WO2022108117A1 - Structure sphéroïde d'organoïde cérébral-motoneurones et procédé de fabrication s'y rapportant - Google Patents

Structure sphéroïde d'organoïde cérébral-motoneurones et procédé de fabrication s'y rapportant Download PDF

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WO2022108117A1
WO2022108117A1 PCT/KR2021/014097 KR2021014097W WO2022108117A1 WO 2022108117 A1 WO2022108117 A1 WO 2022108117A1 KR 2021014097 W KR2021014097 W KR 2021014097W WO 2022108117 A1 WO2022108117 A1 WO 2022108117A1
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organoid
spheroid
brain
neuron
culture
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최정우
박수정
손주형
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서강대학교산학협력단
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.
  • Tissue-specific organoid culture technology is a cutting-edge field that is currently receiving the most spotlight in stem cell research, and has unlimited potential in regenerative medicine and new drug research, such as intractable disease models, patient-specific drug screening platforms, and in vitro models for new drug development. can be expanded.
  • the present inventors fabricated a structure through cerebral organoid-motoneuron spheroid coupling and measured changes in bioelectrical signals. As a result, it was confirmed that the electrical signals were amplified by caffeine, known as a cranial nerve stimulant.
  • an object of the present invention is to provide a method for preparing a brain organoid-neuron spheroid structure comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
  • Another object of the present invention is to provide a brain organoid-neuron spheroid structure comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid.
  • Another object of the present invention is a brain organoid, and a brain organoid comprising one or more neuronal spheroids bound to the lower end of the brain organoid - a drug screening method comprising the step of treating a target drug to a neuron spheroid structure will provide
  • Another object of the present invention relates to the use of brain organoid-neuron spheroid constructs for drug screening.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the cerebral organoid-motoneuron spheroid structure according to the present invention is a cerebral organoid-motoneuron spheroid structure. indicates.
  • the present inventors prepared a co-culture chip in which each culture medium of cerebral organoids and motor neuron spheroids can be independently supplied to prepare such a cerebral organoid-motoneuron spheroid structure, and the structure was prepared using this .
  • One aspect of the present invention is a method for preparing a brain organoid-neuron spheroid construct comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
  • organ is also called an organ-like organ, and by re-aggregating/recombining stem cells or cells isolated from an organ through three-dimensional culture, organization similar to an actual organ and reproduction of the function of a living organ are possible. It can be used as a model for various studies such as diseases.
  • spheroid is a three-dimensional aggregate of cells, which prevents spontaneous differentiation and facilitates differentiation of a desired lineage. In particular, since it is easy to induce differentiation into nerve cells in 3D, many studies are proceeding with nerve differentiation using spheroids.
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • the co-culturing step is performed in a co-culture chip
  • the co-culture chip is a co-culture chamber; a brain organoid culture reservoir connected by an independent channel to the co-culture chamber; and a neuron spheroid culture reservoir connected to an independent channel in the co-culture chamber.
  • the co-culturing step may be performed by placing one or more neuron spheroids in the co-culture chamber and then adding brain organoids.
  • the channel connected to the brain organoid culture reservoir may have a higher position connected to the co-culture chamber than the channel connected to the neuron spheroid culture reservoir.
  • the brain organoid culture medium is, for example, in a medium mixed with DMEM-F12 and Neurobasal medium, N2 supplement (supplement), insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 2-mercaptoethanol (2-mercaptoethanol) and vitamin A (vitamin A) containing 1X B27 supplement may be included, but is not limited thereto.
  • Brain-derived neurotrophic factor (BDNF) and Glial cell-Derived Neurotrophic Factor (GDNF) may be added to the culture medium of the neuronal spheroids in StemPro hESC medium, but is limited thereto it is not
  • the co-culture chamber connection position of the channel through which the culture solution is introduced from the neuron spheroid culture medium reservoir is lower than the brain organoids is located in Accordingly, the neuronal spheroid culture is used for the culture of the neuronal spheroids underneath the brain organoids.
  • the co-culture chamber connection position of the channel through which the culture medium is introduced from the brain organoid culture medium reservoir is located above the brain organoid. Accordingly, brain organoid cultures are used for culturing brain organoids and not for culturing neuronal spheroids underneath brain organoids.
  • a combined structure can be prepared while independently using a culture medium suitable for each culture.
  • the manufacturing method may further include a brain organoid differentiation step of differentiating human induced pluripotent stem cells (hiPSCs) into brain organoids.
