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WO2025169902A1 - Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales - Google Patents

Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales

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Publication number
WO2025169902A1
WO2025169902A1 PCT/JP2025/003513 JP2025003513W WO2025169902A1 WO 2025169902 A1 WO2025169902 A1 WO 2025169902A1 JP 2025003513 W JP2025003513 W JP 2025003513W WO 2025169902 A1 WO2025169902 A1 WO 2025169902A1
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cells
glycan
binding molecule
undifferentiated
neural
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Japanese (ja)
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陽樹 小高
浩章 舘野
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National Institute of Advanced Industrial Science and Technology AIST
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells.
  • Non-Patent Document 1 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).
  • Non-Patent Document 2 a technique for detecting tumorigenic undifferentiated neural progenitor cells by PET imaging using a probe [ 18 F]FEDAC specific to translocator protein 18 kDa (TSPO) expressed on the outer mitochondrial membrane of undifferentiated neural progenitor cells has been reported (Non-Patent Document 2).
  • glycan markers present on the cell surface. Because cell surface markers are easy to detect, they enable highly efficient and accurate cell identification. However, no glycan 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.
  • the present invention aims to easily and accurately detect and remove undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells.
  • the present invention provides a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, the method 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
  • steps (a1) and (b1) may be performed simultaneously or sequentially in any order
  • steps (a2) and (b2) may be performed simultaneously or sequentially in any order.
  • the glycan-binding molecule is preferably a lectin or an antibody.
  • the glycan-binding molecule is preferably conjugated to a detectable label.
  • the glycan-binding molecule is preferably conjugated to a cytotoxic substance.
  • the pluripotent stem cells are preferably iPS cells, and more preferably human iPS cells.
  • the undifferentiated neural progenitor cells express NES, PAX6, VIM, and SOX1.
  • the mesenchymal-like cells express NES, ACTA2, and PDGFRB.
  • the method of the present invention makes it possible to specifically detect or remove undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells. Therefore, the method of the present invention is useful for preparing highly safe neural cell populations for transplantation and for developing high-quality assay systems using highly pure neural cell populations.
  • FIG. 1 is a schematic diagram showing the schedule for producing an iPS cell-derived neural cell population.
  • FIG. 2 shows fluorescent immunostained images (top) of iPS cells (iPSCs), neural progenitor cells (NPCs), and neuronal populations (neurons) using an anti-TUJ1 antibody, and Hoechst stained images (bottom).
  • FIG. 3 shows UMAP plots of RNA expression information and sugar chain reactivity information of iPSCs, NPCs, and neurons.
  • FIG. 4 is a UMAP plot of RNA expression information and sugar chain reactivity information for neuron only.
  • FIG. 1 is a schematic diagram showing the schedule for producing an iPS cell-derived neural cell population.
  • FIG. 2 shows fluorescent immunostained images (top) of iPS cells (iPSCs), neural progenitor cells (NPCs), and neuronal populations (neurons) using an anti-TUJ1 antibody, and Hoechst stained images (bottom).
  • FIG. 3 shows UMAP plots of
  • FIG. 5 is a heat map showing the expression profiles of marker genes in mature neurons (mNeurons), immature neurons (imNeurons), undifferentiated NPCs (undiffNPCs), and mesenchymal-like cells (MCs).
  • FIG. 6 is a dot plot showing the reactivity of mNeuron, imNeuron, undiffNPC, and MC with various lectins.
  • FIG. 7 is a violin plot showing the amount of rAAL binding and the amount of FUT10 gene expression in mNeurons, imNeurons, undiffNPCs, and MCs.
  • FIG. 8 is a violin plot showing the amount of rLSLN binding in mNeurons, imNeurons, undiffNPCs, and MCs, and the expression levels of B4GALT1 and B3GNT2 genes.
  • FIG. 9 shows fluorescent immunostained images of neurons using anti-SSEA1 antibody and anti-nestin antibody (top) and Hoechst stained images (bottom).
  • FIG. 10 shows a fluorescent stained image (top) of a neuron stained with Cy3-labeled rLSLN and a Hoechst stained image (bottom).
