Axioloid-based modular bottom-up in vitro model of human post-gastrulation development

Abstract

It is an object of the present disclosure to provide a new three-dimensional cellular aggregate. In order to achieve the object above, the present disclosure provides a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation. The present disclosure also provides a method for producing the cellular aggregate from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate consisting of a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate consisting of the mesodermal cell and the ectodermal cell to induce the cellular aggregate.

Description

AXIOLOID-BASED MODULAR BOTTOM-UP IN VITRO MODEL OF HUMAN POST-GASTRULATION DEVELOPMENT

[0001] The present disclosure relates to a three-dimensional cellular aggregate termed axioloid generated in vitro from a pluripotent stem cell and to a method for producing the same.

[0002] The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches, a system that robustly recapitulates the human embryo somitogenesis process in vitro in both space and time, including its characteristic morphogenetic features including the oscillatory expression of segmentation clock genes and the related sequential formation of epithelial somites along the anterior to posterior axis of the embryo, remains largely missing.

[0003] It is an object of the present disclosure to provide a new three-dimensional cellular aggregate.

[0004] In order to achieve the object above, the present disclosure provides a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation.

[0005] The present disclosure also provides a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure.

[0006] The present disclosure further provides a method for producing the cellular aggregate from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate comprising (preferably, consisting of) the mesodermal cell and the ectodermal cell to induce the cellular aggregate.

[0007] The present disclosure can provide a new three-dimensional cellular aggregate.

[0008] Figure 1. Morphological and molecular characterization of axioloid v2.0. a, 409B2 iPSC line derived Axioloid v1.0 at 120h of culture with upper panel showing a bright field image, and the lower panel shows immunofluorescence staining of SOX2 (green), TBXT (blue), MEOX1 (red), and phalloidin (white). b, 201B7 iPSC line derived axioloid with neural tube-like structure only, upper panel shows a bright field image at 120h of culture, and lower panel shows immunofluorescence staining at 144h with PAX6 (green), SOX2 (red), MEOX1 (blue), and phalloidin (white). c, TJP1-mEGFP iPSC line derived axioloid V2.0 at 120h of culture. Upper panel shows a bright field image, and lower panels shows immunofluorescence staining of SOX2 (red, middle panel), MEOX1 (red, lowest panel), TBXT (blue), ZO-1 (green) and Phalloidin (white). d, Effect of the initial cell aggregate size, ranging from 250 to 1000 mTag-RFP cells (left to right), on the morphology of axioloid v2.0. e, Morphological classification of axioloid v2.0, based on the position of the somites relatively to the neural tube. Nt, neural tube-like structure; So, somite. Scale bars are 200 μm.

[0009] Figure 2. Time-lapse imaging of human PSC derived axioloid v2.0. a-c, Time-lapse images, from 72 hours to 122 hours of culture with 5h interval between each image of a, a neural tube-like structure derived from the 201B7 iPSC line, b, an mTag-RFP line derived axioloid v2.0., c, a TJP1-mEGFP line derived axioloid v2.0. Blue arrowhead points at neural tube-like structures, white at somites, yellow at neural tube-like structure lumen. d, Images captured at different angles from the 3D reconstitution of a TJP1-mEGFP axioloid v2.0. The dotted yellow lines highlight the posterior to anterior merging of the posterior multiple neural tube-like structure lumens into a single anterior lumen. A red arrowhead indicates the fusion point. Scale bars are 200 μm.

[0010] Figure 3. WTC11-WT PSC line-dependent axioloid v2.0 morphogenesis. a, Representative bright-field (BF) images and b, c morphological outcome quantification of the differentiation matrix of the WTC11-WT PSC line induced in conditions containing variable FGF (10 / 20 ng / mL), and CHIR (2.5 / 3 / 3.5 / 4 / 4.5 / 5 μM) concentrations, and with initial 3D aggregates of 200 or 400 cells, at 120h of culture. Morphological outcomes are classified based on the differentiation balance between neural and mesodermal commitment of cell aggregates. Abbreviations used are FN: fail neuronal, NT: neural tube, NTV2: neural tube / axioloid v2.0, V2V1: axioloid v2.0 / axioloid v1.0, V1: axioloid v1.0 and FM: failed mesoderm. Scale bar is 200 μm.

[0011] Figure 4. Characterization of WTC11-WT PSC line axial morphogenesis potential. a,b, representative bright-field and fluorescence images at 5-hour intervals of axioloid v2.0 between 72h and 120h of culture, derived from a, WTC11-WT PSC (FGF10 CHIR 3.5 μM, 200 cells) and b, WTC11-TJP1-mEGFP (FGF20, CHIR 3.5 μM, 400 cells) PSC lines. c,d, Immunofluorescence-based characterization of WTC11-WT PSC-derived axioloid v2.0 for c, SOX2 (green) CDX2 (red) and PAX6 (gray), and d, TBX6 (green), CDX2 (red), and MEOX1 (gray) expression at 120h of culture. Scale bar is 200 μM.

[0012] Figure 5. Live imaging-based characterization of a WTC11-CTNNB1-mEGFP PSC reporter line derived axioloid v2.0 (FGF20 CHIR 5.5 μM, 400 cells) between 72h and 120h of culture. Scale bar is 200 μm.

[0013] Figure 6. Hybridization chain reaction (HCR) based characterization of axioloid v2.0 morphogenesis. a,b, Merged images of whole-mount in situ hybridization chain reaction (HCR) stainings of WTC11-WT PSC-derived axioloid v2.0 (200 cell aggregates, FGF10, CHIR3.5 μM) at 120h of culture for a, MESP2 (Blue) SOX2 (green) and MEOX1 (red) and b, PCDH8 (yellow), TBX18 (magenta), UNCX (Cyan). Scale bar is 200 μM.

[0014] Figure 7. 201B7-WT PSC line-dependent axioloid v2.0 morphogenesis. a, Representative bright-field images and b,c morphological outcome quantification of the differentiation matrix of the 201B7-WT PSC line induced in conditions containing variable FGF (10 / 20), and CHIR (2.5 / 3 / 3.5 / 4 / 4.5 / 5 μM) concentrations, and with initial 3D aggregates of 200 or 400 cells at 120h of culture. Cell aggregates’ morphological outcomes are classified based on the differentiation balance between neural and mesodermal commitment. Abbreviations used are FN: fail neuronal, NT: neural tube, NTV2: neural tube / axioloid v2.0, V2V1: axioloid v2.0 / axioloid v1.0, V1: axioloid v1.0 and FM: failed mesoderm. Scale bar is 200 μm.

[0015] Figure 8. Characterization of 201B7-WT PSC-line derived axioloids. a,c, Live imaging-based characterization of axioloid morphogenesis between 72 and 120h of culture and representative bright-field images at 5-hour intervals with a, a neural tube axioloid (FGF20 CHIR 3.5 μM, 300 cells) and b, axioloid v2.0 (FGF20, CHIR 4 μM, 300 cells), c, axioloid v1.0 (FGF20, CHIR 4 μM, 300 cells). d,e, Immunofluorescence-based characterization, at 120h of culture, of d, a neural tube axioloid for SOX2 (green) CDX2 (red) and PAX6 (gray) expression, and e, an axioloid v1.0 for TBX6 (green), CDX2 (red), and MEOX1 (gray). Scale bar is 200 μM.

[0016] Figure 9. Schematic representation of the machine learning-based morphological classifier. A trained ResNet50V2 model was used to classify the morphologies observed in the WTC11-WT and 201B7-WT PSC line differentiation matrix. For each line, 1296 bright-field images were manually annotated by one experienced researcher (ground truth). The dataset was then split, 864 images were used to train the machine learning-based classifier, and 432 images were used to validate its ability to distinguish the different morphologies in the WTC11-WT and 201B7-WT differentiation matrix.

[0017] Figure 10. Comparison of Human vs machine-based WTC11-WT PSC line differentiation matrix morphological classification. a, Overview of the different morphological outcomes observed in the differentiation matrix of the WTC11-WT PSC-line. b, Comparison of the accuracy of our machine learning-based classification method compared to manual annotation done by experienced and inexperienced laboratory members. c,d, Overview of the distributions of WTC11-WT differentiation matrix morphological outcomes done c, manually (ground truth) d, or using ResNet50V2 machine learning-based annotations.

[0018] Figure 11. Comparison of Human vs machine-based 201B7-WT PSC line differentiation matrix morphological classification. a, Overview of the different morphological outcomes observed in the differentiation matrix of the 201B7-WT PSC-line. b, Comparison of the accuracy of our machine learning-based classification method compared to manual annotation done by experienced and inexperienced laboratory members. c,d, Overview of the distributions of 201B7-WT differentiation matrix morphological outcomes done c, manually (ground truth) d, or using ResNet50V2 machine learning-based annotations.

[0019] Figure 12. Reporter-based analysis of axioloid v2.0 differentiation kinetic. a, Live imaging-based characterization of RUES2-GLR derived axioloid v2.0, between 72h and 120h of culture, and representative bright-field images at 5-hour intervals with SOX2 (yellow) and TBXT (cyan). b, Bright-field image of RUES2-GLR (FGF5 400 cells) differentiation under different CHIR concentrations, c, corresponding kinetics of SOX2 (orange) and TBXT (blue) gene expression were measured every day and plotted as a mean relative fluorescence intensity. Scale bar 200 μm.

[0020] Figure 13. Reporter-based analysis of axioloid v2.0 differentiation kinetic. a, Live imaging-based characterization of a PAX3-Venus PSC reporter line derived axioloid v2.0, between 72h and 120h of culture and corresponding representative bright-field and fluorescence images, extracted at 5 hours intervals. b, Bright-field images of the PAX3-Venus PSC reporter line differentiation matrix (FGF15 400 cells) under different CHIR concentrations, c, corresponding kinetics of PAX3 (green) gene expression were measured every day and plotted as a mean relative fluorescence intensity. Scale bar 200 μm.

[0021] Figure 14. Live imaging-based characterization of the segmentation clock in axioloid v2.0. a, Time series of a HES7-Achilles PSC reporter line (HES7-T2A-Achilles-NLS-CL1-PEST) derived axioloid v2.0 (FGF 20, CHIR 3.5, 200 cells). b, Corresponding kymograph and c, measurements of HES7-Achilles fluorescence intensity over time. Scale bars, 200 μm. AU, arbitrary units.

[0022] Figure 15. Reporter-based analysis of axioloid morphogenesis. a,b confocal imaging of a PSC triple reporter line for HES7 (Yellow) / MESP2 (red) / UNCX (yellow) (HES7-T2A-Achilles-NLS-CL1-PEST, MESP2-T2A-H2B-mCherry, Achilles-NLS-T2A-UNCX) derived a, axioloid v2.0 and b, axioloid v1.0, stained with phalloidin (blue). Scale bar is 200 μm.

[0023] Figure 16. Schematic overview of the axioloid v2.1 generation protocol. Axioloids v2.1 were generated by combining a, neural biased 409B2-GFP (200cells) and mesoderm biased 201B7-mCherry (300 cells) 3D aggregates b, at day 2 after initial aggregation. c, Resulting structures contain predominantly GFP neural-tube-like structure and mCherry somites.

[0024] Figure 17. Assembly of Neural and Mesodermal Biased 3D aggregates: a, b, Epi-fluorescence imaging of the matrigel embedded axioloids v2.1 and corresponding control neural tube and mesodermal biased 3D aggregates. [Description of Embodiments]

[0025] Figure 18. Antero-posterior and dorso-ventral patterning of axioloid v2.0 neural tube-like structures. a-c, immunofluorescence staining of mTag-RFP axioloid v2.0 with a, OTX2 (red: left panel, white: right panel), PAX2 (green), PAX6 (yellow) and SOX2 (in blue and cyan), and mTag-RFP (white: left panel, red: right panel). mTag-RFP iPSC line derived axioloid v2.0, b, before, or c, after, neural tube-like structure ventralization using 500 nM SAG treatment for 8 days, stained for the dorsal neural tube marker PAX3 and PAX6, and ventral neural tube markers FOXA2, NKX6.1, LHX1, OLIG2, ISLET1, and pan-neural tube marker SOX2. Scale bars are 200 μm.

[0026] Figure 19. Differentiation potential of axioloid v2.0. a, Time-lapse images of mTag-RFP derived axioloid v2.0 derived neural crest migration between 168h and 190h of culture. Red arrowhead follows the migration of a single cell. b, corresponding images of migrating neural crest at day 14 of culture c, Immunostaining of mTag-RFP derived axioloid v2.0. at day 14 with PAX7 (green), SOX10 (white), mTag-RFP (red). Note that there is very little overlap between the SOX10-expressing cells migrating away from the axioloid and the resident PAX7 cells. d, Immunostaining of mTag-RFP derived axioloid v2.0 for PAX3 (green), FOXD3 (white) and mTag-RFP (red). e, Time-lapse imaging, between 176h and 181.5h of culture, of a single neurite extending from an mTag-RFP axioloid v2.0. Red arrowhead indicates the front of the extending neurite. f, Differentiated Neuronal cells at day 14, pink lines and arrows in right panel highlights the branching pattern of the neurites. g, and h, immunofluorescence of mTag-RFP derived axioloid v2.0 at day 14 of culture stained with g, SOX2 (white) and NF-H (green) and mTag-RFP (red) and h, SOX2 (white), TUJ1 (green), and mTag-RFP (red). i, Dermomyotome differentiation of somites in mTag-RFP derived axioloid v2.0 with PAX3 and PAX7 (green) and MEOX1 (white), and mTag-RFP (red). j, Skeletal muscle differentiation in an axioloid v2.0. Left panel shows filiform and parallel cell soma extending outward from mTag-RFP axioloid v2.0. Right panel shows immunostaining of ACTN2 (red) and DAPI (blue). Axioloids v2.0 in a-h, were embedded in Matrigel-containing medium in an 8-well chamber glass slide at 120h and then allowed to further differentiate for 14 days before staining. Axioloids v2.0 in i and j were first cultured for 144h, then went through dermomyotome induction for 3 days and finally were allowed to differentiate on a glass slide for 14 or 21 days respectively. Scale bars are 200 μm.

[0027] Figure 20. Valproic acid-induced neural tube defects in axioloid v2.0. a, Immunostaining of control and 1mM VPA-treated (72h to 144h) TJP1-mEGFP derived axioloid v2.0 at 144h stained with MEOX1 (cyan), PAX6 (magenta) and SOX2 (white), and mTJP1-mEGFP (yellow) marks the lumen of the neural tube-like structure and somitocoels. b, Representative image of a 3D reconstructed control and VPA treated axioloid v2.0 stained with SOX2 (yellow) and ZO-1 (green). Scale bars are 200 μm.

[0028] DETAILED DESCRIPTION The present disclosure relates to a three-dimensional cellular aggregate termed axioloid generated in vitro from a pluripotent stem cell and to a method for producing the same.

[0029] In one aspect, the present disclosure is a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation.

[0030] In another aspect, the present disclosure is a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure.

[0031] In the present disclosure, the "lower" is used to intend a group having less number and / or amount of a subject compared with criterion, unless otherwise provided.

[0032] In the present disclosure, the "higher" is used to intend a group having more number and / or amount of a subject compared with criterion, unless otherwise provided.

[0033] In the present disclosure, suitable example of "one or more” may be the number of 1 to 6, in which the preferred one may be the number of 1 to 3.

[0034] In the present disclosure, the "marker" means a nucleic acid, a gene, a polypeptide, or a protein that is expressed to a different extent in a target cell. When the marker is a positive marker, the different extent means increased expression compared to the cells to be compared with. When the marker is a negative marker, the different extent means reduced expression compared to the cells to be compared with.

[0035] In the present disclosure, the "pluripotent cell" means a cell capable of differentiating into ectodermal, mesodermal, and endodermal cells. The pluripotent cell may also be referred to as a pluripotent stem cell when the pluripotent cell is capable of self-replication.