  • hiPSCs human induced pluripotent stem cells
  • human induced pluripotent stem cell refers to four specific genes (Oct4, Sox2, Klf4, c-Myc) that cause dedifferentiation into adult somatic cells that have been differentiated to thereby introduce pluripotency such as embryonic stem cells.
  • Oct4, Sox2, Klf4, c-Myc specific genes that cause dedifferentiation into adult somatic cells that have been differentiated to thereby introduce pluripotency such as embryonic stem cells.
  • the brain organoid differentiation step is a neurodifferentiation induction step of embedding the spheroid having the germ layer formed in the cells cultured in the nerve induction medium into MatriGel, the brain for neuronal differentiation from the embedded spheroid. It may consist of a patterning step of culturing in an organoid differentiation medium, and a final differentiation step of brain tissue producing brain organoids by inducing final differentiation of brain tissue when a neuroepithelial bud is formed.
  • the manufacturing method may further include a neuron spheroid differentiation step of differentiating human neural stem cells into neuronal spheroids.
  • Another aspect of the present invention is a brain organoid-neuron spheroid construct comprising a brain organoid and one or more neuronal spheroids bound to the underside of the brain organoid.
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • Another aspect of the present invention is a drug screening method comprising the step of treating a target drug to a brain organoid-neuron spheroid construct comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid. .
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • the target drug may be a candidate substance for the prevention or treatment of brain diseases causing movement disorders including Parkinson's disease or Huntington's disease, and various brain nervous system diseases.
  • the drug screening method in the present invention may be performed by deriving an analysis result using a multi-channel electrode array (MEA) system for the brain organoid-neuron spheroid structure after the treatment of the target drug.
  • MEA multi-channel electrode array
  • multi-channel electrode array system is a device that simultaneously measures the action potential of a nerve cell with multiple electrodes. By measuring the change in the action potential of brain organoids, electrophysiological variation analysis, neuronal ion channel It can be used for analysis of changes, signal transduction rate, and signal transduction detection between nerve cells.
  • the drug screening method may be performed for the purpose of testing the toxicity of a target drug.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the structure can be prepared by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to contact an appropriate culture medium. And from this, it is possible to observe the change in the electrical signal of the motor neuron according to a specific substance that stimulates the cerebrum, so it can be effectively used for drug screening or toxicity test for the prevention or treatment of brain diseases that cause movement disorders.
  • 1a is a schematic diagram showing the differentiation process of cerebral organoids according to an embodiment of the present invention.
  • Figure 1b is a photograph showing the tissue change according to the differentiation progress of cerebral organoids according to an embodiment of the present invention.
  • 1C is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by fluorescence immunostaining method.
  • 1D is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by a three-dimensional fluorescence immunostaining method.
  • Figure 2a is a schematic diagram showing the differentiation process of motor neuron spheroids according to an embodiment of the present invention.
  • Figure 2b is a photograph confirming the differentiation result of the motor neuron spheroid according to the embodiment of the present invention by the fluorescence immunostaining method.
  • Figure 2c is a photograph confirming the gene expression level by performing a polymerase chain reaction (PCR) as a result of differentiation of motor neuron spheroids according to an embodiment of the present invention.
  • PCR polymerase chain reaction
  • Figure 3a is a schematic diagram of the manufacturing process and application examples of the co-culture chip manufactured to combine cerebral organoids and motor neuron spheroids.
  • Figure 3b is a photo of the implementation of the co-cultured chip fabricated to combine cerebral organoids and motor neuron spheroids.
  • Figure 4a is a graph showing the signal results appearing as a result of processing caffeine to the cerebral organoids attached to the multi-electrode array (Microelectrode array; MEA) substrate.
  • Figure 4b is a graph showing the signal results appearing as a result of the caffeine treatment on the motor neuron spheroids attached to the MEA substrate.
  • Figure 4c is a graph showing the signal results appearing as a result of caffeine treatment on the cerebral organoid-motoneuron spheroid structure attached to the MEA substrate.
  • a brain organoid comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids - a method for preparing a neuron spheroid construct.
  • % used to indicate the concentration of a specific substance is (weight/weight)% solid/solid, (weight/volume)%, and (weight/volume)% for solid/solid, and Liquid/liquid is (vol/vol) %.
