Axioloid: A stem cell model for human axis formation

Abstract

The present disclosure relates to a method using pluripotent stem cells (PSCs) to reconstitute axis development in vitro, and to a method for producing the same. The present disclosure provides three-dimensional cell aggregates called "axioloids" that are generated in vitro from pluripotent stem cells and are composed of mesodermal cells polarized along the anterior-posterior and apical-basolateral axes. The cell aggregates can reconstitute various aspects of somitogenesis and axis development, including axial elongation, segmentation, epithelial somitogenesis and patterning (the formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions. The axioloids can also be used to induce various cell lineages and functional cells, and can be used as a platform for modeling and reconstituting human embryonic development, disease, and evolution. The axioloids can be further utilized for, among other things, teratogenic and toxicological evaluation of chemicals, production and testing of cell therapy products, research of congenital and acquired human diseases, and evaluation of ongoing and future therapeutic approaches.

Description

Detailed Description of the Invention

[0001] (Technical field) The present disclosure relates to three-dimensional cellular aggregates, called axioloids, generated in vitro from pluripotent stem cells, and methods for their production.

[0002] (Background technology) Vertebrate somite formation is a well-studied process in model organisms but has remained largely unexplored in humans due to ethical and technical constraints. Pluripotent stem cell (PSC)-based approaches 1-5 Despite recent advances by , there are still very few systems that spatially and temporally recapitulate the human embryonic somitogenesis process in vitro, including characteristic morphogenetic features such as oscillatory expression of segmentation clock genes and sequential formation of epithelial somites along the embryo's anterior-posterior axis.

[0003] (Summary of the invention) It is an object of the present disclosure to provide novel three-dimensional cell aggregates.

[0004] To achieve the above objectives, the present disclosure provides three-dimensional cell aggregates generated in vitro from pluripotent stem cells comprising mesodermal cells, said cell aggregates having polarity in the anterior-posterior or rostro-caudal axis and the apical-basolateral axis, and said cell aggregates capable of reconstituting various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions.

[0005] The present disclosure can provide novel three-dimensional cell aggregates. [Brief description of the drawings]

