to the guidance given in the present disclosure.

[0091] Uses

[0092] The in-vitro cell population of arcuate nucleus cell types of the present invention may find useful applications in medicine, such as cell therapy, for example of neurodegenerative diseases.

[0093] Thus in some embodiments of the in-vitro cell population of arcuate nucleus cell types according to the present invention, said population is for use in medicine, such as in the treatment of neurodegenerative diseases.

[0094] The in-vitro cell population of arcuate nucleus cell types of the present invention may also find useful applications in research, disease modelling, or drug screening, as models of the ARC cells of a single type of cells, or models recapitulating the cell diversity of the ARC. The skilled person will know that the hypothalamus cell populations are critical to many metabolic processes such as thermoregulation, sleep and appetite. The in-vitro cell population of arcuate nucleus cell types of the present invention may find applications for example in the study of these processes, or in the drug screening of drugs affecting these processes.

[0095] Thus, in other embodiments, said population is for use in drug screening, such as screening of drugs in the field of metabolic diseases, appetite disorders, sleeping disorders, or disorders impacting thermoregulation.

[0096] Methods for identifying a population of arcuate nucleus cell types from an in-vitro mixed cell population

[0097] Another aspect of the present invention relates to a method for identifying a population of arcuate nucleus cell types from an in-vitro mixed cell population, said method comprising the step of: a) detecting the expression of one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB.DSCAM, GPC3, SDC2, SLCO1C1, HTR2C, CD99, FAT1, NR3C2, and TRPM3 on single cells of the in-vitro mixed cell population, thereby identifying a population of arcuate nucleus cell types from the in-vitro mixed population.

[0098] A mixed cell population can be a mixed cell population of any type of cells, preferably the mixed cell population comprises or consists of different types of arcuate nucleus cell types, such as a mixed populations of neural progenitor cells, tanycytes and / or mature neurons expressing markers specific to the ARC.

[0099] Positive and / or negative characterization approaches, as described herein in the section In vitro cell populations of arcuate nucleus cell types and markers may be useful in methods for identifying and / or sorting said populations of arcuate nucleus cell types from mixed cell populations.

[0100] Thus, in preferred embodiments, said arcuate nucleus cell type is posterior tuberal progenitor cells and said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4. In other embodiments, said arcuate nucleus cell type is posterior tuberal progenitor cells and said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

[0101] In some embodiments, said arcuate nucleus cell type is neurons, and said one or more cell-surface marker is DSCAM.

[0102] In further embodiments, said arcuate nucleus cell type is tanycytes and said one or more cell-surface markers selected from the group consisting of: GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably said one or more cell-surface markers are selected from the group consisting of: GPC3 and SDC2, even more preferably, said one or more cell-surface markers are GPC3, and / or SDC2, and SLCO1C1.

[0103] In some applications, it is for instance beneficial to detect arcuate nucleus cell type population expressing markers of a tanycyte population, such as the tanycyte markers of the present invention, while detecting the absence of expression of markers of the tuberal progenitors population.

[0104] In preferred embodiments, the method further comprises a step of detecting the absence of expression of the cell-surface markers PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB on single cells of the mixed cell population.

[0105] In some applications, it is for instance beneficial to detect arcuate nucleus cell type population expressing markers of the tuberal progenitor and / or neuron population, such as the tuberal progenitor and / or neuron population markers of the present invention, while detecting the absence of expression of markers of the tanycyte population.

[0106] In other embodiments, the method further comprises a step of detecting the absence of expression of the cell-surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3 on single cells of the mixed cell population

[0107] In some applications, it is for instance beneficial to detect arcuate nucleus cell type population expressing markers of the neuron population, such as the neuron population markers of the present invention, while detecting the absence of markers of the tanycyte and / or tuberal progenitor population. In further embodiments, the method further comprises a step of detecting the absence of expression of the cell-surface marker DSCAM on single cells of the mixed cell population.

[0108] The skilled person will appreciate that the detection of positive and negative markers can be performed simultaneously, or one after the other, depending on the application. Therefore in some embodiments, the step of detecting the absence of expression of the cell-surface marker is performed before, simultaneously, and / or after, the step of detecting the expression of the one or more cell-surface markers.

[0109] The skilled person will know that the detection of expression of the cell-surface markers may be performed by labelling cells expressing said surface markers.

[0110] In preferred embodiments, the method further comprises a step of labelling cells expressing the one or more cell-surface markers.

[0111] The skilled person will know that cell labelling methods include, but are not limited to immunocytochemistry (ICC), in situ hybridization, fluorescent protein tagging, magnetic cell sorting, and biotinylation techniques. The skilled person will know that nanobodies, also known as VHH or single-domain antibodies, which are small antigen-binding fragments derived from the variable domain of heavy-chain-only antibodies found in camelids, may be beneficially used in cell labelling techniques. Microbodies, protein fusions to domain 3 of the IgG heavy chain Fc region (lgG-CH3) may also be used. Thus, in even more preferred embodiments, the labelling is with one or more labelled binding members arranged to bind the one or more cell surface markers, preferably the labelled binding members are fluorescently labelled antibody(ies), nanobodies or microbodies specific for the one or more cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for the one or more cell-surface marker.

[0112] Sorting a population of arcuate nucleus cell types from an in-vitro mixed cell population

[0113] For some applications, it may be beneficial that the methods of identification of populations of arcuate nucleus cell types of the present invention further comprise a step of sorting the cells thus identified. Therefore, in further embodiments, the methods of further comprises a step of sorting from said mixed cell population the cells in which said expression of cell-surface markers is detected. Said methods may therefore also be referred to as methods for sorting populations of arcuate nucleus cell types from a mixed cell population.