  • FIG. 9 shows fluorescent immunostained images of neurons using anti-SSEA1 antibody and anti-nestin antibody (top) and Hoechst stained images (bottom).
  • FIG. 10 shows a fluorescent stained image (top) of a neuron stained with Cy3-labeled rLSLN and a Hoechst stained image (bottom).
  • FIG. 11 is a histogram showing the results of flow cytometry analysis of SSEA1-positive cells in iPSC-derived neuronal cell populations that were untreated (control) or treated (IR700-SSEA1) with IR700-labeled anti-SSEA1 antibody.
  • FIG. 12 shows the results of flow cytometric gating of rLSLN+/PDGFRB+ cells in iPSC-derived neuronal cell populations untreated (control) or treated with LSL (LSL 0.3 ⁇ g/mL or 1 ⁇ g/mL).
  • the present invention provides a method for detecting or removing undifferentiated cells remaining in a neural cell population induced from pluripotent stem cells, the method 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 bound by the first glycan-binding molecule in step (a1), wherein the cells bound by the first glycan-binding molecule 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 bound by the second glycan-binding molecule in
  • the method of this embodiment targets a neural cell population induced from pluripotent stem cells.
  • 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.
  • 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.
  • pluripotent stem cells can be prepared from any tissue or cell according to methods known in the art.
  • 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), or the American Type Culture Collection (ATCC).
  • the term "neuronal cell population” refers to a cell population that is obtained by inducing differentiation of pluripotent stem cells into neurons and that is substantially composed of neurons. Therefore, the neural cell population of this embodiment may, for example, contain neurons at a ratio of at least 50%, preferably 80% or more.
  • a neuronal 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.
  • a basal medium such as DMEM/F-12 medium, N2 medium, or a mixture thereof
  • a BMP signaling pathway inhibitor such as dorsomorphin
  • TGF signaling pathway inhibitor such as SB431542
  • Wnt signaling pathway inhibitor such as XAV-939.
  • undifferentiated cells refer to cells that are resistant to neural induction and retain pluripotency (the ability to differentiate into cells other than neural cells) and self-renewal ability even under neural induction conditions. Therefore, in this embodiment, undifferentiated cells are specifically undifferentiated neural progenitor cells or mesenchymal-like cells that remain after neural induction of pluripotent stem cells.
  • 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.
  • undifferentiated cell markers such as stemness markers, proliferative cell markers, neural progenitor cell markers, and mesenchymal stem cell markers.
  • stemness markers such as stemness markers, proliferative cell markers, neural progenitor cell markers, and mesenchymal stem cell markers.
  • undifferentiated neural progenitor cells may also include neural stem cells.
  • 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).
  • the mesenchymal-like cells preferably express, for example, NES, ACTA2 (smooth muscle ⁇ 2 actin gene), and PDGFRB (platelet-derived growth factor receptor ⁇ 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).
  • NES smooth muscle ⁇ 2 actin gene
  • PDGFRB platelet-derived growth factor receptor ⁇ gene
  • the undifferentiated neural progenitor cells and mesenchymal-like cells in this embodiment do not express neuronal markers or synaptic markers.
  • Neuronal markers include, for example, 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 ⁇ 3 gene).
  • synaptic markers examples 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).
  • Marker expression can be analyzed using known techniques such as RT-PCR, Western blotting, and flow cytometry.
  • glycan-binding molecules are used to identify undifferentiated cells. Because the glycans displayed on cell surfaces vary depending on the type and state of the cell, undifferentiated cells can be identified based on the binding between glycans specifically present on the surface of undifferentiated cells and the corresponding glycan-binding molecules.
  • 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 Gal ⁇ 1-4(Fuc ⁇ 1-3)GlcNAc.
  • LacNAc is a glycan consisting of a structure represented by Gal ⁇ 1-4GlcNAc.
  • either 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.
  • both (a) and (b) are performed, (a) and (b) may be performed sequentially or simultaneously.
  • (a) and (b) are performed sequentially, the order is not particularly limited, and (b) may be performed after (a), or vice versa.
  • the "glycan-binding molecule" in this embodiment may be any molecule that can recognize 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 antibody.