[0006] [Figure 1]Figure 1 shows the generation of axioloids from human pluripotent stem cells. a, Schematic diagram of the human iPS cell-derived axioloid induction protocol. Different color codes for the main steps of the induction protocol: yellow represents small molecule treatment, blue represents suspension culture, and purple represents the Matrigel (MG) embedding stage; the abbreviations used, WNT, FGF, and TGFβi, represent CHIR99021, bFGF, and SB431542, respectively. b and c, Time-lapse live imaging of human axioloid induction. b, Representative bright-field images of axioloids during outgrowth at 24 h, 48 h, and 72 h, and images of axioloids after MG embedding at 96 and 120 h of culture. c, Sequential images of axioloid formation at 5-h intervals from 74 to 119 h (excerpted from Supplementary Video 1). Colored arrows highlight the process of segment formation at each time point, with yellow arrowheads indicating regions where somite segmentation is ongoing and red arrowheads highlight regions where segmentation is complete. d, Periodicity of somite segmentation based on live imaging observations (N = 4, n = 9). e, Length (anterior-posterior) of axioloid at 24 h, 48 h, 72 h, and 96 and 120 h with and without MG embedding (N = 3, n = 18). f and g, Immunofluorescence staining and signal quantification of MG-embedded axioloid at 96 and 120 h. f, Merged channel image of axioloid stained in grey for F-actin (phalloidin), green for TBXT (BRA), and red for MEOX1 (corresponding single channel image is shown in Fig. 6i). g, Corresponding quantification of TBXT (green line) and MEOX1 (red) signal intensity along the posterior-to-anterior axis (N = 3, n = 9). h and i, Immunofluorescence staining of MG-embedded axioloids at 96 and 120 h. Images shown are representative of three independent experiments. h, F-actin (phalloidin) in grey, TBX6 in blue, and SOX2 in red. i, F-actin (phalloidin) in grey, TBXT in green, and SOX2 in red. J and k, HCR staining of MG-embedded axioloids at 96 h. Overlaid channel images are representative of two independent experiments. j, HCR staining showing MSGN1 in cyan, TCF15 in magenta, and RIPPLY2 in yellow.k, HCR staining showing RIPPLY2 in blue, LFNG in green, and HES7 in red; white arrowheads highlight the striped staining of LFNG in the posterior part of each somite. l and m, HCR staining of MG-embedded axioloids at 96 h showing MESP2 in yellow, UNCX in cyan, and TBX18 in magenta, and corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak. Numbered red arrows pinpoint the striped pattern of TBX18 observed in the posterior part of each somite. n to q, Time course from 72 to 112 h of an iPSC-derived cell line (201B7 Luc) expressing HES7:luciferase. n, Quantification of the total amount of HES7:luciferase signal in axioloids with and without MG (N = 6, n = 22) and o, Periodicity was measured as the time interval between successive HES7:luciferase signal peaks. p, Right, Kymograph along the line indicated in the left image of an axioloid embedded in MG. q, Periodicity of the HES7:luciferase signal measured in p (N = 4, n = 8). Scale bar, 200 μm. [Diagram 2]Figure 2 shows scRNA-seq characterization of human axioloid. a-c, UMAP projections of scRNA-seq datasets of human axioloid after 48, 72, 96, and 120 h. a, Samples, b, c, are color-coded by identified clusters. MG-free and MG-supplemented samples are included at 96 and 120 h. Arrows in c indicate RNA velocities. d, Time-dependent changes in the proportion of cell types in axioloid. For 96 and 120 h, only MG-plus samples are shown. e, Expression levels of selected genes are shown in UMAP plots. f, Single-cell expression profiles of identified marker genes for each cell cluster except E-SM2, M-SM2, and putative apoptotic cells. The top 50 (or less) genes with high rates of change are shown. Putative apoptotic cells are excluded. g, UMAP plots of axioloids (MG) from 96 h cultures, colored by identified clusters in g and by pseudotime in h. Arrows in g indicate RNA velocities. Two runs are combined in this analysis. i, h Expression patterns of marker genes after 96 h in MG samples along with pseudotime ranks. IM-like and EC-like cells are excluded. Abbreviations used: TB (tailbud), E-TB (early tailbud), M-TB (mid tailbud), L-TB (late tailbud), PSM (presomitic mesoderm), E-PSM (early presomitic mesoderm), APSM (prepresomitic mesoderm), E-APSM (early prepresomitic mesoderm), N-SM (neoplasmic mesoderm), SM1 (somitic mesoderm 1), E-SM 1 (early somitic mesoderm 1), SM2 (somitic mesoderm 2), E-SM2 (early somitic mesoderm 2), M-SM1 (mid somitic mesoderm 1), M-SM2 (mid somitic mesoderm 2), L-SM (late somitic mesoderm), IM-like (intermediate mesoderm-like), EC-like (endothelial cell-like), MG (Matrigel). [Diagram 3]Figure 3 shows signaling pathways and MG effects in axioloids. a-d, Integration of HCR staining images and signal quantification of MG-embedded axioloids at 96 and 120 h. Images shown are representative of at least three independent experiments. a, HCR staining for MESP2 in blue, FGF8 in green, and b, corresponding quantification along the posterior-to-anterior axis of signal intensity normalized to the position of the WNT3a signal peak (96 h: N=3, n=9 and 120 h: N=4, n=10). c, HCR staining for CYP26A1 in cyan, ALDH1A2 in magenta, and RIPPLY2 in yellow, and d, corresponding quantification along the posterior-to-anterior axis of signal intensity normalized to the position of the CYP26A1 signal peak (96 h: N=3, n=8 and 120 h: N=3, n=9). e, UMAP plots in two trials of 96 h axioloids with and without MG after MNN integration of four samples. Note that EC-like cells appear only with MG, as indicated by the black arrow. f, Volcano plots in PSM and SM. Red and blue dots indicate genes that were up- and downregulated by MG, respectively, in both replicates. g, Log2 fold changes of expressed genes in both replicate experiments that were consistently up- or downregulated by MG in SM are shown. h, Expression levels of identified genes in SM are compared between samples. Abbreviations used: TB (tail bud), PSM (presomitic mesoderm), aPSM (anterior somitic mesoderm), N-SM (neo-somitic mesoderm), SM (somitic mesoderm), EC-like (endothelial-like), MG (Matrigel). Scale bar 200 μm. [Figure 4]Figure 4 shows RA signaling and HOX coding in axioloids. a and b, Brightfield images of axioloids 96 and 120 h after embedding in b, MG and RAL, or a, MG and RA. c, Immunofluorescence staining of axioloids embedded in MG and RA after 120 h stained for F-actin (phalloidin) in grey, FN in green, MEOX1 in red, and TBXT (BRA) in blue: overlaid channel images are shown (taken from Supplementary Video 4), extracted from the central part of a z-stack that was denoised using AI-based software. d, e, HCR staining of axioloids embedded in MG after 96 h (left image) and 120 h (right image). MESP2 is shown in yellow, UNCX in cyan, and TBX18 in magenta, with corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak after 96 h (top) and 120 h (bottom). Green and red arrows pinpoint the TBX18 stripes observed in the posterior part of each somite at 96 and 120 h, respectively. f, and g, UMAP projections of axioloid after 96 and 120 h, colored by sample f, and colored by clusters annotated in f. All four conditions (MG, MG+RAL, MG+RA) are included for each time point. g, RNA velocities are indicated by arrows. h-j, UMAP projections of integrated scRNA-seq profiles of axioloid and human embryos. Axioloid after 96 and 120 h with MG and retinal, and embryos with CS12 are analyzed. h, Axioloid cell clusters are colored. i, Cell groups in the embryo are colored. j, The origin of each part of the embryo is indicated. k, For each axioloid cluster and embryo cluster, Pearson's correlation coefficients were calculated based on the distribution of cell numbers assigned to the clusters defined in the integrated dataset.l, Visualization of spatial distribution of ACTB, TBXT, HES7, TBX6, MESP2, RIPPLY2, MEOX1, and TCF15 transcripts in sections embedded in axioloids in MG+RAL after 96 h using HybISS, and m, corresponding heatmap plots (n=3) showing average gene expression along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak. n, Heatmap showing distribution of HOXC gene expression based on pseudotime analysis of RNA-seq of axioloids embedded in MG. o, and p, HOXC cluster analysis in axioloids embedded in MG+RAL after 96 h. o, Top panel, Analysis of the epigenetic landscape of HOXC loci profiled with antibodies against H3K4me3 (green) and H3K27me3 (red) by the CUT&Tag method. Bottom panel, Visualization of spatial distribution of HOXC transcripts by HybISS analysis of HOXC clusters. p, Heatmap plots of HybISS data in s showing average HOXC cluster gene expression along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak (n=3). Scale bar, 200 μm. [Diagram 5]Figure 5 shows molecular and functional characterization of patient-like axioloids. Panels a-g show data for HES7 KO1, panels hn show data for HES7R25W MT1, and panels ou show data for MESP2 KO1. a, h, o, Brightfield images of forming patient-like axioloids at 72 h, 96 h, and 120 h, extracted from Supplementary Videos 9, 11, and 13, respectively (check if applicable, do not modify image). b, i, p, Axioloid length along the posterior to anterior axis at 24, 48, 72, 96 and 120 hours for the corresponding cell lines HES7 KO1 (N=3, n=18), HES7 KO2 (N=2, n=12), HES7R25W MT1 (N=3, n=18), HES7R25W MT2 (N=3, n=18), MESP2 KO1 (N=3, n=17), MESP2 KO2 (N=3, n=15). c, j, q, Immunofluorescence staining and signal quantification of MG+RAL embedded axioloids after 120 hours. Top panel, composite of channel images of axioloid stained in grey for F-actin (phalloidin), green for FN, red for MEOX1, and blue for TBXT (BRA). Lower panels, corresponding quantification of TBXT (green line) and MEOX1 (red) signal intensity along the posterior-to-anterior axis for HES7 KO1 (N = 3, n = 10), HES7R25W MT1 (N = 4, n = 9), MESP2 KO1 (N = 3, n = 12). d, k, and r, HCR staining and signal quantification of MG+RAL embedded axioloids after 120 h. Upper panels, MESP2 (yellow), UNCX (cyan), TBX18 (magenta); lower panels, corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak for HES7 KO1 (N = 3, n = 9), HES7R25W MT1 (N = 3, n = 8), MESP2 KO1 (N = 3, n = 7).e, l, and s, Time course from 72 to 112 h of HES7:luciferase expressing hiPSC cell line (201B7 Luc) for HES7 KO1 (N = 4, n = 12), HES7 KO2 (N = 4, n = 12), HES7R25W MT1 (N = 3, n = 9), HES7R25W MT2 (N = 3, n = 9), MESP2 KO1 (N = 4, n = 12), MESP2 KO2 (N = 4, n = 12). f, m, and t, Kymographs of axioloids embedded in MG+RAL in f, HES7 KO1, m, HES7R25W MT1, and t, MESP2 KO1. g, n, and u, Mean measurements of HES7:luciferase signal over time for HES7 KO1 (N=4, n=8), HES7 KO2 (N=5, n=9), HES7R25W MT1 (N=6, n=12), HES7R25W MT2 (N=6, n=11), MESP2 KO1 (N=4, n=9), and MESP2 KO2 (N=4, n=9). Scale bar, 200 μm. [Figure 6]Figure 6 shows the morphological and molecular characteristics of human axioloids. af, a Brightfield images of elongating axioloids at 24, 48, and 72 h, and subsequent images of axioloids after MG embedding at 96 and 120 h (b, c without MG embedding). d and e, Representative brightfield images of MG-embedded axioloids at 96 and 120 h in two cell lines, d, 409B2 (N=3, n=128 axioloids) and e, 201B7 Luc (N=3, n=60 axioloids), categorized based on morphology (Cat#1: straight, minimal curvature, well-defined segments; Cat#2: curved, but well-defined segments; Cat#3: very curvilinear, but segment boundaries still distinguishable; Cat#4: not properly elongated or completely collapsed; no clear segment boundaries). f, Sequential images of axioloid formation at 5-h intervals from 74 to 119 h (extracted from Supplementary Video 1). Colored arrowheads indicate the process of segment formation at each time point, with yellow arrowheads indicating regions where somitogenesis is ongoing and green arrowheads indicating regions where segmentation is complete. g, Periodicity of somite segmentation based on live-cell imaging observations (N = 3, n = 9). h, Axioloid length (anterior-posterior) at 24 h, 48 h, 72 h, and 96 and 120 h with and without MG implantation (N = 3, n = 18). iq, Immunofluorescence staining and corresponding quantification. i, j, m, n, p, m, Representative images of axioloids stained in grey for F-actin (phalloidin), green for TBXT, and red for MEOX1 with MG embedding at 72 h, i, j, 96 h and 120 h, and n, p, 96 h and 120 h without MG embedding. k, o, p, k, Corresponding quantification along the posterior to anterior axis of TBXT (green line) and MEOX1 (red) signal intensity at 96 h (N=3, n=14) and 120 h (N=4, n=16) with MG embedding, and o, q, 96 h (409B2, N=3, n=9 and 201B7, N=3, n=15) and 120 h (409B2, N=3, n=9 and 201B7, N=3, n=11) without MG embedding.l, Somite lengths were measured based on phalloidin staining performed in i and j in both cell lines 409B2 (N = 3, n = 9) and 201B7 Luc (N = 3, n = 9). Scale bar, 200 μm. [Figure 7]Figure 7 shows assessment of apical-basal polarity, developmental protein and gene expression patterns, craniocaudal patterning, oscillatory activity of HES7 and traveling wavefronts of segmentation in human axioloids embedded in MG. ad, Immunofluorescence staining of MG-embedded axioloids. a, b, High magnification images (X63) of single segments at 120 h with staining for F-actin (phalloidin) in grey, aPKC and FN in green, and MEOX1 in red in axioloids derived from a, 409B2 and b, 201B7 Luc iPS cell lines; images are representative of two independent experiments. c, Image showing F-actin (phalloidin) in grey, TBX6 in blue, SOX2 in red; d, Image showing F-actin (phalloidin) in grey, TBXT in green, SOX2 in red. ep, HCR staining and quantification of the corresponding signals (e and f, HCR staining for MSGN1 (cyan), TCF15 (magenta), RIPPLY2 (yellow); g and h, HCR staining for RIPPLY2 (blue), LFNG (green), HES7 (red); purple arrowheads highlight the striped staining pattern of LFNG in the posterior part of each somite. Images shown are representative of two independent experiments. i, HCR staining of MG embedded axioloids at 96 and 120 h, respectively, in two different cell lines (i, j, 409B2 (N=4, n=10 and N=3, n=9), k, l, 201B7 Luc (N=3, n=9 and N=3, n=10)) and MG non-embedded axioloids at 96 h (m, n, 409B2 (N=3, n=12) and o, p, 201B7 Luc (N=3, n=11)). The corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak are shown in Fig. 1. c, g, i, Single channel images shown in the upper row correspond to the merged channel images shown in Fig. 1 h-j. q, r, Annotated sequential images of the forming axioloid with the HES7:luciferase signal overlaid in green (adapted from Supplementary Video 2).Colored dotted lines indicate the anteriormost position reached by the HES7 oscillatory wave of gene expression, and identically colored arrowheads indicate the change in this position over time (yellow for first oscillation, red for second oscillation, blue for third oscillation, orange for fourth oscillation). r, Images at 24 h show that the position of each HES7:luciferase expression wavefront corresponds to the area of ​​the formed segment. s, Time-lapse measurements of the average HES7:luciferase intensity (N=4, n=8). Scale bars in a and b are 50 μm, others 200 μm. [Figure 8] Figure 8 shows single-cell RNA-seq analysis of human axioloids. a, UMAP projections of scRNA-seq data from axioloids at 48, 72, 96, and 120 h, color-coded by estimated cell cycle phase (G1, G2M, S). b, G2M.Score and S.Score of cells in each cluster in Fig. 2b. c, Percentage of cell types within axioloids with and without MG (at 96 and 120 h). d, Average expression levels of ribosomal protein genes in each cluster in Fig. 2b. e, Shift in TB marker gene expression over time. f, Expression levels of TBXT and SOX2 in each cell are plotted for three TB clusters (E-TB, M-TB, and L-TB, representing early, middle, and late tailbud, respectively). [Figure 9]Figure 9 shows expression gradients of components of FGF, WNT and RA signaling pathways in human axioloids embedded in MGs. ac, Pseudo-temporal representation of transcripts related to FGF, WNT and RA signaling pathways upon exposure of MGs to human axioloids at 96 h of culture (24 h after embedding in MGs). Gene expression patterns arranged along pseudo-temporal rank order. Includes pseudo-temporal expression patterns of effectors and negative regulators of all three pathways. di, HCR staining images and signal quantification after 96 and 120 h of MG-embedded axioloids from d and g, 409B2, e, f, h and i, 201B7 Luc iPSC lines. Images shown are representative of three independent experiments. d and e, HCR staining in blue for MESP2, green for FGF8 and red for WNT3a. f, Corresponding quantification of signal intensity normalized to the position of the WNT3a signal peak along the posterior to anterior axis (96 h: N = 3, n = 8 and 120 h: N = 3, n = 7). g and h, HCR staining for CYP26A1 in cyan, ALDH1A2 in magenta, and RIPPLY2 in yellow, and i, corresponding quantification of signal intensity normalized to the position of the CYP26A1 signal peak along the posterior to anterior axis (96 h: N = 3, n = 6 and 120 h: N = 3, n = 7). Scale bar 200 μm. [Figure 10]Figure 10 shows single-cell RNA-seq analysis: Identification of DEGs associated with exposure of axioloids to MG. a, UMAP projection of two combined trials of axioloids 96 h after MG addition, colored by the clusters in Fig. 2g. b, UMAP projection of two combined trials of axioloids 96 h after MG addition, colored by the clusters in Fig. 2b. Note that b includes only one trial. c, Expression levels of indicated genes on the same UMAP plot in a. d, Mean expression levels of identified EC-like marker genes in each cluster in Fig. 2b. e, Enrichment analysis of upregulated genes in SM in the presence of MG, using Hallmark gene set and KEGG dataset. f and g, Differentially expressed genes between axioloids with and without MG at 96 h in PSM and TB. [Figure 11]Figure 11 shows an assessment of the morphological effects of retinoid signaling on human axioloid. a and b, Brightfield images of axioloids at 96 and 120 h after embedding in MG (Matrigel) alone, MG+RAL (retinal), MG+ROL (retinol), or MG+RA (retinoic acid) for 409B2 (a) and 201B7 (b) Luc. c, Sequential images showing axioloid elongation in 5-h intervals from 74 to 119 h (taken from Supplementary Video 3). Colored arrowheads highlight the process of segmentation at each time point, with yellow arrowheads identifying areas where somitogenesis is ongoing, whereas red for 409B2 or green for 201B7 highlight areas where segmentation has been completed. d and e, Immunofluorescence high magnification images (X63) of single somites of axioloids cultured for 120 h in MG+RAL, showing F-actin (phalloidin) in grey, aPKC, CDH2, LAMC1 and FN from top to bottom in green, and MEOX1 in red in d, 409B2, and e, 201B7 Luc iPS cell lines. Images are representative of two different experiments. f, Brightfield images of axioloids without MG embedding at 120 h after addition of RAL only, ROL only, and RA only (all without MG) in 409B2 (top) and 201B7 Luc (bottom). g and h, Representative bright-field images of MG+RAL-embedded axioloids at 96 and 120 h in two cell lines g (409B2, N = 5, n = 128 axioloids) and h (201B7 Luc, 96 h N = 3, n = 91 axioloids and 120 h N = 3, n = 92 axioloids), categorised based on morphology (Cat#1: straight, little curvature, well-defined somites; Cat#2: curved, but well-defined somites; Cat#3: very curved, but somite-somites boundaries are still distinguishable; Cat#4: neither properly elongated nor completely collapsed; somite-somites boundaries are not clearly defined). i and j, axioloid length measurements at 96 and 120 h after implantation in MG only, MG+RAL, MG+ROL, or MG+RA for i, 409B2, and j, 201B7 Luc.k and l, Periodicity of somite segmentation based on live cell imaging observations for k, 409B2 (N = 3, n = 11) and l, 201B7 Luc (N = 3, n = 9), respectively. m and n, Total number of somites in axioloids embedded in MG+RAL or MG+ROL or MG+RA at 120 h for m, 409B2, or embedded in MG+RAL or MG+RA at 120 h for n, 201B7 Luc. Scale bars in d and e are 50 μm, others 200 μm. [Figure 12]Figure 12 shows molecular characterization of human axioloids exposed to agonists or inhibitors of retinoic acid (RA) signaling. a,b, 409B2 at 96 h (N=4, n=10) and 120 h (N=4, n=9); c,d, 201B7 Luc at 96 h (N=4, n=13) and 120 h (N=4, n=13); representative images of immunofluorescence staining (F-actin (phalloidin) in grey, TBXT in green, and MEOX1 in red) of axioloids embedded in MG+RAL, and corresponding quantification of signal intensity. Immunofluorescence data of axioloids embedded in MG+RAL (e, f, 409B2 at 96 hr (N=4, n=15) and 120 hr (N=3, n=12); and g, h, 201B7 Luc at 96 hr (N=3, n=15) and 120 hr (N=4, n=16)). ip, representative images of HCR staining with MESP2 in yellow, UNCX in cyan, and TBX18 in magenta, and corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak in MG+RAL-embedded axioloids (i, j, 409B2 at 96 hr (N=3, n=9) and 120 hr (N=3, n=9); k, l, 201B7 Luc at 96 hr (N=3, n=9) and 120 hr (N=3, n=9)). In situ hybridization data of axioloids embedded in MG+RA (m, n, 409B2, 96 h (N=3, n=9) and 120 h (N=3, n=5); and o, p, 201B7 Luc, 96 h (N=2, n=5) and 120 h (N=2, n=5)). q, Immunofluorescence staining of 409B2 axioloids embedded in MG+RAL at 96 h (top) and 120 h (bottom), showing F-actin (phalloidin) in grey, TBXT in blue, and SOX2 in red. Images shown are representative of three independent experiments. Scale bar 200 μm. r and s, Representative images of immunofluorescence staining of axioloids embedded in MG+RAL+BMS493 (201B7 Luc) at 120 h of culture, showing F-actin (phalloidin) in grey, TBXT in green, and MEOX1 in red, and corresponding quantification of signal intensity (N = 3, n = 11).t and u, Representative images of HCR staining with MESP2 in yellow, UNCX in cyan, and TBX18 in magenta, and corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak in axioloids embedded in MG+RAL+BMS493 (201B7 Luc) at 120 h of culture (N = 2, n = 6). [Figure 13] Figure 13 shows single-cell RNA-seq-based assessment of RA signaling effects on MG embedded in human axioloids. a, Integrated UMAP projection of single-cell transcriptome profiles of axioloids in all four conditions (control, MG only, MG+RAL (retinal), MG+RA (retinoic acid)) at both 96 and 120 h. b, c, Expression changes with MG+RAL (retinal) and MG+RA (retinoic acid) compared to MG only condition for consistently up- or down-regulated genes at 96 and 120 h in both SM b, TB c. d, Enrichment analysis of down-regulated genes in SM with retinal (RAL) added to MG. Hallmark and KEGG datasets were used. e, Expression changes compared to control (no MG) samples are shown for different conditions (RAL or RA added) at both 96 and 120 h. The genes shown here are the DEGs identified in Fig. 3g (96 h MG). fh, Expression levels of the indicated genes under different conditions of SM in axioloids at 96 h and 120 h. [Figure 14]Figure 14 shows the formation of midlines and bilateral somites in human axioloids. a and e, Sequential images (taken from Supplementary Video 5) showing the elongation of axioloids at 8-10 h intervals from 72 to 120 h. a, The red dotted arrows indicate the formation and elongation of the superficial midline along the posterior-to-anterior axis of axioloids embedded in MG+RAL. e, The red arrowheads highlight and indicate the initiation of somites splitting into two bilaterally symmetric structures in axioloids embedded in MG+ROL. b-d, Left, Brightfield images of axioloids (409B2) at 120 h, with the area enclosed by the red dotted line being the magnified area in the right image. b, Axioloid embedded in MG+RAL showing prominent midline highlighted by the dotted double arrow in the right image. c and d show embedded axioloids of MG+RAL and MG+RA, respectively, with a single bilateral somite in c and multiple bilateral somites in d highlighted by red dotted lines. f, h, i, k, Immunofluorescence staining showing F-actin (phalloidin) in grey, TBXT (BRA) in blue, FN in green and MEOX1 in red. g and j, HCR staining of human axioloid (409B2) at 120 hours showing MESP2 in yellow, UNCX in cyan and TBX18 in magenta. Axioloids show a single bilateral somite in f and g and multiple bilateral somites in h and k. i, j and k correspond to magnified views of merged channel images in f, g and h, respectively. Scale bars 200 μm. [Figure 15]Figure 15 shows morphological and molecular (scRNA-seq) comparison of human axioloid with human CS11 and CS12 embryos. a and b, 3D modeling based on phalloidin staining and z-stack images of a, 409B2 and b, 201B7 Luc at 120 hours embedded in MG+RAL, and somite volume measurements. c and d, 3D modeling based on OPT stack single images and image stacks, and somite volume measurements of eight posterior somites of CS11 human embryos (OPT data for human embryos obtained from Human Developmental Biology Resource (HDBR)). ef, UMAP projection of scRNA-seq dataset of human embryo CS12. e, Color coding based on clustering, f, Color coding based on cell annotation by Xu et al., g, Color coding based on sample origin. h, i, Average expression levels of indicated genes in each cluster. h, Genes shown are marker genes for the annotated clusters in Xu et al., i are marker genes for axioloid. jm, UMAP projections of integrated scRNA-seq profiles of axioloid and human embryos, colored by j, their origin (axioloid or embryo), k, the defined cluster, l, axioloid specimens (96 h or 120 h), and m, cell type (annotated by Xu et al.). [Figure 16]Figure 16 shows the evaluation of the HOX code in human axioloids. ac, Pseudo-chronological representation of the expression of HOXA, HOXB and HOXD cluster-related genes in human MG-exposed axioloids at 96 h of culture (24 h after embedding in MG); gene expression patterns are arranged in pseudo-chronological order. d, f and h, Top, Analysis of the epigenetic landscape at HOXA, HOXB and HOXD loci profiled with antibodies against H3K4me3 (green) and H3K27me3 (red) by the CUT&Tag method. Bottom, Visualization of the spatial distribution of HOXA, HOXB and HOXD transcripts using HybISS analysis of all members of the HOX cluster at 96 and 120 h for axioloids cultured in MG+RAL. Heatmap plots of HybISS data shown in e, g and i, d, f, h show the average HOXA, HOXB, and HOXD cluster gene expression along the posterior-to-anterior axis in MG+RAL axioloids at 96 and 120 h, normalized to the position of the MESP2 signal peak (n = 3). [Figure 17]Figure 17 shows expression gradients of components of FGF, WNT, and RA signaling pathways in human axioloids visualized and quantified by HCR and HybISS. a, a, b, e, f, HCR whole-mount in situ hybridization images and signal quantification at 96 and 120 hours in MG+RAL-embedded axioloids from 409B2 and c, d, g, h, 209B7 Luc iPSC lines. Images shown are representative of three independent experiments. a and c, HCR staining showing FGF8 in green, WNT3a in red, and MESP2 in blue. b and d, Corresponding quantification of signal intensity along the posterior-to-anterior axis normalized to the position of the WNT3a signal peak (409B2 96h: N=4, n=7 and 120h: N=3, n=6, 201B7 Luc 96h: N=3, n=9 and 120h: N=3, n=8). e and g, HCR staining showing CYP26A1 in cyan, ALDH1A2 in magenta, and RIPPLY2 in yellow. f and h, Corresponding quantification of signal intensity along the posterior-to-anterior axis normalized to the position of the CYP26A1 signal peak (409B2 96h: N=4, n=10 and 120h: N=3, n=7, 201B7 Luc 96h: N=3, n=8 and 120h: N=3, n=10). Scale bar 200 μm. il, HybISS-based visualization and quantification of spatial distribution of FGF / WNT and RA signaling pathway transcripts in human axilloids at 96 h (top) and 120 h (bottom) of culture in MG+RAL. i and j, HybISS images and quantification of spatial expression of FGF3, FGF4, FGF8, FGF17, WNT3a and WNT5b. k and l, HybISS images and quantification of spatial expression of ALDH1A2, CYP26A1 and RDH10. [Figure 18]Figure 18 shows the regulation of RA, FGF, WNT and Notch signaling in human axioloid. Quantification of the total amount of HES7:luciferase signal over time in axioloid+ / -MG with ar, a, b, +RAL (retinal) (N=5, n=17 and N=5, n=20), c, d, +ROL (retinol) (N=3, n=12 and N=5, n=20), e, f, +RA (retinoic acid) (N=6, n=22 and N=5, n=20) and the corresponding measurement period as the time interval between successive HES7:luciferase signal peaks. Kymographs of HES7:luciferase signals in axioloids (g, MG+RAL embedded axioloids (N=4, n=6, data same as shown in Figure 4 g, n, u); h, MG+RA embedded axioloids (N=4, n=8) and corresponding i, average signal, and j, periodicity measurements; k, MG+RAL+DMSO embedded axioloids (N=3, n=6); l, MG+RAL+BMS493 embedded axioloids (N=3, n=6) and corresponding m, average signal, and n, periodicity measurements). Time course of HES7:luciferase signal in MG+RAL axioloids. o, effect of addition of BMS493 (N=3, n=9), r, effect of addition of DAPT (N=3, n=9), PD173074 (N=3, n=9), and XAV939 (N=3, n=9) compared to DMSO (N=3, n=9). p, Comparison of the total number of somites of axioloids embedded in MG+RAL alone or in MG+RAL supplemented with DMSO or BMS493 or DAPT, PD173074, XAV939 (N = 3, all n = 9), and the length of axioloids at 96 and 120 h after addition of DMSO (N = 3, n = 24 for p, N = 3, n = 9 for t), BMS493 (N = 3, n = 9), DAPT or PD173074 and XAV939 (N = 3, n = 9). s, Representative brightfield images of axioloids embedded in MG+RAL supplemented with DMSO or BMS493 or DAPT or PD173074 and XAV939. Scale bar is 200 μm. [Figure 19]Figure 19 shows morphological, molecular, and functional characterization of axioloids derived from patient-like iPSCs harboring mutations in HES7 and MESP2. Panels ad show data for HES7 KO2, eh show data for HES7R25W MT2, and il show data for MESP2 KO2. a, h, o, Brightfield sequential images of forming axioloids at 72, 96, and 120 hours. Extracted from Supplementary Videos 9, 11, and 13, respectively. b, f, and j, Immunofluorescent staining and signal quantification of MG+RAL-embedded axioloids at 120 hours. Top, merged images of channels of axioloids stained with F-actin (phalloidin) in grey, FN in green, and MEOX1 in red and TBXT (BRA) in blue. Bottom, corresponding quantification of TBXT (green line) and MEOX1 (red) signal intensity along the posterior-to-anterior axis for HES7 KO2 (N=3, n=9), HES7R25W MT2 (N=3, n=9), and MESP2 KO2 (N=3, n=12). c, g, and k, HCR staining (MESP2 in yellow, UNCX in cyan, and TBX18 in magenta) and signal quantification of MG+RAL-embedded axioloids at 120 hours, and corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak for HES7 KO2 (N=3, n=8), HES7R25W MT2 (N=3, n=8), and MESP2 KO2 (N=3, n=9). d, h, and l, Kymographs of HES7 oscillatory activity for d, HES7 KO2, h, HES7R25W MT2, and l, MESP2 KO2 axioloids embedded in MG+RAL. Scale bars are 200 μm. [Figure 20]Figure 20 shows the morphogenetic characteristics of axioloids embedded only in +MG and the scRNA-seq expression profile of human axioloids. a, Representative brightfield images of MG-embedded axioloids (three independent experiments, n = 60 axioloids) at 96 and 120 h derived from 201B7 Luc cell line, categorized based on morphology (Cat#1: straight, minimal curvature, clear segments; Cat#2: curved, but clear segments; Cat#3: very curvilinear, but segment boundaries still distinguishable; Cat#4: not properly elongated or completely collapsed; segment boundaries not clear). Cat#1 corresponds to Fig. 7b, 96 h, CTL. b, scRNA-seq expression profile of genes identified as marker genes for each cell cluster shown in Fig. 2b (except E-SM2, M-SM2, and putative apoptotic cells). The top 50 (or less) genes with high fold changes are shown. Scale bar is 200 μm. [Figure 21]Figure 21 shows the characterization of retinoid-treated human axioloids. a, Brightfield images of axioloids cultured for 120 h without MG implantation after addition of RAL alone, ROL alone, and RA alone (all without MG) in 409B2 (top) and 201B7 Luc (bottom). b and c, Representative brightfield images of +MG+RAL embedded axioloids at 96 and 120 h in two different cell lines b, 409B2 (five independent experiments, n = 128 axioloids, mean ± SD) and c, 201B7 Luc hiPSC line (96 h, three independent experiments, n = 91 axioloids and 120 h, three independent experiments, n = 92 axioloids, mean ± SD), categorized based on morphology (Cat#1: straight, little curvature, well-defined somites; Cat#2: curved, but well-defined somites and somites; Cat#3: very curved, but somites and somites boundaries are still distinguishable; Cat#4: not properly elongated or completely collapsed, somites and somites boundaries are not clearly defined). Representative images of immunofluorescence staining (F-actin (phalloidin) in grey, TBXT in cyan, and MEOX1 in red) of 409B2-derived axioloids embedded at 96 h (three independent experiments, n = 9 axioloids) and 120 h (three independent experiments, n = 9 axioloids); fg, +MG+RAL, 96 h (four independent experiments, n = 10 axioloids) and 120 h (four independent experiments, n = 9 axioloids); hi, +MG+RA409B2, 96 h (four independent experiments, n = 15 axioloids) and 120 h (four independent experiments, n = 12 axioloids) and corresponding quantitative values ​​of signal intensity.jm, Representative images of HCR staining (UNCX shown in cyan, TBX18 in magenta, and MESP2 in yellow) of 409B2-derived axioloids embedded in +MG+RAL for 96 h (three independent experiments, n = 9 axioloids) and 120 h (three independent experiments, n = 9 axioloids) (jk), and of 409B2-derived axioloids embedded in +MG+RA for 96 h (three independent experiments, n = 9 axioloids) and 120 h (four independent experiments, n = 8 axioloids) (lm), with corresponding signal intensity measurements along the posterior-to-anterior axis normalized to the position of the MESP2 signal peak. The single channel images shown in j correspond to the merged channel images shown in Figure 4f. Lines in e, g, i, k and m correspond to the mean, error bands represent the 95% confidence interval above and below the 2.5th and 97.5th percentiles of each data point. Scale bar, 200 μm. au, arbitrary units. [Figure 22]Figure 22 shows midline and bilateral somitogenesis in human axioloids. a and b, Sequential images of human axioloid (409B2) elongation from 72 to 120 h at 8–10 h intervals (taken from Supplementary Video 5). a, Red dotted arrows indicate the formation and extension of a superficial midline along the posterior-to-anterior axis of an axioloid embedded in +MG +RAL. b, Red arrows indicate the onset of bilateral somitogenesis in an axioloid embedded in +MG +ROL. c-e, Left, bright-field image of an axioloid (409B2) 120 h later, with the area outlined in red dotted lines being the area magnified in right. c, +MG+RAL-embedded axioloid showing an obvious midline highlighted by an arrowhead in the right image. c and d, Axioloids embedded in +MG+RAL and +MG+RA, respectively, with a single bilateral somite in c and multiple bilateral somites in d highlighted by red arrowheads. fk, Immunofluorescence and in situ-based evaluation of axioloids with bilateral somites treated with +MG+RAL and +MG+RA. F, h, i, k, Immunofluorescence staining of F-actin (phalloidin) in grey, TBXT (BRA) in cyan, MEOX1 in red, and FN1 in yellow. g and j, HCR staining of human axioloids (409B2) at 120 h, with UNCX in cyan, TBX18 in magenta, and MESP2 in yellow. Axioloids show a single bilateral somite in f and g and multiple bilateral somites in h and k. i, j, k correspond to magnified views of merged channel images in f, g, and h, respectively. Images shown in ch are representative of three independent experiments. Scale bar is 200 μm. [Figure 23]Figure 23 shows HOX gene expression in axioloids in medium supplemented with +MG only. a, Visualization of spatial distribution of ACTB, TBXT, HES7, TBX6, MESP2, RIPPLY2, MEOX1, TCF15 transcripts in sections of axioloids from 409B2 embedded in +MG+RAL after 96 hours using HybISS, and b, corresponding heatmap plot showing average gene expression along the posterior-to-anterior axis normalized relative to the position of the MESP2 signal peak (n=3 axioloids). cj, Visualization and quantification of HOX loci based on HybISS. c,e,g,i, Visualization of spatial distribution of HOXA, HOXB, HOXC and HOXD transcripts using HybISS analysis of all members of the HOX cluster using 96- and 120-hour sections of axioloids cultured in MG only. d, f, h and j, Heatmap plots of HybISS data shown in c, e, g and i showing the average expression of HOXA, HOXB, HOXC and HOXD cluster genes along the posterior to anterior axis of axioloids (n = 3 axioloids) at 96 and 120 h, cultured in MG medium only. [Figure 24]Figure 24 shows expression gradients of components of FGF, WNT, and RA signaling pathways in human axioloids visualized and quantified by HCR and HybISS. a, a, b, e, f, HCR whole mount in situ hybridization images and signal quantification at 96 and 120 hours in +MG+RAL embedded axioloids from 409B2 and c, d, g, h, 209B7 iPS cell lines. Images shown are representative of three independent experiments. a and c, HCR staining showing FGF8 in green, WNT3A in red, and MESP2 in blue. b and d, Corresponding quantification of signal intensity normalized to the position of the WNT3a signal peak along the posterior-to-anterior axis (409B2 96 h: 4 independent experiments, n = 7 axioloids and 120 h: 3 independent experiments, n = 6 axioloids, 201B7 Luc 96 h: 3 independent experiments, n = 9 axioloids and 120 h: 3 independent experiments, n = 8 axioloids). e and g, HCR staining with cyan for CYP26A1, magenta for ALDH1A2 and yellow for RIPPLY2. f and h, corresponding quantification along the posterior-to-anterior axis of signal intensity (409B2 96 h: 4 independent experiments, n = 10 axioloids and 120 h: 3 independent experiments, n = 7 axioloids, 201B7 Luc 96 h: 3 independent experiments, n = 8 axioloids and 120 h: 3 independent experiments, n = 10 axioloids). Lines in b, d, f and h correspond to the mean values, error bands represent the 95% confidence intervals, above and below which indicate the 2.5th and 97.5th percentiles of each data point. Scale bars are 200 μm. il, HybISS-based visualization and quantification of the spatial distribution of FGF / WNT and RA signaling pathway transcripts in human 409B2-derived axioloids at 96 h (top) and 120 h (bottom) of culture in +MG+RAL. i and j, HybISS images and quantification of spatial expression of FGF3, FGF4, FGF8, FGF17, WNT3A and WNT5B. k, l, HybISS images and quantification of spatial expression of ALDH1A2, CYP26A1 and RDH10.Lines in j and l correspond to the mean and error bands represent the SD of each data point (n = 3 axioloids). au is arbitrary unit. [Diagram 25] Figure 25 shows the effect of alternative culture media on axioloid morphogenesis. Brightfield images of 10% MG+RAL embedded axioloids at 96 and 120 hours, generated in a, ba, NDiff227 or b, RMPI medium supplemented with B27 with (RPMI+) or without (RPMI-) retinol. Scale bar, 200 μm. [Figure 26] Figure 26 shows the effect of bFGF, CHIR99421 (WNT agonist) and SB431542 (TGFβ inhibitor) concentrations on axioloid induction and morphogenesis. ac Brightfield images of 10% MG+RAL embedded axioloids at 96 and 120 hours created with different concentrations of a, bFGF, b, CHIR99421, c, SB431542 in two cell lines 409B2 (top) and 201B7 Luc (bottom). Scale bar is 200 μm. [Figure 27] Figure 27 shows the effect of FGF8b and A-83-01 (TGFβ inhibitor) on axioloid morphogenesis. ab Brightfield images of axioloids embedded in 10% MG+RAL for 96 and 120 hours, generated with different concentrations of a FGF8b and b A-83-01. Scale bar 200 μm. [Figure 28] Figure 28 shows the effect of ECM-rich components and TTNBP (a retinoid agonist) on axioloid morphogenesis. ab Brightfield images of axioloids embedded in a, medium containing RAL and 5% MG or 10% MG or 10% Cultrex or 10% Geltrex or 10% ECMGel, and b, medium containing 10% MG and ROL or RAL or RA or TTNPB for 96 and 120 h. Scale bar 200 μm.