[0036] In the present disclosure, the "ectodermal cell" means a cell destined to be capable of differentiating into neural tissues such as nerves; epithelial tissues such as epidermis; and the like if there is a developmentally appropriate stimulation, and is a cell expressing an ectodermal cell marker to be described below.

[0037] In the present disclosure, the "mesodermal cell" means a cell destined to be capable of differentiating into connective tissue, such as bones, cartilages, blood vessels, lymphatic vessels and the like; muscle tissues; and the like if there is a developmentally appropriate stimulation, and is a cell expressing a mesodermal cell marker to be described below.

[0038] In the present disclosure, the "endodermal cell" means a cell destined to be capable of differentiating into thymus; digestive organs such as stomach, intestine, liver, and the like; respiratory organs such as trachea, bronchi, lungs, and the like; and urinary organs such as bladder, urethra, and the like; and the like if there is a developmentally appropriate stimulation, and is a cell expressing an endodermal cell marker to be described below.

[0039] In the present disclosure, the "three-dimensional cellular aggregate" means a structure in which cells are aggregated in three dimensions. The three-dimensional culture aggregate is different from, for example, a two-dimensional cellular aggregate (cell sheet) obtained in a planar culture, and forms a three-dimensional structure by, for example, accumulating cells in a thickness direction.

[0040] In the present disclosure, the sequence information of a protein or a nucleic acid encoding the same (e.g., DNA or RNA) described herein is available from Protein Data Bank, UniProt or Genbank.

[0041] The present disclosure refers to a cellular aggregate (hereinafter also referred to as "axioloid") generated in vitro from one or more pluripotent stem cells, a method for producing such cellular aggregate, and the cell derived, obtained or obtainable from the cellular aggregate.

[0042] Certain aspect of the cellular aggregate (cell aggregate) described in the present disclosure includes being a three-dimensional cellular aggregate generated in vitro from pluripotent stem composed of mesodermal cells (including primitive streak and presomitic mesoderm cells) and ectodermal cells, wherein the cellular aggregate has a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis or can obtain, induce, or acquire a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis, and the cellular aggregate can reconstitute various aspects of a neural tube formation.

[0043] In one another aspect, the cellular aggregate described in the present disclosure includes being a polarized three-dimensional cellular aggregate generated in vitro from pluripotent stem composed of mesodermal cell and ectodermal cell, wherein the cellular aggregate has a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis or can obtain, induce, or acquire a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis, and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure.

[0044] In another aspect, the cellular aggregate described in the present disclosure includes being a polarized three-dimensional cellular aggregate generated in vitro from pluripotent stem cell-derived mesodermal cells, ectodermal cell and progenitor cells, wherein the cellular aggregate has a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis or can obtain, induce, or acquire a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis, and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure.

[0045] In another aspect, a cellular aggregate of the present disclosure is a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, composed of mesodermal cells and ectodermal cells, wherein the cellular aggregate has a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis or can obtain, induce, or acquire a polarity along its antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis.

[0046] In the present disclosure, the dorso‐ventral axis may be defined by a dorsal region and a ventral region. A cell in the dorsal region has, for example, a higher or lower expression of one or more markers as compared to a cell in the ventral region.

[0047] In the present disclosure, the dorsal region may have the neural tube-like structure. In the present disclosure, a cell in a neural tube-like structure may have an expression of at least one marker selected from the groups consisting of a dorsal markers PAX3, PAX6 and PAX7 and ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2. In this case, the expression level of the ventral markers may be much lower than that of the dorsal markers.

[0048] In the present disclosure, the antero-posterior axis may be defined by an anterior (rostral) region and a posterior (caudal) region. A cell in the anterior region has, for example, a higher or lower expression of one or more markers as compared to a cell in the posterior region. The cellular aggregate of the present disclosure may show anterior-posterior morphological and molecular organization that mimics the one seen in in vivo human and vertebrate embryos, with MEOX1+ somitic mesoderm (SM), a HES7+ / TBX6+ presomitic mesoderm (PSM) and a TBXT+ / SOX2+ tailbud-like (TB) structure in the posterior-most part of the axioloids which contains a neuromesodermal progenitor (NMP) cell population.

[0049] In the present disclosure, a cell in the anterior region may have a lower expression of one or more markers as compared to a cell in the posterior region. In this case, for example, the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2, preferably TBXT, SOX2, TBX6, HES7, MEOX1 and TCF15, more preferably TBXT. One type or two or more types of them may be used as the marker.

[0050] In the present disclosure, a cell in the anterior region may have a higher expression of one or more markers as compared to a cell in the posterior region. In this case, for example, the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10. The anterior region may include somitic mesoderm (SM) and head mesoderm-like cells.

[0051] In the present disclosure, the posterior (caudal) region may include tailbud (TB) like-cells.

[0052] In the present disclosure, a cell in the anterior region may have an expression of OTX2 and PAX2.

[0053] In the present disclosure, a cell in an intermediate region between the anterior region and the posterior region may have an expression of PAX6.

[0054] In the present disclosure, the cellular aggregate may have a polarity in an apical-basolateral axis.

[0055] In the present disclosure, the apical-basolateral axis may be defined by an apical region and a basolateral region. A cell in the apical region has, for example, a higher or lower expression of one or more markers as compared to a cell in the basolateral region.

[0056] The mesodermal cell expresses one or more markers (mesodermal cell marker) selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR, for example. One type or two or more types of them may be used as the marker.

[0057] In the present disclosure, a cell in the tube-like structure may have an expression of PAX3, PAX6, PAX7, SOX2 and OTX2

[0058] The cellular aggregate of the present disclosure can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, TBX18, UNCX, ALDH1A2 and RDH10.

[0059] The cellular aggregate of the present disclosure can form a somite or a somite-like structure under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, TBX18, UNCX, ALDH1A2 and RDH10.

[0060] The somitogenic culture condition is, for example, a presence of a gel and / or a matrix and a retinoic acid, a retinoic acid precursor or its derivative and / or a retinoic acid receptor (RAR) agonist.

[0061] The somitogenic culture condition is, for example, a presence of a gel and / or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or its derivative and / or a retinoic acid receptor (RAR) agonist.

[0062] The RAR agonist includes, for example, Vitamin A, retinol, retinal, , 9-cis retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, AGN193078, TTNN(Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and / or Re80, preferably, retinal or retinol.

[0063] The retinoid includes, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid,13-cis-retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AM555S, CD2314, CD437, CD666, CD2325, SR11254, SR11364, TTNN(Ro19-0645), CD-270, CD271, SR3985, and / or Ch55 preferably, retinal or retinol.

[0064] The cellular aggregate is, for example, embedded in the gel or the matrix, or disposed inside the gel or the matrix.

[0065] The matrix includes, for example, an extracellular matrix. The extracellular matrix includes, for example, collagen, laminin, fibronectin, vitronectin, gelatin and / or entactin. The matrix can be used at various concentrations (e.g., 1%, or 5%, or 10% or 20%) per volume of utilized embedding media. The concentration is preferably 5%, more preferably 10%. One type or two or more types of them may be used as the extracellular matrix.

[0066] The gel includes, for example, a hydrogel. The gel includes, for example, a basement membrane matrix. The basement membrane matrix includes, for example, one or more group comprising laminin, collagen, fibronectin, gelatin, vitronectin, heparan sulphate proteoglycan and / or entactin. One type or two or more types of them may be used as the basement membrane matrix. The gel includes, for example, an acrylamide gel, an arginine gel, an agarose gel and / or a polyethylene glycol hydrogel with various biomechanical properties.

[0067] In the present disclosure, the somite or the somite-like structure may include an anterior (rostral) portion and / or a posterior (caudal) portion in the antero-posterior axis. An anterior portion cell has, for example, a higher or lower expression of one or more markers as compared to a posterior portion cell.

[0068] The anterior (rostral) portion cell may have a higher expression of one or more markers than the posterior portion cell. In this case, for example, the one or more markers are selected from the group consisting of TBX18 and ALDH1A2, and is preferably TBX18. One type or two or more types of them may be used as the marker.

[0069] The anterior portion cell may have a lower expression of one or more markers as compared to the posterior portion cell. In this case, for example, the one or more markers are selected from the group consisting of UNCX and LNFG, and is preferably UNCX. One type or two or more types of them may be used as the marker.

[0070] The cellular aggregate of the present disclosure may include anterior paraxial / presomitic mesoderm (aPSM). The aPSM means an area on the posterior (caudal) side of the axioloid located between PSM and SM. The aPSM can be defined by the expression of one or more makers selected from a group of markers consisting of MESP2, RIPPLY2, RIPPLY1 and PCDH8, preferably MESP2.

[0071] In the present disclosure, the somite is formed, for example, in a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours or a cycle of 4 to 6 hours. The somite is formed preferably in a cycle of 3.5 to 6.6 hours, more preferably in a cycle of 4 to 6 hours.

[0072] In the present disclosure, the length of the somite in the antero-posterior axis is, for example, 30 to 200 μm, 50 to 150 μm, 60 to 140 μm, 70 to 130 μm or 80 to 120 μm. The length of the somite is preferably 30 to 200 μm, more preferably 80 to 120 μm.

[0073] The cellular aggregate may include pluripotent stem cells.

[0074] In the present disclosure, the pluripotent stem cell expresses one or more markers (pluripotent stem cell marker) selected from the group consisting of OCT4, SOX2, NANOG, ABCG2, CRIPTO, FOXD3, Connexin43, Connexin45, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, LIN28 and REX1. One type or two or more types of them (preferably OCT4, more preferably NANOG) may be used as the marker.

[0075] The proportion of the pluripotent stem cell in the cellular aggregate is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.

[0076] The proportion of the pluripotent stem cell defined by the expression of NANOG in the cellular aggregate is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.

[0077] The cellular aggregates do not substantially include, for example, an endodermal cell.

[0078] The proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.

[0079] The endodermal cell expresses one or more markers (endodermal cell marker) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1, and preferably GATA6 and / or SHH, for example. One type or two or more types of them (preferably FOXA2, more preferably GATA6) may be used as the marker.

[0080] The proportion of the ectodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.

[0081] The ectodermal cell expresses one or more markers (ectodermal cell marker) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1, and preferably OTX2, for example. One type or two or more types of them (preferably SOX1, more preferably OTX2) may be used as the marker.

[0082] The proportion of the mesodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.

[0083] In one aspect, the proportion of the mesodermal cell and the ectodermal cell in the cellular aggregate can be controlled by changing the concentration of Wnt agonist added to the culture medium.

[0084] In the cellular aggregate, an expression of a segmentation clock gene may be subjected to gene oscillation. The gene oscillation means, for example, that the expression level of the target gene is periodically oscillating in space and time. The gene oscillation is also referred to as, for example, a gene expression oscillation.

[0085] The segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7, and is preferably HES7. One type or two or more types of them (preferably LFNG, more preferably HES7) may be used as the segmentation clock gene.

[0086] The cycle of the gene oscillation is, for example, a cycle of 3 to 7 hours, preferably a cycle of 3.5 to 6.6 hours or more preferably a cycle of 4 to 6 hours.

[0087] The pluripotent stem cell used for derivation of axioloids is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, a whale, an armadillo, a tenrec, an elephant, a sea lion, a snake, a gecko, a chicken and the like. Non-human animal pluripotent stem cells also include monotreme species, such as a platypus or an echidna and marsupial species, such as an opossum, a kangaroo, a wombat or the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificially engineered pluripotent stem cell.

[0088] The pluripotent stem cell may be, for example, a pluripotent stem cell in which a gene or genomic sequence is modified. Examples of the modification include introduction of a mutation into a coding or non-coding gene or genome region, repair of a coding or non-coding (regulatory) gene or genome mutation, introduction of a foreign gene or non-coding (regulatory) genome region, and knockout of a gene or genome region. The modification of the gene or genomic sequence can be performed using, for example, genome editing techniques such as ZFN, TALEN, CRISPR / Cas systems and the like; gene recombination techniques; and the like.

[0089] In the present disclosure, the cellular aggregate includes, for example, at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The number of the cellular aggregate is, for example, 100 to 100000 cells, 200 to 100000 cells, 300 to 100000 cells, 400 to 500000 cells, 600 to 100000 cells, 700 to 100000 cells, 800 to 100000 cells, 900 to 100000 cells, 1000 to 90000 cells, 1500 to 80000 cells, 2000 to 70000 cells, 2500 to 60000 cells, 5000 to 50000 cells, 10000 to 50000 cells, 15000 to 50000 cells, 20000 to 50000 cells, 30000 to 50000 cells or 40000 to 50000 cells. The number of the cellular aggregate is preferably at least 50 cells, more preferably 100 to 1000 cells.

[0090] In the present disclosure, the cellular aggregate has, for example, a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. The length of the cellular aggregate is, for example, 0.05 to 10 mm, 0.1 to 9 mm, 0.2 to 8 mm, 0.3 to 7 mm, 0.4 to 6 mm, 0.5 to 5 mm, 0.6 to 4 mm, 0.7 to 3 mm, 0.8 to 2 mm or 0.9 to 1 mm. The length of the cellular aggregate is preferably 0.05mm to 10mm, more preferably at least 1mm.

[0091] In a further aspect, the method of the present disclosure is a method for producing a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, including the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate including (preferably, consisting of) a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell to induce a three-dimensional cellular aggregate, wherein the three-dimensional cellular aggregate is the cellular aggregate according to the present disclosure.

[0092] In the present disclosure, the cellular aggregate may further comprise a notochord-like structure.

[0093] In a further aspect, the present disclosure is a method for producing the cellular aggregate from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate comprising (preferably, consisting of) a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate comprising (preferably, consisting of) the mesodermal cell and the ectodermal cell to induce the cellular aggregate, wherein the three-dimensional cellular aggregate is the cellular aggregate (‘axioloid’) according to the present disclosure.

[0094] In the method of the present disclosure, the "culture" can be carried out, for example, using a medium optionally supplemented with a factor. In the method of the present disclosure, the medium may be replaced during the culture period.

[0095] The medium can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM, Medium199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI1640, Fischer's medium, a Neurobasal Medium (manufactured by Thermo Fisher Scientific), a stem cell culture medium (e.g., mTeSR-1 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), CDM-PVA, StemFit (registered trademark) , AK02N (manufactured by ReproCELL Inc.), StemPRO (registered trademark) hESC SFM (manufactured by Life Technologies), E8 (manufactured by Life Technologies), Essential 6 (manufactured by Thermo Fisher scientific) and mixed media thereof. The medium may be supplemented with serum or may be serum-free. The medium may contain, for example, serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for ES-cell culture), N2 supplement (manufactured by Invitrogen), B27 supplement (manufactured by Invitrogen), fatty acids, insulin, collagen progenitor, trace elements, 2-mercaptoethanol, 3'-thiol glycerol and the like. Further, the medium may contain additives such as lipids, amino acids, L-glutamine, Glutamax (manufactured by Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like. When a growth culture is performed using the structure, the medium is preferably a stem cell culture medium to which NEAA, glutamic acid and an antibiotic are added.

[0096] The culture conditions in the culture may adopt, for example, common conditions of cell culture. As a specific example, the culture temperature is, for example, 25℃ to 40℃, preferably 30℃ to 40℃ or more preferably about 37℃. The carbon dioxide concentration during the culture is 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is carried out, for example, in a wet environment.

[0097] The culture conditions implemented during the culture may adopt, for example, common conditions of cell culture. As a specific example, the culture temperature is, for example, 25℃ to 40℃, preferably 30℃ to 40℃ or more preferably about 37℃. The carbon dioxide concentration during the culture is 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is carried out, for example, in a wet environment.

