Method for detecting or removing undifferentiated cells remaining in neural cell population

Abstract

Provided is a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, the method comprising: (1) a step for bringing a sugar chain-binding molecule into contact with the neural cell population; and (2) a step for detecting or removing cells having the sugar chain-binding molecule bound thereto. In the method, (a) the sugar chain-binding molecule binds to Lewis X, and the undifferentiated cells are undifferentiated neural progenitor cells, or (b) the sugar chain-binding molecule binds to N-acetyllactosamine or poly-N-acetyllactosamine, and the undifferentiated cells are mesenchymal-like cells.

Description

Method for detecting or eliminating undifferentiated cells remaining in a neural cell population

[0001] The present invention relates to a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells.

[0002] Methods for inducing differentiation of pluripotent stem cells into specific cell types have been developed, and various types of cells prepared from pluripotent stem cells are used in regenerative medicine and drug discovery. However, cell populations prepared from pluripotent stem cells typically contain residual undifferentiated cells, posing a risk of tumorigenicity for transplantation. Furthermore, cell assay systems used in drug discovery face the problem of reduced purity of target cells due to contamination with undifferentiated cells. Therefore, there is a need for a technology to detect and remove residual undifferentiated cells from cell populations prepared from pluripotent stem cells.

[0003] It is known that neural cell populations induced from pluripotent stem cells are contaminated with undifferentiated cells such as neural progenitor cells and mesenchymal cells that are resistant to neural induction, and techniques for detecting or removing them have been developed. For example, it has been reported that treating neural cell populations induced from pluripotent stem cells with a gamma-secretase inhibitor can promote the maturation of the neural cell population and suppress tumorigenicity after transplantation (Non-Patent Document 1). In addition, a probe specific for translocator protein 18 kDa (TSPO) expressed on the outer mitochondrial membrane of undifferentiated neural progenitor cells [ １８ A technique for detecting tumorigenic undifferentiated neural progenitor cells by PET imaging using [F]FEDAC has been reported (Non-Patent Document 2).

[0004] On the other hand, it is known that the type and state of cells can be identified based on glycosylation markers present on the cell surface. Because cell surface markers are easy to detect, they enable highly efficient and accurate cell identification. However, no glycosylation markers specific to undifferentiated cells remaining in neural cell populations induced from pluripotent stem cells have been reported, nor have any techniques for detecting and removing undifferentiated cells based on such markers been reported.

[0005] Okubo, T. et al. , Stem Cell Reports, 2016; (4): 649-663 Tanimoto, Y. et al. , Stem Cells Transl. Med. , 2020;9(4):465-477

[0006] The present invention has been made with the aim of easily and highly accurately detecting and removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells.

[0007] As a result of extensive research, the present inventors have succeeded in identifying sugar chains that are specifically present on the surface of undifferentiated cells, thereby completing the present invention.

[0008] That is, according to one embodiment, the present invention provides a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, comprising: (a1) a step of contacting the neural cell population with a first glycan-binding molecule, wherein the first glycan-binding molecule binds to Lewis X; (a2) a step of detecting or removing cells to which the first glycan-binding molecule has bound in step (a1), wherein the cells to which the first glycan-binding molecule has bound are undifferentiated neural progenitor cells; and / or (b1) a step of contacting the neural cell population with a second glycan-binding molecule, wherein the second glycan-binding molecule binds to N-acetyllactosamine or poly-N-acetyllactosamine; and (b2) a step of detecting or removing cells to which the second glycan-binding molecule has bound in step (b1), wherein the cells to which the second glycan-binding molecule has bound are mesenchymal-like cells.

[0009] In the above method, steps (a1) and (b1) may be performed simultaneously or sequentially in any order, and steps (a2) and (b2) may be performed simultaneously or sequentially in any order.

[0010] The sugar chain-binding molecule is preferably a lectin or an antibody.

[0011] The carbohydrate-binding molecule is preferably conjugated to a detectable label.

[0012] The carbohydrate-binding molecule is preferably conjugated to a cytotoxic substance.

[0013] The pluripotent stem cells are preferably iPS cells, and more preferably human iPS cells.

[0014] Preferably, the undifferentiated neural progenitor cells express NES, PAX6, VIM and SOX1.

[0015] The mesenchymal-like cells preferably express NES, ACTA2 and PDGFRB.

[0016] The method of the present invention enables specific detection or removal of undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, and is therefore useful for preparing a highly safe neural cell population for transplantation and for developing a high-quality assay system using a highly pure neural cell population.