[0007] (Detailed Description) The present disclosure relates to three-dimensional cellular aggregates, called axioloids, generated in vitro from pluripotent stem cells, and methods for their production.

[0008] The present disclosure also relates to three-dimensional cell aggregates generated in vitro from pluripotent stem cells comprising mesodermal cells, said cell aggregates having polarity in the anterior-posterior or rostrocaudal axis and the apical-basolateral axis, said cell aggregates being capable of reconstituting various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions.

[0009] The present disclosure also relates to three-dimensional cell aggregates generated in vitro from pluripotent stem cells, comprising mesodermal cells and / or progenitor cells, said cell aggregates having polarity in the anterior-posterior or rostrocaudal axis and the apical-basolateral axis, said cell aggregates being capable of reconstituting various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions.

[0010] The present disclosure also relates to three-dimensional cell aggregates generated in vitro from pluripotent stem cells comprising mesodermal cells, said cell aggregates having polarity in the anterior-posterior or rostro-caudal axis and the apical-basolateral axis, and wherein the percentage of mesodermal cells in said cell aggregates 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 cell number.

[0011] The present disclosure also relates to a method for producing a three-dimensional cell aggregate generated in vitro from pluripotent stem cells, comprising the steps of: (a) culturing pluripotent stem cells to induce a three-dimensional cell aggregate comprising mesodermal cells; and (b) culturing the cell aggregate comprising mesodermal cells to induce a three-dimensional cell aggregate, wherein the three-dimensional cell aggregate is a cell aggregate.

[0012] The present disclosure also relates to a method for producing a precursor cell or a differentiated cell, comprising the step of culturing the cell aggregates and inducing a precursor cell or a differentiated cell selected from the group consisting of: (a) a neuro-mesodermal cell or a precursor cell thereof; (b) a muscle cell or a precursor cell thereof; (c) a bone cell or a precursor cell thereof; (d) a chondrocyte or a precursor cell thereof; (e) a tenocyte or a precursor cell thereof; and (f) an endothelial or hematopoietic cell or a precursor cell thereof.

[0013] The present disclosure also relates to a method for producing a precursor cell or a differentiated cell, comprising the step of culturing the cell aggregates and inducing a precursor cell or a differentiated cell selected from the group consisting of: (a) a neural-mesodermal cell or a precursor cell thereof; (b) a muscle cell or a precursor cell thereof; (c) a bone cell or a precursor cell thereof; (d) a chondrocyte or a precursor cell thereof; (e) a tenocyte or a precursor cell thereof; and (f) an endotome or endothelial or hematopoietic cell or a precursor cell thereof; (g) an adipocyte or a precursor cell thereof, including white, beige and brown cells; (h) a dermal cell or a precursor cell thereof; and (i) a neural tube cell or a precursor cell thereof.

[0014] The present disclosure relates to a method of evaluating a test substance, comprising the steps of: culturing the test substance in the presence of a three-dimensional cellular aggregate; and evaluating the three-dimensional cellular aggregate after said culturing, wherein the three-dimensional cellular aggregate is a cellular aggregate of the present disclosure.

[0015] The present disclosure also relates to a method for evaluating gene or genome function, comprising the steps of: preparing pluripotent stem cells having a modified test gene or test genome; generating three-dimensional cell aggregates from the pluripotent stem cells; and evaluating the three-dimensional cell aggregates after culture, wherein the generation of the three-dimensional cell aggregates is performed by the method of the present disclosure.

[0016] The present disclosure also relates to a method for evaluating gene function or genomic sequence function, comprising the steps of: preparing pluripotent stem cells in which a test gene or test genomic sequence has been modified; generating three-dimensional cell aggregates from the pluripotent stem cells; and evaluating the three-dimensional cell aggregates after culture, wherein the generation of the three-dimensional cell aggregates is performed by the method of the present disclosure.

[0017] In this disclosure, "low" is used to connote a lower number and / or amount of a subject compared to a reference, unless otherwise stated.

[0018] In this disclosure, "high" is used to connote a greater number and / or amount of an object compared to a reference, unless otherwise stated.

[0019] In the present disclosure, suitable examples of "one or more" can be numbers from 1 to 6, and preferred numbers can be 1 to 3.

[0020] In this disclosure, "marker" refers to a nucleic acid, gene, polypeptide, or protein that is expressed to a different extent in a target cell. When the marker is a positive marker, the different extent refers to an increased expression compared to the cell to be compared. When the marker is a negative marker, the different extent refers to a decreased expression compared to the cell to be compared.

[0021] In this disclosure, "pluripotent cells" refers to cells that can differentiate into ectodermal, mesodermal, and endodermal cells. Pluripotent cells are also called pluripotent stem cells when the pluripotent cells are capable of self-renewal.

[0022] In the present disclosure, "ectodermal cells" refers to cells that are destined to differentiate into neural tissue such as nerves, epithelial tissue such as epidermis, etc., when given appropriate developmental stimuli, and are cells that express ectodermal cell markers described below.