[0098] In the method of the present disclosure, the step (a) may comprise: (a2) culturing the pluripotent stem cell in a medium containing an FGF (such as a basic FGF), a transforming growth factor β (TGFβ) inhibitor, a Rho-associated protein kinase (ROCK) inhibitor and a Wnt agonist (such as a GSK3β inhibitor) to induce the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell.

[0099] In the method of the present disclosure, the step (a) may comprise before the step (a2): (a1) culturing the pluripotent stem cell in a medium containing a fibroblast growth factor (FGF) (such as a basic FGF) and a Wnt agonist (such as a glycogen synthase kinase 3β (GSK3β) inhibitor) to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell.

[0100] In one aspect of the present invention, the step (a1) is preferably conducted by the two-dimensional culture.

[0101] In one aspect of the present invention, the step (a2) is preferably conducted by the three-dimensional culture.

[0102] In one aspect of the present invention, the desired cellular aggregate can be produced even if the pluripotent stem cells are subjected to the three-dimensional culture in the step (a2) without going through the two-dimensional culture in the step (a1).

[0103] In the method of the present disclosure, the step (a) may comprise: (a4) culturing the pluripotent stem cell in a medium containing an FGF (such as a basic FGF), a TGFβ inhibitor and a Wnt agonist (such as a GSK3β inhibitor) to induce the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell.

[0104] In the method of the present disclosure, the step (a) may comprise before the step (a4): (a3) culturing the pluripotent stem cell in a medium containing an FGF (such as a basic FGF), a ROCK inhibitor and a Wnt agonist (such as a GSK3β inhibitor) to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell.

[0105] In one aspect of the present invention, the step (a3) is preferably conducted by the two-dimensional culture.

[0106] In one aspect of the present invention, the step (a4) is preferably conducted by the three-dimensional culture.

[0107] In one aspect of the present invention, the desired cellular aggregate can be produced even if the pluripotent stem cells are subjected to the three-dimensional culture in the step (a4) without going through the two-dimensional culture in the step (a3).

[0108] In the method of the present disclosure, the step (b) may comprise: (b1) culturing the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell in a medium containing a retinoid, a retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or a matrix to induce neuronal differentiation.

[0109] In the method of the present disclosure, the step (b) may further comprise: (b2) culturing the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell in a medium containing an SHH pathway agonist in the presence of a gel or a matrix to induce neuronal differentiation.

[0110] The method of the present disclosure may further comprise the steps of: (c) culturing the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell in a medium containing a GSK3β inhibitor, a bone morphogenetic protein 4 (BMP4) and hedgehog inhibitor to induce dermomyotome differentiation; and optionally (d) culturing the cellular aggregate derived, obtained or obtainable from step (c) in a medium in the presence of a gel or a matrix to induce myogenic differentiation.

[0111] In the method of the present disclosure, the FGF may be a basic FGF.

[0112] In the method of the present disclosure, the FGF is, for example, bFGF (FGF2).

[0113] The concentration of bFGF in the medium is, for example, 1 to 1000 ng / ml or preferably 10 to 1000 ng / ml.

[0114] In the method of the present disclosure, the Wnt agonist may be a GSK3β inhibitor or Wnt.

[0115] In the method of the present disclosure, the concentration of the Wnt agonist (such as a GSK3β inhibitor) may be 4 μM or less.

[0116] In case of using CHIR99021 as the GSK3β inhibitor, when inducing a neural tube-like structure, for example, CHIR99021 concentration ranges from 2 to 3 μM. In case of using CHIR99021 as the GSK3β inhibitor, when inducing both a neural tube-like structure and a somite structure, for example, CHIR99021 concentration ranges from 3 to 4 μM. In case of using CHIR99021 as the GSK3β inhibitor, when inducing a somite structure, for example, CHIR99021 concentration ranges from 4 to 6 μM.

[0117] In the method of the present disclosure, the GSK3β inhibitor may be, for example, a substance that inhibits the kinase activity of GSK3β protein (e.g., phosphorylation ability to β catenin), specifically, an indirubin derivative such as BIO (GSK-3 β inhibitor IX: 6-bromoindirubin 3'-oxime) or the like; a maleimide derivative such as SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione or the like; a phenyl α bromomethylketone compound such as GSK-3β inhibitor VII (4-dibromoacetophenone) or the like; a cell membrane penetrating phosphorylated peptide such as L803-mts (GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2) or the like; CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile); an expression suppressing the nucleic acid molecule of GSK3β protein (siRNA, shRNA, antisense or the like) and the like. The GSK3β inhibitor is preferably CHIR99021, because of its high selectivity to GSK3β. The GSK3β inhibitor is commercially available, for example, from Calbiochem, Biomol and the like. In the present disclosure, a WNT agonist including recombinant WNT proteins such as WNT3a as well as other canonical and non-canonical WNT agonists can be used instead of the GSK3β inhibitor.

[0118] The concentration of the GSK3β inhibitor in the medium is, for example, 1 nmol / l (also referred to as “M” hereinafter) to 1000 μmol / l, preferably 10 nmol / l to 100 μmol / l or more preferably 100 nmol / l to 100 μmol / l.

[0119] In the method of the present disclosure, the TGFβ inhibitor is a substance that inhibits SMAD mediated signaling caused by binding of TGFβ to a receptor. Examples of the TGFβ inhibitor include a substance that inhibits binding to the ALK family, which is a TGFβ acceptor, and a substance that inhibits phosphorylation of SMAD by the ALK family. Specific examples of the TGFβ inhibitor include Lefty-1(NCBI Accession Number: NM-010094 (mouse), NM-020997 (human), SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridine-2-yl)-1H-imidazol-2-yl)benzamide), SB202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB505124 (2-(5-Benzo[1,3dioxol]-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories) and A-83-01 (WO2009 / 146408), and SB431542 is preferred.

[0120] The concentration of the TGFβ inhibitor in the medium is, for example, 1 nmol / l to 1000 μmol / l, preferably 10 nmol / l to 100 μmol / l or more preferably 100 nmol / l to 100 μmol / l.

[0121] In the method of the present disclosure, the ROCK inhibitor is a substance that can suppress the function of the Rho-kinase (ROCK). Examples of the ROCK inhibitor include Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), Fasudil / HA1077 (5-(1, 4-Diazepane-1-sulfonyl)isoquinoline), H-1152 ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine), Wf-536 ((+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide) and an expression suppressing the nucleic acid molecule of ROCK protein (siRNA, shRNA, antisense, etc.), and Y-27632 is preferred.

[0122] The concentration of the ROCK inhibitor in the medium is, for example, 1 nmol / l to 50 μmol / l, 10 nmol / l to 40 μmol / l, 50 nmol / l to 30 μmol / l, 100 nmol / l to 25 μmol / l, 500 nmol / l to 20 μmol / l or 750 nmol / l to 15 μmol / l, and is preferably 1 nmol / l to 40 μmol / l, more preferably 1 nmol / l to 15 μmol / l.

[0123] In the method of the present disclosure, the RAR agonist is, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, AGN193078, TTNN(Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and / or Re80, preferably, retinal or retinol.

[0124] In the method of the present disclosure, the retinoid includes, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, 13-cis-retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AM555S, CD2314, CD437, CD666, CD2325, SR11254, SR11364, TTNN(Ro19-0645), CD-270, CD271, SR3985, and / or Ch55 preferably, retinal or retinol.

[0125] The concentration of the retinoic acid, the retinoic acid precursor or derivative and / or the retinoic acid receptor (RAR) agonists in the medium is, for example, 1 nmol / l to 1000 μmol / l, preferably 10 nmol / l to 100 μmol / l or more preferably 100 nmol / l to 100 μmol / l.

[0126] The concentration of the retinoid, retinoic acid, the retinoic acid precursor or derivative and / or the retinoic acid receptor (RAR) agonists in the medium is, for example, 1 nmol / l to 1000 μmol / l, preferably 10 nmol / l to 100 μmol / l or more preferably 100 nmol / l to 100 μmol / l.

[0127] In the methods of the present disclosure, for example, the GSK3β inhibitor is CHIR99021, the FGF is bFGF, the TGFβ inhibitor is SB431542 and / or the ROCK inhibitor is Y-27632.

[0128] In the method of the present disclosure, the SHH pathway agonist may be SAG.

[0129] In the present disclosure, the hedgehog inhibitor may be SHH inhibitor (GDC0449).

[0130] In the method of the present disclosure, for example, the matrix includes an extracellular matrix.

[0131] The matrix includes, for example, an extracellular matrix. The extracellular matrix includes, for example, collagen, laminin, fibronectin, vitronectin, gelatin and / or entactin. One type or two or more types of them may be used as the extracellular matrix.

[0132] In the method of the present disclosure, the gel includes, for example, a hydrogel. The gel includes, for example, a basement membrane matrix. The basement membrane matrix includes, for example, fibronectin, laminin, collagen, vitronectin, gelatin, heparan sulphate proteoglycan and / or entactin. One type or two or more types of them may be used as the basement membrane matrix. The gel includes an acrylamide gel, an arginine gel, an agarose gel and / or a polyethylene glycol hydrogel. The gel includes preferably an agarose gel, more preferably a polyethylene glycol hydrogel.

[0133] In the method of the present disclosure, the concentration of the gel and / or the matrix in the medium is, for example, a concentration capable of three-dimensional culture, and can be appropriately set according to the type of the gel and the matrix. As a specific example, the concentration of the gel and / or the matrix in the medium is, for example, 0.01 to 50% (v / v), 0.1 to 25% (v / v), 1 to 20% (v / v) or 5 to 10% (v / v). The concentration is preferably 0.01 to 50% (v / v), more preferably 5 to 10% (v / v).

[0134] The mesodermal cell expresses one or more markers (mesodermal cell marker) selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2, for example. One type or two or more types of them may be used as the marker.

[0135] In the method of the present disclosure, for example, the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell substantially does not include an endodermal cell.

[0136] The proportion of the endodermal cell in the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 10% or less, more preferably 1% or less.

[0137] The endodermal cell expresses one or more markers (endodermal cell marker) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1, and preferably GATA6 and / or SHH, for example. One type or two or more types of them may be used as the marker.

[0138] The proportion of the ectodermal cell in the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell is, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.

[0139] The ectodermal cell expresses one or more marker (ectodermal cell marker) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2 and BARX1, and preferably OTX2, for example. One type or two or more types of them may be used as the marker.

[0140] The proportion of the mesodermal cell in the cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell is, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.

[0141] In one aspect, the proportion of the mesodermal cell and the ectodermal cell in the cellular aggregate can be controlled by changing the concentration of Wnt agonist added to the culture medium.

[0142] The number of days of culture of the step (a) is, for example, 0.1 to 10 days, 0.25 to 6 days, 0.25 to 4 days, 0.5 to 3 days or 0.5 to 2 days. The number is preferably 0.1 to 10 days, more preferably 0.5 to 2 days.

[0143] The number of days of culture of the step (a1) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.

[0144] The number of days of culture of the step (a2) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.

[0145] The number of days of culture of the step (a3) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.

[0146] The number of days of culture of the step (a4) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.

[0147] The number of days of culture of the step (b) is, for example, 0.1 to 10 days, 1 to 9 days, 2 to 8 days or 3 to 7 days. The number is preferably 0.1 to 10 days, more preferably 2 to 8 days.

[0148] The number of days of culture of the step (b1) is, for example, 0.1 to 7 days preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.

[0149] The number of days of culture of the step (b2) is, for example, 0.1 to 7 days preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.

[0150] The number of days of culture of the step (c) is, for example, 0.1 to 10 days, 1 to 9 days, 2 to 8 days or 3 to 7 days. The number is preferably 0.1 to 10 days, more preferably 2 to 8 days.

[0151] The number of days of culture of the step (d) is, for example, 0.1 to 10 days, 1 to 9 days, 2 to 8 days or 3 to 7 days. The number is preferably 0.1 to 10 days, more preferably 2 to 8 days.

[0152] In the method of the present disclosure, for example, the pluripotent stem cell is one or more suitable dissociated pluripotent stem cell(s) or one or more suitable cell(s) suspension containing any pluripotent stem cell(s).

[0153] In the method of the present disclosure, the pluripotent stem cell is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, an elephant, a sea lion, a snake, a gecko, a chicken, and the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificial pluripotent stem cell.

[0154] In the method of the present disclosure, the pluripotent stem cell is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, a whale, an armadillo, a tenrec, an elephant, a sea lion, a snake, a gecko, a chicken and the like. Non-human animal pluripotent stem cells also include monotreme species, such as a platypus or an echidna and marsupial species, such as an opossum, a kangaroo, a wombat or the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificially engineered pluripotent stem cell.

[0155] The cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell includes, for example, at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The cellular aggregate includes preferably at least 50 cells, more preferably at least 100 to 1000 cells.

[0156] The cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell is made of, for example, at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The cellular aggregate includes preferably at least 50 cells, more preferably at least 100 to 1000 cells.

[0157] The cellular aggregate including (preferably, consisting of) the mesodermal cell and the ectodermal cell has, for example, a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. The cellular aggregate has preferably at least 0.05 mm, more preferably at least 0.5 mm.

[0158] In another aspect, the present disclosure provides a cell obtained from the three-dimensional cellular aggregate of the present disclosure.

[0159] In yet another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, including the step of: culturing the three-dimensional cellular aggregate of the present disclosure to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (h): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endothelial or hemogenic cell or a progenitor cell thereof; (g) a neural crest cell or a progenitor cell thereof; and (h) a neuronal cell or a progenitor cell thereof.

[0160] In yet another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, including the step of: culturing the three-dimensional cellular aggregate of the present disclosure to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof.

[0161] The method for inducing the differentiated cell and the progenitor cell thereof of (a) to (h) can be carried out in the same manner as, for example, a method for inducing each differentiated cell and a progenitor cell thereof from the mesodermal cell.

[0162] The production method of the present disclosure may include, for example, the step of inducing a three-dimensional cellular aggregate from a pluripotent stem cell prior to the inducing. In this case, for example, the inducing of the three-dimensional cellular aggregate may be performed by the method for producing the three-dimensional cellular aggregate of the present disclosure.

[0163] The production method of the present disclosure may include, for example, the step of forming a three-dimensional cellular aggregate from a pluripotent stem cell prior to the induction. In this case, for example, the formation of the three-dimensional cellular aggregate may be performed by the method for producing the three-dimensional cellular aggregate of the present disclosure.

[0164] In another aspect, the present disclosure provides a method for evaluating a test substance, including the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the three-dimensional cellular aggregate through and / or after the culturing, wherein the three-dimensional cellular aggregate is the cellular aggregate of the present disclosure.

[0165] The type of the test substance is not particularly limited, and examples thereof include a protein, an antibody, a peptide, a nucleic acid molecule, a sugar chain, a lipid, an organic low molecular weight compound, an inorganic low molecular weight compound, a bacterial releasing substance, a fermentation product, a cell extract, a vacuole culture supernatant, a plant extract and an animal tissue extract. One type or two or more types of them (preferably an organic low molecular weight compound, more preferably a protein) may be used as the test substance.

[0166] In the evaluation, for example, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate.

[0167] The culture is, for example, a culture under a segmentation culture condition. The segmentation culture conditions can be referred to, for example, the above description. The culture conditions may adopt, for example, the common conditions of cell culture.

[0168] During the evaluation, for example, a test substance that changes somitogenesis of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the somitogenesis process of the cellular aggregate. During the evaluation, for example, a test substance that changes a neural tube formation of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the neural tube formation process of the cellular aggregate.

[0169] The culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (h): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endothelial or hemogenic cell or a progenitor cell thereof; (g) a neural crest cell or a progenitor cell thereof; and (h) a neuronal cell or a progenitor cell thereof

[0170] The culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof.

[0171] During the evaluation, for example, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h).