[0114] As described herein for the arcuate nucleus cell types of the present invention, and for the methods for identifying a population of arcuate nucleus cell types from an in-vitro mixed cell population, positive and / or negative characterization approaches, may be useful in steps of sorting specific populations of arcuate nucleus cell types from mixed cell populations.

[0115] Thus, in other embodiments, the method further comprises a step of sorting from said mixed cell population the cells in which said absence of cell-surface marker expression is detected.

[0116] The skilled person will appreciate that the steps of sorting based on positive and negative markers can be performed simultaneously, or one after the other, depending on the application.

[0117] Thus, in some embodiments, the step of sorting from said mixed cell population the cells in which said expression of cell-surface markers is detected is performed before, during, and / or after, the step of sorting from said mixed cell population the cells in which said absence of cell-surface marker expression is detected.

[0118] Thus in some embodiments, the methods of the present invention comprise the steps of: a) detecting the expression of one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1, HTR2C, CD99, FAT1 , NR3C2, and TRPM3 on single cells of the in-vitro mixed cell population, b) optionally detecting the absence of expression of: the cell-surface markers: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB, or the cell surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3 and / or the cell-surface marker DSCAM on single cells of the mixed cell population c) sorting from said mixed cell population the cells in which said expression of cell-surface markers is detected. d) optionally sorting from said mixed cell population the cells in which said absence of cell-surface marker expression is detected

[0119] The same combinations of markers described herein as useful in applications to detect arcuate nucleus cell type population expressing markers of the tuberal progenitor and / or neuron population, neuron population or tanycyte population, while detecting the absence of expression markers of other populations, are applicable to the methods comprising the positive and optional negative sorting steps described hereinabove.

[0120] The skilled person will appreciate that further steps of quality control of the sorted cells, such as verification of the expression of the markers in the cell populations obtained after sorting, may be performed by methods known in the art following the sorting steps.

[0121] Expression detection and sorting techniques

[0122] The skilled person will appreciate that several methods exist in the field for detecting the expression of genes, transcripts of genes, and encoded proteins thereof. The skilled person will thus know that gene detection may be performed by, but not limited to, next generation sequencing (NGS), quantitative PCR (qPCR), reverse Transcription PCR (RT-PCR), or quantitative reverse Transcription PCR (qRT-OCR), RNA Sequencing (RNA-seq) such as single cell or single nucleus RNA sequencing, In situ Hybridization (ISH). The skilled person will know that protein detection may be performed by, but not limited to immunocytochemistry (ICC) approaches, flow cytometry, Western Blotting, Enzyme-Linked Immunosorbent Assay (ELISA) or mass spectrometry. In some preferred embodiments, such as embodiments of the methods comprising sorting steps, the step of detection of the expression of the one or more cell-surface markers is performed by immunocytochemistry or flow cytometry.

[0123] The skilled person will appreciate that several approaches are available to perform cell detection and / or sorting steps, such as flow cytometry, fluorescence-activated cell sorting (FACS), Magnetic-activated cell sorting (MACS), Immunomagnetic cell separation, Microfluidic-based cell sorting.

[0124] In other preferred embodiments, the step of detection of the expression and / or the step of sorting the cells is performed by flow cytometry, such as fluorescence activated cell sorting (FACS), Magnetic-Activated Cell Sorting (MACS), or microfluidic cell sorting.

[0125] The skilled person would appreciate that the percentage of cells in a population being positive for a marker may vary depending on the method used to detect said marker. In other words, the skilled person would appreciate that if two or more approaches, for example flow cytometry and transcriptom ics approaches, are used to detect the expression of a specific marker in the same cell population, the percentage of cells in the population that is determined to be marker-positive may differ between approaches. In one embodiment, the method used to determine the percentage of cells in the population that are marker-positive is flow cytometry.

[0126] In one embodiment, the method used to determine the percentage of cells in the population that are marker-positive is transcriptomics.

[0127] Mixed cell population and sorted arcuate nucleus cell type population

[0128] As described for the in-vitro cell populations of arcuate nucleus cell types herein in the section In vitro cell populations of arcuate nucleus cell types and markers, different in- vitro mixed populations used in the methods of the present invention may also present different percentage of expression of the cell surface markers, due to different origins, such as, but not limited to, populations being obtained from different differentiation protocols, or for example in the case of stem cell differentiation protocols, using different stem cells cell lines as starting material for the differentiation. Thus, some embodiments of the methods of the present invention: a) the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; b) the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; c) the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the in-vitro mixed population; d) the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; e) the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; f) the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; g) the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; h) the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; i) the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 14%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; j) the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; k) the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of the in-vitro mixed population; l) the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; m) the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; n) the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; o) the cell surface marker FAT1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; p) the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population; and / or q) the cell surface marker TRPM3 is expressed in at least 1 %, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population.

[0129] The skilled person would appreciate that following the steps of identification and sorting of the methods of the present invention, the percentage of expression of the cell surface markers in the populations of arcuate cell types obtained would be typically higher than in the mixed cell population said population of arcuate cell type was sorted from. In some aspects, the methods of the present invention comprising sorting steps may therefore be referred to as methods for enriching a mixed cell population in cell populations of arcuate nucleus cell types. In further embodiments, said methods may comprise a step of comparing the percentage of expression of the markers before and after the sorting step(s).