[0023] In the present disclosure, "mesodermal cells" refer to cells that are destined to differentiate into connective tissues such as bone, cartilage, blood vessels, and lymphatic vessels, muscle tissue, etc., if developmentally appropriate stimuli are provided, and are cells that express mesodermal cell markers described below.

[0024] In the present disclosure, "mesodermal cells" refer to cells that are destined to differentiate into connective tissues such as bone, cartilage, fat, blood vessels, lymphatic vessels, muscle tissue, etc., if developmentally appropriate stimuli are provided, and are cells that express mesodermal cell markers described below.

[0025] In the present disclosure, "endodermal cells" refers to cells that are destined to differentiate, upon receipt of appropriate developmental stimuli, into digestive organs such as the thymus, stomach, intestines, and liver, respiratory organs such as the trachea, bronchi, and lungs, and urinary organs such as the bladder and urethra, and express endodermal cell markers described below.

[0026] In the present disclosure, the term "three-dimensional cell aggregate" refers to a structure in which cells are aggregated in three dimensions. The three-dimensional cell aggregate is different from, for example, a two-dimensional cell aggregate (cell sheet) obtained by flat culture, and forms a three-dimensional structure by, for example, accumulating cells in the thickness direction.

[0027] In this disclosure, sequence information for the proteins described herein or the nucleic acids (eg, DNA or RNA) encoding them is available from the Protein Data Bank, UniProt or Genbank.

[0028] The present disclosure refers to cellular aggregates (hereinafter also referred to as "axioloids") generated in vitro from one or more pluripotent stem cells, methods for producing said cellular aggregates, and cells derived, obtained, or obtainable from said cellular aggregates.

[0029] Certain aspects of the cell aggregates (cell aggregates) of the present disclosure include being three-dimensional cell aggregates generated in vitro from pluripotent stem cells comprised of mesodermal cells (including primitive streak and presomitic mesodermal cells), wherein said cell aggregates have polarity along their anterior-posterior and apical-basolateral axes or can acquire, induce or obtain polarity along their anterior-posterior and apical-basolateral axes, and said cell aggregates can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions. The anterior-posterior axis may also be referred to as the rostral-caudal axis.

[0030] Certain aspects of the cell aggregates (cell aggregates) of the present disclosure include being three-dimensional cell aggregates generated in vitro from pluripotent stem cell-derived mesodermal cells (including primitive streak and presomitic mesodermal cells), wherein said cell aggregates have polarity along their anterior-posterior and apical-basolateral axes or can acquire, induce or obtain polarity along their anterior-posterior and apical-basolateral axes, and said cell aggregates can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions. The anterior-posterior axis may also be referred to as the rostral-caudal axis.

[0031] In one other aspect, the cell aggregates of the present disclosure include polarized three-dimensional cell aggregates generated in vitro from pluripotent stem cells comprised of mesodermal cells, wherein said cell aggregates have polarity along their anterior-posterior and apical-basolateral axes or are capable of acquiring, inducing or acquiring polarity along their anterior-posterior and apical-basolateral axes, and said cell aggregates are capable of forming one or more somites or somite-like structures under somitogenic culture conditions.

[0032] In other embodiments, the cell aggregates of the present disclosure include polarized three-dimensional cell aggregates generated in vitro from pluripotent stem cell-derived mesodermal cells and progenitor cells, wherein said cell aggregates have polarity along their anterior-posterior and apical-basolateral axes or are capable of acquiring, inducing or acquiring polarity along their anterior-posterior and apical-basolateral axes, and said cell aggregates are capable of forming one or more somites or somite-like structures under somitogenic culture conditions.

[0033] In other embodiments, the cell aggregates of the present disclosure are three-dimensional cell aggregates made in vitro from pluripotent stem cells consisting of mesodermal cells, wherein said cell aggregates have polarity along their anterior-posterior and apical-basolateral axes or can acquire, induce, or obtain polarity along their anterior-posterior and apical-basolateral axes, and the percentage of mesodermal cells in said cell aggregates 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 cell number. The percentage is preferably at least 50%, and more preferably at least 90%.

[0034] In the present disclosure, the anterior-posterior axis may be defined by an anterior (cephalic) region and a posterior (caudal) region, with anterior region cells having, for example, higher or lower expression of one or more markers compared to posterior region cells.

[0035] In the present disclosure, the anterior region cells may have lower expression of one or more markers compared to the posterior region cells.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 or more types thereof can be used as markers.

[0036] In the present disclosure, the anterior region cells may have higher or lower expression of one or more markers compared to the posterior region cells. 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.

[0037] In the present disclosure, the posterior (tail) region may comprise tailbud (TB)-like cells.

[0038] In the present disclosure, the apical-basolateral axis can be defined by an apical region and a basolateral region, where the apical region of a cell has higher or lower expression of one or more markers selected from the group consisting of aPKC, CDH2, Ezrin, and ZO1, compared to the basolateral region of a cell.

[0039] In the present disclosure, the apical region of a cell may have a lower expression of one or more markers compared to the basolateral region of a cell, where the one or more markers are selected from the group consisting of, for example, fibronectin, collagen, and laminin, one or more types of which may be used as markers.

[0040] In the present disclosure, the apical region of a cell may have higher expression of one or more markers compared to the basolateral region of a cell. In this case, the one or more markers are selected from the group consisting of, for example, CDH2, aPKC, Ezrin, ZO1 and F-actin, preferably CDH2 and / or aPKC. One or more types thereof may be used as markers.

[0041] In the present disclosure, for example, the percentage of mesodermal cells in the cell aggregates 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 cell number, preferably at least 50%, more preferably at least 90%.

[0042] The mesodermal cells express one or more markers (mesodermal cell markers) selected from the group consisting of, for example, BRA, MIXL1, NODAL, WNT3a, WNT5a, WNT5b, DLL1, CYP26A1, TBX6, HES7, MSGN1, RIPPLY1, RIPPLY2, MESP1, MESP2, MEOX1, TCF15, TBX18, UNCX, ALDH1A2, RDH10 and FLK1 / KDR. One or more types thereof can be used as markers.

[0043] The mesodermal cells express one or more markers (mesodermal cell markers) selected from the group consisting of, for example, BRA, SOX2, MIXL1, NODAL, WNT3a, WNT5a, WNT5b, DLL1, CYP26A1, TBX6, HES7, MSGN1, RIPPLY1, RIPPLY2, MESP1, MESP2, MEOX1, TCF15, TBX18, UNCX, ALDH1A2, RDH10 and FLK1 / KDR. One or more types thereof can be used as markers.

[0044] The cell aggregates of the present disclosure can reconstitute various aspects of somitogenesis and axial development, including, for example, axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock, under somitogenic culture conditions, where the somites and somite-like structures may be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, TBX18, UNCX, ALDH1A2, and RDH10.

[0045] The cell aggregates of the present disclosure can reconstitute various aspects of somitogenesis and axial development, including, for example, axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions, where the somites and somite-like structures can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, PAX3, TBX18, UNCX, ALDH1A2, and RDH10.

[0046] The cell aggregates of the present disclosure can form somites or somite-like structures under, for example, somitogenic culture conditions, which 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.

[0047] The cell aggregates of the present disclosure can form somites or somite-like structures under, for example, somitogenic culture conditions, which can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, PAX3, TBX18, UNCX, ALDH1A2, and RDH10.

[0048] Said somitogenic culture conditions are, for example, the presence of a gel and / or matrix and retinoic acid, a retinoic acid precursor or derivative thereof and / or a retinoic acid receptor (RAR) agonist.

[0049] The somitogenic culture conditions are, for example, the presence of a gel and / or matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative thereof, and / or a retinoic acid receptor (RAR) agonist.

[0050] The RAR agonist includes, for example, vitamin A, retinol, retinal, 9-cis retinoic acid, all-trans 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.

[0051] The retinoids include, for example, vitamin A, retinol, retinal, 9-cis retinoic acid, 13-cis retinoic acid, all-trans 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, and preferably include retinal or retinol.

[0052] The cell aggregates may, for example, be embedded in or disposed within a gel or matrix.

[0053] The matrix includes, for example, an extracellular matrix. The matrix includes, for example, collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. The matrix can be used at various concentrations (for example, 1%, or 5%, or 10% or 20%) per volume of the embedding media used. The concentration is preferably 5%, more preferably 10%. One or more types thereof can be used as the extracellular matrix.

[0054] The gel includes, for example, hydrogel. The gel includes, for example, basement membrane matrix. The basement membrane matrix includes, for example, one or more of the following groups: laminin, collagen, fibronectin, gelatin, vitronectin, heparan sulfate proteoglycan, and / or entactin. One or more types thereof can be used as the basement membrane matrix. The gel includes, for example, acrylamide gel, arginine gel, agarose gel, and / or polyethylene glycol hydrogel, which have various biomechanical properties.

[0055] In the present disclosure, the somites or somite-like structures can include anterior (rostral) and / or posterior (caudal) regions in the anterior-posterior axis, with anterior cells having, for example, higher or lower expression of one or more markers compared to posterior cells.

[0056] Anterior (head) cells may have higher expression of one or more markers compared to the posterior cells. In this case, the one or more markers are selected from the group consisting of, for example, TBX18 and ALDH1A2, preferably TBX18. One or more types thereof may be used as markers.

[0057] The anterior cells may have a lower expression of one or more markers compared to the posterior cells. In this case, the one or more markers are selected from the group consisting of, for example, UNCX and LNFG, preferably UNCX. One or more types thereof may be used as markers.

[0058] The cell aggregates of the present disclosure may include anterior lateral / anterior mesoderm (aPSM). aPSM refers to the posterior (caudal) region of the axioloid located between the PSM and the SM. The aPSM may be defined by expression of one or more markers selected from the group of markers consisting of MESP2, RIPPLY2, RIPPLY1 and PCDH8, preferably MESP2.

[0059] In the present disclosure, the somites are 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 formation of the somites is preferably in a cycle of 3.5 to 6.6 hours, and more preferably in a cycle of 4 to 6 hours.

[0060] In the present disclosure, the length of the somite in the anterior-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, and more preferably 80 to 120 μm.

[0061] The cell aggregates may comprise pluripotent stem cells.

[0062] In the present disclosure, the pluripotent stem cells express one or more markers (pluripotent stem cell markers) selected from the group consisting of, for example, 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 or more types thereof (preferably OCT4, more preferably NANOG) can be used as a marker.

[0063] In the present disclosure, the pluripotent stem cells express one or more markers (pluripotent stem cell markers) 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 or more types thereof (preferably OCT4, more preferably NANOG) can be used as a marker.

[0064] The percentage of pluripotent stem cells in the cell aggregates 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 cell number. The percentage is preferably 1%, more preferably less than 1%.

[0065] The percentage of pluripotent stem cells defined by the expression of NANOG in the cell aggregates 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 cell number. The percentage is preferably 1%, more preferably less than 1%.

[0066] The cell aggregates may, for example, be substantially free of endodermal and / or ectodermal cells.

[0067] The percentage of endoderm cells in the cell aggregates 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 cell number, preferably 1%, more preferably less than 1%.

[0068] The endoderm cells express one or more markers (endodermal cell markers) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1, preferably, for example, GATA6 and / or SHH. One or more types thereof (preferably, FOXA2, more preferably, GATA6) can be used as the marker.

[0069] The percentage of ectodermal cells in the cell aggregates 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 cell number. The percentage is preferably 1%, and more preferably less than 1%.

[0070] The ectodermal cells express one or more markers (ectodermal cell markers) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1, preferably, for example, OTX2. One or more types thereof (preferably, SOX1, more preferably, OTX2) can be used as a marker.

[0071] In the cell aggregates, the expression of segmental clock genes can be subject to genetic oscillation, which means that, for example, the expression level of a target gene oscillates periodically in space and time. The genetic oscillation is also referred to as, for example, gene expression oscillation.

[0072] The segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3, and HES7, and preferably HES7. One or more types thereof (preferably LFNG, more preferably HES7) can be used as the segmentation clock gene.

[0073] The cycle of the gene oscillation is, for example, a 3 to 7 hour cycle, preferably a 3.5 to 6.6 hour cycle, or more preferably a 4 to 6 hour cycle.

[0074] The pluripotent stem cells used for induction of axioloids are, for example, human pluripotent stem cells or non-human animal pluripotent stem cells. Examples of non-human animals include amniotes, such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, apes, dolphins, elephants, sea lions, snakes, geckos, chickens, etc. The pluripotent stem cells are, for example, embryonic stem cells or induced pluripotent stem cells.

[0075] The pluripotent stem cells used for induction of axioloids are, for example, human pluripotent stem cells or non-human animal pluripotent stem cells. Examples of non-human animals include amniotes, such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, apes, dolphins, whales, armadillos, tenrecs, elephants, sea lions, snakes, geckos, chickens, etc. Non-human animal pluripotent stem cells also include monotremes, such as platypuses or echidnas, and marsupials, such as opossums, kangaroos, wombats, etc. Pluripotent stem cells are, for example, embryonic stem cells or artificially modified pluripotent stem cells.

[0076] The pluripotent stem cell may be, for example, a pluripotent stem cell whose gene or genome is modified. Examples of the modification include the introduction of mutations into coding or non-coding genes or genome regions, the repair of coding or non-coding (regulatory) genes or genome mutations, the introduction of foreign genes or non-coding (regulatory) genome regions, and the knockout of genes or genome regions. The modification of genes or genomes may be carried out using, for example, genome editing techniques such as ZFN, TALEN, CRISPR / Cas system; recombinant gene technology; and the like.

[0077] The pluripotent stem cell may be, for example, a pluripotent stem cell whose gene or genome sequence has been modified. Examples of the modification include the introduction of mutations into coding or non-coding genes or genome regions, the repair of coding or non-coding (regulatory) genes or genome mutations, the introduction of foreign genes or non-coding (regulatory) genome regions, and the knockout of genes or genome regions. The modification of genes or genome sequences may be carried out using, for example, genome editing techniques such as ZFN, TALEN, CRISPR / Cas system; recombinant gene technology; and the like.

[0078] In the present disclosure, the cell aggregates may comprise, 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 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 cell aggregates is, for example, 100 to 100,000 cells, 200 to 100,000 cells, 300 to 100,000 cells, 400 to 500,000 cells, 600 to 100,000 cells, 700 to 100,000 cells, 800 to 100,000 cells, 900 to 100,000 cells, 1000 to 90,000 cells, 1500 to 80,000 cells, 2000 to 70,000 cells, 2500 to 60,000 cells, 5000 to 50,000 cells, 10,000 to 50,000 cells, 15,000 to 50,000 cells, 20,000 to 50,000 cells, 30,000 to 50,000 cells, or 40,000 to 50,000 cells. The cell aggregate preferably contains at least 50 cells, and more preferably 100 to 1000 cells.

[0079] In the present disclosure, the cell aggregate has a length of, for example, 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 cell 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 cell aggregate is preferably 0.05 to 10 mm, and more preferably at least 1 mm.

[0080] In a further aspect, the method of the present disclosure is a method of producing a three-dimensional cell aggregate generated in vitro from pluripotent stem cells, comprising the following steps: (a) culturing the pluripotent stem cells to induce a three-dimensional cell aggregate comprising mesodermal cells; and (b) culturing the cell aggregate comprising mesodermal cells to induce a three-dimensional cell aggregate, wherein the three-dimensional cell aggregate is a cell aggregate of the present disclosure.

[0081] In a further aspect, the method of the present disclosure is a method for producing a three-dimensional cell aggregate generated in vitro from pluripotent stem cells, comprising the following steps: (a) culturing pluripotent stem cells in two-dimensional or three-dimensional culture to induce three-dimensional cell aggregates comprising mesodermal cells; and (b) culturing the cell aggregates comprising mesodermal cells to induce three-dimensional cell aggregates, wherein the three-dimensional cell aggregates are cell aggregates ("axioloids") according to the present disclosure.

[0082] In the methods of the present disclosure, "cultivation" may be performed, for example, using a medium supplemented with factors as desired. In the methods of the present disclosure, the medium may be replaced during the culture period.