[0172] During the evaluation, for example, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i).

[0173] In the evaluation method, the evaluation is an evaluation using a control in which the test substance is not present as a reference.

[0174] During the evaluation, the method of evaluation uses a control in which the test substance is not present as a reference.

[0175] The culture is generation of a three-dimensional cellular aggregate from a pluripotent stem cell, and the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.

[0176] The culture refers to the generation of a three-dimensional cellular aggregate from a pluripotent stem cell, where the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate described in the present disclosure.

[0177] In another aspect, the present disclosure provides a method for evaluating gene function or genome function, including the steps of: preparing a pluripotent stem cell which a test gene or a test genome is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate through and / or after the culturing, wherein the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.

[0178] In another aspect, the present disclosure provides a method for evaluating gene function or genomic sequence function, including the steps of: engineering a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate through and / or after the culturing, wherein the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.

[0179] During the evaluation, for example, a test gene or a test genome (coding and non-coding genome regions) that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genome (coding and non-coding genome regions) that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate.

[0180] During the evaluation, for example, a test gene or a test genomic sequence (coding and non-coding genome regions) that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence (coding and non-coding genome regions) that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate.

[0181] The function evaluation method of the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition. In this case, during the evaluation, a test gene or a test genome (coding and non-coding genome regions) that changes somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate is evaluated as a candidate gene or a candidate genome (coding or non-coding) that modifies, promotes or suppresses the somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate. The function evaluation method of the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a neural tube formation culture condition. In this case, during the evaluation, a test gene or a test genome (coding and non-coding genome regions) that changes a neural tube formation is evaluated as a candidate gene or a candidate genome (coding or non-coding) that modifies, promotes or suppresses the neural tube formation.

[0182] The method to evaluate the function in the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition. In this case, during the evaluation, a test gene or a test genomic sequence (coding and non-coding genome regions) that changes somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence (coding or non-coding) that modifies, promotes or suppresses the somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate. The method to evaluate the function in the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a neural tube formation culture condition. In this case, during the evaluation, a test gene or a test genomic sequence (coding and non-coding genome regions) that changes neural tube formation is evaluated as a candidate gene or a candidate genomic sequence (coding or non-coding) that modifies, promotes or suppresses the neural tube formation.

[0183] In the present disclosure, the gene may be any gene and the example thereof is a gene related to human diseases (e.g., HES7). The test genes may be a gene having gene mutation related to human diseases (e.g., HES7R25Wand spondylocostal dysostosis).

[0184] In the function evaluation method of the present disclosure, the genome is an exon region, an intron region, a promoter region, an enhancer region and / or a non-coding region of the genome.

[0185] In the function evaluation method of the present disclosure, the genomic sequence is an exon region, an intron region, a promoter region, an enhancer region and / or a non-coding region of the genome.

[0186] In one aspect, the present invention is disclosed as follows: [Item 1] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation. [Item 2] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure. [Item 3] The cellular aggregate according to item 1 or 2, wherein the dorso‐ventral axis is defined by a dorsal region and a ventral region, and the dorsal region has a neural tube-like structure. [Item 4] The cellular aggregate according to item 3, wherein a cell in the neural tube-like structure has an expression of at least one marker selected from the groups consisting of dorsal markers PAX3, PAX6 and PAX7 and ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2. [Item 5] The cellular aggregate according to any one of items 1 to 4, wherein the antero-posterior axis is defined by an anterior region and a posterior region, and i) a cell in the anterior region has a lower expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2; or ii) a cell in the anterior region has a higher expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10. [Item 6] The cellular aggregate according to item 5, wherein the posterior region comprises a tailbud (TB) like-cell. [Item 7] The cellular aggregate according to any one of items 1 to 4, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in the anterior region has an expression of OTX2 and PAX2. [Item 8] The cellular aggregate according to any one of items 1 to 4, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in an intermediate region between the anterior region and the posterior region has an expression of PAX6. [Item 9] The cellular aggregate according to any one of items 1 to 8, wherein the cellular aggregate has a polarity in an apical-basolateral axis. [Item 10] The cellular aggregate according to any one of items 1 to 9, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 11] The cellular aggregate according to item 2, wherein a cell in the neural tube-like structure has an expression of PAX3, PAX6, PAX7, SOX2 and OTX2. [Item 12] The cellular aggregate according to any one of items 1 to 11, wherein the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition. [Item 13] The cellular aggregate according to item 12, wherein the somitogenic culture condition is a presence of a gel or a matrix and a retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist; or the somitogenic culture condition is a presence of a gel or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist. [Item 14] The cellular aggregate according to item 13, wherein the RAR agonist comprises retinal or retinol; the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist; the retinoid comprises retinal or retinol; or the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist. [Item 15] The cellular aggregate according to item 13 or 14, wherein the cellular aggregate is embedded in the gel or the matrix or disposed inside the gel or the matrix. [Item 16] The cellular aggregate according to any one of items 13 to 15, wherein the matrix includes an extracellular matrix. [Item 17] The cellular aggregate according to item 16, wherein the extracellular matrix includes collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. [Item 18] The cellular aggregate according to any one of items 13 to 17, wherein the gel includes a hydrogel. [Item 19] The cellular aggregate according to any one of items 13 to 18, wherein the gel includes a basement membrane matrix. [Item 20] The cellular aggregate according to item 19, wherein the basement membrane matrix includes laminin, collagen, heparan sulphate proteoglycan and / or entactin. [Item 21] The cellular aggregate according to any one of items 13 to 20, wherein the gel includes an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. [Item 22] The cellular aggregate according to any one of items 12 to 21, wherein the somite or the somite-like structure comprises an anterior portion and a posterior portion in the antero-posterior axis, and an anterior portion cell has a higher or lower expression of one or more markers as compared to a posterior portion cell. [Item 23] The cellular aggregate according to item 22, wherein the anterior portion cell has a higher expression of one or more markers than the posterior portion cell, and the one or more markers are selected from the group consisting of TBX18 and ALDH1A2. [Item 24] The cellular aggregate according to item 22 or 23, wherein the anterior portion cell has a lower expression of one or more markers as compared to the posterior portion cell, and the one or more markers are selected from the group consisting of UNCX and LNFG. [Item 25] The cellular aggregate according to any one of items 12 to 24, comprising: anterior paraxial / presomitic mesoderm (PSM), wherein the anterior PSM has an expression of one or more makers selected from group consisting of MESP1, MESP2, RIPPLY1, RIPPLY2, and PCDH8, preferably MESP2. [Item 26] The cellular aggregate according to any one of items 12 to 25, wherein the somite is formed in a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours, or a cycle of 4 to 6 hours. [Item 27] The cellular aggregate according to any one of items 1 to 26, comprising: the pluripotent stem cell. [Item 28] The cellular aggregate according to item 27, wherein a proportion of the pluripotent stem cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 29] The cellular aggregate according to any one of items 1 to 28, substantially not comprising an endodermal cell. [Item 30] The cellular aggregate according to item 29, wherein a proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 31] The cellular aggregate according to item 29 or 30, wherein the endodermal cell expresses a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. [Item 32] The cellular aggregate according to any one of items 29 to 31, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 33] The cellular aggregate according to any one of items 29 to 32, wherein the ectodermal cell expresses a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2, and BARX1. [Item 34] The cellular aggregate according to any one of items 12 to 33, wherein an expression of a segmentation clock gene is subjected to gene oscillation. [Item 35] The cellular aggregate according to item 34, wherein the segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7. [Item 36] The cellular aggregate according to item 34 or 35, wherein a cycle of the gene oscillation is a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours, or a cycle of 4 to 6 hours. [Item 37] The cellular aggregate according to any one of items 1 to 36, wherein the pluripotent stem cell is a human pluripotent stem cell. [Item 38] The cellular aggregate according to any one of items 1 to 37, wherein the pluripotent stem cell is an embryonic stem cell or an artificial pluripotent stem cell. [Item 39] The cellular aggregate according to any one of items 1 to 38, comprising: at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells. [Item 40] The cellular aggregate according to any one of items 1 to 39, which has a length of at least 0. 05 mm, at least 0. 1 mm, at least 0. 2 mm, at least 0. 3 mm, at least 0. 4 mm, at least 0. 5 mm, at least 0. 6 mm, at least 0. 7 mm, at least 0. 8 mm, at least 0. 9 mm, or at least 1 mm. [Item 41] The cellular aggregate according to any one of items 1 to 40, further comprising a notochord-like structure. [Item 42] A method for producing the cellular aggregate according to any one of items 1 to 41 from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate comprising a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate comprising the mesodermal cell and the ectodermal cell to induce the cellular aggregate according to any one of items 1 to 41. [Item 43] The method according to item 42, wherein the step (a) comprises: (a1) culturing the pluripotent stem cell in a medium containing a fibroblast growth factor (FGF) and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell; and (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium containing an FGF, a transforming growth factor β (TGFβ) inhibitor, a Rho-associated protein kinase (ROCK) inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 44] The method according to item 42, wherein the step (a) comprises: (a3) culturing the pluripotent stem cell in a medium containing an FGF, a ROCK inhibitor and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell; and (a4) culturing the cells derived, obtained or obtainable from step (a3) in a medium containing an FGF, a TGFβ inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 45] The method according to item 43 or 44, wherein the concentration of the Wnt agonist is 4 μM or less. [Item 46] The method according to any one of items 42 to 45, wherein the step (b) comprises: (b1) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a retinoid, a retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 47] The method according to item 46, wherein the step (b) further comprises: (b2) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a SHH pathway agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 48] The method according to any one of items 42 to 47, further comprising the steps of: (c) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a Wnt agonist, a bone morphogenetic protein 4 (BMP4) and hedgehog inhibitor to induce dermomyotome differentiation; and optionally (d) culturing the cellular aggregate derived, obtained or obtainable from step (c) in a medium in the presence of a gel or a matrix to induce myogenic differentiation. [Item 49] The method according to any one of items 42 to 48, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 50] The method according to any one of items 42 to 49, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 51] The method according to any one of items 46 to 50, wherein the RAR agonist is retinal or retinol; the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist; the retinoid is retinal or retinol; or the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist. [Item 52] The method according to any one of items 46 to 51, wherein the Wnt agonist is CHIR99021, the FGF is bFGF, the TGFβ inhibitor is SB431542, and / or the ROCK inhibitor is Y-27632. [Item 53] The method according to any one of items 46 to 52, wherein the matrix includes an extracellular matrix. [Item 54] The method according to item 53, wherein the extracellular matrix includes collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. [Item 55] The method according to any one of items 46 to 54, wherein the gel includes a hydrogel. [Item 56] The method according to any one of items 46 to 55, wherein the gel includes a basement membrane matrix. [Item 57] The method according to item 56, wherein the basement membrane matrix includes laminin, collagen, heparan sulphate proteoglycan, and / or entactin. [Item 58] The method according to any one of items 46 to 57, wherein the gel includes an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. [Item 59] The method according to any one of items 46 to 58, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 60] The method according to any one of items 46 to 59, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 61] The method according to any one of items 46 to 60, wherein the cellular aggregate comprising the mesodermal cell substantially does not comprise an endodermal cell. [Item 62] The method according to item 61, wherein a proportion of the endodermal cell in the cellular aggregate comprising the mesodermal cell is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 63] The method according to item 61 or 62, wherein the endodermal cell expresses a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. [Item 64] The method according to any one of items 61 to 63, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 65] The method according to any one of items 61 to 62, wherein the ectodermal cell expresses a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2, and BARX1. [Item 66] The method according to any one of items 61 to 65, wherein the pluripotent stem cell is a dissociated pluripotent stem cell or a cell suspension containing a pluripotent stem cell. [Item 67] The method according to any one of items 61 to 66, wherein the pluripotent stem cell is a human pluripotent stem cell. [Item 68] The method according to any one of items 61 to 67, wherein the pluripotent stem cell is an embryonic stem cell or an artificial pluripotent stem cell. [Item 69] The method according to any one of items 61 to 68, wherein the cellular aggregate comprising the mesodermal cell and the ectodermal cell comprises at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells. [Item 70] The method according to any one of items 61 to 69, wherein the cellular aggregate comprising the mesodermal cell and the ectodermal cell has a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. [Item 71] A cell obtained from the cellular aggregate according to any one of items 1 to 41. [Item 72] A method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate according to any one of items 1 to 41 to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof. [Item 73] The method according to item 72, comprising the step of: inducing a three-dimensional cellular aggregate from a pluripotent stem cell prior to the inducing, wherein the cellular aggregate inducing is performed by the method according to any one of items 42 to 70. [Item 74] A method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the cellular aggregate after the culturing, wherein the cellular aggregate is the cellular aggregate according to any one of items 1 to 41, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate. [Item 75] The method according to item 74, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate. [Item 76] The method according to item 75, wherein the culture is a culture under a segmentation culture condition. [Item 77] The method according to item 76, wherein during the evaluation, a test substance that changes somitogenesis of the cellular aggregate is selected as a candidate substance that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate. [Item 78] The method according to item 77, wherein the culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (h): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endothelial or hemogenic cell or a progenitor cell thereof; and (g) a neural crest cell or a progenitor cell thereof; and (h) a neuronal cell or a progenitor cell thereof; or the culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof. [Item 79] The method according to item 78, wherein during the evaluation, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h).; or during the evaluation, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i). [Item 80] The method according to any one of items 74 to 79, wherein the evaluation is an evaluation using a control in which the test substance is not present as a reference. [Item 81] The method according to item 80, wherein the culture is generation of a three-dimensional cellular aggregate from a pluripotent stem cell, and the generation of the cellular aggregate is carried out by the method according to any one of items 42 to 70. [Item 82] A method for evaluating gene function or genomic sequence function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culturing, wherein the generation of the cellular aggregate is carried out by the method according to any one of items 42 to 70. [Item 83] The method according to item 82, wherein during the evaluation, a test gene or a test genome that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genome that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate; or during the evaluation, a test gene or a test genomic sequence that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate. [Item 84] The method according to item 82 or 83, comprising the step of: culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition, wherein during the evaluation, a test gene or a test genome that changes somitogenesis of the cellular aggregate is evaluated as a candidate gene or a candidate genome that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate; or culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition, wherein during the evaluation, a test gene or a test genomic sequence that changes somitogenesis of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate. [Item 85] The method according to any one of items 82 to 84, wherein the genome is an exon region, an intron region, a promoter region, an enhancer region, and / or a non-coding region of the genome.; or the genomic sequence is an exon region, an intron region, a promoter region, an enhancer region, and / or a non-coding region of the genome.