  • Example 1 Preparation of cerebral organoids from human induced pluripotent stem cells
  • hiPSC human induced pluripotent stem cells
  • NIP laminin-based iMatrix-511
  • NIP laminin-based iMatrix-511
  • hiPSC NCRM5AS1-iCAGcGFP.c9; National Stem Cell Regeneration Center, Korea Centers for Disease Control and Prevention, 1382D2; RIKEN BioResource Research Center purchase, 201B7; ATCC purchase
  • TrypLE Select (Gibco, Cat #. 12563-011) was treated to separate the cells into single cells. The separated cells were resuspended after centrifugation, and spheroids were prepared by dispensing at 100,000 cells/well in an ultra-low attachment 96 well plate (Corning®, Cat #. CLS7007).
  • the produced spheroids are about 500 to 600 ⁇ m in diameter, in DMEM-F12 with 1XN2 supplement, 1X GlutaMAX supplement, 1X MEM-NEAA and 1 ⁇ g/ml heparin in a neural induction medium containing 4-5 Incubated for one day.
  • the germ layer (Germ layer) was formed by embedding the spheroid in MatriGel (MatriGel) to induce nerve differentiation.
  • the embedded spheroids were mixed with DMEM-F12 and Neurobasal medium at a 1:1 ratio with 0.5X N2 supplement, 62.5 ⁇ l of insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 10 ⁇ M 2-mercaptoethanol (2-mercaptoethanol) and vitamin A removed 1X B27 supplement added cerebral organoid differentiation medium (Cerebral organoid differentiation medium) was cultured for about 5 days and patterned.
  • 1X B27 supplement is replaced with a vitamin A-containing one in the cerebral organoid differentiation medium to induce differentiation for about 15 days to grow cerebral tissue and finally differentiate to complete cerebral organoids.
  • Fluorescent immunostaining was performed to confirm the expression of specific markers in the prepared cerebral organoids.
  • the cerebral organoids were fixed with 4% paraformaldehyde for more than 24 hours, and washed three times with PBS (phosphate-buffered saline) containing 0.2% Triton X 100. Thereafter, non-specific binding of the antibody was prevented by blocking with PBS containing bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Antibodies such as FOXG1 and PROX1, which are markers of cerebral organoids, TUJ1, which are neuronal markers, and SOX2, which are stem cell markers, were each treated at a concentration of 1:100 and maintained at 4°C for more than 24 hours.
  • cerebral organoid markers FOXG1 and PROX1, neuronal cell marker TUJ1, stem cell marker SOX2, etc. were stained and fluorescence was observed with a confocal microscope, and cerebral organoids was confirmed to be well formed.
  • Example 3 Preparation of motor neuron spheroids from human neural stem cells
  • Motor neuron spheroids were obtained by dispensing human neural stem cells at 5.0 X 10 4 cells/well in an ultra-low attachment 96 well U-bottom plate (Corning®, Cat #. CLS7007) and cultured for 24 hours. Then, in StemPro hESC medium (Gibco, Cat #. A1000701), retinoic acid (RA) 50 ⁇ M, secretory signal factor (sonic hedgehog) 200 ng/ml, basic fibroblast growth factor (bFGF) A medium for motor neuron differentiation containing 8 ng/ml and 10 ng/ml of activin A was replaced by 100 ⁇ l per well.
  • StemPro hESC medium StemPro hESC medium (Gibco, Cat #. A1000701)
  • RA retinoic acid
  • secretory signal factor sonic hedgehog
  • bFGF basic fibroblast growth factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cell-derived neurotrophic factor
  • Fluorescence immunostaining was performed to confirm the expression of specific markers of the prepared motor neuron spheroids. It was performed in the same manner as in Example 2, except that the antibody of TUJ1, a neuron marker and islet1, a motor neuron marker, was used in a ratio of 1:100, respectively.
  • cDNA was synthesized using the Superscript III kit, and PCR was performed using the FlexCycler2 system device. PCR was performed using PAX6 and SOX1 as undifferentiated markers, ChAT, HB9 and islet1 as motor neuron differentiation markers, and GAPDH primer to confirm the initial amount of cDNA.
  • Example 5 Induction of cerebral organoid-motoneuron spheroid binding
  • a structure was manufactured by combining the prepared cerebral organoids and motor neuron spheroids, and a co-culture chip (3D engineered nervous system, 3D-ENS) including two culture medium reservoirs and a co-culture chamber for culturing them was prepared.
  • the co-culture chip was manufactured by assembling an upper plate and a lower plate formed using 3D printing technology.
  • the co-culture chip was prepared for the purpose of minimizing the stimulus to be applied to the other tissue during the induction of connective tissue because the culture medium for each tissue is different.