[0083] The medium may be prepared using a medium used for culturing animal cells as the basal medium. Examples of basal media include IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 medium, RPMI 1640, Fisher-Scher medium, Neurobasal Medium (manufactured by Thermo Fisher Scientific), 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), StemPRO (registered trademark) hESC SFM (manufactured by Life Technologies), E8 (manufactured by Life Technologies), Essential 6 (manufactured by Thermo Fisher Scientific), and the like. Scientific) and mixed media thereof. The medium may be supplemented with serum or may be serum-free. The medium may contain serum substitutes such as albumin, transferrin, knockout serum substitute (KSR) (serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. Furthermore, the medium may contain additives such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. When the growth culture is performed using the construct, the medium is preferably a stem cell culture medium supplemented with NEAA, glutamic acid, and antibiotics.

[0084] The culture conditions for the culture may be, for example, common conditions for cell culture. As a specific example, the culture temperature is, for example, 25°C to 40°C, preferably 30°C to 40°C, and more preferably about 37°C. The carbon dioxide concentration during the culture is, for example, 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is, for example, performed in a humid environment.

[0085] The culture conditions implemented during the culture may be, for example, common conditions for cell culture. As a specific example, the culture temperature is, for example, 25°C to 40°C, preferably 30°C to 40°C, and more preferably 37°C. The carbon dioxide concentration during the culture is 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is, for example, performed in a humid environment.

[0086] The method of the present disclosure may, for example, include the following steps: (a1) culturing the pluripotent stem cells in a medium comprising a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., basic FGF (bFGF), to initiate commitment to primitive streak and mesoderm fates and / or induce mesoderm cells (preferably in two-dimensional culture); (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium comprising a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., bFGF, and a TGFβ inhibitor, e.g., SB-431542, and a ROCK inhibitor, to induce three-dimensional cell aggregates comprising said mesodermal cells; and, optionally, (b2) culturing said three-dimensional cell aggregates comprising mesodermal cells in a medium comprising retinoic acid, a retinoic acid precursor or derivative thereof and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or matrix, and inducing morphogenesis and / or self-organization of the three-dimensional cell aggregates, and / or the three-dimensional cell aggregates.

[0087] The method of the present disclosure may, for example, include the following steps: (a1) culturing the pluripotent stem cells in a medium containing a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., bFGF, to initiate commitment to primitive streak and mesoderm fates and / or induce mesoderm cells (preferably in two-dimensional culture (flat culture)); (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium comprising a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., bFGF, and a TGFβ inhibitor, e.g., SB-431542, and a ROCK inhibitor, to induce three-dimensional cell aggregates comprising said mesodermal cells; (b1) culturing the three-dimensional cell aggregates comprising said mesodermal cells in a medium free of a WNT agonist, e.g., a GSK3β inhibitor, an FGF agonist, e.g., bFGF, a TGFβ inhibitor, e.g., SB-431542, and a ROCK inhibitor; and, optionally, (b2) culturing said three-dimensional cell aggregates comprising mesodermal cells (primitive streak and lateral / anterior mesoderm) in a medium comprising retinoic acid, a retinoic acid precursor or derivative thereof and / or a retinoic acid receptor (RAR) in the presence of a gel or matrix, and inducing morphogenesis and / or self-organization of the three-dimensional cell aggregates.

[0088] The method of the present disclosure may, for example, include the following steps: (a1) culturing the pluripotent stem cells in a medium containing a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., bFGF, to initiate commitment to primitive streak and mesoderm fates and / or induce mesoderm cells (preferably in two-dimensional culture (flat culture)); (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium comprising a WNT agonist, e.g., a GSK3β inhibitor, and an FGF agonist, e.g., bFGF, and a TGFβ inhibitor, e.g., SB-431542, and a ROCK inhibitor, to induce three-dimensional cell aggregates comprising said mesodermal cells; (b1) culturing the three-dimensional cell aggregates comprising said mesodermal cells in a medium free of a WNT agonist, e.g., a GSK3β inhibitor, an FGF agonist, e.g., bFGF, a TGFβ inhibitor, e.g., SB-431542, and a ROCK inhibitor; and, optionally, (b2) culturing said three-dimensional cell aggregates comprising mesodermal cells (primitive streak and lateral / anterior mesoderm) in a medium comprising a retinoid, retinoic acid, a retinoic acid precursor or derivative thereof and / or a retinoic acid receptor (RAR) in the presence of a gel or matrix, and inducing morphogenesis and / or self-organization of the three-dimensional cell aggregates.

[0089] The disclosed methods include, for example, culturing three-dimensional cell aggregates comprising said mesodermal cells embedded in or disposed inside a gel or matrix in a medium comprising retinoic acid, a retinoic acid precursor or derivative thereof, and / or a retinoic acid receptor (RAR) agonist to induce three-dimensional cell aggregates.

[0090] The disclosed method includes, for example, culturing three-dimensional cell aggregates comprising said mesodermal cells embedded in or disposed inside a gel or matrix in a medium comprising a retinoid, retinoic acid, a retinoic acid precursor or derivative thereof, and / or a retinoic acid receptor (RAR) agonist to induce the three-dimensional cell aggregates.

[0091] In steps (a1), (a2) and / or (b1), the FGF is, for example, bFGF (FGF2).

[0092] The concentration of bFGF in the medium is, for example, 1 to 1000 ng / ml, or preferably 10 to 1000 ng / ml.

[0093] In steps (a1), (a2) and / or (b1), the GSK3β inhibitor may be, for example, a substance that inhibits the kinase activity of GSK3β protein (e.g., the ability to phosphorylate β-catenin), particularly, an indirubin derivative such as BIO (GSK3β inhibitor IX: 6-bromoindirubin 3′-oxime) and 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) and the like; a phenyl α bromomethyl ketone compound such as SK-3β inhibitor VII (4-dibromoacetophenone) and the like; L803-mts (GSK-3β peptide inhibitor; The GSK3β inhibitor may be a cell membrane permeable phosphorylated peptide such as Myr-N-GKEAPPAPPQSpP-NH2; CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile); a nucleic acid molecule suppressing the expression of GSK3β protein (such as siRNA, shRNA, antisense, etc.). The GSK3β inhibitor is preferably CHIR99021 because of its high selectivity for GSK3β. GSK3β inhibitors are commercially available, for example, from Calbiochem, Biomol, etc. In the present disclosure, WNT agonists (including recombinant WNT proteins such as WNT3a) and other classical and non-classical WNT agonists can be used instead of GSK3β inhibitors.

[0094] The concentration of the GSK3β inhibitor in the culture medium is, for example, 1 nmol / l (hereinafter sometimes referred to as "M") to 1000 μmol / l, preferably 10 nmol / l to 100 μmol / l, or more preferably 100 nmol / l to 100 μmol / l.

[0095] In step (a2) and / or (b1), the TGFβ inhibitor is a substance that inhibits SMAD-mediated signal transduction caused by TGFβ binding to a receptor. Examples of TGFβ inhibitors include substances that inhibit the binding of TGFβ to the ALK family, which is a TGFβ receptor, and substances that inhibit the phosphorylation of SMAD by the ALK family. Specific examples of TGFβ inhibitors include Lefty-1 (NCBI accession numbers: NM-010094 (mouse), NM-020997 (human), SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide), SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB505124 (2-(5-benzo[1,3-dioxol-5-yl]-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), NPC30345, SD2080, SD2089, SD2086, SD2085, SD2084, SD2086, SD2089, SD2088, SD2089 ... (from Scios), LY2109761, LY364947, LY580276 (from Lilly Research Laboratories) and A-83-01 (WO2009 / 146408), with SB431542 being preferred.

[0096] 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.

[0097] In step (a2) and / or (b1), the ROCK inhibitor is a substance that can suppress the function of Rho-kinase (ROCK). Examples of ROCK inhibitors 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 expression inhibitors of nucleic acid molecules of ROCK protein (siRNA, shRNA, antisense, etc.), with Y-27632 being preferred.

[0098] 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, preferably 1 nmol / l to 40 μmol / l, and more preferably 1 nmol / l to 15 μmol / l.

[0099] In step (b2), the RAR agonist is, for example, vitamin A, retinol, retinal, 9-cis Retinoic acid, all-trans 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.

[0100] In step (b2), the retinoid may, for example, be vitamin A, retinol, retinal, 9-cis retinoic acid, 13-cis retinoic acid, all-trans 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, and preferably, retinal or retinol.

[0101] The concentration of retinoic acid, a retinoic acid precursor or derivative thereof, and / or a retinoic acid receptor (RAR) agonist 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.

[0102] The concentration of the retinoid, retinoic acid, retinoic acid precursor or derivative thereof, and / or retinoic acid receptor (RAR) agonist 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.

[0103] 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.

[0104] In step (b2), for example, the matrix includes an extracellular matrix.

[0105] The matrix may, for example, include an extracellular matrix. The extracellular matrix may, for example, include collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. One or more types thereof may be used as the extracellular matrix.

[0106] In step (b2), 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 sulfate proteoglycan, and / or entactin. One or more types thereof 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 preferably includes an agarose gel, more preferably a polyethylene glycol hydrogel.

[0107] In step (b2), the concentration of the gel and / or matrix in the medium is, for example, a concentration that allows three-dimensional culture, and can be appropriately set based on the type of gel and matrix. As a specific example, the concentration of the gel and / or 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).

[0108] In step (b2), the concentration of the gel and / or matrix in the medium is, for example, a concentration that allows three-dimensional culture, and can be appropriately adjusted based on the type of gel and matrix. As a specific example, the concentration of the gel and / or 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).

[0109] In the methods of the present disclosure, for example, the percentage of mesodermal cells in a cell aggregate comprising mesodermal cells 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 cell number, preferably at least 50%, more preferably at least 90%.

[0110] The mesodermal cells express one or more markers (mesodermal cell markers) selected from the group consisting of, for example, TBXT, SOX2, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2. One or more types thereof can be used as a marker.

[0111] In the methods of the present disclosure, for example, the cell aggregates comprising mesodermal cells are substantially free of endodermal and / or ectodermal cells.

[0112] The percentage of endoderm cells in the cell aggregate containing mesoderm cells 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 percentage is preferably 10% or less, and more preferably 1% or less.

[0113] The endoderm cells express one or more markers (endodermal cell markers) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1, preferably, for example, GATA6 and / or SHH. One or more types thereof can be used as the marker.

[0114] The proportion of ectodermal cells in the cell aggregate containing mesodermal cells 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, and more preferably 1% or less.

[0115] The ectodermal cells express one or more markers (ectodermal cell markers) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2 and BARX1, preferably, for example, OTX2. One or more types thereof can be used as markers.

[0116] The ectodermal cells express one or more markers (ectodermal cell markers) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1, preferably OTX2, for example. One or more types thereof can be used as markers.

[0117] The number of days for culture in 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 of days is preferably 0.1 to 10 days, and more preferably 0.5 to 2 days.

[0118] The number of days for culture in step (a1) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days, or more preferably 0.5 to 2 days.

[0119] The number of days for culture in step (a2) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days, or more preferably 0.5 to 2 days.

[0120] The number of days for culture in 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 of days is preferably 0.1 to 10 days, and more preferably 2 to 8 days.

[0121] The number of days for culture in step (b1) is, for example, 0.1 to 7 days, preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.

[0122] The number of days for culture in step (b2) is, for example, 0.1 to 7 days, preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.

[0123] In step (a1), for example, the pluripotent stem cells are one or more suitable dissociated pluripotent stem cell(s) or one or more suitable cell(s) suspensions comprising any pluripotent stem cell(s).

[0124] In step (a1), the pluripotent stem cells are, for example, human pluripotent stem cells or non-human animal pluripotent stem cells. Examples of non-human animals include amniotes, such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, apes, dolphins, elephants, sea lions, snakes, geckos, chickens, etc. The pluripotent stem cells are, for example, embryonic stem cells or induced pluripotent stem cells.

[0125] In step (a1), the pluripotent stem cells are, for example, human pluripotent stem cells or non-human animal pluripotent stem cells. Examples of non-human animals include amniotes, such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, apes, dolphins, whales, armadillos, tenrecs, elephants, sea lions, snakes, geckos, chickens, etc. Non-human animal pluripotent stem cells also include monotremes, such as platypuses or echidnas, and marsupials, such as opossums, kangaroos, wombats, etc. The pluripotent stem cells are, for example, embryonic stem cells or artificially modified pluripotent stem cells.

[0126] The cell aggregate containing the mesodermal cells 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 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 cell aggregate preferably includes at least 50 cells, more preferably at least 100 to 1000 cells.

[0127] The cell aggregate containing the mesodermal cells is, for example, composed of 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 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 cell aggregate preferably contains at least 50 cells, more preferably at least 100 to 1000 cells.

[0128] The cell aggregates comprising the mesodermal cells have a length of, for example, 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 cell aggregates preferably have a length of at least 0.05 mm, more preferably at least 0.5 mm.

[0129] In other aspects, the present disclosure provides cells obtained from the three-dimensional cell aggregates of the present disclosure.

[0130] In still another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, comprising the steps of culturing the three-dimensional cell aggregate of the present disclosure and inducing a progenitor cell or a differentiated cell selected from the group consisting of (a) to (f) below: (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) endothelial or hematopoietic cells or their precursor cells.

[0131] In yet another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, comprising the steps of culturing the three-dimensional cell aggregate of the present disclosure and inducing a progenitor cell or a differentiated cell selected from the group consisting of: (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) an endotome or endothelial or hematopoietic cell or a precursor cell thereof; (g) adipocytes or their precursor cells, including white, beige, and brown cells; (h) dermal cells or their precursor cells; and (i) Neural tube cells or their precursor cells.

[0132] The method for inducing the differentiated cells (a) to (f) and their precursor cells can be carried out in the same manner as the method for inducing each differentiated cell and its precursor cell from a mesodermal cell, for example.

[0133] The method for inducing the differentiated cells (a) to (i) and their precursor cells can be carried out in the same manner as the method for inducing each differentiated cell and its precursor cell from a mesodermal cell, for example.

[0134] The manufacturing method of the present disclosure may, for example, include a step of inducing a three-dimensional cell aggregate from pluripotent stem cells before induction. In this case, the induction of the three-dimensional cell aggregate may be performed, for example, by the manufacturing method of the three-dimensional cell aggregate of the present disclosure.

[0135] The manufacturing method of the present disclosure may, for example, include a step of forming a three-dimensional cell aggregate from pluripotent stem cells prior to induction. In this case, the formation of the three-dimensional cell aggregate may be performed, for example, by the manufacturing method of the three-dimensional cell aggregate of the present disclosure.

[0136] In other aspects, the present disclosure provides a method for evaluating a test substance comprising culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the three-dimensional cellular aggregate through and / or after said culturing, wherein said three-dimensional cellular aggregate is a cellular aggregate of the present disclosure.

[0137] The type of test substance is not particularly limited, and includes, for example, proteins, antibodies, peptides, nucleic acid molecules, sugar chains, lipids, organic low molecular weight compounds, inorganic low molecular weight compounds, bacterial release substances, fermentation products, cell extracts, vacuolar culture supernatants, plant extracts, and animal tissue extracts. One or more of these types (preferably, organic low molecular weight compounds, more preferably, proteins) can be used as the test substance.

[0138] In the evaluation, a test substance that alters the polarity of cell aggregates, the morphology of cell aggregates and / or the size of cell aggregates is selected as a candidate substance that, for example, modifies, promotes or inhibits the polarity of cell aggregates, the morphology of cell aggregates and / or the size of cell aggregates.

[0139] The culture is, for example, culture under segmental culture conditions. The segmental culture conditions may be, for example, as described above. The culture conditions may be, for example, common conditions for cell culture.

[0140] During evaluation, for example, test substances that alter the somitogenesis of said cell aggregates are selected as candidate substances that modify, promote, or inhibit the somitogenesis process of the cell aggregates.

[0141] The culture is a culture for inducing a precursor cell or a differentiated cell selected from the group consisting of the following (a) to (f): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) endothelial or hematopoietic cells or their precursor cells.