  • 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, or fully human antibody. Furthermore, the antibody in this embodiment may also include antigen-binding fragments with equivalent glycan recognition ability, such as Fab, F(ab')2, scFv, and nanobody.
  • 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.
  • 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). Lectins in this embodiment may also include partial fragments or recombinant forms that have equivalent glycan recognition ability.
  • 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).
  • RSL Ralstonia solanacearum-derived lectin
  • RSIIL Ralstonia solanacearum-derived lectin 2
  • AAL Aleuria aurantia-derived lectin
  • AOL Aspergillus oryzae-derived lectin
  • LTL Lotus tetragonolobus-derived lectin
  • PAIIL Pseudomonas aeruginosa-derived lectin
  • Examples of lectins and recombinant forms thereof that recognize (poly)LacNAc include Aikawatake mushroom (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).
  • Aikawatake mushroom Laetiporus sulphureus
  • LSLN Aikawatake lectin N-terminal domain recombinant
  • rLSLN Aikawatake lectin N-terminal domain recombinant
  • rLSLN Aikawatake lectin N-terminal domain recombinant
  • rLSLN Aikawatake lectin N-terminal domain
  • Lectins may be prepared by any method known in the art. For example, they may be isolated from the organism from which they originate, or nucleic acids encoding the lectins may be prepared by genetic engineering methods and introduced into host cells such as E. coli for expression. Information on the amino acid sequences of lectins and the nucleic acid sequences encoding them can be obtained from designated databases via portal sites 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.
  • the glycan-binding molecule in this embodiment may be conjugated to a detectable label.
  • 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, and 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.
  • the glycan-binding molecule of this embodiment may be conjugated to a cytotoxic substance instead of or in addition to a detectable label.
  • 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.
  • 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.
  • the detectable label or cytotoxic substance may be conjugated to the glycan-binding molecule directly or via a linker according to any method known in the art.
  • 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.
  • a glycan-binding molecule is brought into contact with a neuronal population.
  • the glycan-binding molecule is added to the culture medium of 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 ⁇ g/mL, preferably 5 to 10 ⁇ g/mL.
  • the incubation time may be, for example, 0.5 to 3 hours, preferably 1 to 2 hours.
  • Cells to which glycan-binding molecules are bound can be detected by any method known in the art, including, but not limited to, fluorescent detection and chemiluminescent detection. In the method of this embodiment, cells to which glycan-binding molecules are bound are preferably detected by fluorescent detection.
  • Cells bound by glycan-binding molecules can be removed by any method known in the art, including, but not limited to, killing with a cytotoxic substance, separation by flow cytometry, and separation by magnetic beads. In the method of this embodiment, cells bound by glycan-binding molecules are preferably removed by a cytotoxic substance.
  • the method of this embodiment distinguishes between differentiated and undifferentiated cells in a neural cell population induced from pluripotent stem cells based on cell surface glycosylation markers. 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.
  • iPS cells 201B7 line
  • iPSCs 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 in all experiments.
  • iPSCs were induced to differentiate into neural progenitor cells (NPCs) using the STEMdiff SMADi Neural Induction Kit (Veritas) according to the accompanying 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.
  • NPCs were detached using Accutase (Innovative Cell Technologies), dispersed into single cells, and then seeded onto plates coated with 0.07% polyethyleneimine (Sigma-Aldrich) and 3.3 ⁇ g/mL laminin (Fujifilm Wako Pure Chemical Industries). They were then cultured in NeuroBasal Medium (Thermo Fisher Scientific) supplemented with 2% B27 supplement (Thermo Fisher Scientific), 1% GlutaMax supplement (Thermo Fisher Scientific), and 5 ⁇ M 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. Thereafter, half of the medium was replaced twice a week.