[0017] Figure 1 is a schematic diagram showing the schedule for generating iPS cell-derived neural cell populations. Figure 2 shows fluorescent immunostained images (top) and Hoechst stained images (bottom) of iPS cells (iPSCs), neural progenitor cells (NPCs), and neural cell populations (neurons) using anti-TUJ1 antibodies. Figure 3 shows UMAP plots of RNA expression and glycan reactivity information for iPSCs, NPCs, and neurons. Figure 4 shows UMAP plots of RNA expression and glycan reactivity information for neurons only. Figure 5 shows heat maps showing the expression profiles of marker genes in mature neurons (mNeurons), immature neurons (imNeurons), undifferentiated NPCs (undiffNPCs), and mesenchymal-like cells (MCs). Figure 6 shows dot plots showing the reactivity of mNeurons, imNeurons, undiffNPCs, and MCs to various lectins. Figure 7 is a violin plot showing the amount of rAAL binding and the expression level of FUT10 gene in mNeuron, imNeuron, undiffNPC, and MC. Figure 8 is a violin plot showing the amount of rLSLN binding and the expression levels of B4GALT1 and B3GNT2 genes in mNeuron, imNeuron, undiffNPC, and MC. Figure 9 is a diagram showing fluorescent immunostained images (top) and Hoechst stained images (bottom) of Neuron using anti-SSEA1 antibody and anti-Nestin antibody. Figure 10 is a diagram showing fluorescent immunostained images (top) and Hoechst stained images (bottom) of Neuron using Cy3-labeled rLSLN. Figure 11 is a histogram showing the results of flow cytometry analysis of SSEA1-positive cells in iPSC-derived neuronal cell populations untreated (control) or treated (IR700-SSEA1) with IR700-labeled anti-SSEA1 antibody. Figure 12 shows the results of flow cytometry gating of rLSLN+ / PDGFRB+ cells in iPSC-derived neuronal cell populations untreated (control) or treated (LSL 0.3 μg / mL or 1 μg / mL).

[0018] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0019] According to a first embodiment, the present invention provides a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, comprising: (a1) a step of contacting the neural cell population with a first glycan-binding molecule, wherein the first glycan-binding molecule binds to Lewis X; (a2) a step of detecting or removing cells to which the first glycan-binding molecule has bound in step (a1), wherein the cells to which the first glycan-binding molecule has bound are undifferentiated neural progenitor cells; and / or (b1) a step of contacting the neural cell population with a second glycan-binding molecule, wherein the second glycan-binding molecule binds to N-acetyllactosamine or poly-N-acetyllactosamine; and (b2) a step of detecting or removing cells to which the second glycan-binding molecule has bound in step (b1), wherein the cells to which the second glycan-binding molecule has bound are mesenchymal-like cells.

[0020] The method of this embodiment targets a neural cell population induced from pluripotent stem cells. Examples of "pluripotent stem cells" include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic germ (EG) cells, pluripotent germ stem (mGS) cells, and Muse cells. The neural cell population targeted by the method of this embodiment may be derived from any pluripotent stem cell, but is preferably derived from ES cells or iPS cells, and more preferably derived from iPS cells.

[0021] The pluripotent stem cells in this embodiment may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, sheep, goats, pigs, cows, monkeys, and humans, and particularly preferably from humans.

[0022] Methods for preparing pluripotent stem cells have been well established (e.g., for iPS cells, see Cell, 2007; 131(5): 861-872, doi: 10.1016 / j.cell.2007.11.019), and pluripotent stem cells can be prepared from any tissue or cell according to methods known in the art. Alternatively, already established iPS cell lines or ES cell lines may be obtained from, for example, the Kyoto University iPS Cell Research Foundation (CiRA_F), the RIKEN BioResource Research Center (RIKEN BRC), the American Type Culture Collection (ATCC), or the like.

[0023] In this embodiment, the term "neuronal cell population" refers to a cell population substantially composed of neurons obtained by inducing differentiation of pluripotent stem cells into neurons. Therefore, the neural cell population of this embodiment may contain, for example, at least 50%, preferably 80% or more, neurons.

[0024] Methods for inducing pluripotent stem cells into neurons have already been well established (e.g., Chambers, S.M. et al., Nat. Biotechnol., 27:275-280, 2009). The neuronal cell population in this embodiment may be prepared from pluripotent stem cells according to any method known in the art. Specifically, a neuronal cell population can be prepared from pluripotent stem cells by culturing the pluripotent stem cells under conditions in which, for example, a basal medium such as DMEM / F-12 medium, N2 medium, or a mixture thereof is appropriately supplemented with a BMP signaling pathway inhibitor such as dorsomorphin, a TGF signaling pathway inhibitor such as SB431542, or a Wnt signaling pathway inhibitor such as XAV-939.

[0025] In this embodiment, "undifferentiated cells" refer to cells that are resistant to neural induction and retain pluripotency (the ability to differentiate into cells other than neurons) and self-renewal ability even under neural induction conditions. Therefore, the undifferentiated cells in this embodiment are specifically undifferentiated neural progenitor cells or mesenchymal-like cells that remain after neural induction of pluripotent stem cells.

[0026] In this embodiment, "undifferentiated neural progenitor cells" and "mesenchymal-like cells" can be defined based on the expression of undifferentiated cell markers, such as stemness markers, proliferative cell markers, neural progenitor cell markers, and mesenchymal stem cell markers. In other words, even cells that morphologically exhibit a shape similar to that of differentiated neural cells may be included in the undifferentiated cells of this embodiment as long as they express undifferentiated cell markers. Furthermore, the undifferentiated neural progenitor cells of this embodiment may also include neural stem cells.