[0130] Thus in some embodiments of the methods of the present invention: a) said population of arcuate nucleus cell type is anterior or posterior tuberal progenitors, and the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; b) said population of arcuate nucleus cell type is anterior or posterior tuberal progenitors and the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; c) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; d) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; e) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; f) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; g) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; h) said population of arcuate nucleus cell type is posterior tuberal progenitors and the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; i) said population of arcuate nucleus cell type is arcuate nucleus neurons and the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 14%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; j) said population of arcuate nucleus cell type is tanycytes and the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; k) said population of arcuate nucleus cell type is tanycytes and the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of said population; l) said population of arcuate nucleus cell type is tanycytes and the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; m) said population of arcuate nucleus cell type is tanycytes and the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; n) said population of arcuate nucleus cell type is tanycytes and the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; o) said population of arcuate nucleus cell type is tanycytes and the cell surface marker FAT1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; p) said population of arcuate nucleus cell type is tanycytes and the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population; and / or q) said population of arcuate nucleus cell type is tanycytes and the cell surface marker TRPM3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population. The skilled person would appreciate that further steps of comparison of the percentage of expression of the markers, such as comparing the expression percentage in the cell population before and after sorting steps may be performed.

[0131] In some embodiments of the method of the present invention, further steps of performing functional validation assays may be performed on the arcuate nucleus cell types identified and / or isolated.

[0132] Kits

[0133] For some applications, it may be beneficial to provide the elements necessary to perform the detection of the expression of the cell surface markers, and / or labelling and / or sorting of the arcuate nucleus cell types under the form of a kit together with instructions on how to perform the methods of the present invention.

[0134] Thus, another aspect of the present invention relates to a kit for isolating the in-vitro population of arcuate nucleus cell types of the present invention, said kit comprising : a) labelled binding members arranged to bind one or more cellsurface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably wherein said binding members are antibody(ies), nanobodies or microbodies specific for said cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for said cellsurface markers; b) Instructions performing the methods of the present invention. Examples

[0135] Example 1

[0136] Single cell analysis of early stem cell-derived ARC progenitors reveals cellular heterogeneity and early tuberal specification events.

[0137] Material and methods

[0138] Cell culturing and neuronal differentiation hESC line RC17 (Roslin Cells, hESC line, RCeO21-A) and was maintained on 1 pg / cm2 laminin-521 (Biolamina, #LN521-02) in IPS-Brew (Miltenyi, #130-104-368). Cells were kept to 70-90% confluency before passaging with 75 pL / cm2 of 0.5 mM EDTA (Thermo Fisher Scientific, #15575020) for approx. 7 min before detachment and replated for 24 hours with 10pM Y-27632 (ROCK inhibitor, Miltenyi, cat. # 130-106-538) with daily media changes thereafter. For differentiation into regionalized hypothalamic progenitors, protocols from Nolbrant et al., 2017 were adapted. hESCs were kept until 70-90% confluent then dissociated with 0.5mM EDTA and plated at 10K / cm2 in 2 pg / cm2 laminin-111 (Biolamina, #LN111-02) coated plates. All day 0 cells, regardless of regionality, received 10 pM Y-2763 for 24 hours and neuroectodermal lineages were initiated using 10 pM SB413542 (Miltenyi, #130-106-543), and 100 ng / mL Noggin (Miltenyi, #130-103-456), diluted in N2 basal medium from day 0 to 9. For the PVN v1 CHIR is added to the N2 medium from day 0 to 9 at 0.3 pM whilst a low concentration and delayed addition of 150ng / ml of SHH is added from day 6 to 14 to enhance dorsal identity of the cells. For the v1 ARC protocol, cells were ventralized with high SHH of 300 ng / mL from day 0 to 9. Rostral diencephalic positional identify was encouraged with 0.3 pM CHIR between day 2 to 9 and tuberal induction was incurred by 50 ng / mL BMP4 from day 6 to 14. ARC v2 protocol found the use of BMP4 at day 5 to be optimal for ARC patterning and we identified IGFBP3 as an enhancer of mature ARC fates. All protocols received media change every 2 days. By day 11, early progenitors were very confluent, cells were dissociated in 75 pL / cm2 accutase (ThermoFisher, #A11105-01) for 10 minutes at 37 and transferred at a density of 800k / cm2 in B27 medium, containing 0.2 mM ascorbic acid (Sigma-Merck, #A4403-100MG), 20 pg / mL BDNF (Miltenyi, #130-096-286), and 10 pM 69Y-27632 and plated into onto Lam111 coated plates. Day 16 of the differentiation, regionalised progenitors are replated one final time at 700k / cm2 to Lam521 coated plates for maturation and long term culturing in B27 containing 0.2 mM ascorbic acid 20 pg / mL BDNF, 500 pM dibutyryl-cAMP (Sigma- Merck, #D0627-1G), 1 pM DAPT (Miltenyi, #130-110-489), 10 ng / mL GDNF (Miltenyi, #130-098-449), and 10 pM Y-27632 For the generation for spheroids, patterned day 16 hypothalamic progenitors were thawed and diluted in pre-warmed DMEM / F12 with 5% KOSR. Cells were counted, centrifuged at 400 xg for 5 min and resuspended in maturation medium (B27 medium [MACS Neuromedium containing 2% NB21, 1% Glutamax, Penicillin / Streptomycin] complemented with 10 pM Y- 27632 (ROCKi), 20 ng / mL BDNF, 0.2 mM ascorbic acid, 500 pM Db-cAMP and 1 pM DAPT) at 250 000 cells / mL. The cell suspension was transferred to an ultralow attachment Il-bottom 96- well plate (Costar) at 200 pL / well, centrifuged at 100 xg for 5 min and maintained at 37 °C with 5% CO2. 75% of maturation medium without ROCKi was changed every 2-3 days until termination of the experiment