  • PBS D-PBS(-)
  • primary antibody solution anti-TUJ1 antibody (Merck Millipore, MAB1637), diluted 1/500
  • secondary antibody Cy3-labeled anti-mouse IgG (Jackson ImmunoResearch), diluted 1/2000
  • Hoechst 33342 Dojindo Chemical Industries
  • scGR-seq analysis of iPSC-derived neuronal populations Non-neuronal glycan markers remaining in iPSC-derived neuronal populations were identified using single-cell glycan and RNA sequencing (scGR-seq) (Minoshima, F. et al., 2021; iScience, 24(8):102882). iPSCs and NPCs were detached and dispersed into single cells using Accutase. Neurons were detached and dispersed into single cells using Accutase supplemented with 50 units/mL papain (Worthington Biochemical) and 0.25 mg/mL L-cysteine (Sigma-Aldrich).
  • the DNA barcodes in the supernatant were amplified for 20 cycles by PCR using i5/i7 index primers (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).
  • An RNA library for scGR-seq was prepared using the GenNext TM RamDA-seq TM Single Cell Kit according to the accompanying protocol.
  • 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 DNA barcode sequence data was analyzed using the Barcode DNA counting system (https://github.com/bioinfo-tsukuba/barcode-dna-counting-system) to obtain raw count data for each lectin.
  • 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.
  • UMAP Uniform Manifold Approximation and Projection
  • the data from the three iPSC, NPC, and neuron samples were classified by sample and displayed in a UMAP plot.
  • UMAP_1 and UMAP_2 represent orthogonal components obtained by dimensionally compressing the RNA and glycan data.
  • the clusters for each cell population were distributed without overlapping.
  • 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 neuron (mNeuron), immature neuron (imNeuron), undifferentiated NPC (undiffNPC), and mesenchymal-like cells (MC).
  • 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 undiffNPCs and MCs. Neural stem cell marker genes (Stemness in the figure) and proliferating cell marker genes (Proliferation) were highly expressed in undiffNPCs, suggesting that undiffNPCs possess 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.
  • iPSC-derived neural cell populations contain sub
  • Figure 6 shows the results of dot plots of lectin reactivity (binding ratio) for each subcluster for lectins that showed significant (p ⁇ 0.05) differences in reactivity between subclusters.
  • the size of the circle indicates the prevalence of lectin reactivity (the percentage of cells with non-zero values), and the gray scale indicates the intensity of reactivity (the average of the values across all cells).
  • the lectin with the highest fold change relative to other subclusters was rAAL (recombinant Aleuria aurantia lectin).
  • rAAL is a fucose-binding lectin
  • the lectin with the highest fold change relative to other subclusters was rLSLN (recombinant Laetiporus sulphureus lectin N-terminal domain). Since rLSLN has a high affinity for LacNAc/polyLacNAc, this suggests an increase in the expression levels of LacNAc/polyLacNAc-containing glycans and enzymes involved in their synthesis in MC.
  • Figure 7 shows the results of comparing the amount of rAAL bound in each subcluster and the expression level of FUT10, an ⁇ 1,3-fucosyltransferase gene involved in the synthesis of fucose-containing glycans. It was confirmed that rAAL was bound to undiffNPC in large amounts, and that FUT10 was highly expressed in undiffNPC. FUT10 is known to be primarily involved in the synthesis of LeX, and these results suggest that the expression level of LeX is increased in undiffNPC.
  • Figure 8 shows the results of comparing the amount of rLSLN bound in each subcluster and the expression levels of B4GALT1, a ⁇ 1,4-galactosyltransferase gene involved in the synthesis of LacNAc/polyLacNAc-containing glycans, and B3GNT2, a ⁇ 1,3-N-acetylglucosaminyltransferase involved in the elongation of polyLacNAc chains. 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 glycans is enhanced in MC.
  • IR700 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.
  • 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.
  • An iPSC-derived neuronal cell population supplemented with medium without IR700-labeled anti-SSEA1 antibody served as a control.
  • the medium was then replaced with one without IR700-labeled anti-SSEA1 antibody, and the cells were irradiated with near-infrared light (10 mW/ cm2 ) for 10 minutes. After incubation in a CO2 incubator at 37°C for 3 hours, cells were detached using Accutase and collected. Ghost Dye TM Violet 450 (Cytek Biosciences, 1/1000 dilution) was added to 1 x 106 cells 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).