[0187] In another aspect, the present invention is disclosed as follows: [Item 1a] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation. [Item 2a] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure. [Item 3a] The cellular aggregate according to item 1a or 2a, wherein the dorso‐ventral axis is defined by a dorsal region and a ventral region, and the dorsal region has a neural tube-like structure. [Item 4a] The cellular aggregate according to item 3a, wherein a cell in the neural tube-like structure has an expression of at least one marker selected from the groups consisting of dorsal markers PAX3, PAX6 and PAX7 and ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2. [Item 5a] The cellular aggregate according to any one of items 1a to 4a, wherein the antero-posterior axis is defined by an anterior region and a posterior region, and i) a cell in the anterior region has a lower expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2; or ii) a cell in the anterior region has a higher expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10. [Item 6a] The cellular aggregate according to item 5a, wherein the posterior region comprises a tailbud (TB) like-cell. [Item 7a] The cellular aggregate according to any one of items 1a to 4a, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in the anterior region has an expression of OTX2 and PAX2. [Item 8a] The cellular aggregate according to any one of items 1a to 4a, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in an intermediate region between the anterior region and the posterior region has an expression of PAX6. [Item 9a] The cellular aggregate according to any one of items 1a to 8a, wherein the cellular aggregate has a polarity in an apical-basolateral axis. [Item 10a] The cellular aggregate according to any one of items 1a to 9a, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 11a] The cellular aggregate according to item 2a, wherein a cell in the neural tube-like structure has an expression of PAX3, PAX6, PAX7, SOX2 and OTX2. [Item 12a] The cellular aggregate according to any one of items 1a to 11a, wherein the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition. [Item 13a] The cellular aggregate according to item 12a, wherein the somitogenic culture condition is a presence of a gel or a matrix and a retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist; or the somitogenic culture condition is a presence of a gel or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist. [Item 14a] The cellular aggregate according to item 13a, wherein the RAR agonist comprises retinal or retinol; the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist; the retinoid comprises retinal or retinol; or the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist. [Item 15a] The cellular aggregate according to item 13a or 14a, wherein the cellular aggregate is embedded in the gel or the matrix or disposed inside the gel or the matrix. [Item 16a] The cellular aggregate according to any one of items 13a to 15a, wherein the matrix includes an extracellular matrix. [Item 17a] The cellular aggregate according to item 16a, wherein the extracellular matrix includes collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. [Item 18a] The cellular aggregate according to any one of items 13a to 17a, wherein the gel includes a hydrogel. [Item 19a] The cellular aggregate according to any one of items 13a to 18a, wherein the gel includes a basement membrane matrix. [Item 20a] The cellular aggregate according to item 19a, wherein the basement membrane matrix includes laminin, collagen, heparan sulphate proteoglycan and / or entactin. [Item 21a] The cellular aggregate according to any one of items 13a to 20a, wherein the gel includes an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. [Item 22a] The cellular aggregate according to any one of items 12a to 21a, wherein the somite or the somite-like structure comprises an anterior portion and a posterior portion in the antero-posterior axis, and an anterior portion cell has a higher or lower expression of one or more markers as compared to a posterior portion cell. [Item 23a] The cellular aggregate according to item 22a, wherein the anterior portion cell has a higher expression of one or more markers than the posterior portion cell, and the one or more markers are selected from the group consisting of TBX18 and ALDH1A2. [Item 24a] The cellular aggregate according to item 22a or 23a, wherein the anterior portion cell has a lower expression of one or more markers as compared to the posterior portion cell, and the one or more markers are selected from the group consisting of UNCX and LNFG. [Item 25a] The cellular aggregate according to any one of items 12a to 24a, comprising: anterior paraxial / presomitic mesoderm (PSM), wherein the anterior PSM has an expression of one or more makers selected from group consisting of MESP1, MESP2, RIPPLY1, RIPPLY2, and PCDH8, preferably MESP2. [Item 26a] The cellular aggregate according to any one of items 12a to 25a, wherein the somite is formed in a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours, or a cycle of 4 to 6 hours. [Item 27a] The cellular aggregate according to any one of items 1a to 26a, comprising: the pluripotent stem cell. [Item 28a] The cellular aggregate according to item 27a, wherein a proportion of the pluripotent stem cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 29a] The cellular aggregate according to any one of items 1a to 28a, substantially not comprising an endodermal cell. [Item 30a] The cellular aggregate according to item 29a, wherein a proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 31a] The cellular aggregate according to item 29a or 30a, wherein the endodermal cell expresses a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. [Item 32a] The cellular aggregate according to any one of items 29a to 31a, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 33a] The cellular aggregate according to any one of items 29a to 32a, wherein the ectodermal cell expresses a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2, and BARX1. [Item 34a] The cellular aggregate according to any one of items 12a to 33a, wherein an expression of a segmentation clock gene is subjected to gene oscillation. [Item 35a] The cellular aggregate according to item 34a, wherein the segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7. [Item 36a] The cellular aggregate according to item 34a or 35a, wherein a cycle of the gene oscillation is a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours, or a cycle of 4 to 6 hours. [Item 37a] The cellular aggregate according to any one of items 1a to 36a, wherein the pluripotent stem cell is a human pluripotent stem cell. [Item 38a] The cellular aggregate according to any one of items 1a to 37a, wherein the pluripotent stem cell is an embryonic stem cell or an artificial pluripotent stem cell. [Item 39a] The cellular aggregate according to any one of items 1a to 38a, comprising: at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells. [Item 40a] The cellular aggregate according to any one of items 1a to 39a, which has a length of at least 0. 05 mm, at least 0. 1 mm, at least 0. 2 mm, at least 0. 3 mm, at least 0. 4 mm, at least 0. 5 mm, at least 0. 6 mm, at least 0. 7 mm, at least 0. 8 mm, at least 0. 9 mm, or at least 1 mm. [Item 41a] The cellular aggregate according to any one of items 1a to 40a, further comprising a notochord-like structure. [Item 42a] A method for producing the cellular aggregate according to any one of items 1a to 41a from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate consisting of a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate consisting of the mesodermal cell and the ectodermal cell to induce the cellular aggregate according to any one of items 1a to 41a. [Item 43a] The method according to item 42a, wherein the step (a) comprises: (a2) culturing the pluripotent stem cell in a medium containing a fibroblast growth factor (FGF), a transforming growth factor β (TGFβ) inhibitor, a Rho-associated protein kinase (ROCK) inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 44a] The method according to item 43a, wherein the step (a) further comprises before the step (a2): (a1) culturing the pluripotent stem cell in a medium containing an FGF and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell. [Item 45a] The method according to item 42a, wherein the step (a) comprises: (a4) culturing the pluripotent stem cell in a medium containing an FGF, a TGFβ inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 46a] The method according to item 45a, wherein the step (a) further comprises before the step (a4): (a3) culturing the pluripotent stem cell in a medium containing an FGF, a ROCK inhibitor and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell. [Item 47a] The method according to any one of items 43a to 46a, wherein the concentration of the Wnt agonist is 4 μM or less. [Item 48a] The method according to any one of items 42a to 47a, wherein the step (b) comprises: (b1) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a retinoid, a retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 49a] The method according to item 48a, wherein the step (b) further comprises: (b2) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a SHH pathway agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 50a] The method according to any one of items 42a to 49a, further comprising the steps of: (c) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a Wnt agonist, a bone morphogenetic protein 4 (BMP4) and hedgehog inhibitor to induce dermomyotome differentiation; and optionally (d) culturing the cellular aggregate derived, obtained or obtainable from step (c) in a medium in the presence of a gel or a matrix to induce myogenic differentiation. [Item 51a] The method according to any one of items 42a to 50a, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 52a] The method according to any one of items 42a to 51a, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 53a] The method according to any one of items 48a to 52a, wherein the RAR agonist is retinal or retinol; the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist; the retinoid is retinal or retinol; or the retinoid, comprises, retinoic acid, a retinoic acid precursor such as retinal or retinol or a RAR agonist. [Item 54a] The method according to any one of items 48a to 53a, wherein the Wnt agonist is CHIR99021, the FGF is bFGF, the TGFβ inhibitor is SB431542, and / or the ROCK inhibitor is Y-27632. [Item 55a] The method according to any one of items 48a to 54a, wherein the matrix includes an extracellular matrix. [Item 56a] The method according to item 55a, wherein the extracellular matrix includes collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. [Item 57a] The method according to any one of items 48a to 56a, wherein the gel includes a hydrogel. [Item 58a] The method according to any one of items 48a to 57a, wherein the gel includes a basement membrane matrix. [Item 59a] The method according to item 58a, wherein the basement membrane matrix includes laminin, collagen, heparan sulphate proteoglycan, and / or entactin. [Item 60a] The method according to any one of items 48a to 59a, wherein the gel includes an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. [Item 61a] The method according to any one of items 48a to 60a, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 62a] The method according to any one of items 48a to 61a, wherein the mesodermal cell expresses a marker selected from the group consisting of TBXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1 and FLK1 / KDR. [Item 63a] The method according to any one of items 48a to 62a, wherein the cellular aggregate comprising the mesodermal cell substantially does not comprise an endodermal cell. [Item 64a] The method according to item 63a, wherein a proportion of the endodermal cell in the cellular aggregate comprising the mesodermal cell is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 65a] The method according to item 63a or 64a, wherein the endodermal cell expresses a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. [Item 66a] The method according to any one of items 63a to 65a, wherein a proportion of the ectodermal cell in the cellular aggregate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. [Item 67a] The method according to any one of items 63a to 66a, wherein the ectodermal cell expresses a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2, and BARX1. [Item 68a] The method according to any one of items 63a to 67a, wherein the pluripotent stem cell is a dissociated pluripotent stem cell or a cell suspension containing a pluripotent stem cell. [Item 69a] The method according to any one of items 63a to 68a, wherein the pluripotent stem cell is a human pluripotent stem cell. [Item 70a] The method according to any one of items 63a to 69a, wherein the pluripotent stem cell is an embryonic stem cell or an artificial pluripotent stem cell. [Item 71a] The method according to any one of items 63a to 70a, wherein the cellular aggregate consisting of the mesodermal cell and the ectodermal cell comprises at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells. [Item 72a] The method according to any one of items 63a to 71a, wherein the cellular aggregate consisting of the mesodermal cell and the ectodermal cell has a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. [Item 73a] A cell obtained from the cellular aggregate according to any one of items 1a to 41a. [Item 74a] A method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate according to any one of items 1a to 41a to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; (i) a neural tube cell or a progenitor cell thereof; (j) a neural crest cell or a progenitor cell thereof; and (k) a neuronal cell or a progenitor cell. [Item 75a] The method according to item 74a, comprising the step of: inducing a three-dimensional cellular aggregate from a pluripotent stem cell prior to the inducing, wherein the cellular aggregate inducing is performed by the method according to any one of items 42 to 72. [Item 76a] A method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the cellular aggregate after the culturing, wherein the cellular aggregate is the cellular aggregate according to any one of items 1a to 41a, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate. [Item 77a] The method according to item 76a, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate. [Item 78a] The method according to item 77a, wherein the culture is a culture under a segmentation culture condition. [Item 79a] The method according to item 78a, wherein during the evaluation, a test substance that changes somitogenesis of the cellular aggregate is selected as a candidate substance that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate. [Item 80a] The method according to item 79a, wherein the culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (h): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endothelial or hemogenic cell or a progenitor cell thereof; and (g) a neural crest cell or a progenitor cell thereof; and (h) a neuronal cell or a progenitor cell thereof; or the culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof. [Item 81a] The method according to item 80a, wherein during the evaluation, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (h).; or during the evaluation, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i). [Item 82a] The method according to any one of items 76a to 81a, wherein the evaluation is an evaluation using a control in which the test substance is not present as a reference. [Item 83a] The method according to item 82a, wherein the culture is generation of a three-dimensional cellular aggregate from a pluripotent stem cell, and the generation of the cellular aggregate is carried out by the method according to any one of items 42a to 72a. [Item 84a] A method for evaluating gene function or genomic sequence function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culturing, wherein the generation of the cellular aggregate is carried out by the method according to any one of items 42 to 72. [Item 85a] The method according to item 84a, wherein during the evaluation, a test gene or a test genome that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genome that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate; or during the evaluation, a test gene or a test genomic sequence that changes a polarity of the cellular aggregate, a shape of the cellular aggregate, and / or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence that modifies, promotes, or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate, and / or the size of the cellular aggregate. [Item 86a] The method according to item 84a or 85a, comprising the step of: culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition, wherein during the evaluation, a test gene or a test genome that changes somitogenesis of the cellular aggregate is evaluated as a candidate gene or a candidate genome that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate; or culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition, wherein during the evaluation, a test gene or a test genomic sequence that changes somitogenesis of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence that modifies, promotes, or suppresses the somitogenesis of the cellular aggregate. [Item 87a] The method according to any one of items 84a to 86a, wherein the genome is an exon region, an intron region, a promoter region, an enhancer region, and / or a non-coding region of the genome.; or the genomic sequence is an exon region, an intron region, a promoter region, an enhancer region, and / or a non-coding region of the genome.

[0188] In a preferred aspect, the present invention is disclosed as follows: [Item 1b] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation. [Item 2b] A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure. [Item 3b] The cellular aggregate according to item 1b or 2b, wherein the dorso‐ventral axis is defined by a dorsal region and a ventral region, and the dorsal region has a neural tube-like structure, and a cell in the neural tube-like structure has an expression of at least one marker selected from the groups consisting of dorsal markers PAX3, PAX6 and PAX7 and ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2. [Item 4b] The cellular aggregate according to any one of items 1b to 3b, wherein the antero-posterior axis is defined by an anterior region and a posterior region, and i) a cell in the anterior region has a lower expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2; or ii) a cell in the anterior region has a higher expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10. [Item 5b] The cellular aggregate according to item 4b, wherein the posterior region comprises a tailbud (TB) like-cell. [Item 6b] The cellular aggregate according to any one of items 1b to 3b, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in the anterior region has an expression of OTX2 and PAX2. [Item 7b] The cellular aggregate according to any one of items 1b to 3b, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in an intermediate region between the anterior region and the posterior region has an expression of PAX6. [Item 8b] The cellular aggregate according to any one of items 1b to 7b, wherein the cellular aggregate has a polarity in an apical-basolateral axis. [Item 9b] The cellular aggregate according to any one of items 1b to 8b, wherein the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition. [Item 10b] The cellular aggregate according to item 9b, wherein the somitogenic culture condition is a presence of a gel or a matrix and a retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist; or the somitogenic culture condition is a presence of a gel or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist. [Item 11b] The cellular aggregate according to item 9b or 10b, wherein the somite or the somite-like structure comprises an anterior portion and a posterior portion in the antero-posterior axis, and an anterior portion cell has a higher or lower expression of one or more markers as compared to a posterior portion cell. [Item 12b] The cellular aggregate according to any one of items 9b to 11b, comprising: anterior paraxial / presomitic mesoderm (PSM), wherein the anterior PSM has an expression of one or more makers selected from group consisting of MESP1, MESP2, RIPPLY1, RIPPLY2, and PCDH8, preferably MESP2. [Item 13b] The cellular aggregate according to any one of items 1b to 12b, comprising: the pluripotent stem cell, wherein a proportion of the pluripotent stem cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 14b] The cellular aggregate according to any one of items 1b to 13b, substantially not comprising an endodermal cell, wherein a proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. [Item 15b] The cellular aggregate according to any one of items 9b to 14b, wherein an expression of a segmentation clock gene is subjected to gene oscillation., wherein the segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7. [Item 16b] The cellular aggregate according to any one of items 1b to 15b, wherein the pluripotent stem cell is a human pluripotent stem cell. [Item 17b] The cellular aggregate according to any one of items 1b to 16b, comprising: at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells. [Item 18b] The cellular aggregate according to any one of items 1b to 17b, which has a length of at least 0. 05 mm, at least 0. 1 mm, at least 0. 2 mm, at least 0. 3 mm, at least 0. 4 mm, at least 0. 5 mm, at least 0. 6 mm, at least 0. 7 mm, at least 0. 8 mm, at least 0. 9 mm, or at least 1 mm. [Item 19b] The cellular aggregate according to any one of items 1b to 18b, further comprising a notochord-like structure. [Item 20b] A method for producing the cellular aggregate according to any one of items 1b to 19b from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate consisting of a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate consisting of the mesodermal cell and the ectodermal cell to induce the cellular aggregate according to any one of items 1b to 19b. [Item 21b] The method according to item 20b, wherein the step (a) comprises: (a2) culturing the pluripotent stem cell in a medium containing a fibroblast growth factor (FGF), a transforming growth factor β (TGFβ) inhibitor, a Rho-associated protein kinase (ROCK) inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 22b] The method according to item 21b, wherein the step (a) further comprises before the step (a2): (a1) culturing the pluripotent stem cell in a medium containing an FGF and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell. [Item 23b] The method according to item 22b, wherein the step (a) comprises: (a4) culturing the pluripotent stem cell in a medium containing an FGF, a TGFβ inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell. [Item 24b] The method according to item 23b, wherein the step (a) further comprises before the step (a4): (a3) culturing the pluripotent stem cell in a medium containing an FGF, a ROCK inhibitor and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell. [Item 25b] The method according to any one of items 21b to 24b, wherein the concentration of the Wnt agonist is 4 μM or less. [Item 26b] The method according to any one of items 20b to 25b, wherein the step (b) comprises: (b1) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a retinoid, a retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 27b] The method according to item 26b, wherein the step (b) further comprises: (b2) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a SHH pathway agonist in the presence of a gel or a matrix to induce neuronal differentiation. [Item 28b] The method according to any one of items 20b to 27b, further comprising the steps of: (c) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a Wnt agonist, a bone morphogenetic protein 4 (BMP4) and hedgehog inhibitor to induce dermomyotome differentiation; and optionally (d) culturing the cellular aggregate derived, obtained or obtainable from step (c) in a medium in the presence of a gel or a matrix to induce myogenic differentiation. [Item 29b] A cell obtained from the cellular aggregate according to any one of items 1b to 19b. [Item 30b] A method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate according to any one of items 1b to 19b to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; (i) a neural tube cell or a progenitor cell thereof; (j) a neural crest cell or a progenitor cell thereof; and (k) a neuronal cell or a progenitor cell. [Item 31b] A method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the cellular aggregate after the culturing, wherein the cellular aggregate is the cellular aggregate according to any one of items 1b to 19b, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate. [Item 32b] A method for evaluating gene function or genomic sequence function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culturing, wherein the generation of the cellular aggregate is carried out by the method according to any one of items 20b to 28b.