[0139] Single-cell RNA sequencing (scRNA-seq) and Single nucleus RNA sequencing (snRNA-Seg) scRNA-seq as well as snRNA-seq were performed using the 10X Genomics v3.1 technology (following supplier instructions) combined with CiteSeq Hashtagging (Stoeckius et al., 2017)

[0140] Seguence alignment and sample assignment

[0141] The raw cDNA libraries from all single-cell / nuclei samples were processed using CellRanger count pipeline v7.1.0 (Zheng et al., 2017) by aligning the day 16 reads against the human reference genome GRCh38-2020-A, and the day 25, 50, and 70 reads against a recently published reference with optimized genome annotations (Pool et al., 2023). The obtained gene count matrices were corrected with CellBender v0.3.0 (Fleming et al., 2023) to remove ambient RNA. The CellRanger BAM file for D16 data was processed with STARsolo v2.7.10b (Kaminow et al., 2021) to generate intronic and exonic counts required for RNA velocity. CITE-seq-Count v1.45 was used to identify the hashtag oligo (HTO) tags from the cell / nucleus hashing FASTQ files. To assign the sample-of-origin for each cell the HTO and RNA count matrices, generated as described above, were further processed in R. For D16 data HTODemux function from Seurat v4.1.0 (Hao et al., 2021) was used for sample assignment, whereas for the remaining timepoints the HTO demultiplexing was done with an optimized cell classification strategy implemented in COMUNEQAID pipeline (https: / / github.com / CBMR-Single-Cell-OmicsPlatform / COMUNEQAID). Sinqle-cell / nuclei data processing and cluster annotation

[0142] The cells classified as doublets or negatives were excluded from the further analysis. The resulting count matrices were filtered and processed with Scanpy v1.9.5 (Wolf et al., 2018). Genes occurring in less than three cells, and cells with high mitochondrial content or outliers in gene counts, were removed from each batch individually by manually adjusted thresholds. Furthermore, to address bias arising from metabolism related factors ribosomal (RPS / RPL genes) and mitochondrial genes (MT- genes) were excluded from downstream analysis. The resulting data was log-normalized, and the day 25 batches and the day 50 and 70 batches were concatenated together. Leading to three different datasets referred to as d16, d25, and d50_d70, each of them processed and analyzed separately. The top 2000 variable genes were identified using batch-aware feature selection (d16 batch_key=None, d25 and d50_d70 batch_key = reactionlD). The d16 data underwent count depth regression, scaling, and dimensionality reduction (PCA and LIMAP). The cells sharing transcriptional similarities were clustered together with the Leiden algorithm (resolution^.85). The FOXG1 + cells, corresponding to forebrain contamination, not captured by the initial clustering, were distinguished from the clusters corresponding to anterior tuberal progenitors, ARC precursors, and neuronal precursors. To annotate the maturing neurons with higher resolution the cells in STMN2+ clusters were extracted and re-clustered

[0143] (resolution^.7). The d25 batches and d50_d70 batches were integrated with FastMNN implemented in batchelor package v1.14.0 (Haghverdi et al., 2018) using sequencing lane (reactionlD) as a batch key. After excluding an outlier cluster containing cells with high total counts and few differentially expressed genes, the d25 data was clustered (resolution 0.95). Followed by extraction and re-clustering of OTP+ clusters to distinguish an OTP- cell population. The integrated d50_d70 dataset was clustered (resolution= 1.025) and annotated with two different annotation resolutions low and high. To further investigate the cellular heterogeneity in d50_d70 data the RAX- / LHX8- clusters corresponding to ARC neurons and DIO2+ clusters corresponding to tanycytes were extracted. For both datasets, the top 2000 highly variable genes were computed (batch_key=reactionlD) followed by FastMNN batch integration and clustering (neurons resolution^.292 and tanycytes resolution^.14). From the neuronal dataset cluster containing the AgRP+ cells was further divided into two clusters through re-clustering to achieve an AgRP-specific population. An STMN2+ outlier cluster was excluded from the tanycyte dataset before downstream analysis. Unless for tanycyte data, which was not annotated, the resulting clusters in all the described datasets were identified and annotated based on our previous knowledge and markers described in the literature. Clusters deemed to be transcriptionally similar were merged. These processed datasets (d16, d25, d50_d70, d50_d70_tanycytes, d50_d70_neurons) formed the basis of the rest of the analysis.

[0144] Day 16 data developmental analysis

[0145] To unravel the differentiation direction of day 16 cells RNA velocity analysis was conducted using the stochastic model from scVelo vO.2.5 (Bergen et al., 2020) Python package. The resulting velocity stream was visualized on LIMAP embedding (Figure 1 B).