[0142] The culture is a culture for inducing a precursor cell or a differentiated cell selected from the group consisting of the following (a) to (i): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) an endotome or endothelial or hematopoietic cell or a precursor cell thereof; (g) adipocytes or their precursor cells, including white, beige, and brown cells; (h) dermal cells or their precursor cells; and (i) Neural tube cells or their precursor cells.

[0143] During the evaluation, for example, a test substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a)-(f) is selected as a candidate substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a)-(f).

[0144] During the evaluation, for example, a test substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a) to (i).

[0145] In the above-mentioned evaluation method, the evaluation is performed using a control that does not contain the test substance as a reference.

[0146] The evaluation method uses a control that does not contain the test substance during the evaluation as a reference.

[0147] The culture is the production of a three-dimensional cell aggregate from pluripotent stem cells, and the production of the three-dimensional cell aggregate is carried out by the method for producing a three-dimensional cell aggregate of the present disclosure.

[0148] The culturing refers to the production of three-dimensional cell aggregates from pluripotent stem cells, and the production of the three-dimensional cell aggregates is carried out by the method for producing three-dimensional cell aggregates disclosed herein.

[0149] In other aspects, the present disclosure provides a method for evaluating gene function or genome function, comprising the steps of: preparing pluripotent stem cells having a modified test gene or test genome; generating three-dimensional cell aggregates from said pluripotent stem cells; and evaluating said three-dimensional cell aggregates through and / or after culture, wherein the generation of the three-dimensional cell aggregates is performed by a method for producing a three-dimensional cell aggregate of the present disclosure.

[0150] In other aspects, the present disclosure provides a method for evaluating gene function or genomic sequence function, comprising the steps of: preparing pluripotent stem cells having a modified test gene or test genomic sequence; generating three-dimensional cell aggregates from said pluripotent stem cells; and evaluating said three-dimensional cell aggregates through and / or after culture, wherein the generation of the three-dimensional cell aggregates is performed by a method for producing a three-dimensional cell aggregate of the present disclosure.

[0151] During evaluation, test genes or test genomes (coding and non-coding genomic regions) that alter the polarity of the cellular aggregates, the morphology of the cellular aggregates and / or the size of the cellular aggregates are evaluated as candidate genes or genomes that, for example, modify, promote or inhibit the polarity of the cellular aggregates, the morphology of the cellular aggregates and / or the size of the cellular aggregates.

[0152] During evaluation, test genes or test genomic sequences (coding and non-coding genomic regions) that alter the polarity of the cellular aggregates, the morphology of the cellular aggregates and / or the size of the cellular aggregates are evaluated, for example, as candidate genes or candidate genomic sequences that modify, promote or inhibit the polarity of the cellular aggregates, the morphology of the cellular aggregates and / or the size of the cellular aggregates.

[0153] The disclosed functional evaluation method includes, for example, culturing in the presence of three-dimensional cell aggregates under somitogenic culture conditions, wherein during evaluation, test genes or test genomes (coding and non-coding genomic regions) that alter somitogenesis, including axial elongation, segmentation, epithelialization, or oscillation of the segmentation clock, of the cell aggregates are evaluated as candidate genes or genomes that modify, promote, or suppress somitogenesis, including axial elongation, segmentation, epithelialization, or oscillation of the segmentation clock, of the cell aggregates.

[0154] The disclosed functional evaluation method includes, for example, culturing in the presence of three-dimensional cell aggregates under somitogenic culture conditions, wherein during evaluation, test genes or test genomic sequences (coding and non-coding genomic regions) that alter somitogenesis, including axial elongation, segmentation, epithelialization, or oscillation of the segmentation clock, of the cell aggregates are evaluated as candidate genes or candidate genomic sequences that modify, promote, or suppress somitogenesis, including axial elongation, segmentation, epithelialization, or oscillation of the segmentation clock, of the cell aggregates.

[0155] In the present disclosure, the gene can be any gene, examples of which include genes associated with human diseases (e.g., HES7). The test gene can be a gene mutation associated with a human disease (e.g., HES7 R25W and spondylocostal dysostosis).

[0156] 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.

[0157] 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.

[0158] The following preparation methods and examples are provided for the purpose of illustrating the present invention in more detail, but the scope of the present invention is not limited thereto.

[0159] (Example) The present disclosure will be specifically described below with reference to examples. However, it should be noted that the present disclosure is in no way limited to the embodiments described in the following examples.

[0160] summary The process of vertebrate somitogenesis has been well studied in model organisms but remains poorly understood in humans due to ethical and technical constraints. Despite recent advances in pluripotent stem cell (PSC)-based approaches,1-5 However, a system that robustly recapitulates human somitogenesis both spatially and temporally remains to be found. Here, a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis is introduced, termed the axioloid, which not only accurately captures the oscillatory dynamics of the segmentation clock but also captures the morphological and molecular features of segment formation and sequential somitogenesis in vitro. The axioloid displays proper anterior-posterior patterning of the forming segments and a robust anterior-posterior FGF / WNT signaling gradient and retinoic acid (RA) signaling components. An unexpected critical role of RA signaling in stabilizing segment formation is identified, and distinct but synergistic effects of RA and extracellular matrix (ECM) on somite formation and epithelialization are shown. Importantly, comparative analysis shows striking similarities of the axioloid to the human embryo, further validated by the presence of the HOX code in the axioloid. Finally, the utility of the axioloid system was demonstrated to study the pathogenesis of human congenital spinal disorders using patient-like iPSCs harboring mutations in HES7 and MESP2, which revealed disease-associated phenotypes including loss of epithelial somitogenesis and abnormalities in rostral patterning. These results suggest that the axioloid is a promising novel platform for studying axis development and disease in humans.

[0161] introduction Previously, we were able to reconstitute the human segmentation clock in vitro. 1,4,6 However, these systems lacked the ability to form proper axial segmental organization, a central feature of any vertebrate, limiting their utility in understanding how higher-order tissue organization and more advanced stages of human embryonic development occur. The in vitro addition of extracellular matrix (ECM) molecules promoted the formation of higher-order tissue structures in organoids, and mouse pluripotent stem cell-derived gastrocnemiocytes (GSCs) were able to form pluripotent stem cell–derived gastrocnemiocytes (GSCs) in vitro. 2 and trunk-like structures 3It has been shown that the signaling pathway helps the formation of the axial nucleus, and we attempted to establish a single germ layer, mesoderm-based 3D model of human axial development using human iPS cells (induced pluripotent stem cells) and ECM (extracellular matrix). iPS cells were stepwise exposed to signals promoting primitive streak (PS) and presomitic mesoderm (PSM) fates (Fig. 1a and Experimental Procedure). Mesoderm aggregates, which naturally break bilateral symmetry and elongate (Fig. 1b, Fig. 6a, b), were then embedded in 10% Matrigel (MG), an ECM-rich culture supplement, and the emergence of spatiotemporally coordinated segments was observed along the anterior-posterior axis of these growing, normally curved structures. This structure was named axioloid (Fig. 1b, Fig. 6a-e). Axioloids were derived from two different human iPS cell lines to ensure reproducibility and were assessed for morphological, molecular and functional properties.

[0162] Results and Discussion Axioloids exhibit morphological and molecular characteristics of vertebrate embryo axes and tails To determine the similarity of axioloids to vertebrate embryonic axes and tails, their morphological and molecular characteristics were first evaluated. In axioloids, segments appeared at intervals of approximately 4–5 h (Fig. 1c, d; Fig. 6f, g; Supplementary Video 1), and exposure to MG significantly increased their elongation by contraction elongation, with axioloids reaching a total length of approximately 1000–1400 μm at 120 h in culture (Fig. 1e; Fig. 6h). These polar axis structures could be further subdivided into a posterior terminal region expressing TBXT (also known as Brachury) and an anterior somitic mesoderm (SM) region positive for MEOX1 (Fig. 1f, g; Fig. 6i–k). In the SM region of each axioloid, segments formed every 80–140 μm (Fig. 6l). The tail tip of the axioloid was strongly positive for TBXT, and its expression was reduced along the axis of the adjacent PSM. This is similar to expression in the tail bud (TB) and PSM of developing amniote embryos. 7Interestingly, in the absence of MG, polar patterns of TBXT and MEOX1 expression were observed in axioloids even after 72, 96, and 120 h of culture, suggesting that MG is not required for the initial establishment of polar expression patterns (Fig. 6m-q). Although early somitogenesis was regular, fully epithelialized somite-like structures with clear apical-basal polarity and a central somite cavity, as seen in developing embryos, were rarely observed in axioloids (Fig. 7a, b). Segments formed by exposure to MG in MEOX1+ SM regions of human axioloids were characterized by the apical accumulation of actin, indicative of the initiation of the epithelialization process (Fig. 1f), but the epithelialization process within each segment was incomplete and largely disorganized (Fig. 7a, b).

[0163] Further evaluation of gene and protein expression patterns in axioloids embedded in MG revealed striking similarities to anatomically and functionally localized molecular signatures described for the developing tail of vertebrate embryos. The posterior-most TB region, positive for TBXT and SOX2, was clearly distinct (Fig. 1h, Fig. 7c) and adjacent to the presomitic mesoderm (PSM) region, clearly distinguished by the region-specific expression of TBX6, HES7, and MSGN1 (Fig. 1i-k, Fig. 7d-h). Further comparison of axioloids stained for TBXT, SOX2, and TBX6 revealed the presence of TBXT+ and SOX2+ double positive cells in the TB, which could be distinguished from TBX6+ but SOX2-negative PSM cells (Fig. 1h, i, Fig. 7c, d). Furthermore, between 96 and 120 h of axioloid culture, the TBX6+ PSMs in axioloids decreased in size, whereas the SOX2+ population in TBs increased in size, indicating that this may be related to the slowing of axis elongation and segment formation in axioloids after 120 h of culture (Fig. 1h, i; Fig. 7c, d).

[0164] Besides the TB and PSM, we could also distinguish a narrow RIPPLY2 marking the anterior PSM (aPSM) region (Fig. 1j, k; Fig. 7e-g), which further differentiated the MEOX1+ and TCF15-expressing somitic mesoderm (SM) region rostrally, and then the rostral-most MEOX1+ region of presomitic mesoderm (Fig. 1f, j; Fig. 7e, f). LFNG (lunatic fringe), a master regulator of Notch signaling, was also expressed in the PSM, aPSM, and SM regions of human axillary lamina, with a striped expression pattern in the SM similar to that reported in vertebrate embryos. 8-10 In the emergent segment of the SM aspect of the axillary lamina, the anterior part of the aPSM expresses MESP2, while the UNCX 11 and TBX18 12 We found striped expression of the anterior-posterior polarity genes UNCX and TBX18, indicating that proper anterior-posterior axis identity is established within the forming axioloid segments (Fig. 1l,m; Fig. 7i-l). Interestingly, UNCX and TBX18 were expressed in the SM region of the axioloid even in the absence of MG, but in a disorganized manner and without a clear head-to-tail pattern (Fig. 7m-p). This indicates that the axioloid shares morphological and molecular characteristics of the vertebrate embryo axis and tail, although it lacks a notochord and neural tube.

[0165] Axioloids recapitulate the traveling wave-like oscillatory activity of the segmentation clock. A universal and central feature of somitogenesis is the oscillatory activity of the segmentation clock, a molecular oscillator and gene regulatory network centered on Notch signaling that is activated throughout the growing tail of vertebrate embryos and is thought to control the pace and size of somites that form. 13-15 To determine the dynamics of oscillatory activity within the human axioloid, a reporter system previously used to characterize the human segmentation clock in vitro was used. 1 Well-studied segmentation clock genes 1,4,16,17Reproducible oscillatory activity of HES7, with a periodicity of approximately 4-5 h, was clearly observed in the axilloid, regardless of the presence of MG (Fig. 1n, o). Furthermore, HES7 showed robust traveling wave-like expression, with a periodicity of 4-5 h, in the anterior direction along the PSM from the TB and PSM regions at the posterior end (Fig. 1p, q, Fig. 7q-s, Supplementary Video 2). 1,4,6 In contrast, we observed that distinct segment formation in human axioloid occurs in a remarkable synchronization with the segmentation clock. In the anterior PSM region of the axioloid, segments form every 4–5 h, and this region overlaps and coincides with the wavefront of HES7 oscillatory activity (Figure 7q-s; Supplementary Video 2). This data indicates that in human axioloid, both processes, segmentation of the segmentation clock and traveling wave-like oscillatory activity, are coupled and occur in a spatiotemporally coordinated manner; such a close association has only been reported in the embryonic tail of a vertebrate model organism. 18,19 .

[0166] Single-cell RNA-seq analysis of human axioloids To further understand the functional properties of the model system and prospectively characterize the dynamic changes in cellular composition and molecular complexity of the human axilloid, we performed time-resolved single-cell RNA-seq (scRNA-seq) analysis of this system at different stages (48 h, 72 h, 96 h, 120 h) and conditions (+ / -MG). Our analysis revealed the presence of multiple dynamically changing cell populations that may correspond to distinct mesodermal cell populations that emerge during vertebrate embryonic axis and tail development, including tail bud (TB), presomitic mesoderm (PSM), anterior presomitic mesoderm (aPSM), somitic mesoderm (SM), hemangioblast / endothelial-like (EC-like) cells, etc. (Fig. 2a, b). These cell populations could be further classified into hypothetical subpopulations based on cell cycle status or emergence time, or maturation state during in vitro culture (Fig. 8a, b). RNA kinetics analysis revealed a major differentiation pathway in which early TB and early PSM cells predominated after 48 h in culture, giving rise to the SM population that constituted the majority of cells after 96 and 120 h in culture (Fig. 2b-d). Interestingly, the presence or absence of MG did not significantly affect the differentiation pathway or distribution of pre-existing or newly arising cell populations within the axioloid (Fig. 2d, Fig. 8c). Many genes previously reported to be differentially expressed in anatomical compartments and cell populations that constitute the mesoderm aspect of the embryonic tail were identified and shown to match specific expression profiles in human axioloid (Fig. 2e, f). SM cells could be classified into six distinct clusters based on their expression profiles, some of which showed specific high-level expression of various ribosomal proteins, including RPL38, and mutation of RPL38 in mice has been shown to cause homeotic changes in the axial skeleton. 20(Fig. 2f, Fig. 8d). We observed that some genes highly expressed in early TB, such as TBXT, CYP26A1, WNT3a, and FGF8, were down-regulated in late cultures, whereas SOX2 was strongly expressed in late TB. This suggests that there may be a change in the number and function of TBXT and SOX2 double-positive neuromesodermal progenitor cells (NMPs) present in human axioloid TBs, and is consistent with our previous observation of decreased TBXT staining in SOX2+ TBs of 120-h human axioloids (Fig. 2f, Fig. 8e, f, Fig. 1h, i, Fig. 7c, d). RNA velocity and pseudotime analysis of scRNAseq data from sequential sectioning of MG-embedded human axioloids in 96-h cultures revealed the presence of a major differentiation pathway originating from the TB and leading to SM cells via the PSM and aPSM, consistent with the posterior-to-anterior spatial distribution of these cell populations within human axioloids ( Figure 2 g-i ).

[0167] Axioloids establish appropriate FGF / WNT gradients and express retinoic acid signaling components The vertebrate embryonic tail is characterized by opposing gradients of FGF / WNT and retinoic acid (RA), which are thought to be involved in establishing a "wave front" during somitogenesis. 21-23 We next investigated whether a similar gradient exists within the human axioloid. Pseudotemporal analysis of scRNA-seq data, which was in good agreement with the anterior-posterior organization and spatial distribution of major cell populations seen in the axioloid, was used to predict the expression patterns of multiple FGF-, WNT-, and RA-signaling-related transcripts within the axioloid (Fig. 9a-c). Two genes reported to be associated with wavefronts during somitogenesis 24-26To validate the predicted spatial expression patterns of FGF8 and WNT3a, HCR-based in situ hybridization was used, revealing a clear posterior-to-anterior gradient in their expression in human axioloid. Both genes were most strongly expressed in the posterior TB and PSM regions, with expression gradually decreasing throughout the PSM toward the aPSM region where MESP2 expression was found (Fig. 3a, b; Fig. 9d-f). The expression of RA signaling-related molecules in the axioloid was then examined, focusing on ALDH1A2, an enzyme involved in the synthesis of RA from retinal (RAL), and CYP26A1, an RA metabolizing enzyme. High levels of localized expression of CYP26A1 were detected in the TB, while high expression of ALDH1A2 was observed in the SM region of human axioloid, with expression highest anterior to the aPSM-specific expression of RIPPLY2, which gradually decreased along the posterior-to-anterior axis (Fig. 3c, d; Fig. 9g-i). Furthermore, ALDH1A2 was also expressed in the SM region of human axioloid, which is consistent with that reported in mice. 27 , formed a stripe-like expression pattern, with higher levels of expression in the rostral half of the forming segment (Fig. 3c, d; Fig. 9g–i).