[0027] In this embodiment, the undifferentiated neural progenitor cells preferably express, for example, NES (nestin gene), PAX6 (paired box 6 gene), VIM (vimentin gene), and SOX1 (SRY-related HMG box 1 gene), and more preferably further express one or more markers selected from the group consisting of SOX2 (SRY-related HMG box 2 gene), HES5 (Hes family bHLH transcription factor 5 gene), ID4 (DNA-binding inhibitor 4 gene), PCNA (proliferating cell nuclear antigen gene), and MKI67 (KI-67 antigen gene). In this embodiment, the mesenchymal-like cells preferably express, for example, NES, ACTA2 (smooth muscle α2 actin gene), and PDGFRB (platelet-derived growth factor receptor β gene), and more preferably further express one or more markers selected from the group consisting of PAX6, VIM, MCAM (melanoma cell adhesion molecule gene), DES (desmin gene), NT5E (5′-nucleotidase gene), and ENG (endoglin gene).

[0028] On the other hand, it is more preferable that the undifferentiated neural progenitor cells and mesenchymal-like cells in this embodiment do not express neuronal markers and synaptic markers, such as CD24 (signal transduction factor CD24 gene), DCX (doublecortin gene), GAP43 (growth-associated protein 43 gene), MAPT (microtubule-associated protein tau gene), STMN2 (stathmin 2 gene), and TUBB3 (tubulin β3 gene). Examples of synaptic markers include NRXN1 (neurexin 1 gene), NRXN2 (neurexin 2 gene), NRXN3 (neurexin 3 gene), SHANK1 (SH3 and multiple ankyrin repeat protein 1), SHANK2 (SH3 and multiple ankyrin repeat protein 2), SHANK3 (SH3 and multiple ankyrin repeat protein 3), SYT1 (synaptotagmin 1 gene), SYT2 (synaptotagmin 2 gene), SYT3 (synaptotagmin 3 gene), and STX1A (syntaxin 1A gene).

[0029] Marker expression can be analyzed by known techniques such as RT-PCR, Western blotting, and flow cytometry.

[0030] In the method of this embodiment, a glycan-binding molecule is used to identify undifferentiated cells. Since the glycans displayed on the cell surface differ depending on the type and state of the cell, undifferentiated cells can be identified based on the binding of glycans specifically present on the surface of undifferentiated cells to glycan-binding molecules.

[0031] In the method of this embodiment, (a) a glycan-binding molecule that binds to Lewis X (LeX, also known as SSEA1 or CD15) is used to identify undifferentiated neural progenitor cells, and (b) a glycan-binding molecule that binds to N-acetyllactosamine (LacNAc) or poly-N-acetyllactosamine (polyLacNAc) is used to identify mesenchymal-like cells. LeX is a glycan consisting of a structure represented by Galβ1-4(Fucα1-3)GlcNAc. LacNAc is a glycan consisting of a structure represented by Galβ1-4GlcNAc.

[0032] In the method of this embodiment, either one or both of (a) LeX-based detection or elimination of undifferentiated neural progenitor cells and (b) (poly)LacNAc-based detection or elimination of mesenchymal-like cells can be performed, and preferably both can be performed. When both (a) and (b) are performed, (a) and (b) may be performed sequentially or simultaneously. When (a) and (b) are performed sequentially, the order is not particularly limited, and (b) may be performed after (a), or vice versa.

[0033] The "glycan-binding molecule" in this embodiment may be any molecule capable of recognizing LeX or (poly)LacNAc, including, but not limited to, proteins such as lectins or antibodies, and nucleic acids such as aptamers. The glycan-binding molecule in this embodiment may preferably be a lectin or an antibody.

[0034] The "antibody" in this embodiment may be either a polyclonal antibody or a monoclonal antibody, and the monoclonal antibody may be any of a mouse antibody, chimeric antibody, humanized antibody, and fully human antibody. Furthermore, the antibody in this embodiment may also include antigen-binding fragments having equivalent glycan-recognition ability, such as Fab, F(ab')2, scFv, and nanobody.

[0035] Methods for preparing antibodies are well established, and anti-LeX antibodies and anti-(poly)LacNAc antibodies can be prepared according to any method known in the art. Anti-LeX antibodies and anti-(poly)LacNAc antibodies are commercially available, and they may be used in the method of this embodiment.

[0036] "Lectin" is a general term for proteins other than antibodies that recognize and bind to glycans, and is found in a wide range of organisms, from animals to plants, fungi, and viruses. In the method of this embodiment, any type of lectin derived from any organism can be used as long as it can recognize LeX or (poly)LacNAc with high accuracy. Lectins in this embodiment may include not only those that specifically bind to LeX or (poly)LacNAc, but also those that can bind to other glycans but bind to LeX or (poly)LacNAc with sufficiently high affinity (e.g., at least twice as high). Furthermore, lectins in this embodiment may also include partial fragments or recombinant forms that have equivalent glycan recognition ability.