[0189] The following Preparations and Examples are given for the purpose of illustrating the present invention in more detail. However, the scope of the present invention is not limited to the following.

[0190] The present disclosure will be described specifically below with reference to examples. It is to be noted, however, that the present disclosure is by no means limited to embodiments described in the following examples.

[0191] INTRODUCTION 1)Technological basis of this discovery We previously described the generation of a pluripotent stem cell-derived mesodermal 3D model of human early embryonic axial development [1]. Axioloids are generated using a stepwise protocol that involves the gradual commitment of human pluripotent stem cells to a primitive streak identity under 2D culture conditions using FGF and WNT pathway activation, followed by paraxial mesoderm fate induction in 3D (upon aggregation) using FGF- and WNT-pathway activation in combination with TGFB pathway inhibition. Following these induction steps, upon further culture, the resulting 3D cellular structures are able to spontaneously break symmetry and start elongating, forming bean-shaped structures that upon addition of an ECM rich compound along with retinoids (Vitamin A or its derivatives) to the culture medium results in axial elongation and the sequential emergence of epithelialized segments morphologically, molecularly and functionally similar to forming somites of the human embryo (Fig. 1 a).

[0192] 2)Aim and brief scientific background of this invention We sought to modify the previously described axioloid protocol to recapitulate additional aspects of early human embryonic development. Using the original axioloid protocol we hypothesized that it will be possible to “add” additional anatomical structures and functional cell types which emerge during human axial development. This includes for example the selective or simultaneous emergence of the embryonic neural tube alone or together with forming somites, but can be extended to other functionally relevant early embryonic structures such as the notochord, neural crest, primitive gut, heart and early brain. We here show proof of concept that this can be achieved using the emerging human neural tube as an example.

[0193] Axioloids show anterior-posterior morphological & molecular organization that mimics the one seen in in vivo human and vertebrate embryos, with MEOX1+ somitic mesoderm (SM), a HES7+ / TBX6+ presomitic mesoderm (PSM) and a TBXT+ / SOX2+ tailbud-like (TB) structure in the posterior-most part of the axioloids which contains a neuromesodermal progenitor (NMP) cell population. NMP cells are characterized as a proliferative cell population that have the ability to contribute to both mesodermal and neuronal cell fates, and as a consequence has a critical role for neural tube and somite formation and sustaining axial elongation of the developing embryo [2].

[0194] 3)Nomenclature From this point onward we will be referring to the previous model of human axial development (Yamanaka, Hamidi et al., 2023 Nature) briefly described above as ‘axioloid v1.0’. We will refer to the new model described hereafter as ‘axioloid v2.0’. The term axioloid, when used alone, without any version number, will refer to both existing (v1.0 and v2.0) and future versions of our PSC-based model of embryo axial development and morphogenesis.

[0195] RESULTS & CLAIMS 1)ESTABLISHMENT OF ‘AXIOLOID V2.0’ - Axioloid v2.0 recapitulates core aspects of human neural development and somitogenesis Here we describe the “axioloid v2.0” in vitro embryo model that recapitulates, besides the somitic component, an elongated and epithelialized neural tube-like SOX2+ and PAX6+ structure which develops either independently (Fig. 1 b) or together with MEOX1+ segmented epithelialized somites emerging both from a single tail-bud like structure at the posterior end of the axioloid (Fig. 1 c-e).

[0196] - WNT pathway modulates NMP cell state and fate in ‘axioloid v2.0’ In accordance with previous reports, we identified that level of WNT pathway activation is a critical parameter determining the propensity of in vitro-derived NMP progenitors to contribute to either the neuronal or somitic cellular identity in our system [3]. Modulation of WNT pathway activation levels was achieved by modulating the GSK3B inhibitor (CHIR99021) concentration either alone or in conjunction with adjustment of the utilized bFGF concentration. We further found that initial cell state including cellular aggregate size to be a critical factor regulating the potency of WNT activation and the differentiation balance of the emerging NMPs in our system (Fig. 1 d).

[0197] - WNT dependent modulation of axioloid v2.0 morphologies Human PSC-derived axioloids v2.0 can span a large spectrum of morphologies with the extremes reflecting a trend toward either increased neuronal differentiation propensity resulting in an epithelialized elongated neural tube-like structure with an underdeveloped or totally absent somitic compartment under low WNT activation levels (Fig. 1 b, d right image, Fig. 2 a), or increased somitic mesoderm differentiation propensity resulting in structures characterized by well-defined epithelialized segments and circular somites alone or together with an epithelialized neural tube under higher levels of WNT activation (Fig. 1 c, d left panel, e, Fig. 2 b and c).

[0198] - Axioloid v2.0 recapitulates aspects of human embryonic development & axial morphogenesis Axioloid v2.0 generated under high WNT pathway activation conditions can be classified into three different overall categories based on their similarity with embryonic axial architecture. Two of these categories are characterized by the presence of a single stream of somites located either in the inner or outer curvatures of the forming neural tube-like structure. The third one on the other hand presents bilateral somites flanking the emerging neural tube-like structure (Fig. 1 e). Besides the somitic component, the morphogenesis of the neural tube-like structure of axioloids v2.0 (Fig. 1 b and Fig. 2 a) presents high similarity with the formation of the NMP-derived posterior neural tube in the embryo, commonly referred to as secondary neurulation [4]. Indeed, de novo lumenogenesis like events, which correspond to the formation of a rostral single continuous lumen through fusion of multiple small caudal focal lumens can be frequently observed in axioloids v2.0 (Fig. 2 b-d).

[0199] 2)ESTABLISHMENT OF EMBRYONIC AXIS IN AXIOLOID V2.0 NEURAL TUBE-LIKE TISSUES - Antero-posterior patterning of axioloid v2.0 neural tube-like structures The establishment of embryonic body axes, including the antero-posterior axis, ensures the correct formation and positioning of the differentiating cell types during morphogenesis, and is mediated by the action of patterning genes like HOX genes. In the central nervous system, the neural tissue develops first with anterior characteristics, before subsequently acquiring a posterior (spinal cord) identity[5]. Analysis of the expression of antero-posterior markers in the neural tube-like structures of axioloids v2.0 revealed that PAX6 is expressed in an intermediate area between the posterior-most and anterior-most part of the forming neural tube-like structures, whereas the rostral markers OTX2 and PAX2 are expressed in the anterior part of axioloids v2.0 at 144h (Fig. 18 a), indicating that in addition to posterior neural fates axioloid v2.0 possess anterior neural fates cell types in a position-dependent manner.

[0200] - Dorso-ventral patterning of axioloid v2.0 neural tube-like structures In the embryo the caudal region of the neural tube will develop to form the spinal cord and pattern along a dorso-ventral axis through the antagonizing action of two opposing signaling center and anatomical structures: the dorsal roof-plate secreting BMPs and WNTs and the ventral floor-plate secreting Sonic Hedgehog (SHH) [6]. In axioloid v2.0 neural tube-like structures express dorsal markers PAX3, PAX6 and PAX7, and very low levels of the ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2 [7] (Fig. 18 b). This can be explained by the fact that axioloid v2.0 do usually not contain any notochord-equivalent responsible in the embryo for the establishment of a SHH secretion center. Ectopic addition of a SHH pathway agonist (SAG) to axioloid v2.0 culture in 2D conditions results in a global ventralization of the neural tissues forming in axioloid v2.0 as exemplified by the increased expression of the ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2 (Fig. 18 c). Axioloid which contain a notochord-like structure in addition to a neural tube-like structure and forming somites is a good example for further ‘expanded’ and ‘enhanced’ versions of our model systems. Promising preliminary results of ongoing experiments in our lab indicate that notochord associated cells can be indeed induced in axioloid v2.0.

[0201] 3)AXIOLOID V2.0 NEURAL TISSUES CONTRIBUTE TO EMBRYO MORPHOGENESIS - Induction of neural tube derivatives in axioloid v2.0: Neural Crest Cells Neural crest cells are a transitory multipotent cell population that originate in the dorsal region of the neural tube and have very high migratory capacity allowing them to spread throughout the embryo, colonizing and contributing to a large number of tissues of both ectodermal, endodermal and mesenchymal origin. The various cell fates of neural crest cells depend mostly on their anterior-posterior origin along the emerging neural tube during early embryonic development. This will determine the tissues they migrate to and settle in. Examples are among others sensory and enteric neurons, Schwann cells, glial cells, cartilage and bone cells, tendons and connective tissue, melanocytes, endocrine or even adipose cells [8]. To assess the presence of neural crest cells in the neural tube-like component of our model we cultured axioloid v2.0 under 2D culture conditions. Upon attachment to the culture substrate a stream of cells migrating outward from the neural tube-like structures in axioloid v2.0 (Fig. 19 a, b) with some individual cells having a high migration potential could be observed (Fig. 19 a, b). Molecular characterization of these cells using immunofluorescence showed that the early neural crest marker PAX7 was expressed predominantly inside attached axioloid v2.0 neural tube-like tissues [9], whereas migrating cells lost PAX7 expression and acquired the neural crest specific markers including SOX10

[0010] (Fig. 19 c), FOXD3 marking the neural / glial precursor

[0011] and PAX3

[0012] (Fig. 19 d).

[0202] - Neural tube derivatives in the developing embryo: Neuronal Differentiation Besides neural crest cells, the embryonic neural tube gives rise a variety of neuronal cell types. Dorso-ventral and anterior-posterior patterning results in the specification of various regions of the central nervous system (CNS). This regionalization process is accompanied by the differentiation of the neuroepithelial cells constituting the neural tube into numerous types of neuronal progenitors and mature neurons

[0013] . Using a similar approach as previously described, live imaging of 2D cultured axioloid v2.0, indicated the emergence of very thin and long filamentous cytoplasmic cellular extensions branching from cell soma (Fig. 19 e, f), and expressing neuronal markers such as neurofilament protein (NF-H) (Fig. 19 g). Further investigation revealed the expression of the neural marker class III β-tubulin (TUJ1) (Fig. 19 h) confirming the ability of axioloid v2.0 neural tube-derived neuroepithelial cells to give rise to mature neurons and neuronal cell types, and underlining the utility of axioloid v2.0 to model various aspects of human neural development including neural tube formation and differentiation.

[0203] 4)DERMOMYOTOME DIFFERENTIATION OF AXIOLOID V2.0 SOMITES During embryonic morphogenesis, signals emanating from the notochord and the neural tube floor plate, including SHH, induce the specification of the ventral half of neighboring somites into sclerotome cells, which later give rise to various structures, including the cartilage and bone of the forming vertebral column, ribs, tendons, ligaments and joints from the emerging syndetome, endotome, and arthrotome compartments of the patterning somites. On the opposite side, the dorsal aspect of somites, under the influence of WNT and BMP4 signaling secreted from the surface ectoderm and lateral plate mesoderm, differentiate into dermomyotome cells, resulting in epithelialized cells that subsequently undergo a delamination step before specifying into skeletal muscle lineage among other lineages, including cells that contribute to the dermis or fat (foremost brown fat) during development. We induced dermomyotome differentiation of axioloid v2.0 (at 144h of culture) by culturing them in 2D conditions for an additional 3 day in the presence of BMP4, WNT activator (CHIR99021) and a SHH inhibitor (GDC0449). Resulting axioloid v2.0 expressed dermomyotome markers PAX3 and PAX7 (Fig. 19 i). We also succeeded with the further differentiation of these cells into ACTN2 expressing human skeletal muscle cells by switching to a medium containing horse serum and N2 supplement for 21 days (Fig. 19 j).

[0204] 5)MODELING HUMAN DISEASES WITH AXIOLOID V2.0 E.G., NEURAL TUBE DEFECTS During embryogenesis, various and complex morphogenetic processes involved in the formation, elongation and closure of the neural tube can lead, if perturbed, to severe birth defects of the CNS, generally referred to as neural tube defects (NTD). These defects include anencephaly and spina bifida which both are believed to result from incomplete closure of the neural tube lumen. The multifactorial nature of the mechanism underlying human NTDs, including both genetic and environmental components, is still poorly understood. Nevertheless, several nongenetic environmental risk factors have been identified. Among these intake during pregnancy of antiepileptic drugs such as valproic acid (VPA) or Carbamazepine (CBZ) have been directly linked to higher risk of severe birth defects and NTD occurrence

[0014] . Aiming to establish an in vitro model of human NTDs, exposure of axioloid v2.0 to 1mM of VPA from the embedding phase resulted in abnormal epithelialization of the somites and of the neural tube-like structure (Fig. 20 a). Further analysis using 3D reconstructed z-stack images unveiled the presence of discontinuous multi-lumen segmented neural tube-like structures in VPA-exposed axioloid v2.0 (Fig. 20 b). Although preliminary, these results highlight the ability of axioloid v2.0 to recapitulate aspects of NTDs and the potential of our model to understand the molecular pathology of congenital disorders or to screen for the teratogenic or toxic effect of exogeneous factors including drugs and other epigenetic and environmental factors.

[0205] DISCUSSION 1)PUTATIVE APPLICATIONS OF THE AXIOLOID V2.0 MODEL The ability of axioloid v2.0 to robustly recapitulate both the neural and somitic components of embryonic axial development, expands the possible applications of the axioloid v1.0 model described previously [1]. Axioloid v2.0 can thus be used to study a broad array of biological questions not only limited to the development of the nervous system, but also to the intertwined parallel development of the neural and somitic tissues and their derivatives during embryo morphogenesis both in physiological and pathological conditions.

[0206] A. Potential applications of the axioloid v2.0 model to the study of human embryonic development and organogenesis include but are not limited to: The process of secondary neurulation The study of the antero-posterior patterning of the body axis (neural tube and somites) The processes of CNS and peripheral nervous system formation and patterning The process of neural tube dorso-ventral patterning The processes of formation, delamination and migration of the neural crest cells The processes of neural crest differentiation The process of bone / cartilage / tendon / ligament / joint formation & differentiation The process of skeletal muscle, dermis and (brown) fat differentiation The establishment of the electrical and chemical synaptic connections including but not limited to the axosomatic (neuromuscular synapses), axo-axonic, or axo-dendritic synapses The development of mesodermal and other germ-layer organs and tissues including but not limited to formation and differentiation of heart, visceral organs, kidney, early brain, blood etc.