[0146] Results

[0147] After establishing an optimised v2 ARC protocol from hESCs (Figure 1A), the inventors performed single cell RNA sequencing analysis of 4 batches of day 16 stem cell-derived ARC progenitors in a single 10X experiment using CiteSeq Hashtagging (Stoeckius et al., 2017). From analysis of the 10X data, cell clusters were annotated (Figure 1 B). The data showed that the progenitor cells (i.e. the SOX2-positive, ST / W / V2-negative population of cells) were divided into two main clusters: one cluster of anterior tuberal progenitors expressing S0X2, RAX, NKX2-1 and SHH, and one cluster of posterior tuberal progenitors expressing S0X2, RAX, NKX2-1, TBX3 and FGF10 while being S / 7 / 7-negative (Figure 1 B and C). The inventors identified 6 novel cell surface markers enriched in the posterior tuberal progenitor cell population: SEMA5A, PTPRM, PCDH9, BRINP3, CNTN6 and ERBB4 (Figure 1 D and Table 1), and two other cell surface markers which were expressed in the entire anterior and posterior tuberal population: EDRNB and PTPRT (Figure 1E and Table 1). The inventors next studied scRNAseq data from day 25 of the same batches of v2 ARC protocol cells to allow greater resolution of the data. From this, a clear neuronal population marked by ST / W / V2and an early tanycyte population marked by S0X2 and DIO2 were identified (Figure 2A, B and Table 1). From this, DSCAM was identified as a cell surface marker expressed specifically in the neuronal population (Figure 2C and Table 1, and Figure 3), and SLCO1C1, GPC3, HTR2C, CD99, SDC2, FAT1, NR3C2 and TRPM3 were identified as cell surface markers enriched specifically in the early tanycyte population (Figure 2D and Table 1). Next, single nucleus RNAseq analysis of mature ARC cultures at day 50 and 70 (Figure 2E and F) was performed, maturing the same batches of ARC cells as were used in the original day 16 and day 25 analyses in Figure 1B and 2A. This analysis showed a large neuronal population (STMN2+) and a smaller mature tanycyte population (DIO2+) in the day 50+70 dataset (Figure 2E and D). From this analysis, we confirmed that the 8 novel tanycyte markers identified at day 25 SLCO1C1, GPC3, HTR2C, CD99, SDC2, FAT1, NR3C2 and TRPM3) were also enriched in the mature tanycyte population at day 50-70 (Figure 2F)

[0148] Conclusion

[0149] 6 novel cell surface markers enriched in posterior tuberal progenitor cell population were identified: SEMA5A, PTPRM, PCDH9, BRINP3, CNTN6 and ERBB4, and two other cell surface markers which were expressed in the entire anterior and posterior tuberal population were also identified : EDRNB and PTPRT. DSCAM was identified as a cell surface marker expressed specifically in the neuronal population SLCO1C1, GPC3, HTR2C, CD99, SDC2, FAT1, NR3C2 and TRPM3 were identified as cell surface markers enriched specifically in the early tanycyte population. The 8 novel tanycyte markers identified at day 25 (SL.CO1C1, GPC3, HTR2C, CD99, SDC2, FAT1, NR3C2 and TRPM3) were also found to be enriched in the mature tanycyte population at days 50-70.

[0150] Example 2

[0151] Purification of human in vitro derived arcuate nucleus cell types by CD99, GPC3 and DSCAM

[0152] Materials and methods

[0153] The materials and methods are as in Example 1 , with additional methods described below.

[0154] Fluorescence-activated cell sorting (FACS)

[0155] To test the potential of the identified cell surface candidates to sort populations of arcuate nucleus cell types from an in-vitro mixed cell population, FACS was used. Day 25 ARC cells were dissociated to a single cell suspension using 75 pL / cm2accutase (ThermoFisher, #A11105-01) and resuspended in FACS buffer containing PBS- / - with 1 % BSA (Miltenyi Biotec #130-091-376) and Pen / Strep / Neo (Thermo Fisher #15640055). The cell suspension was then incubated with antibodies for each of the three cell surface candidates CD99 (Biolegend #371305), GPC3 (R&D systems #FAB2119A) and DSCAM (R&D systems #AF3315) separately, for 30 minutes on ice. After that, cells were washed two times by centrifuging at 400 x g for 5 minutes and resuspending in FACS buffer. For DSCAM, a secondary antibody incubation was done with AF647 anti-goat (ThermoFisher Scientific #A-21447). Samples were finalized by adding the live / dead discriminator DAPI (1 :10.000 from 1 mg / mL ThermoFisher Scientific #62248). Single colour controls of DAPI stained only and secondary antibody stained only were used to set the gating. Based on these controls, the positive and negative populations of CD99, GPC3 and DSCAM stained cells were sorted into 5 mL FACS tubes. Each cell suspension was centrifuged at 1000 x g for 5 minutes and the pelleted cells were lysed in RLT plus buffer (QIAGEN #74034) containing 10% of p- mercaptoethanol (Millipore#444203). Samples were stored at -80°C until RNA extraction.

[0156] RNA extraction and quantification and analysis

[0157] The gene expression profiles of hESC-derived neuronal cultures were analysed via RTqPCR. Approx 300-500K cells were lysed at either day 16 progenitor stage or mature stage using 350 pL of RLT buffer (QIAGEN, #74034) containing 0.5 mM betamercaptoethanol (ThermoFisher, #31350010). RNA isolation was performed using a QIAcube using the RNeasy plus micro kit (Qiagen, #74034). Approx 1 pg of RNA was used for cDNA synthesis using Maxima first strand cDNA synthesis kit (ThermoFisher, #K1642) using random hexamer primers. cDNA was diluted in 250 pl EB buffer (Qiagen, #19086) and stored at -20°C degrees. Due to the high-throughput primer panel and large number of differentiations, SYBR green (Roche, #04887352001), forward (FWD) and reverse (REV) primers (SEQ ID NOs. 1 to 36) and subsequent cDNA sample were pipetted and mixed using iDOT liquid handler and analysed by Light Cycler 480 II instrument (Roche, #05015243001) with a 40 cycles program - 60°C for 60 s annealing / elongation step and 95°C, 30s denaturation. The average CT of technical duplicates was used to finalize the relative expression and this is normalized to a reference consisting of the average expression of 3 replicates of undifferentiated H9 and RC17 cells. Genes of interest we normalised to both GAPDH and ACTB.