[0037] Examples of lectins that recognize LeX include Ralstonia solanacearum-derived lectin (RSL), Ralstonia solanacearum-derived lectin 2 (RSIIL), Aleuria aurantia-derived lectin (AAL), Aspergillus oryzae-derived lectin (AOL), Lotus Tetragonolobus-derived lectin (LTL), and Pseudomonas aeruginosa-derived lectin (PAIIL). Examples of lectins and recombinant forms thereof that recognize (poly)LacNAc include Aikawatake (Laetiporus sulphureus)-derived lectin (LSL), Aikawatake lectin N-terminal domain recombinant (rLSLN), cellular slime mold (Dictyostelium dicodeum)-derived lectin (Discoidin II), human-derived galectin 3 (rGal3C), and tomato (Lycopersicon esculentum)-derived lectin (LEL).

[0038] Lectins may be prepared by any method known in the art. For example, they may be isolated from the organisms from which they originate, or nucleic acids encoding the lectins may be prepared by genetic engineering and introduced into host cells such as Escherichia coli for expression. Information on the amino acid sequences of lectins and the nucleic acid sequences encoding them can be obtained from a designated database via a portal site such as the GlyCosmos Portal (https: / / glycosmos.org / ). For example, for LSL, UniProt ID: Q7Z8V1 and GenBank ID: AB112940.1 are available. The above-mentioned lectins and their recombinant forms are also commercially available, and these may be used in the method of this embodiment.

[0039] The glycan-binding molecule in this embodiment may be conjugated to a detectable label. By using a glycan-binding molecule conjugated to a detectable label, undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells can be easily detected or removed, preferably detected. Detectable labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, metal particles, etc. The detectable label in this embodiment may preferably be a fluorescent dye or an enzyme.

[0040] Furthermore, the glycan-binding molecule of this embodiment may be conjugated to a cytotoxic substance instead of or in addition to a detectable label. By using a glycan-binding molecule conjugated to a cytotoxic substance, undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells can be easily detected or removed, preferably removed. Examples of cytotoxic substances include, but are not limited to, anticancer drugs such as methotrexate, cyclosporine, and cisplatin; cytotoxic peptides such as diphtheria toxin, Pseudomonas exotoxin A, saporin, and ricin; radionuclides such as iodine-131, rhenium-186, indium-111, and yttrium-90; and photosensitizers such as phthalocyanine derivatives (e.g., IRDye700DX), merocyanine derivatives (e.g., merocyanine-540), and chlorin derivatives (e.g., talaporfin). The cytotoxic substance of this embodiment may preferably be a photosensitizer.

[0041] The detectable label or cytotoxic substance may be conjugated to the carbohydrate-binding molecule directly or via a linker according to any method known in the art. For example, if the carbohydrate-binding molecule is an antibody or a lectin, the detectable label or cytotoxic substance may be conjugated to any one or more positions of the antibody or lectin, for example, to either or both of the N-terminus and C-terminus.

[0042] In the method of this embodiment, a glycan-binding molecule is contacted with a neuronal population. To contact the glycan-binding molecule with a neuronal population, the glycan-binding molecule is added to a culture medium for the neuronal population and incubated for a certain period of time. The glycan-binding molecule may be added at a final concentration of, for example, 1 to 10 μg / mL, preferably 5 to 10 μg / mL. The incubation time may be, for example, 0.5 to 3 hours, preferably 1 to 2 hours.

[0043] Next, the cells to which the glycan-binding molecule binds are detected or removed. In this embodiment, the term "removal" (and grammatical variations thereof) includes not only the complete removal of undifferentiated cells from a neuronal population, but also a reduction in the number or proportion of undifferentiated cells.

[0044] Cells to which glycan-binding molecules are bound can be detected by any method known in the art, including, but not limited to, fluorescence detection and chemiluminescence detection. In the method of this embodiment, cells to which glycan-binding molecules are bound are preferably detected by fluorescence.

[0045] Cells to which glycan-binding molecules are bound can be removed by any method known in the art, including, but not limited to, killing with a cytotoxic substance, separation by flow cytometry, separation by magnetic beads, etc. In the method of this embodiment, cells to which glycan-binding molecules are bound are preferably removed by a cytotoxic substance.

[0046] The method of this embodiment distinguishes differentiated cells from undifferentiated cells in a neural cell population induced from pluripotent stem cells based on a cell surface glycan marker, and therefore, the method of this embodiment makes it possible to easily and accurately detect and remove undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells.

[0047] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0048] <1. Preparation of iPS cell-derived neural cell population> (1-1) Neural differentiation of iPS cells Human iPS cells (201B7 line) (hereinafter simply referred to as "iPS cells" or "iPSCs") were obtained from RIKEN BRC. iPS cells were cultured on plates coated with Matrigel human ES cell-optimized matrix (Corning) using mTeSR+ medium (Veritas). iPSCs with passage numbers less than 60 were used for all experiments.