[0168] Matrigel only promotes segmentations in vitro, but does not stabilize them Our data showed that although MG promotes axis elongation and initial segment formation, it is likely insufficient to maintain or stabilize these segments. To better understand the role of MG in human axioloid elongation and segmentation, we compared scRNA-seq data of axioloids cultured with and without MG. Analysis revealed that after 96 h of axioloid culture, hemangioblasts and endothelial cell-like (EC-like) cell populations emerged in a MG-dependent manner (Fig. 3e, Fig. 10a-d). Matrigel 3 Stem cell-like structures generated from mouse pluripotent stem cells in the presence of also reported to contain similar hemangioblast- or endothelial-like populations, suggesting a role for evolutionarily conserved ECM components in the generation of vascular progenitor cells known to emerge during somitogenesis. 28 .

[0169] We next performed a more detailed analysis of the scRNA-seq data (Fig. 3f, g), focusing on differentially expressed genes (DEGs) in PSM and SM cells. Gene enrichment analysis confirmed increased expression of genes related to epithelial-mesenchymal transition (EMT) in MG-exposed axioloid SM cells, whereas decreased expression of another set of genes related to EMT in MG-exposed PSM cells (Fig. 3f, g; Fig. 10e, f; Supplementary Table 2). MG-exposed SM cells showed increased expression of DKK1, SFPR1, FST, and ID2, negative regulators of the WNT and TGFβ signaling pathways, respectively (Fig. 3g, h; Fig. 10e). These genes have been reported to be involved in somitic mesoderm development and are known to be expressed in migratory mesenchymal cells. 29-31 Basement membrane-associated ECM components, including FN1, FN2, and VIM1, were upregulated in SM cells of axioloids cultured in the presence of MG (Fig. 3g, h). Exposure to MG also led to changes in gene expression in the TB, although these changes were smaller than those in the PSM and SM of human axioloids (Fig. 10f, g). These results, in particular, showed an increase in MG-associated EMT indicators in SM cells, and an increase in TCF15 32 or PAX3 33 The reduction of epithelialization-related genes such as MG1, MG2, and MG3 suggests that the effect of MG on human axioloids is not simple, but that it may promote or hinder molecular processes related to segmentation and epithelialization. This is in good agreement with the observation that exposure to MG promotes axial elongation and early distinct somitogenesis, but subsequently fails to stabilize the somites and is insufficient to establish well-organized and fully epithelialized somites in the axioloids.

[0170] RA signaling is required for somite stability and epithelialization We next explored factors that may stabilize the somite segmentation process and promote epithelialization. Although molecules related to RA synthesis, such as ALDH1A2 and RDH10, were specifically expressed in this system, the precursors retinol (ROL) and retinal (RAL) were not present in the culture conditions. The inability of axioloids to generate RA de novo in this way raises the question of the function of RA signaling in axioloids. To elucidate this question, we added either directly RA or its precursors, ROL or RAL, to the in vitro system between 72 and 120 h of MG embedding. Remarkably, we observed that the presence of RA molecules dramatically improved the stability and epithelialization of the forming segments within MG-embedded axioloids after 96 and 120 h (Figure 4a, b; Figure 11a-c; Supplementary Video 3). MG+RA axioloids sequentially formed fully epithelialized somites with properly formed apical-basal polarity and central somite lumen, which could not be achieved by MG or RA alone (Figure 4c; Figure 11d, e). Despite a clear effect on epithelialization, which could not be achieved by RA signaling alone (Fig. 11f), the characteristics of these axioloids, i.e. overall morphology, total length, periodicity of segment formation after 48 h of implantation or number of segments formed, were not altered by the addition of RA to the system (Fig. 11g-n). When we assessed the impact of RA signaling on key protein and gene expression patterns within axioloids, including TBXT and MEOX1, or UNCX and TBX18, these molecular signatures were largely unchanged or even improved in RA- and RAL-treated axioloids, indicating a clear craniocaudal identity of the forming epithelial somites (Fig. 4d, e, Fig. 12a-p, Supplementary Video 4). In MG+RA axioloids, we observed similar changes in size and expression of TBXT+ SOX2+ TBs as in MG-only axioloids (Fig. 12q, see also Fig. 1h, Fig. 7c). Furthermore, we found that simultaneous inhibition of RA signaling by the pan-RAR inverse agonist BMS493, while allowing RAL-mediated RA synthesis in axioloids, strongly inhibited somitogenesis and epithelialization, reversing the RA effect ( Figure 12r-u ; Supplementary Video 5 ).

[0171] To further explore the functional regulation of RA in axioloids and changes in somitogenesis, axioloid samples supplemented with RA and RAL at 96 and 120 h were analyzed by scRNA-seq analysis. A clear separation of SM and TB clusters based on the presence or absence of RA and RAL was observed, but the overall identity of the cell clusters was stable and still consistent with each other (Fig. 4f, g; Fig. S13a). Comparing the different conditions, many of the DEGs observed in axioloids upon addition of MG alone, including the upregulation of WNT (SFRP1, DKK1) or TGFβ (FST, ID2) signaling and the downregulation of epithelialization-related transcription factors TFC15 and PAX3, were attenuated by the addition of RA or RAL (Fig. S13b-f). This suggests the intriguing possibility that the effects of MG are balanced by the presence of RA activity in the system, suggesting that both MG-mediated initiation of axis elongation and somitogenesis, and RA-mediated stabilization and epithelialization of the forming somites, are required for proper establishment and progression of somitogenesis in human axioloids. Furthermore, many of the known and novel targets of RA signaling in the context of human segmentation and somitogenesis were identified, including multiple RA targets in TB and SM (Fig. S13b, c). Retinoid / RA signaling homeostasis-related genes, such as RBP1, DHRS3, CYP26A1, or CRABP2, were also upregulated by RA in both TB and SM, suggesting the existence of a negative feedback loop controlling RA production and utilization in this system (Fig. S13e, g). Several transcription factors, including TCF15, MEOX1, PAX3, PBX1, ZIC3, NR2F1, and MEIS2, which are expressed in somites and known to be important for axis development in model organisms, were also upregulated in MG-embedded human axioloids exposed to RAL / RA (Fig. 13e, h).

[0172] It has been reported that RA deficiency in embryos generally leads to reduced somites and asymmetric somitogenesis rather than a strong epithelialization-related phenotype. 21,27,34, the finding that RA plays a key role in somitogenesis and epithelialization is surprising. Moreover, for symmetry and bilateral symmetry in this system, a single axis was typically observed in axioloids treated with MG and RA, where epithelial somites sequentially formed with a single central somite cavity. Interestingly, axioloids frequently exhibited a superficial sulcus or midline-like structure starting from the PSM and running through most of the forming segment (Fig. 14a, b), but usually remained superficial and did not separate the segment into two completely separated somites (Fig. 14a, b, Supplementary Video 5a). However, axioloids sometimes contained fully segmented true somites, each with a central somite cavity in the middle, sometimes as an isolated pair of fully segmented somites or rarely as a succession of two or more somite pairs along the AP axis of the axioloid (Fig. 14c-e, Supplementary Video 5b). These bipartites usually exhibited normal protein and gene expression patterns, including proper anterior-posterior polarity (Fig. 14f-k).

[0173] Human axioloid mimicking properties of human embryos Somitogenesis is a hallmark of vertebrate embryos, and the number of somites allows us to roughly assess the age of the embryo. Carnegie stage (CS) 10 human embryos are characterized by 4–12 pairs of somites, suggesting that the 96- and 120-h-old axioloids are at least at comparable stages. Comparing the dimensions of axioloid somites with those of CS10 and 11 embryos, we find that they are similar in shape and size (Figure 15a-d). Furthermore, in both axioloid and human embryos, we found that the earlier formed (older) somites close to the anterior anastomosis are larger, whereas the newly formed somites in the caudal region are smaller in volume. Notably, in human axioloids, the posterior somites are noticeably reduced in size (Figure 15a, b; Supplementary Video 6). Thus, the high degree of morphological similarity observed between in vitro-derived axioloids and in vivo human embryos strikingly suggests that the mesoderm alone, in the absence of other germ layers, is able to robustly self-organize and give rise to the appropriately sized somites that constitute the body axis of the early embryo.

[0174] To further compare the morphogenetic and molecular features of axioloids with those of real human embryos, we utilized the recently uploaded scRNA-seq datasets of human embryos CS12 to CS16. 35 For comparison, we reanalyzed the data from CS12 embryos and performed a fusion analysis based on mutual nearest neighbors (MNN). 36 We performed a multi-step analysis (Fig. S15e-m) and found that the majority of cells present in the human axioloid closely matched those identified in CS12 human embryos (Fig. S4h-k, S15h-m). As expected, endodermal, neuroectodermal, and other unrelated mesodermal cell populations identified in human embryos (e.g., lateral plate mesoderm (LPM), blood) did not match the human axioloid (Fig. S4i, k). On the other hand, the axioloid showed strong overlap with paraaxial mesodermal and mesodermal cell populations associated with axis development, including populations labeled as somitic and head mesoderm, found in CS12 human embryos (Fig. S4i, k, S15h-m).

[0175] We also found that a portion of the hemangioblast / EC-like cells derived from the axilloid coincided with cells marked as endothelial cells in the human CS12 dataset (Fig. 4i, k), while TB cells in the axilloid overlapped with cells marked as neural progenitors (Fig. 4h-k, Fig. S15e, f, h, i). The latter population coincided with cells derived mainly from the lower half of the embryo, and may therefore be similar to the SOX2 / TBXT double-positive neuromesodermal progenitors (NMPs) present in the tail of human embryos (Fig. 4j).

[0176] Presence of a HOX code in human axioloids. Having established a robust in vitro system to model axis development using both MG and RA, the next step was to ascertain whether there is a HOX coding in human axioloids, i.e., spatiotemporal regulated expression of HOX genes. Spatial transcriptomics based on scRNA-seq data and HybISS. 38Combining these data sets, we recapitulated the spatial expression of key TB, PSM, and SM markers (Fig. 4l,m) and assessed the expression of the four HOX clusters and associated HOX genes in human axioloid. The pseudotemporal analysis-based predictions of the HOX code in axioloid corresponded well with the actual spatial distribution of HOX gene expression along the anterior-posterior axis of human axioloid (Fig. 4n, Fig. 16a-c). A caudal degeneration of HOXC genes was observed, especially in the HOXC cluster, consistent with the results obtained in mouse embryos (Fig. 4n-p). Combining these data sets with CUT & Tag experiments to assess the promoting and inhibitory epigenetic landscape of axioloid, we found a clear association between the switching of HOX gene expression in axioloid and active and repressive chromatin markings within each of the four HOX clusters, supporting the concept of a HOX code in human axioloid, similar to previous findings in human and mouse gastroids (Fig. 4n-p, Fig. 16d-i).

[0177] Regulation of signaling pathways in human axioloids Based on the morphogenetic and molecular similarities between axioloids and actual human embryos, we investigated whether our latest model system (MG+RAL) could be repeatedly used to investigate the role of signaling pathways in the human somitogenesis process. Using HCR-based in situ hybridization, we observed that axioloids cultured in the presence of MG+RAL exhibited clear expression gradients of FGF8 and WNT3a in their TB and PSM regions, similar to axioloids cultured in the presence of Matrigel alone (Fig. S17a-d). RA signaling-related genes ALDH1A2 and CYP26A1 were also specifically expressed in the SM and TB regions, respectively (Fig. S17e-h). To visualize and validate the spatial expression profiles of these and additional FGF / WNT and RA signaling pathway members predicted to be present in axioloids from pseudotime analysis of scRNAseq data, we applied HybISS-based spatial transcriptomics (Fig. S17i-l). The results showed that FGF3, FGF4, FGF17, WNT5a and WNT5b showed progressive expression in the axioloid, similar to the expression of FGF8 and WNT3a, whereas RDH10 was expressed similarly to ALDH1A2 in the SM region of MG+RAL-treated axioloid, consistent with previous reports in vertebrate embryos (Figure 17i-l).

[0178] Since RA signaling has a strong effect on somitogenesis and epithelialization, we next investigated whether it also affects the oscillation and traveling-wave-like activity of the segmentation clock within the axioloid. We found that RA, RAL, and ROL had little effect on oscillatory activity, including periodicity, of the segmentation clock gene HES7, with or without MG, and traveling-wave-like expression was strongly present in all treated axioloids (Fig. 18a-j, Supplementary Video 7a). Inhibition of RA signaling by BMS493 also did not affect the oscillatory activity of the segmentation clock gene HES7 (Fig. 18k-o, Supplementary Video 7b). Despite the apparent deleterious effect of BMS493 on epithelialization and somitogenesis, the overall length of BMS493-treated axioloids was stable (Fig. 18p-r). These findings suggest that the segmentation clock and axis elongation can be uncoupled from RA-dependent epithelial somite formation.

[0179] The next step was to modulate the FGF, WNT and Notch signaling pathways in human axioloids via small molecules. The observed changes in the segmentation clock were consistent with previously reported in vitro 1,4,40 or in vivo 41 As expected, Notch inhibition by DAPT quickly attenuated and abolished oscillatory activity of the axioloid, whereas FGF and especially WNT inhibition had less profound effects on the segmentation clock (Fig. 18s). Morphology of treated axioloids was affected, albeit to a lesser extent than RA inhibition, resulting in a reduction in somite number after 120 h in all three cases (Fig. 18p, t, u). Surprisingly, FGF inhibition had the most dramatic effect on the overall length of the axioloid after 120 h (Fig. 18p, u; Supplementary Video 8), consistent with the results observed in classical embryo models. 42,43 These results suggest that FGF signaling is involved in axioloid axial elongation, similar to that in the control of axioloid axial elongation.

[0180] Modeling human spinal disease using axioloids Finally, we investigated whether axioloids could be used to model genetically related disorders of the human spine. 44 or MESP2 45Focusing on this, we generated axioloids using patient iPSC lines that introduced loss-of-function mutations in the coding regions of genes known to be associated with spinal segmentation abnormalities (SDVs) and assessed their morphological, molecular and functional characteristics. Two different HES7 knockout iPSC lines were used and showed similar phenotypes: despite obvious axial elongation, they showed a marked loss of segmentation and epithelial somitogenesis (Fig. 5a, b; Fig. 19a). Despite the apparent lack of segments and somites, polarity protein expression of TBXT and MEOX1 remained nearly normal (Fig. 5c; Fig. 19b; Supplementary Video 9). In HES7 KO axioloids, we further observed a clear loss of anterior-posterior axial patterning, indicated by the disappearance of the stripe-like expression pattern of UNCX and TBX18 (Fig. 5d; Fig. 19c). The oscillatory activity of the segmentation clock in these patient axioloids with deleted HES7 was then assessed and a clear loss of HES7 oscillation was observed. This is consistent with previous model systems 1 This is similar to the oscillatory phenotype observed in HES7 KO PSM cells in ( Figure 5 e-g , Figure 19 d , Supplementary Video 10 ).