[0158] Immunocytochemistry (ICC)

[0159] All 2D cultured cells were fixed in 4% PFA for 15 min at 37°C followed by 3 X washes with phosphate-buffered saline PBS- / - and stored at 4°C until required. For ICC, cells are blocked at room temperature for 1-3 h in blocking buffer (PBS- / - 0.1 %, Triton X-100 and 5% (vol / vol) donkey serum). Primary antibodies (Table 3) we diluted in blocking buffer and incubated overnight at 4°C. Following incubation, cells were washed 3 X in PBS - / - before incubation with 1:200 secondary conjugated fluorophores (Table 2) and DAPI 1 ng / ml for 2 hr room temperature on a shaker. A final 3 X PBS wash was performed before microscopic analysis. Spheroids were fixed with 4% Paraformaldehyde for 30 min at room temperature, washed twice with phosphate- buffered saline (PBS) and blocked for 6 h at 4°C with blocking buffer. Then, they were incubated with primary antibodies diluted in blocking buffer (200 pL / tube) on a shaker at 4°C for 48 h. The primary antibody solution was removed and the samples were washed with 300 pL blocking buffer, once for 2 min and then at 4°C for 6 h. All subsequent steps were performed under the exclusion of light. The secondary antibodies and 2 pg / mL DAPI for nuclear counterstaining were diluted in 200 pL blocking buffer and added to the tissues. The samples were again incubated on a shaker at 4°C for 48 h. Subsequently, the tissues were washed for 2 min and then at 4°C for 6 h on a shaker. After two more washes in PBS, the tissues were transferred to 18-well ibidi chambers and stored at 4°C until visualization.

[0160] Imaging and quantification

[0161] Imaging of ICC of in vitro-derived cells was performed on the Leica AF600 widefield epifluorescence microscope using Leica LAS-X software and for the main figures, the confocal microscope LSM 880 Airyscan, Zeiss. All imaging of staining was also performed using the LSM 880 Airyscan. Images were processed using Fiji (version 2.1.0). Images of transplantations (single and stacks) were acquired by confocal fluorescence (Stellaris, Plan-fluotar 10x / 0.30 Dry or Plan-apochromat 40x / 1.25 GLYC, Leica Microsystems, Wetzlar, Germany), widefield fluorescence (AF6000, plan-fluotar 10x / 0.30 Dry or plan-fluotar 7320x / 0.40 Dry, Leica Microsystems, Wetzlar, Germany) or widefield brightfield microscopy (Leica DM5500, plan-apochromatic 10x / 0.40, DFC450 colour camera). The images were processed using Imaged and brightness / contrast were adjusted using Adobe Photoshop 2024.

[0162] Results

[0163] The specificity of three cell surface candidates to mark their predicted cellular subpopulation in the ARC cultures were tested at day 25. For this purpose, fluorescently labelled antibodies for CD99, GPC3 and DSCAM were used and the analysis by fluorescence-activated cell sorting (FACS) revealed clear positive and negative populations for all three antibodies (Figure 4A, B and C). Upon qRT-PCR analysis of these sorted populations, the transcriptomic signatures confirmed high mRNA levels of the reference tanycyte markers DIO2, CRYM and NFIA in the GPC3 and CD99 positive sorted fraction (Figure 6A), and depletion of such markers in their respective negative sorted fractions. In particular, mRNA of early neuron reference markers OTP and PRDM12 was only present in the GPC3 negative sorted fraction (Figure 6B), confirming the specificity of this cell surface candidate to tanycytes. Furthermore, the DSCAM positive sorted fraction showed very low mRNA values for the tanycyte reference markers DIO2, CRYM and NFIA (Figure 6A), and high mRNA levels of the neuron reference marker PRDM12 (Figure 6B), verifying the specificity of this cell surface candidate to neurons. Additionally, an immunocytochemistry assay on ARC cultures at day 30 showed colocalization of CD99 and GCP3 with the reference tanycyte marker S100b (Figure 5A). The early neuron cell surface candidate DSCAM seems to bind specifically to neuronal cells (Figure 5B).

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[0176] Stoeckius, M., Hafemeister, C., Stephenson, W., Houck-Loomis, B., Chattopadhyay, P.K., Swerdlow, H., Satija, R., and Smibert, P. (2017). Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865-868. https: / / doi.org / 10.1038 / nmeth.4380. Wolf FA, Angerer P, Theis FJ. (2018) SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018; 19(1 ): 15. Published 2018 Feb 6. doi : 10.1186 / s 13059-017- 1382-0

[0177] Xie, Y., & Dorsky, R. I. (2017). Development of the hypothalamus: Conservation, modification and innovation. Development (Cambridge), 144(9), 1588±1599. https: / / doi.Org / 10.1242 / dev.139055

[0178] Yoo, S., Kim, J., Lyu, P., Hoang, T. V., Ma, A., Trinh, V., Dai, W., Jiang, L., Leavey, P., Duncan, L., Won, J.-K., Park, S.-H., Qian, J., Brown, S. P., & Blackshaw, S. (2021). Control of neurogenic competence in mammalian hypothalamic tanycytes. In Sci. Adv (Vol. 7, pp. 3777±3805). https: / / www.science.Org / doi / 10.1126 / sciadv.abg3777

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[0180] Items

[0181] 1. An in-vitro cell population of arcuate nucleus cell types, wherein said population expresses one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3.

[0182] 2. The in-vitro cell population according to item 1 , wherein said population is a posterior tuberal progenitor cells population and wherein said one or more cellsurface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3, PTPRP, EDNRB and ERBB4.