[0049] iPSCs were induced to differentiate into neural progenitor cells (NPCs) using the STEMdiff SMADi Neural Induction Kit (Veritas) according to the attached protocol. NPCs were cultured on plates coated with Matrigel human ES cell-optimized matrix using STEMdiff SMADi Neural Induction Medium (Veritas). NPCs at passage number less than 5 were used for all experiments.

[0050] For differentiation into neurons, NPCs were detached using Accutase (Innovative Cell Technologies) and dispersed into single cells. They were then seeded onto plates coated with 0.07% polyethyleneimine (Sigma-Aldrich) and 3.3 μg / mL laminin (Fujifilm Wako Pure Chemical Industries) and cultured in NeuroBasal Medium (Thermo Fisher Scientific) supplemented with 2% B27 supplement (Thermo Fisher Scientific), 1% GlutaMax supplement (Thermo Fisher Scientific), and 5 μM DAPT (Sigma-Aldrich). On day 7, the medium was replaced with NeuroBasal Plus Medium supplemented with 2% B27 Supplement Plus (Thermo Fisher Scientific) and 1% GlutaMax Supplement, and half of the medium was replaced twice a week thereafter.

[0051] The schedule for generating neural cell populations is outlined in Figure 1. NPCs induced from iPSCs were passaged up to P4, and neural cell populations (hereinafter also referred to as "neurons") were obtained by culturing the NPCs under neuronal differentiation conditions for 21 to 28 days.

[0052] (1-2) Immunocytostaining To confirm differentiation into neurons, immunostaining was performed using a monoclonal antibody against the neuronal marker tubulin β3 (TUBB3, hereafter referred to as "TUJ1"). Cells were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) and washed with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereafter referred to as "PBS"), followed by incubation with PBS containing 1% BSA (Sigma-Aldrich) and 0.2% Triton X-100 (Nacalai Tesque) at room temperature for 30 minutes. After washing three times with D-PBS(-) (hereafter referred to as "PBS"), primary antibody solution (anti-TUJ1 antibody (Merck Millipore, MAB1637), diluted 1 / 500) was added and incubated overnight at 4°C. After washing three times with PBS, a solution of secondary antibody (Cy3-labeled anti-mouse IgG (Jackson ImmunoResearch), diluted 1 / 2000) and Hoechst 33342 (Dojindo Chemical Industries) (1 μg / mL) was added and incubated at room temperature for 1 hour. After washing three times with PBS, images were taken with an inverted fluorescence microscope (IX51, Olympus).

[0053] The results are shown in Figure 2. Compared to iPSCs and NPCs, Neuron showed strong TUJ1 staining and neurite structures. These results demonstrated that a neural cell population could be prepared from iPSCs.

[0054] <2. scGR-seq analysis of iPSC-derived neuronal populations> Glycan markers specific to non-neuronal cells remaining in iPSC-derived neuronal populations were searched for using single-cell glycan / RNA sequencing (scGR-seq) (Minoshima, F. et al., 2021; iScience, 24(8):102882). iPSCs and NPCs were detached using Accutase and dispersed into single cells. Neurons were detached using Accutase supplemented with 50 units / mL papain (Worthington Biochemical) and 0.25 mg / mL L-cysteine ​​(Sigma-Aldrich) and dispersed into single cells. Cells (1 x 10 ５The cells (800 cells) were incubated in 1% BSA / PBS containing 0.5 μg / mL of the DNA oligonucleotide-labeled lectin library (Table 1) at 4°C for 1 hour. The cells were irradiated with 365 nm ultraviolet light (15 W) for 15 minutes using a UV bench lamp (UVP Blak-Ray XX-15L, Analytik Jena). The cells were collected by centrifugation for 30 seconds, and the supernatant containing the DNA barcodes was transferred to a new PCR tube. The cells were then purified by GenNext. ＴＭ RamDA-seq ＴＭ The cells were dissolved in the lysis buffer of the Single Cell Kit (Toyobo). The DNA barcodes in the supernatant were amplified for 20 cycles by PCR using i5 / i7 index primer (New England Biolabs) and NEBNext Ultra II Q5 Master Mix (New England Biolabs). The PCR product was purified using Agencourt AMPure XP (Beckman Coulter). The quality of the purified PCR product was confirmed using MultiNA (Shimadzu Corporation), and the nucleic acid sequence was determined using a MiSeq sequencer (Illumina) (26 bp, paired end). The RNA library for scGR-seq was prepared using GenNext. ＴＭ RamDA-seq ＴＭThe RNA library was prepared using the Single Cell Kit according to the accompanying protocol. The quality of the RNA library was confirmed using MultiNA, and the nucleic acid sequence was determined using Nova-Seq6000 (Illumina) (151 bp, paired-end) and HiSeqX (Illumina) (151 bp, paired-end). The sequence data of the DNA barcodes was analyzed using the Barcode DNA counting system (https: / / github.com / bioinfo-tsukuba / barcode-dna-counting-system), and raw count data for each lectin was obtained. The sc-RNAseq sequence data was analyzed using fastp (version 0.22.0), HISAT2 (version 2.2.0), and StringTie (version 2.1.1) to obtain raw count data mapped to GRCh38. Raw count data derived from three samples (iPSC, NPC, and Neuron), or from Neuron samples alone, was quality-controlled and normalized using the Seurat R package (version 4.0.2). Glycan (DNA barcode) and RNA data were integrated using a weighted nearest neighbor algorithm, and dimensionality was reduced using the nonlinear dimensionality reduction method UMAP (Uniform Manifold Approximation and Projection). Data from the three iPSC, NPC, and Neuron samples were grouped by sample and plotted using a UMAP plot. For the Neuron sample data, clustering analysis (Louvain algorithm) was performed to identify cell subpopulations and classify them into clusters. Differentially expressed genes were then analyzed for each cluster, and lectins with variable reactivity were extracted.