  • the cerebral organoid culture medium storage The height connected to the co-culture chamber was designed to be higher than the height at which the motor neuron spheroid culture medium reservoir was connected to the co-culture chamber.
  • binding was induced by culturing each tissue to be exposed to a specific individual culture medium. Specifically, in performing the coupling of cerebral organoids and motor neuron spheroids using the co-culture chip, motor neuron spheroids were first placed on the lower plate of the co-culture chamber, and brain organoids were introduced.
  • Example 3 As the culture medium flowing into the co-culture chamber through the channel from the cerebral organoid culture medium, the same medium was used in which the 1X B27 supplement was replaced with one containing vitamin A in the cerebral organoid differentiation medium after neuroepithelial embryo formation in Example 1 As the culture medium flowing into the co-culture chamber from the motor neuron spheroid culture medium reservoir through the channel, the motor neuron culture medium used for the maturation of motor neurons in Example 3 was used.
  • Example 6 Confirmation of changes in bioelectrical signals of cerebral organoid-motoneuron spheroid structures
  • each electrical signal was confirmed through a microelectrode array (MEA).
  • MEA substrate was coated with polyimide (PI) and then re-coated with laminin to raise the structure and measure the signal.
  • PI polyimide
  • the motor neuron spheroid was placed in contact with the MEA substrate, and the signal change of the motor neuron spheroid according to the stimulation applied to the cerebral organoid was observed.
  • caffeine known as a cranial nerve stimulant, was treated in cerebral organoids, motor neurons, and cerebral organoid-motoneuron spheroid structures, respectively, and changes in electrical signals caused by caffeine were observed.
  • cerebral organoids were amplified electrical signals by caffeine, but motor neuron spheroids did not change.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.

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Abstract

La présente invention concerne une structure sphéroïde d'organoïde cérébral-motoneurones et un procédé de fabrication s'y rapportant. La structure peut être fabriquée par co-culture d'un organoïde cérébral et de sphéroïdes de motoneurones dans une puce de co-culture qui est conçue pour amener l'organoïde cérébral et les sphéroïdes de motoneurones en contact indépendant avec un milieu de culture approprié. La structure peut être efficacement utilisée pour cribler des médicaments pour la prévention ou le traitement de maladies cérébrales provoquant une perturbation motrice ou pour un essai de toxicité parce que le changement de signal électrique de motoneurones selon des substances spécifiques stimulant le cerveau peut être observé à partir de la structure.
PCT/KR2021/014097 2020-11-17 2021-10-13 Structure sphéroïde d'organoïde cérébral-motoneurones et procédé de fabrication s'y rapportant WO2022108117A1 (fr)

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Title
HA, TAEHYEONG; SON, JOOHYUNG; PARK, SOO JEONG; CHO, HYEON-YEOL; CHOI, JEONG-WOO: "P0436 Fabrication of Human Brain-spinal Cord Assembloid for Electrophysiological Monitoring of Neurochemical-based Neural System Crosstalk", 2021 KSBB FALL MEETING AND INTERNATIONAL SYMPOSIUM; OCTOBER 6-9, 2021, 8 October 2021 (2021-10-08) - 9 October 2021 (2021-10-09), Korea, pages 485, XP009536973 *
JEONG HYE-JIN; JIMENEZ ZULY; MUKHAMBETIYAR KARAKOZ; SEO MINWOOK; CHOI JEONG-WON; PARK TAE-EUN: "Engineering Human Brain Organoids: From Basic Research to Tissue Regeneration", TISSUE ENGINEERING AND REGENERATIVE MEDICINE, SPRINGER SINGAPORE, SINGAPORE, vol. 17, no. 6, 1 January 1900 (1900-01-01), Singapore, pages 747 - 757, XP037310132, ISSN: 1738-2696, DOI: 10.1007/s13770-020-00250-y *
KAWADA JIRO, KANEDA SHOHEI, KIRIHARA TAKAAKI, MAROOF ASIF, LEVI TIMOTHÉE, EGGAN KEVIN, FUJII TERUO, IKEUCHI YOSHIHO: "Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons", STEM CELL REPORTS, CELL PRESS, UNITED STATES, vol. 9, no. 5, 1 November 2017 (2017-11-01), United States , pages 1441 - 1449, XP055927400, ISSN: 2213-6711, DOI: 10.1016/j.stemcr.2017.09.021 *

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