[0183] 3. The in-vitro cell population according to any one of items 1 and 2, wherein said population is a posterior tuberal progenitor cells population and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4. 4. The in-vitro cell population according to any one of the preceding items, wherein said population is a posterior tuberal progenitor cells population and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

[0184] 5. The in-vitro cell population of according to any one of the preceding items, wherein said population is an arcuate nucleus neurons population and wherein said one or more cell-surface markers is DSCAM.

[0185] 6. The in-vitro cell population according to any one of the preceding items, wherein said population is a tanycyte population and wherein said one or more cell surface markers are selected from the group consisting of: GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

[0186] 7. The in-vitro cell population according to item 6, wherein said one or more cellsurface markers are selected from the group consisting of: GPC3 and SDC2.

[0187] 8. The in-vitro cell population according to any one of items 6 to 7, wherein said one or more cell-surface markers are GPC3, and / or SDC2, and SLCO1C1.

[0188] 9. The in-vitro cell population according to any one of items 6 to 8, wherein said population does not express any of the cell-surface markers PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB.

[0189] 10. The in-vitro cell population according to any one of items 1 to 5, wherein said population does not express any of the cell-surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

[0190] 11. The in-vitro cell population according to any one of items 2 to 4 and 6 to 9, wherein said population does not express the cell-surface marker DSCAM.

[0191] 12. The in-vitro cell population according to any one of the preceding items, wherein: a) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and wherein the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population b) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and wherein the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population c) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population d) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population e) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population f) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population g) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population h) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population i) said arcuate nucleus cell type is arcuate nucleus neurons and wherein the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population j) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population k) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of the population l) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population m) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population n) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population o) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker FAT1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population p) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population and / or q) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker TRPM3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population. 13. The in-vitro cell population according to any one of the preceding items, for use in medicine.

[0192] 14. The in-vitro cell population according to any one of the preceding items for use in drug screening, such as metabolic disease drugs screening.

[0193] 15. A method for identifying a population of arcuate nucleus cell types from an in- vitro mixed cell population, said method comprising the step of: b) detecting the expression of one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB.DSCAM, GPC3, SDC2, SLCO1C1, HTR2C, CD99, FAT1 , NR3C2, and TRPM3 on single cells of the in-vitro mixed cell population, thereby identifying a population of arcuate nucleus cell types from the in-vitro mixed population.

[0194] 16. The method according to item 15, wherein said arcuate nucleus cell type is posterior tuberal progenitor cells and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4.

[0195] 17. The method according to any one of items 15 and 16, wherein said arcuate nucleus cell type is posterior tuberal progenitor cells and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

[0196] 18. The method according to any one of items 15 to 17, wherein said arcuate nucleus cell type is neurons, and wherein said one or more cell-surface marker is DSCAM.

[0197] 19. The method according to any one of items 15 to 18, wherein said arcuate nucleus cell type is tanycytes and wherein said one or more cell-surface markers selected from the group consisting of: GPC3, SDC2, SLCO1C1, HTR2C, CD99, FAT1, NR3C2, and TRPM3. 20. The method according to item 19, wherein said one or more cell-surface markers are selected from the group consisting of: GPC3 and SDC2.

[0198] 21. The method according to any one of items 19 and 20, wherein said one or more cell-surface markers are GPC3, and / or SDC2, and SLCO1C1.

[0199] 22. The method according to any one of items 19 to 21 further comprising a step of detecting the absence of expression of the cell-surface markers PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB on single cells of the mixed cell population.

[0200] 23. The method according to any one of items 15 to 18, further comprising a step of detecting the absence of expression of the cell-surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3 on single cells of the mixed cell population

[0201] 24. The method according to any one of items 15 to 17 and 19 to 23, further comprising a step of detecting the absence of expression of the cell-surface marker DSCAM on single cells of the mixed cell population.

[0202] 25. The method according to any one of items 17 to 24, wherein the step of detecting the absence of expression of the cell-surface marker is performed before, simultaneously, and / or after, the step of detecting the expression of the one or more cell-surface markers.

[0203] 26. The method according to any one of items 15 to 25, further comprising a step of labelling cells expressing the one or more cell-surface markers.

[0204] 27. The method according to item 26 wherein the labelling is with one or more labelled binding members arranged to bind the one or more cell surface markers, preferably wherein the labelled binding members are fluorescently labelled antibody(ies), nanobodies or microbodies specific for the one or more cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for the one or more cell-surface marker. 28. The method according to any one of items 15 to 27, further comprising a step of sorting from said mixed cell population the cells in which said expression of cell-surface markers is detected.

[0205] 29. The method according to any one of items 22 to 28, further comprising a step of sorting from said mixed cell population the cells in which said absence of cellsurface marker expression is detected.

[0206] 30. The method according to any one of items 28 to 29, wherein the step of sorting from said mixed cell population the cells in which said expression of cell-surface markers is detected is performed before, during, and / or after, the step of sorting from said mixed cell population the cells in which said absence of cell-surface marker expression is detected.

[0207] 31. The method according to any one of items 15 to 30, wherein the step of detection of the expression of the one or more cell-surface markers is performed by immunocytochemistry or flow cytometry..

[0208] 32. The method according to any one of items 15 to 31 , wherein the step of detection of the expression and / or the step of sorting the cells is performed by flow cytometry, such as fluorescence activated cell sorting (FACS), Magnetic- Activated Cell Sorting (MACS), or microfluidic cell sorting.