[0055] Table 1. List of DNA oligonucleotide-labeled lectins (The abbreviations in the table are as follows.Gal: D-galactose, GalNAc: N-acetyl-galactosamine, Glc NAc: N-acetyl-glucosamine, Fuc: L-fucose, Glc: D-glucose, Sia: Sialic acid, LacNAc: N-acetyl-lactosamine, JOM: J-OIL MILLS, INC., Vector: VECTOR LABORATORIES, INC., Seikagaku: SEIKAGAKU CORP. LABORATORIES, INC., AIST: National Institute of Advanced Industrial Science and Technology, R&D: R&D systems, JIR: Jackson ImmunoResearch. Lectin names beginning with "r" indicate recombinant.

[0056] The analysis results for iPSCs, NPCs, and neurons are shown in Figure 3. In the figure, the X-axis (UMAP_1) and Y-axis (UMAP_2) represent orthogonal components obtained by dimensionally compressing the RNA and glycan data. The clusters for each cell population were distributed without overlapping. Furthermore, cluster analysis was performed on neuron data alone, and the results, depicted in a UMAP plot, are shown in Figure 4. Neurons were classified into four subclusters. Based on their RNA expression patterns, these subclusters were named mature neurons (mNeuron), immature neurons (imNeuron), undifferentiated NPCs (undiffNPCs), and mesenchymal-like cells (MCs).

[0057] Figure 5 shows the expression profiles of marker genes in each cluster. Marker genes expressed in neurons in general (pan-Neuron in the figure) were expressed in mNeuron and imNeuron, whereas synaptic marker genes expressed in mature neurons (Synapse in the figure) were highly expressed in mNeuron. Marker genes expressed in NPCs in general (pan-NPC in the figure) were expressed in undiff NPCs and MCs. Neural stem cell marker genes (Stemness in the figure) and proliferating cell marker genes (Proliferation) were highly expressed in undiff NPCs, suggesting that undiff NPCs have high stemness. Genes indicating mesenchymal properties (Mesenchyme) were highly expressed in MCs. These results revealed that in addition to neurons, iPSC-derived neural cell populations contain subpopulations of undifferentiated NPCs (undiffNPCs) with high stemness and NPCs (MCs) with mesenchymal cell properties.

[0058] For lectins whose reactivity differed significantly (p<0.05) between subclusters, the reactivity (binding ratio) of each lectin to each subcluster was depicted in a dot plot (Figure 6). In the figure, the size of the circle indicates the prevalence of lectin reactivity (the percentage of cells with a non-zero value), and the gray scale indicates the intensity of reactivity (the average of the values ​​across all cells). In undiffNPCs, the lectin with the highest fold change relative to other subclusters was rAAL (recombinant Aleuria aurantia lectin). Because rAAL is a fucose-binding lectin, this suggests increased expression levels of fucose-containing glycans and enzymes involved in their synthesis in undiffNPCs. On the other hand, in MCs, the lectin with the highest fold change relative to other subclusters was rLSLN (recombinant Laetiporus sulphureus lectin N-terminal domain). The high affinity of rLSLN for LacNAc / polyLacNAc suggested an increase in the expression levels of LacNAc / polyLacNAc-containing glycans and enzymes involved in their synthesis in MC.

[0059] Figure 7 shows the results of comparing the amount of rAAL bound in each subcluster and the expression level of FUT10, an α1,3-fucosyltransferase gene involved in the synthesis of fucose-containing glycans. It was confirmed that a large amount of rAAL bound to undiffNPC, and that FUT10 was highly expressed in undiffNPC. FUT10 is known to be mainly involved in the synthesis of LeX, and these results suggest that the expression level of LeX is increased in undiffNPC.

[0060] The results of comparing the amount of rLSLN bound in each subcluster and the expression levels of B4GALT1, a β1,4-galactosyltransferase gene involved in the synthesis of LacNAc / polyLacNAc-containing sugar chains, and B3GNT2, a β1,3-N-acetylglucosaminyltransferase involved in the elongation of polyLacNAc chains, are shown in Figure 8. It was confirmed that rLSLN was bound in large amounts to MC, and that B4GALT1 and B3GNT2 were highly expressed in MC. These results suggest that the synthesis of LacNAc / polyLacNAc-containing sugar chains is enhanced in MC.