[0207] B. Potential applications of the axioloid v2.0 model to the production of cells for cellular therapy: The axioloid v2.0 model has the ability to generate, besides the somitic mesoderm derivatives (e.g. skeletal muscle, cartilage, bone, tendon & ligaments, fat (brown), dermis etc.) covered by the previous application related to axioloid v1.0, also cells originating from neuroepithelial progenitors and neural crest cells including but not limited to: Endocrine cells: This includes chromaffin cells of the adrenal medulla, chief cells of the extra-adrenal paraganglia, and thyroid C cells. Cells of the nervous system: including glia of the sensory, sympathetic, and enteric nervous systems Enteric cells: neural crest derivatives that colonize the forming gut and differentiate into enteric glial cells and neurons (myenteric and submucosal) Ectomesenchyme: Neural crest derivatives that colonize the cranial region and contribute to formation of tissues in the head and neck including bones, muscle, teeth or connective tissue. Of interest they also contribute to the formation of the pharyngeal arches during embryo development and play a role in heart formation including valvulogenesis, and development of the cardiac conduction system. Melanocytes

[0208] C. Potential applications of the axioloid v2.0 model for modeling of human congenital and acquired diseases in human Examples of disorders and diseases related to the neural tube and somitic tissue development includes but are not limited to:

[0209] Neuromuscular disorders Neuromuscular (NMD) disorders include a wide-range of diseases that affect the function of motor and sensory neurons, disrupting the connection between the CNS and PNS and the rest of the body, resulting in the progressive development of a muscular weakness, in some cases, fatal. Types of NMD disorders include but are not limited to:Amyotrophic Lateral Sclerosis (ALS):it is a rare fatal neurological disease that affects motor neurons, and is characterized by a progressive degeneration of nerve cells in the spinal cord and brain.Charcot-Marie-Tooth diseaseMultiple sclerosisMuscular dystrophyMyasthenia gravisMyositisPeripheral neuropathySpinal muscular atrophy

[0210] Neurocristophathies Neurocristopathies (NCP) refer to a broad spectrum of diseases which affect tissues that are formed or contain cells derived from neural crest derivatives. Example of NCPs include but are not limited to: DiGeorge syndrome: It is the result of the deletion of 30 to 40 genes in the middle of the chromosome 22 at the 22q11.2 location, and includes a wide variety of symptoms including congenital heart defects, intellectual disability, and craniofacial anomalies among others.Waardenburg syndromeParagangliomaAxenfeld-Rieger syndrome

[0211] Myopathies Myopathy is a general term regrouping a heterogenous group of diseases which primarily affect skeletal muscle function, typically resulting in the establishment of progressive muscle weakness. Myopathies are generally classified into two groups based on their pathogenesis. The inherited or familial forms linked to recessive genetic mutations in particular genes and the acquired forms which are usually induced by contact with a toxin, drug, or infectious agent.

[0212] Inherited forms include among others:Muscular dystrophies:it includes more that 30 different disorders among which the Duchenne muscular dystrophy (DMD) accounts for more than half of the total cases. DMD is a recessive X-associated disease caused by a mutation in the dystrophin gene resulting in a global muscle disarray and death, and has no known cure.MyotoniaCongenital myopathiesMitochondrial myopathies

[0213] Acquired forms include among others: Dermatomyosis Polymyositis Myositis ossificans

[0214] D. Potential applications of the axioloid v2.0 model as a screening platform of pharmacological compounds

[0215] Toxicity and teratogenicity screening Currently, the evaluation of teratogenicity in preclinical assessments of pharmaceutical compounds primarily relies on animal experiments, often resulting in test compounds falling short of expectations due to species differences

[0015] . Over the years, significant strides have been made in developing and optimizing human cell-based platforms for toxicological testing. For instance, organoid-based models can replicate specific aspects of human embryonic development, making them suitable for high-throughput screening of preclinical tests

[0016] . Nevertheless, these models still lack the complexity seen in various tissue types, which highlights the need for more comprehensive models. Considering the potential for disease modeling of axioloids (Fig. 20) and structural resemblance with actual embryos, axioloids could serve as an efficient screening platform for assessing the teratogenicity and / or toxicity of chemical compounds.

[0216] Therapeutic molecule screening Axioloids V2.0 generated from either patient-like PSC lines, or genetically modified with a mutation known to be involved in the pathogenesis of a particular disease, could be used as a high-content screening platform for the identification of therapeutic compounds for congenital diseases. This could be also applied to chemical compound known or suspected to induce birth defects.

[0217] 2)FUTURE ‘EXPANSION’ AND EXPECTED TECHNOLOGICAL EVOLUTION OF THE AXIOLOID MODEL The successful addition of a neural tube-like structure to the somitic component of axioloid v1.0 seems to be a proof of concept that the axioloid model constitutes the foundation of a modular bottom-up system that can reconstitute different aspects of human embryonic development.

[0218] It is expected that axioloid v2.0 represents a versatile “embryonic scaffold” and “modular “backbone” which can be used to study additional aspects and components of the developing human embryo. It is preferable to propose “axioloid v2.0” as a modular system to build bottom-up different functional and anatomical compartments of the developing embryo in a coordinated, robust and reproducible manner.

[0219] MATERIAL & METHODSCell lines Ten human pluripotent stem cell (hPSC) lines were used in this study, including 409B2 line (RIKEN BRC, HPS0076), 201B7 line (RIKEN BRC, HPS0063), the TJP1-mEGFP reporter line for the ZO-1 gene (AICS-0023), the mTag-RFP reporter line (AICS-0054-091), WTC11, WTC-CTNNB1-EGFP (AICS-0058-067), RUES2-GLR, PAX3-Venus (LUMCi041-A-3) and 201B7-HES7-Achilles lines.

[0220] PSC Culture PSC culture was performed in accordance to previous protocol [1], briefly hiPSCs were maintained in StemFit AK02N complete medium (component A+B+C) with 25U penicillin and 25 μg / mlstreptomycin (Gibco) on iMatrix-511-coated culture vessel in a humidified incubators at 37℃ and 5% CO2. Passage was performed at 50~80% confluency, cells were detached using 0.5X TrypLETMSelect solution (1:1 dilution with 0.5mmol / L of EDTA-PBS) and cultured in complete AK02N medium supplemented with 10 μM Y-27632 (Nacalai, Cat No. 1267954). All cell lines were regularly tested for mycoplasma contamination.

[0221] Axioloid v2.0 induction On day 5 following the cell passaging, plates with approximately 60% cell confluency were induced to differentiation using 2 different approaches, either in 2D conditions for 24h prior to aggregation as previously reported [1] or directly in 3D conditions after detachment and aggregation, in both cases the aggregation step was performed in a medium supplemented with 10 μM of Y27632, both approaches resulted in the generation of axioloid v2.0, nevertheless important cell line differences in overall generation efficiency were observed between each method. Subsequent differentiation was performed in a AK02N+B-C (lacking the component C) medium supplemented with 20 ng / mL of bFGF (Peprotech Cat No. 100-18B) and variable range of CHIR99021 (Nacalai Cat No. 1876444) concentrations depending on the desired outcome. Neural tube-like structures required a low concentration, axioloid v2.0 an intermediate one and axioloid v1.0 required a higher concentration neighboring 5 μM. The concentration range of CHIR99021 required fine tuning in each of the cell line used. The following day differentiation medium was adjusted to contain 20 ng / mL bFGF, 10 μM SB431542 and a concentration of CHIR99021 identical to the one used the previous day. Subsequently, as previously described, medium was changed using AK02N+B-C each of two following days, the third day aggregates were transferred manually in a medium containing 5% Matrigel (MG) (Corning Cat No. 356231) supplemented with 1 μM all-trans Retinal (Sigma Cat No. R2500). Of notice, an alternative simplified embedding approach consisting of removing 150μL of medium from each aggregation well, and adding in a 1:1 ratio 50μL of AK02N+B-C with 10% MG supplemented with 2 μM all-trans Retinal (Sigma Cat No. R2500) to the 50μL of leftover medium directly in the aggregation plate was evaluated and yielded identical results to the previous method.

[0222] Neural differentiation Neuronal and Neural crest differentiation was achieved by culturing axioloidv2.0 at 120h for an additional 8 days in an 8 well glass chamber slides containing N2B27 medium supplemented with 10% MG. For ventralization experiments, 500nM of SAG (Shh pathway Agonist) was added to the medium.

[0223] Dermomyotome and skeletal muscle differentiation Dermomyotome differentiation was achieved by culturing axioloid v2.0 at 144h for an additional 3 days in CDMi medium supplemented with 3 μM of CHIR99021, 50 ng / mL BMP4, 150 nM GDC0449 (SHHi). After that, subsequent myogenic differentiation was achieved by transferring the axioloids in MG-coated 8 chamber slide and culturing them for an additional 21 days in a medium composed of DMEM / F12 (Gibco), 1% insulin-transferrin-selenium (Corning), 1% N2 Supplement (Gibco), 0.2% penicillin / streptomycin (Gibco), 1% L-glutamine (Gibco), 2% horse serum (Sigma-Aldrich).

[0224] Immunostaining procedure Axioloids were manually collected from the 96-well plate and washed three times with 0.1% BSA and 0.25mM EDTA before being fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature (RT). After the fixation axioloids were washed with 0.1% BSA and 0.25mM EDTA 3 times and stored at 4℃ until staining. Fixed axioloids were first permeabilized using PBS-TritonX100 (0,2 to 1%) for 30 min at RT, then washed twice in PBST (1% Tween20) for 5min, blocking was performed using 5% BSA in PBS for 1hr, finally axioloids were incubated overnight at 4oC with primary antibodies (Table 1) diluted in 0.5% BSA-PBST. The following day, axioloids were washed twice in PBST before secondary antibodies and phalloidin (Table 2) incubation in 0.5% BSA-PBST for 3hr at RT. Axioloids were then washed twice in PBS and finally cleared in ScaleS4 overnight at 4℃ prior to imaging.

[0225] Whole-mount in situ hybridization All probes, HCR amplifiers and buffers (hybridization, wash and amplification buffers) were purchased from Molecular Instruments. Human axioloids were collected in microtubes coated with 1% BSA in PBS, washed once with 1% BSA in PBS, incubated in a mixture of Collagenase IV 4μg / mL and dispaseII 100μg / mL for 10min at 37℃, washed with 1% BSA in PBS, and then fixed with 4% PFA in PBS for 1h at room temperature. Samples were then washed three times with PBST (0.1% Tween-20 in PBS) at room temperature 5min each, post-fixed with 100% methanol and stored at -30℃ until further use. Samples were rehydrated by washing with a series of graded 500μl methanol / PBST wash steps (75%, 50% and 25%) for 5min each at room temperature. Samples were then photobleached for 30min under a 20000 lumen LED light source in a solution of PBS containing 27mM of NaOH and 5% of H2O2. After washing in PBST, human axioloid samples were then incubated for 5min with hybridization buffer at room temperature, then for 30min at 37℃. A mixture of probes (8nM final for each) in hybridization buffer was incubated for 30min at 37℃ prior to use. Samples were incubated for 12-16h at 37℃ with probe-containing hybridization buffer. Samples were then washed with probe wash buffer 4 times 15min each at 37℃ followed by washing with 5×SSCT 3 times at room temperature. Next, samples were pre-amplified by incubating at least 30min in probe amplification buffer at room temperature. Amplifier hairpins were prepared by snap-cooling; heating h1 and h2 hairpins separately at 95℃ for 90s and then cooling down at room temperature for 30min in the dark. Hairpin mixtures were prepared at 6nM each by adding h1 and h2 in 250μl of amplification buffer. Axioloid samples were then incubated with the amplifier hairpin-containing solution for 12-16h at room temperature in the dark. Finally, samples were washed with 5×SSCT and PBST, followed by counter staining with DAPI. During each wash and after addition of buffers, probes and hairpin mixtures into samples, tubes were inverted several times (5-20 times) to mix properly. HCR-stained axioloids were stored in 1% BSA in PBST at 4℃ not more than two weeks before imaging. HCR probe design and associated hairpins was as follows: MESP2 (accession NM_001039958.2); TBX18 (accession NM_001080508.3); UNCX (accession NM_001080461.3); SOX2 (NM_003106.4); MEOX1 (NM_004527.4), PCDH8 (NM_002590.4).

[0226] Imaging procedure Brightfield imaging was performed using an Olympus CKX41 inverted microscope with PlanApo 10x 0.75NA objective lens (Olympus). Immunostained samples in ScaleS4 were transferred onto glass-bottom 35mm dishes or 8-well chamber covers (Matsunami) and imaged using a LSM980 confocal microscope (Carl Zeiss Microscopy), a center z-plane of each axioloid was imaged.

[0227] Live imaging Axioloids were transferred into 1% BSA (Nacalai) treated 96-well flat-bottom low attachment plates (Watson) with one axioloid per well in 80μl of AK02N-C based embedding medium supplemented with 5% MG and 1 μM RAL and were cultured in a stage top incubator (Tokai Hit) which was set to 37℃ and 5% CO2. Bright-field and fluorescence live-cell imaging was performed with an inverted Ti2 microscope system (Nikon) using PlanApo λ 10× objective, with the microscope running in autofocus mode, focusing at 10μm intervals over a total range of 190μm. Images were taken every 5min and processed with Fiji. Confocal live imaging was performed using an ImageXpress Micro high-content imaging system with a 10x objective lens.

[0228] Light-sheet microscopy One end of a plastic capillary is sealed by inserting it onto a glass capillary filled with low melting agarose, fixed axioloids resuspended in either PBS alone or PBS-10% MG are then individually transferred into the plastic capillary using a mouth pipette, the capillary is then loaded into the a LUXENDO MuVi-SPIM LS (BRUKER) light-sheet microscopy sample port and the imaging chamber was filled with PBS, before tiled multi-stack images with z-intervals of 2-3μm were acquired with a 20x 1.0 NA objective lens with 33.3x magnification. 3D reconstruction videos of imaged z-stack were generated using the IMARIS (OXFORD) software.

[0229]

[0230]

[0231] 1. Axioloid inductionAxioloid v2.0 generation using classical method (derived from Axioloid V1.0) On day 5 following the initial cell seeding, human pluripotent stem cells (either ESC or iPSC) with approximately 60% confluency are pulsed by treatment with axioloid induction medium which is composed of AK02N+B-C (excluding the C component from the kit) supplemented with 20 ng / mL of bFGF (Peprotech Cat No. 100-18B) and various concentrations of CHIR99021 (Nacalai Cat No.1876444). The specific concentration of CHIR99021 used may vary depending on the desired types of axioloid to be generated, the specific cell line used, and the initial aggregate size, with: - Neural tube-like structures only usually require a CHIR99021 concentration ranging from 2 to 3 μM. - Axioloid v2.0 usually require a CHIR99021 concentration ranging from 3 to 4 μM. - Axioloid v1.0 usually require a CHIR99021 concentration ranging from 4 to 6 μM.

[0232] The next day, cells are washed with PBS and detached (StemProTMAccutase, Thermo Fisher Scientific Cat No. A11105-01), the resulting single cell suspension is counted, and 250, or 500, or 750, or 1000 cells in 50 μL of AK02N+B-C supplemented with 20 ng / mL bFGF, 10 μM SB431542, 10 μM Y27632 and a CHIR99021 concentration identical to the one used the previous day is distributed in each well of a low attachment U shaped 96well plate (Sumitomo Bakelite Cat No.: MS-9096U). The following day 150 μL of AK02N+B-C medium is added to each of the well. The subsequent day 150 μL of medium from each well is replaced with fresh 150 μL of AK02N+B-C medium. The following day, 72h after initial aggregation, the generated cellular structures are embedded in Matrigel following either of the method described hereafter in this document.