[0209] 33. The method according to any one of the preceding items, wherein: a) the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population b) the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population c) the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the in-vitro mixed population d) the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population e) the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population f) the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population g) the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population h) the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population i) the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population j) the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population k) the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of the in-vitro mixed population l) the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population m) the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population n) the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population o) the cell surface marker FAT1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population p) the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population and / or q) the cell surface marker TRPM3 is expressed in at least 1 %, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the in-vitro mixed population. according to any one of the preceding items, wherein: a) said population of arcuate nucleus cell type is anterior or posterior tuberal progenitors, and wherein the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population. b) said population of arcuate nucleus cell type is anterior or posterior tuberal progenitors and wherein the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population c) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population d) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population e) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population f) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population g) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population h) said population of arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population i) said population of arcuate nucleus cell type is arcuate nucleus neurons and wherein the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population j) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population k) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of said population l) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker HTR2C is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population m) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population n) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population o) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker FAT 1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population p) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population and / or q) said population of arcuate nucleus cell type is tanycytes and wherein the cell surface marker TRPM3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of said population. ating the in-vitro population of arcuate nucleus cell types according f items 1 to 14, said kit comprising : a) labelled binding members arranged to bind one or more cellsurface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1, NR3C2, and TRPM3, preferably wherein said binding members are antibody(ies), nanobodies or microbodies specific for said cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for said cellsurface markers; b) Instructions performing the method according to any one of items 15 to 34.

Claims

Claims1 . An in-vitro cell population of arcuate nucleus cell types, wherein said population expresses one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

2. The in-vitro cell population according to claim 1 , wherein said population is a posterior tuberal progenitor cells population and wherein said one or more cellsurface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3, PTPRP, EDNRB and ERBB4, preferably wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4.

3. The in-vitro cell population according to any one of the preceding claims, wherein said population is a posterior tuberal progenitor cells population and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

4. The in-vitro cell population of according to any one of the preceding claims, wherein said population is an arcuate nucleus neurons population and wherein said one or more cell-surface markers is DSCAM.

5. The in-vitro cell population according to any one of the preceding claims, wherein said population is a tanycyte population and wherein said one or more cell surface markers are selected from the group consisting of: GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably wherein said one or more cell surface markers are selected from the group consisting of: GPC3 and SDC2.

6. The in-vitro cell population according to claim 5, wherein said one or more cellsurface markers are GPC3, and / or SDC2, and SLCO1C1.

7. The in-vitro cell population according to any one of claims 4 to 6, wherein said population does not express any of the cell-surface markers PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, and EDNRB.

8. The in-vitro cell population according to any one of claims 1 to 4, wherein said population does not express any of the cell-surface markers GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3.

9. The in-vitro cell population according to any one of claims 2 to 3 and 5 to 6, wherein said population does not express the cell-surface marker DSCAM.

10. The in-vitro cell population according to any one of the preceding claims, wherein: a) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and wherein the cell surface marker EDNRB is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 67%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, b) said arcuate nucleus cell type is anterior or posterior tuberal progenitors and wherein the cell surface marker PTPRT is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, c) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker SEMA5A is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, suchas at least 70%, such as at least 80%, such as at least 86%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, d) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PTPRM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 94%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, e) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker PCDH9 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 73%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, f) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker BRINP3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 41%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, g) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker CNTN6 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population,h) said arcuate nucleus cell type is posterior tuberal progenitors and wherein the cell surface marker ERBB4 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, i) said arcuate nucleus cell type is arcuate nucleus neurons and wherein the cell surface marker DSCAM is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 76%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, j) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker SLCO1C1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, k) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker GPC3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 15%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 99%, such as at least 100% of the cells of the population, l) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker HTR2C is expressed in at least 1%, such as atleast 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, m) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker CD99 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 34%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, n) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker SDC2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 47%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, o) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker FAT 1 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 78%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, p) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker NR3C2 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%,such as at least 72%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population, and / or q) said arcuate nucleus cell type is tanycytes and wherein the cell surface marker TRPM3 is expressed in at least 1%, such as at least 2.5%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 54%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% of the cells of the population.

11. A method for identifying a population of arcuate nucleus cell types from an in- vitro mixed cell population, said method comprising the step of: a) detecting the expression of one or more cell-surface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB.DSCAM, GPC3, SDC2, SLCO1C1, HTR2C, CD99, FAT1, NR3C2, and TRPM3 on single cells of the in-vitro mixed cell population, thereby identifying a population of arcuate nucleus cell types from the in-vitro mixed population.

12. The method according to claim 11 , wherein said arcuate nucleus cell type is posterior tuberal progenitor cells and wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, SEMA5A, CNTN6, PCDH9, BRINP3 and ERBB4, preferably wherein said one or more cell-surface markers are selected from the group consisting of: PTPRM, ERBB4, PTPRP and SEMA5A.

13. The method according to claim 11 , wherein said arcuate nucleus cell type is neurons, and wherein said one or more cell-surface marker is DSCAM.

14. The method according to claim 11 , wherein said arcuate nucleus cell type is tanycytes and wherein said one or more cell-surface markers selected from thegroup consisting of: GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably wherein said one or more cell-surface markers are selected from the group consisting of: GPC3 and SDC2.

15. A kit for isolating the in-vitro population of arcuate nucleus cell types according to any one of claims 1 to 10, said kit comprising : a) labelled binding members arranged to bind one or more cellsurface markers selected from the group consisting of: PTPRM, ERBB4, PTPRP, SEMA5A, PCDH9, BRINP3, CNTN6, EDNRB, DSCAM, GPC3, SDC2, SLCO1C1 , HTR2C, CD99, FAT1 , NR3C2, and TRPM3, preferably wherein said binding members are antibody(ies), nanobodies or microbodies specific for said cell surface markers, or magnetic beads conjugated to antibody(ies), nanobodies or microbodies specific for said cellsurface markers; b) instructions for performing the method according to any one of claims 11 to 14.

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