[0061] 3. Detection of Undifferentiated Cells Based on Glycosylation Markers To verify whether cells belonging to undiffNPCs can be specifically characterized as LeX-positive cells or whether cells belonging to MCs can be specifically characterized as LacNAc / polyLacNAc-positive cells, fluorescent immunostaining was performed as described below.

[0062] (3-1) Immunocytostaining with Anti-SSEA1 Antibody. iPSC-derived neuronal populations were fixed with 4% paraformaldehyde and washed with PBS. After incubation with 1% BSA and 0.2% Triton X-100 / PBS at room temperature for 30 minutes, primary antibodies (anti-SSEA1 antibody (Merck Millipore, MAB4301)) (1 / 100 dilution) and anti-nestin antibody (Merck Millipore, MAB1259) (1 / 1000 dilution) were added and incubated overnight at 4°C. After washing three times with PBS, secondary antibodies (Cy3-labeled anti-mouse IgM (Merck Millipore, AP128C)) (1 / 1000 dilution) and Alexa488-labeled anti-mouse IgG (Invitrogen, A-11001) (1 / 1000 dilution) were added and incubated at room temperature for 1 hour. After washing three times with PBS, a Hoechst 33342 (1 μg / mL) solution was added and incubated at room temperature for 1 hour. After washing once with PBS, the cells were photographed using an inverted fluorescence microscope (IX51, Olympus).

[0063] The results are shown in Figure 9. Cells stained with the anti-SSEA1 antibody had a morphology distinct from that of neurons and were positive for nestin (a neural stem cell / NPC marker). These results demonstrate that undifferentiated NPCs can be detected based on SSEA1(LeX).

[0064] (3-2) Fluorescent staining using labeled rLSLN. iPSC-derived neuronal populations were fixed with 4% paraformaldehyde and washed with PBS. After adding 1% BSA / PBS and incubating at room temperature for 30 minutes, Cy3-labeled rLSLN (10 μg / mL) was added and incubated overnight at 4°C. After washing three times with PBS, Hoechst 33342 (1 μg / mL) solution was added and incubated at room temperature for 1 hour. After washing once with PBS, images were taken with an inverted fluorescence microscope (IX51, Olympus).

[0065] The results are shown in Figure 10. Cells stained with Cy3-labeled rLSLN were flattened and had large nuclei, similar in morphology to mesenchymal cells. These results demonstrate that mesenchymal-like cells can be detected based on LacNAc / polyLacNAc.

[0066] 4. Removal of Undifferentiated Cells Based on Glycosylation Markers (4-1) Removal of Undifferentiated NPCs Using IR700-Labeled Anti-SSEA1 Antibody The photosensitizer IRDye700DX (hereinafter simply referred to as "IR700") is a dye used in photoimmunotherapy. When cells are loaded with a cell surface antigen-specific antibody conjugated with IR700 and irradiated with near-infrared light, the IR700 exerts its local toxicity, selectively killing the antibody-bound cells. We tested whether undifferentiated NPCs could be selectively removed using an IR700-labeled anti-SSEA1 antibody.

[0067] Anti-SSEA1 antibody (STEMCELL Technologies, 60060) was labeled with IR700 using the IRDye700DX Protein Labeling Kit - High MW according to the protocol provided with the kit. Medium containing IR700-labeled anti-SSEA1 antibody (10-20 μg / mL) was added to the iPSC-derived neuronal population, and the cells were incubated at 37°C in CO. ２ The cells were left to stand in an incubator for 1 hour. An iPSC-derived neuronal population supplemented with a medium not containing IR700-labeled anti-SSEA1 antibody served as a control. The medium was then replaced with one not containing IR700-labeled anti-SSEA1 antibody, and the cells were exposed to near-infrared light (10 mW / cm ２ ) for 10 minutes. ２ After leaving the cells in an incubator for 3 hours, the cells were detached with Accutase and collected. ６ Ghost Dye in each cell ＴＭ　 Violet 450 (Cytek Biosciences, 1 / 1000 dilution) was added and incubated on ice for 30 minutes. After washing the cells with 1% BSA / PBS, phycoerythrin (PE)-labeled anti-SSEA1 antibody (BioLegend, 125606) (1 / 20 dilution) was added and incubated on ice for 1 hour. After washing the cells twice with 1% BSA / PBS, the percentage of SSEA1-positive cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter).

[0068] The results are shown in Table 2 and Figure 11. Figure 11 shows the flow cytometry results for condition 4 in Table 2, with the solid line representing the cell population stained with the PE-labeled anti-SSEA1 antibody and the dotted line representing the unstained cell population. Compared to the control, the proportion of SSEA1-positive cells in the cell population treated with the IR700-labeled anti-SSEA1 antibody was reduced by 10 to 30%. These results demonstrate that undifferentiated NPCs can be eliminated using the IR700-labeled anti-SSEA1 antibody.