[0233] Axioloid v2.0 generation using a new simplified method On day 5 following initial cell seeding, human pluripotent stem cells (either ESC or iPSC) with approximately 60% confluency are detached (StemProTMAccutase, Thermo Fisher Scientific Cat No. A11105-01), the resulting single cell suspension is counted, and 250, or 500, or 750, or 1000 cells in 50μL of AK02N+B-C supplemented with 20 ng / mL of bFGF (Peprotech Cat No. 100-18B), 10 mM of Y27632 and various concentrations of CHIR99021 (Nacalai Cat No.1876444) are distributed in each well of a low attachment U shaped 96 well plate (Sumitomo Bakelite Cat No.: MS-9096U). The specific concentration of CHIR99021 used may vary depending on the desired types of axioloid to be generated, the specific cell line used, and the initial aggregate size, with: - Neural tube-like structures only usually require a CHIR99021 concentration ranging from 2 to 3 μM. - Axioloid v2.0 usually require a CHIR99021 concentration ranging from 3 to 4 μM. - Axioloid v1.0 usually require a CHIR99021 concentration ranging from 4 to 6 μM.

[0234] The following day 50μL of medium supplemented with 20 ng / mL bFGF, 20 μM SB431542, and a CHIR99021 concentration identical to the one used the previous day is added in each well of a 96well plate. The next day 50μL of medium is removed from each well and replaced with 150 μL of AK02N+B-C medium. The subsequent day 150 μL of medium from each well is replaced with 150 μL of fresh AK02N+B-C medium. The following day, 96h after initial aggregation, the generated cellular structures are embedded in Matrigel following either of the method described hereafter in this document.

[0235] Assembly based strategy for Axioloid v2.1 derivation Following the new simplified method, we induced axioloids from the 409B2-GFP and 201B7-mCherry lines starting with 200 and 300 cell respectively, in conditions containing 10 μM of bFGF and concentrations of CHIR99021 at 3.5 / 4 μM for 409B2-GFP (neural induction) and 4.5 / 5 μM for 201B7-mCherry (mesoderm induction. The following day 50 μL of medium supplemented with 10 ng / mL SB431542, and identical concentration of CHIR99021 and bFGF to the one used the previous day is added in each well of a 96 well plate. The next day, two aggregates from each line were assembled in the same well. Resulting assembly based axioloid v2.1 were embedded using the direct simplified embedding method and cultured up to 120h similarly to the axioloid v2.0 protocol.

[0236] 2. Axioloid embeddingClassical embedding method Generated cellular aggregates are individually transferred into the well of a flat bottom suspension culture 96 well plate containing 80 μL of a solution prepared by combining AK02N+B-C with 2.5 to 5% cold Matrigel (kept at 4℃), 1 μM all-trans Retinal (Sigma Cat No. R2500), and optionally 500nM of SAG to induce the ventralization of the developing neural tube-like structures. The embedded axioloids are cultured for an additional 24, 48 or 72 hours before fixation or further differentiation steps.

[0237] Direct simplified embedding method The embedding procedure consists of removing 150 μL of medium from each well and replace it with 50 μL of a solution prepared by combining AK02N+B-C with 5 to 10% cold Matrigel (kept at 4℃), 2 μM all-trans Retinal (Sigma Cat No. R2500), and optionally 1mM of SAG to induce the ventralization of the developing neural tube-like structures. The embedded axioloids are cultured for an additional 24, 48 or 72 hours before fixation or further differentiation steps.

[0238] 3. Differences between axioloid v1.0 and v2.0 induction methods A. Human pluripotent stem cells (either ESC or iPSC) derived axioloid v2.0 can be generated with high efficiency using a new simplified method that does not require an initial induction step in 2D culture.

[0239] B. The extended duration of culture in 3D condition, prior to embedding, of human pluripotent stem cells (either ESC or iPSC) derived axioloid v2.0

[0240] C. Generation of neural tube-like structures, axioloid v2.0 or axioloid v1.0 require different levels of WNT pathway activation that can be achieved by varying CHIR99021 concentration: - Neural tube-like structures usually require a CHIR99021 concentration ranging from 2 to 3 μM. - Axioloid v2.0 usually require a CHIR99021 concentration ranging from 3 to 4 μM. - Axioloid v1.0 usually require a CHIR99021 concentration ranging from 4 to 6 μM. Prior patent focusing on axioloid v1.0 suggested an optimal range of 4 to 6 μM of CHIR99021

[0241] D. On top of that, required WNT pathway activation levels can also be fined tuned, by varying the concentration of bFGF used in the system at a given CHIR99021 concentration with: - Lower bFGF concentrations shifting the induction efficiency toward the neural fate. - Higher bFGF concentrations shifting the induction efficiency toward mesoderm fate.

[0242] E. In contrast to prior patented model, the axioloid v1.0, that had the ability to generate somite derivatives, axioloid v2.0 can generate both somite and neural tube derivatives including neural lineages and neural crest lineages.

[0243] Periodicity measurement The periodicity of HES7 oscillations was determined from time-series fluorescence intensity data collected through live imaging. Axioloids were transferred 72 hours after the initial setup (48 hr post-aggregation) into 96-well plates containing 80 μl of embedding medium. The culture plates with axioloids were incubated at 37℃ and 5% CO2. Bright-field and fluorescence (YFP filter) live-cell imaging was performed with an inverted Ti2 microscope system (Nikon) using PlanApo λ 10× objective. Oscillations were monitored for 48 hours, with each well recorded for at 5min intervals.

[0244] Axioloid v2.0 differentiation matrix Axioloid v2.0 were induced from various PSC lines starting with cells aggregates of 200, 300 or 400 cells in conditions containing bFGF concentrations of 10, 15 or 20 ng / ml and CHIR concentrations of 2.5, 3, 3.5, 4, 4.5, or 5 μM using the new simplified induction and direct embedding methods. Resulting structures were then imaged at 120h using an Olympus CKX41 inverted microscope with PlanApo 10x 0.75NA objective lens (Olympus).

[0245] Machine learning-based morphological evaluation of axioloids To evaluate the morphology of axioloids, a trained ResNet50V2 model was used to classify the morphological patterns observed in the differentiation matrix of WTC11-WT and 201B7-WT PSC lines. A total of 1,296 bright-field images from each line were manually annotated by a single experienced researcher to serve as ground truth. The dataset was split, with 864 images used for training the machine learning-based classifier and 432 images reserved for validating its accuracy in distinguishing the morphological variations in the WTC11-WT and 201B7-WT differentiation matrices.

[0246] References 1. Yamanaka, Y., et al., Reconstituting human somitogenesis in vitro. Nature, 2023. 614(7948): p. 509-520. 2. Tzouanacou, E., et al., Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Dev Cell, 2009. 17(3): p. 365-76. 3. Gouti, M., et al., In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biol, 2014. 12(8): p. e1001937. 4. Gonzalez-Gobartt, E., et al., Cell intercalation driven by SMAD3 underlies secondary neural tube formation. Dev Cell, 2021. 56(8): p. 1147-1163 e6. 5. Tour, E., et al., Gbx2 interacts with Otx2 and patterns the anterior-posterior axis during gastrulation in Xenopus. Mech Dev, 2002. 112(1-2): p. 141-51. 6. Le Dreau, G. and E. Marti, Dorsal-ventral patterning of the neural tube: a tale of three signals. Dev Neurobiol, 2012. 72(12): p. 1471-81. 7. Zannino, D.A. and C.G. Sagerstrom, An emerging role for prdm family genes in dorsoventral patterning of the vertebrate nervous system. Neural Dev, 2015. 10: p. 24. 8. Le Douarin, N.M. and E. Dupin, Multipotentiality of the neural crest. Curr Opin Genet Dev, 2003. 13(5): p. 529-36. 9. Basch, M.L., M. Bronner-Fraser, and M.I. Garcia-Castro, Specification of the neural crest occurs during gastrulation and requires Pax7. Nature, 2006. 441(7090): p. 218-22. 10. Kelsh, R.N., Sorting out Sox10 functions in neural crest development. Bioessays, 2006. 28(8): p. 788-98. 11. Thomas, A.J. and C.A. Erickson, FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. Development, 2009. 136(11): p. 1849-58. 12. Wu, M., et al., Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice. J Clin Invest, 2008. 118(6): p. 2076-87. 13. Alvarez-Buylla, A., J.M. Garcia-Verdugo, and A.D. Tramontin, A unified hypothesis on the lineage of neural stem cells. Nat Rev Neurosci, 2001. 2(4): p. 287-93. 14. Copp, A.J. and N.D. Greene, Neural tube defects--disorders of neurulation and related embryonic processes. Wiley Interdiscip Rev Dev Biol, 2013. 2(2): p. 213-27. 15. Akhtar, A., The flaws and human harms of animal experimentation. Camb Q Healthc Ethics, 2015. 24(4): p. 407-19. 16. Li, M., et al., Advanced human developmental toxicity and teratogenicity assessment using human organoid models. Ecotoxicol Environ Saf, 2022. 235: p. 113429.

[0247] While the present disclosure has been described above with reference to exemplary embodiments and examples, the present disclosure is by no means limited thereto. Various changes and modifications that may become apparent to those skilled in the art may be made in the configuration and specifics of the present disclosure without departing from the scope of the present disclosure.

[0248] Patents, patent applications, and references cited in the present specification are incorporated herein in their entirety by reference, as if fully and specifically set forth herein.

Claims

1. A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate can reconstitute various aspects of a neural tube formation.

2. A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, consisting of a mesodermal cell and an ectodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and a dorso‐ventral axis; and the cellular aggregate has differentiation ability to form a neural tube-like structure or comprises a neural tube-like structure.

3. The cellular aggregate according to claim 1 or 2, wherein the dorso‐ventral axis is defined by a dorsal region and a ventral region, and the dorsal region has a neural tube-like structure, and a cell in the neural tube-like structure has an expression of at least one marker selected from the groups consisting of dorsal markers PAX3, PAX6 and PAX7 and ventral markers ISLET1, OLIG2, NKX6.1, LHX1 and FOXA2.

4. The cellular aggregate according to any one of claims 1 to 3, wherein the antero-posterior axis is defined by an anterior region and a posterior region, and i) a cell in the anterior region has a lower expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2; or ii) a cell in the anterior region has a higher expression of one or more markers as compared to a cell in the posterior region, and the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10.

5. The cellular aggregate according to claim 4, wherein the posterior region comprises a tailbud (TB) like-cell.

6. The cellular aggregate according to any one of claims 1 to 3, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in the anterior region has an expression of OTX2 and PAX2.

7. The cellular aggregate according to any one of claims 1 to 3, wherein the antero-posterior axis is defined by an anterior region and a posterior region; and a cell in an intermediate region between the anterior region and the posterior region has an expression of PAX6.

8. The cellular aggregate according to any one of claims 1 to 7, wherein the cellular aggregate has a polarity in an apical-basolateral axis.

9. The cellular aggregate according to any one of claims 1 to 8, wherein the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition.

10. The cellular aggregate according to claim 9, wherein the somitogenic culture condition is a presence of a gel or a matrix and a retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist; or the somitogenic culture condition is a presence of a gel or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist.

11. The cellular aggregate according to claim 9 or 10, wherein the somite or the somite-like structure comprises an anterior portion and a posterior portion in the antero-posterior axis, and an anterior portion cell has a higher or lower expression of one or more markers as compared to a posterior portion cell.

12. The cellular aggregate according to any one of claims 9 to 11, comprising: anterior paraxial / presomitic mesoderm (PSM), wherein the anterior PSM has an expression of one or more makers selected from group consisting of MESP1, MESP2, RIPPLY1, RIPPLY2, and PCDH8, preferably MESP2.

13. The cellular aggregate according to any one of claims 1 to 12, comprising: the pluripotent stem cell, wherein a proportion of the pluripotent stem cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells.

14. The cellular aggregate according to any one of claims 1 to 13, substantially not comprising an endodermal cell, wherein a proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells.

15. The cellular aggregate according to any one of claims 9 to 14, wherein an expression of a segmentation clock gene is subjected to gene oscillation., wherein the segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7.

16. The cellular aggregate according to any one of claims 1 to 15, wherein the pluripotent stem cell is a human pluripotent stem cell.

17. The cellular aggregate according to any one of claims 1 to 16, comprising: at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells, or at least 50000 cells.

18. The cellular aggregate according to any one of claims 1 to 17, which has a length of at least 0. 05 mm, at least 0. 1 mm, at least 0. 2 mm, at least 0. 3 mm, at least 0. 4 mm, at least 0. 5 mm, at least 0. 6 mm, at least 0. 7 mm, at least 0. 8 mm, at least 0. 9 mm, or at least 1 mm.

19. The cellular aggregate according to any one of claims 1 to 18, further comprising a notochord-like structure.

20. A method for producing the cellular aggregate according to any one of claims 1 to 19 from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate consisting of a mesodermal cell and an ectodermal cell; and (b) culturing the cellular aggregate consisting of the mesodermal cell and the ectodermal cell to induce the cellular aggregate according to any one of claims 1 to 19.

21. The method according to claim 20, wherein the step (a) comprises: (a2) culturing the pluripotent stem cell in a medium containing a fibroblast growth factor (FGF), a transforming growth factor β (TGFβ) inhibitor, a Rho-associated protein kinase (ROCK) inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell.

22. The method according to claim 21, wherein the step (a) further comprises before the step (a2): (a1) culturing the pluripotent stem cell in a medium containing an FGF and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell.

23. The method according to claim 22, wherein the step (a) comprises: (a4) culturing the pluripotent stem cell in a medium containing an FGF, a TGFβ inhibitor and a Wnt agonist to induce the cellular aggregate including the mesodermal cell and the ectodermal cell.

24. The method according to claim 23, wherein the step (a) further comprises before the step (a4): (a3) culturing the pluripotent stem cell in a medium containing an FGF, a ROCK inhibitor and a Wnt agonist to initiate their commitment toward a primitive streak and mesodermal fate, and / or induce the mesodermal cell and the ectodermal cell.

25. The method according to any one of claims 21 to 24, wherein the concentration of the Wnt agonist is 4 μM or less.

26. The method according to any one of claims 20 to 25, wherein the step (b) comprises: (b1) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a retinoid, a retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or a matrix to induce neuronal differentiation.

27. The method according to claim 26, wherein the step (b) further comprises: (b2) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a SHH pathway agonist in the presence of a gel or a matrix to induce neuronal differentiation.

28. The method according to any one of claims 20 to 27, further comprising the steps of: (c) culturing the cellular aggregate including the mesodermal cell and the ectodermal cell in a medium containing a Wnt agonist, a bone morphogenetic protein 4 (BMP4) and hedgehog inhibitor to induce dermomyotome differentiation; and optionally (d) culturing the cellular aggregate derived, obtained or obtainable from step (c) in a medium in the presence of a gel or a matrix to induce myogenic differentiation.

29. A cell obtained from the cellular aggregate according to any one of claims 1 to 19.

30. A method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate according to any one of claims 1 to 19 to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; (f) an endotome or endothelial or hemogenic cell or a progenitor cell thereof; (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; (i) a neural tube cell or a progenitor cell thereof; (j) a neural crest cell or a progenitor cell thereof; and (k) a neuronal cell or a progenitor cell.

31. A method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the cellular aggregate after the culturing, wherein the cellular aggregate is the cellular aggregate according to any one of claims 1 to 19, wherein during the evaluation, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and / or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and / or the size of the cellular aggregate.

32. A method for evaluating gene function or genomic sequence function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culturing, wherein the generation of the cellular aggregate is carried out by the method according to any one of claims 20 to 28.

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