  • PE phycoerythrin
  • Figure 11 shows the flow cytometry results for condition 4 in Table 2, with the solid line representing the cell population stained with 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 IR700-labeled anti-SSEA1 antibody was reduced by 10-30%. These results demonstrate that undifferentiated NPCs can be removed using IR700-labeled anti-SSEA1 antibody.
  • rLSLN 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.
  • rLSLN was labeled with R-phycoerythrin (PE) using the R-Phycoerythrin Labeling Kit- NH (Dojindo Laboratories) to prepare PE-labeled rLSLN.
  • PE R-phycoerythrin
  • iPSC-derived neuronal populations were treated with medium containing LSL (0.3-3 ⁇ g/mL) and incubated in a CO2 incubator at 37°C for 1 hour.
  • An iPSC-derived neuronal population treated with medium without LSL served as a control.
  • Cells were detached and collected using Accutase.
  • Ghost Dye TM Violet 450 (1/1000 dilution) was added to 1 x 106 cells and incubated on ice for 30 minutes. After washing 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.
  • the cells were washed twice with 1% BSA/PBS, and the ratio of rLSLN+/PDGFRB+ cells was analyzed using a flow cytometer CytoFLEX (Beckman Coulter).
  • Figure 12 shows the results of flow cytometry under conditions 7 and 8 in Table 3.
  • the vertical axis (PE-rLSLN) indicates the amount of PE-labeled rLSLN bound
  • 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 to the control, the proportion of rLSLN/PDGFRB cells in the cell population treated with 1 ⁇ g/mL LSL was reduced by 29 to 50%.

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Abstract

Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales induites à partir de cellules souches pluripotentes, le procédé comportant les étapes suivantes : (1) une étape de mise en contact d'une molécule liant la chaîne de sucre avec la population de cellules neurales ; et (2) une étape de détection ou d'élimination des cellules présentant la molécule liant la chaîne de sucre. Selon le procédé, (a) la molécule liant la chaîne de sucre se lie à la Lewis X, et les cellules indifférenciées sont des cellules progénitrices neurales indifférenciées, ou (b) la molécule liant la chaîne de sucre se lie à la N-acétylactosamine ou à la poly-N-acétylactosamine, et les cellules indifférenciées sont des cellules de type mésenchymateux.
PCT/JP2025/003513 2024-02-05 2025-02-04 Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales Pending WO2025169902A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010516241A (ja) * 2007-01-18 2010-05-20 スオメン プナイネン リスティ，ヴェリパルベル 新規の特異的細胞結合剤
WO2014126146A1 (fr) * 2013-02-14 2014-08-21 独立行政法人産業技術総合研究所 Procédé d'élimination de cellules indifférenciées
JP2016216387A (ja) * 2015-05-19 2016-12-22 公立大学法人名古屋市立大学 未分化細胞のアポトーシス誘導剤
WO2018190357A1 (fr) * 2017-04-11 2018-10-18 国立研究開発法人産業技術総合研究所 Procédé d'immobilisation de lectine
JP2019100940A (ja) * 2017-12-06 2019-06-24 東ソー株式会社 未分化細胞を高感度に検出および回収する方法
JP2021151206A (ja) * 2020-03-24 2021-09-30 国立研究開発法人産業技術総合研究所 糖鎖を解析する方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010516241A (ja) * 2007-01-18 2010-05-20 スオメン プナイネン リスティ，ヴェリパルベル 新規の特異的細胞結合剤
WO2014126146A1 (fr) * 2013-02-14 2014-08-21 独立行政法人産業技術総合研究所 Procédé d'élimination de cellules indifférenciées
JP2016216387A (ja) * 2015-05-19 2016-12-22 公立大学法人名古屋市立大学 未分化細胞のアポトーシス誘導剤
WO2018190357A1 (fr) * 2017-04-11 2018-10-18 国立研究開発法人産業技術総合研究所 Procédé d'immobilisation de lectine
JP2019100940A (ja) * 2017-12-06 2019-06-24 東ソー株式会社 未分化細胞を高感度に検出および回収する方法
JP2021151206A (ja) * 2020-03-24 2021-09-30 国立研究開発法人産業技術総合研究所 糖鎖を解析する方法

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