[0069] Table 2. Experimental conditions and depletion efficiency (IR700-labeled anti-SSEA1 antibody / undifferentiated NPCs)

[0070] (4-2) Removal of MCs using LSL LSL, a lectin derived from Aikawatake mushroom, is a full-length form of LSLN and has the same carbohydrate-recognition ability as LSLN and cytotoxicity derived from the C-terminal domain (J. Biol. Chem., 2003; 278(42):40455-63). We tested whether LSL could be used to selectively remove mesenchymal-like cells (MCs).

[0071] A nucleic acid sequence encoding the recombinant N-terminal domain (1-149 aa) of LSL (RCSB PDB No. 1W3A) (rLSLN, SEQ ID NO: 1) was inserted into the pET-27b vector to obtain the rLSLN expression vector (rLSLN-pET27b). BL21-CodonPlus(DE3)-RIL was transformed with rLSLN-pET27b, and rLSLN was expressed and purified by standard procedures. R-Phycoerythrin Labeling Kit-NH ２ rLSLN were labeled with R-phycoerythrin (PE) using a Dojindo Chemical Laboratory (Dojindo Laboratories) to prepare PE-labeled rLSLN. A medium containing LSL (0.3-3 μg / mL) was added to the iPSC-derived neuronal population, and the cells were incubated at 37°C in CO ２ The cells were left to stand in an incubator for 1 hour. An iPSC-derived neuronal population supplemented with a medium containing no LSL was used as a control. The cells were detached using Accutase and collected. 1 × 10 ６ Ghost Dye in each cell ＴＭ　Violet 450 (1 / 1000 dilution) was added and incubated on ice for 30 minutes. After washing the cells with 1% BSA / PBS, PE-labeled rLSLN (1 / 100 dilution) and APC-labeled anti-PDGFRB antibody (BioLegend, 323608) (1 / 20 dilution) were added and incubated on ice for 1 hour. After washing the cells twice with 1% BSA / PBS, the ratio of rLSLN+ / PDGFRB+ cells was analyzed using a flow cytometer, CytoFLEX (Beckman Coulter).

[0072] The results are shown in Table 3 and Figure 12. Figure 12 shows the results of flow cytometry under conditions 7 and 8 in Table 3. In the figure, the vertical axis (PE-rLSLN) indicates the amount of PE-labeled rLSLN bound, and the horizontal axis (APC-PDGFRB) indicates the amount of APC-labeled anti-PDGFRB antibody bound. "APC-A, PE-A subset" indicates the rLSLN-positive and PDGFRB-positive cell population (rLSLN+ / PDGFRB+ cells). Compared with the control, the proportion of rLSLN+ / PDGFRB+ cells in the cell population treated with 1 μg / mL of LSL was reduced by 29 to 50%. Furthermore, the cytotoxic activity of LSL was significantly reduced in high-density cell populations (0.5 to 0.875 x 10 ６ cells / cm ２ ) (Table 3, conditions 3 and 4), and was inhibited in low-density cell populations (0.25-1.68 × 10 ５ cells / cm ２ ) showed higher removal efficiency than LSL (Table 3, conditions 7 and 8). These results demonstrated that mesenchymal-like cells can be removed using LSL and that removal efficiency can be further increased in low-density culture.

[0073] Table 3. Experimental conditions and removal efficiency (LSL / MC)

Claims

1. A method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, comprising: (a1) a step of contacting the neural cell population with a first glycan-binding molecule, wherein the first glycan-binding molecule binds to Lewis X; (a2) a step of detecting or removing cells to which the first glycan-binding molecule has bound in step (a1), wherein the cells to which the first glycan-binding molecule has bound are undifferentiated neural progenitor cells; and / or (b1) a step of contacting the neural cell population with a second glycan-binding molecule, wherein the second glycan-binding molecule binds to N-acetyllactosamine or poly-N-acetyllactosamine; and (b2) a step of detecting or removing cells to which the second glycan-binding molecule has bound in step (b1), wherein the cells to which the second glycan-binding molecule has bound are mesenchymal-like cells.

2. The method of claim 1, wherein steps (a1) and (b1) are performed simultaneously or sequentially in any order, and steps (a2) and (b2) are performed simultaneously or sequentially in any order.

3. The method according to claim 1 or 2, wherein the carbohydrate-binding molecule is a lectin or an antibody.

4. The method according to any one of claims 1 to 3, wherein the carbohydrate-binding molecule is conjugated to a detectable label.

5. The method according to any one of claims 1 to 4, wherein the carbohydrate-binding molecule is conjugated to a cytotoxic substance.

6. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells are iPS cells.

7. The method of claim 6, wherein the iPS cells are human iPS cells.

8. The method of any one of claims 1 to 7, wherein the undifferentiated neural progenitor cells express NES, PAX6, VIM, and SOX1.

9. The method of any one of claims 1 to 8, wherein the mesenchymal-like cells express NES, ACTA2, and PDGFRB.

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