[0181] A similar phenotype was observed in axioloids derived from patient-derived iPSC lines containing a point mutation (rs113994160: c.73C>T) in HES7, a pathogenic missense mutation R25W in the helix-loop-helix domain of HES7 that has been reported to cause spinal segmental malformation (SDV)44 (Fig. 5h-n, Fig. 19e-h, Supplementary Videos S11 and 12). These HES7 R25W The axial elongation of axioloids appeared to be slightly increased compared to iPSC-derived axioloids from healthy donors (Fig. 5h, i; Fig. S19e). R25W HES7 from MT1 and MT2 R25WAxioloids showed a clear loss of anterior-posterior axis patterning (Fig. 5k, Fig. 19g), and normal expression of TB and SM markers TBXT and MEOX1 combined with loss of HES7 oscillations (Fig. 5j, Fig. 19f, Supplementary Video 11), which is in line with the findings in HES7 KO axioloids and previously reported HES7 oscillations. R25W iPSC-derived PSM cells 1 This was consistent with the oscillatory phenotype of (Figures 5l-n, Figure 19h, Supplementary Video 12).

[0182] Second, pathogenic mutations have been reported in patients with SDV. 45 The impact of loss of aPSM, a transcription factor associated with MESP2, was evaluated. Patient-like axioloids were derived from MESP2 knockout iPSC lines (MESP2 KO1 and MESP2 KO2) and morphological, molecular and functional characteristics were again assessed. MESP2 KO axioloids elongated normally but lacked segments or epithelial somites (Fig. 5o, p; Fig. 19i). In contrast to the almost normal expression pattern of TBXT and MEOX1, a severely impaired anterior-posterior axis patterning was observed in MESP2 KO axioloids (Fig. 5q, Fig. 19j; Supplementary Video 13), accompanied by a lack of striped expression of UNCX and low levels of expression of TBX18 (Fig. 5r, Fig. 19k). This in vitro "human phenotype" is strikingly similar to that reported in MESP2 KO mice. 46,47 , showing that axioloids can reconstitute complex genetic phenotypes in vitro. In contrast to HES7, deficiency of MESP2 did not lead to a loss of HES7 oscillatory activity in axioloids (Figure 5s-u, Figure 19l, Supplementary Video 14). This data indicates that axioloids will not only provide valuable insights into normal human axis development, but also contribute to our understanding of the pathophysiology of diseases of the human spine.

[0183] In summary, a pluripotent stem cell-derived 3D mesoderm model of human somitogenesis has been established and characterized in detail, which allows reconstituting various aspects of human somitogenesis and axis development in vitro. The axioloid recapitulates complex developmental processes including axis elongation, segmentation, and epithelialization up to oscillation of the segmentation clock, and shares molecular and morphometric features with the tail and axis of the developing human embryo. A bottom-up approach reveals that a primitive, proximal axial mesoderm has a remarkable self-organizing capacity that allows it to give rise to the basic somitic structures of the human embryo, even in the absence of other germ layers. We also uncover a critical role for RA signaling in the morphogenetic processes accompanying somitogenesis and somitogenesis within the axioloid, suggesting that RA supplementation, especially in combination with ECM components, may also improve the morphogenetic properties of other in vitro model systems of human and non-human early embryonic development.

[0184] Bottom-up experimental approaches demonstrate that complex developmental phenomena such as somitogenesis can be decomposed and analyzed into distinct “components” of developmental principles that are usually intricately coupled and cannot be easily separated in vivo. The axioloid, a self-organizing in vitro model of human axis development, now allows such components to be individually assessed and manipulated at the molecular, cellular, and morphogenetic levels. Further iterations of this model system will likely incorporate as yet “undiscovered” anatomical structures, such as the notochord and mesodermal tube, which will allow for the assessment of subsequent stages of somite development and differentiation, including somitic differentiation, such as the partitioning of somites into sclerotomes, dermomyotomes, and other functional derivatives. As a surrogate model of the human embryonic tail, the axioloid can recapitulate key features of human somitogenesis, making it an exciting new platform for studying axis development and disease in the human context.

[0185] method Human iPS cell culture In this study, we used two human pluripotent stem cell (hiPSC) lines derived from healthy donors, 409B248 and 201B749, the latter primarily in the form of a HES7 reporter line as previously described. 1 Disease modeling used patient iPS cells carrying disease mutations in HES7 and MESP2 generated by CRISPR-Cas9-based gene editing. 1 Human iPS cells were maintained on dishes coated with iMatrix-511 silk (Nippi) in StemFit AK02N (Reprocell) medium supplemented with 50 U penicillin and 50 mg / ml streptomycin (Gibco). StemFit AK02N (Reprocell) medium consists of three components, A, B, and C, which were mixed and used for standard hiPS cell maintenance culture in a humidified incubator at 37°C and 5% CO2. The iPS cell lines used were regularly tested and reported to be negative for mycoplasma contamination.

[0186] Induction of human axioloid Human iPS cells were cultured on iMatrix511 silk (Nippi)-coated dishes in 6-well plates at 1.3 × 10 4 The cells were seeded at a density of 1000 cells / well and used at 60% confluence. All subsequent induction steps were performed using AK02N (Reprocell) medium without component C (AK02N-C). First, hiPS cells were cultured in bFGF (20 ng ml -1 The cells were pulsed for 24 h with a combination of CHIR99021 (5 μM) and the WNT agonist CHIR99021 (5 μM). The concentration of CHIR99021 may need to be adjusted depending on the iPS cell line used, but is typically in the range of 3–5 μM. 24 h after the first pulse, the cells were dissociated with Accutase (Thermo Fisher Scientific) and resuspended in CHIR99021 (5 μM), basic FGF (20 ng ml -1), TGFβ inhibitor SB431542 (10 μM), and ROCK inhibitor Y27632 (10 μM) were added to each well of AK02N-C-based aggregation medium, and 500 cells / well were cultured for 24 h in 96-well U-bottom low-attachment plates (Sumilon). 24 h after aggregation, 150 μl of AK02N-C medium was added to each well, and 24 h later, the medium was replaced again with 150 μl of fresh AK02N-C medium. 48 h after aggregation, axolotls were transferred one by one to BSA-treated 96-well flat-bottom low-attachment plates (Watson), embedded in 80 μl of AK02N-C medium containing 10% growth factor-reduced Matrigel (Corning), and further cultured for 24–48 h at 37 °C and 5% CO2. Depending on the experimental setup, retinoic acid signaling molecules, i.e. retinoic acid (RA) (100 nM), retinol (ROL) (10 μM), or all-trans retinal (RAL) (1 μM), were added to the MG-containing medium throughout the embedding stage. Small molecule inhibitors that inhibit FGF (PD173074 (250 nM)), WNT (XAV939 (2 μM)), RA (BMS493 (2.5 μM)), and Notch (DAPT (25 μM)) signaling were also added during the embedding stage to assess their effects on axioloids. For details of the recombinant human proteins, small molecule agonists, and inhibitors used, see Supplementary Table 1.

[0187] Human Embryo Data Digital data of human embryos were obtained from the MRC / Wellcome Trust funded Human Developmental Biology Resource (HDBR, www.hdbr.org) with appropriate maternal written informed consent and approval from the Newcastle and North Tyneside NHS Joint Ethics Committee (REC reference numbers 18 / NE / 0290 and 08 / H0906 / 21+5). HDBR is regulated by the UK Human Tissue Authority (license# 12534) and operates in accordance with the relevant HTA Code of Practice. Embryos were classified as Carnegie stage (CS) 10 (n=1) or CS11 (n=4) based on features observable externally in unfixed specimens (https: / / hdbratlas.org / staging-criteria / carnegie-staging.html). One CS11 embryo (N662) was imaged using optical projection tomography (OPT) 50 The remaining CS10 (13446) and CS11 (CS11-1021, 1177, 1053) embryos were sectioned at 4 μm thickness (20 μm intervals) for CS10 and 7 μm thickness for CS11 (56 μm intervals for 1021 and 1053, 35 μm intervals for 1177), stained with hematoxylin and eosin, and imaged. Data on the embryos used in this paper have been previously published. 51,52 , and / or publicly available on the HDBR atlas website ( https: / / hdbratlas.org / ). Image series of CS11 embryos acquired by OPT were 3D reconstructed, and the area of ​​several visible, identifiable somites was measured by ImageJ software using scale images of stained sections.

[0188] Immunohistochemistry (IHC) Human axioloid specimens were washed twice with 0.1% BSA (Nacalai) in PBS (Takara), fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, and then washed twice again with 0.1% BSA in PBS. Samples were permeabilized with 0.2% Triton X-100 (Nacalai) in PBS for 15 min at room temperature and blocked with 5% BSA in PBS for 1 h at room temperature. Axioloid specimens were stained overnight (12–16 h) at 4°C with primary antibodies diluted in 0.5% BSA in PBST (1% Tween-20 (Nacalai) in PBS). The primary antibodies used in this study were: goat anti-TBXT (TBXT) (1:500, R&D Systems), mouse anti-fibronectin (1:10, DSHB), mouse anti-laminin (γ1) (1:10, DSHB), rabbit anti-MEOX1 (1:500, ATLAS), mouse anti-N-cadherin (1:200, BD Biosciences), mouse anti-PKCζ (1:100, Santa Cruz), rabbit anti-SOX2 (1:400, Cell Signaling), and goat anti-TBX6 (1:500, R&D Systems). Samples were then washed twice with PBST and stained with secondary antibodies and Alexa Fluor 647-conjugated phalloidin (Invitrogen) diluted in PBST with 0.5% BSA for 3 h at room temperature, washed twice with PBST, and counterstained with DAPI for 5 min at room temperature. Secondary antibodies used in this study (all diluted 1:500) were Alexa Fluor 405 donkey anti-goat (Abcam), Alexa Fluor 405 donkey anti-mouse (Abcam), Alexa Fluor 488 donkey anti-goat (Invitrogen), Alexa Fluor 488 donkey anti-mouse (Invitrogen), and Alexa Fluor 555 donkey anti-rabbit (Invitrogen). Stained axioloid specimens were cleared overnight at 4°C in Scale S4 clearing solution before imaging. 53Images were taken using a LSM980 (Carl Zeiss) and Ti2 (Nikon) equipped with a Dragonfly (and / or) or Nikon A1R MP (Multiphoton+N-STORM) fluorescence microscope. For details of primary and secondary antibodies used, see Supplementary Table 2.

[0189] Whole mount in situ hybridization (HCR) All probes, HCR amplifiers, and buffers (hybridization, wash, and amplification buffers) were purchased from Molecular Instruments, and whole-volume fixed in situ hybridization chain reactions (HCRs) were performed as previously described. 54Briefly, human axioloids were collected in microtubes coated with 1% BSA in PBS, washed once with 1% BSA in PBS, and then fixed with 4% PFA in PBS for 1 h at room temperature. They were then washed three times with PBST (0.1% Tween-20 in PBS) for 5 min each at room temperature, post-fixed with 100% methanol, and stored at -30°C. Samples were hydrated by washing with 500 μL of graded concentrations of methanol / PBST washes (75%, 50%, and 25%) for 5 min each at room temperature. Human axioloid specimens were incubated with hybridization buffer for 5 min at room temperature and then at 37°C for 30 min. The mixture of probes (8 nM each) was incubated at 37°C for 30 min before use. The mixture of probes dissolved in hybridization buffer (final concentration of 8 nM each) was incubated at 37°C for 30 min before use. The samples were incubated with hybridization buffer containing the probe for 12–16 h at 37°C. The samples were then washed four times with probe wash buffer for 15 min each at 37°C, followed by three washes with 5xSSCT at room temperature. The samples were then pre-amplified by incubating in probe amplification buffer at room temperature for at least 30 min. Amplified hairpins were prepared by rapid cooling. h1 and h2 hairpins were each heated at 95°C for 90 s, then cooled at room temperature in the dark for 30 min. Hairpin mixtures were prepared by adding h1 and h2 to 250 μL of amplification buffer to 6 nM each. The axioloid specimens were then incubated with the amplified hairpin-containing solution in the dark at room temperature for 12–16 h. Finally, the samples were washed with 5xSSCT and PBST, followed by counterstaining with DAPI. During each wash, and after addition of the buffer, probe, and hairpin mixture to the samples, the tubes were inverted several times (5–20 times) to mix properly. HCR stained axioloids were stored at 4°C in 1% BSA in PBST for up to 2 weeks before imaging. During each wash and after addition of the buffer, probe, and hairpin mixture to the sample, the tube was inverted several times (5-20 times) to ensure proper mixing. HCR stained axioloids were stored at 4°C in 1% BSA in PBST for up to 2 weeks before imaging.The HCR probe designs and associated hairpins were: ALDH1A2 (accession NM_003888.4, hairpin 514-B5); CYP26A1 (accession NM_000783.4, hairpin 594-B4); FGF8 (accession NM_033163.5, hairpin 546- B2); HES7 (accession NM_001165967.2, hairpin 594-B4); LFNG (accession NM_001040167.2, hairpin 488-B1); MESP2 (accession NM_001039958.2, hairpin 647-B3); MSGN1 (accession NM_001105569.3, hairpin 514-B5); PCDH8 (accession NM_002590.4, hairpin 647-B3); RIPPLY2 (accession NM_001 009994.3, hairpin 647-B3); TBX6 (accession NM_004608.4, hairpin 647-B5); TBX18 (accession NM_001080508.3, hairpin 546-B2); TCF15 (accession NM_004609.3, hairpin 594-B4), UNCX (accession NM_001080461.3, hairpin 488-B1), WNT3A (accession NM_033131.4, hairpin 488-B1).

[0190] Hybridization-based in situ sequencing (HybISS) Human axioloids were washed twice with 0.1% BSA in PBS (Nacalai), fixed in 0.4% PFA for 20 min at room temperature, and then washed twice with 0.1% BSA in PBS. Axioloid specimens were then transferred to cryomolds (Sakura Finetek), embedded in cryosectioning compound (Leica), and stored at -80°C until sectioning. Tissues were cryosectioned at 8 μm thickness and collected on glass slides (MAS-01, MATSUNAMI). HybISS was used for cryosectioning as previously reported. 38with slight modifications. Briefly, slides were fixed with 3% formaldehyde for 5 min, washed twice with PBS, permeabilized with 0.1 M HCl for 5 min, and washed twice with PBS. After dehydration through 70% and 100% ethanol, a gasket (SecureSeal hybridization chamber, Grace Bio-Labs) was glued around the tissue. mRNA was reverse transcribed in situ to complementary DNA as follows: tissue was treated with random decamer (5 μM, IDT) at 65°C for 5 min and cooled on ice for 5 min. Tissue was then incubated with in situ reverse transcription mixture containing Superscript IV (20 U / μl, Thermo Fisher Scientific) and random decamer (5 μM) for 10 min at RT, followed by overnight incubation at 42°C. After reverse transcription, tissues were fixed again in 3% formaldehyde for 40 min at room temperature, followed by degrading mRNA with RNaseH (0.4 U / μl, NEB) for 30 min at 37 °C, and padlock probes (PLPs) were hybridized with the remaining cDNA (20 nM of each PLP in 20% formamide) and ligated with Tth ligase (0.5 U / μl, BLIRT) for 90 min at 45 °C. For padlock probe design, we used the Python padlock design software package (https: / / github.com / Moldia / multi_padlock_design) and selected five target sequences per gene with the following parameters: arm length, 15 (20 if no target was found), Tm, low 65, high 75, space between targets, 15. A set of padlock probes for a given gene contains each unique 20-nucleotide (nt) ID sequence and a 20-nt sequence common to all PLPs. After hybridization and ligation of the padlock probe, the cDNA was digested with exonuclease I (0.5 U / μl, Thermo Fisher Scientific) for 3 h at 37°C, followed by rolling circle amplification (RCA) overnight at 30°C using φ29 polymerase (1 U / μl, Monserate).The resulting RCA products (RCPs) were subjected to sequencing by hybridization. Each sequencing round included the processes of bridge probe hybridization (0.2 μM each) in 1X hybridization buffer (2X SSC, 20% formamide), Hoechst staining (1 μg / mL) and detection probe hybridization (0.2 μM each) in 1X hybridization buffer, imaging, and stripping with stripping solution (2X SSC, 65% formamide). The sequences of PLPs, bridge probes, and detection probes are shown in Supplementary Table 3.

[0191] Imaging and sequencing by hybridization Imaging was performed using a standard epifluorescence microscope (Nikon Ti2-E) connected to an LED light source (Lumencor SPECTRA X light engine). Images were acquired with a CMOS camera (ORCA-Flash4.0V3, Hamamatsu) equipped with a CFI Plan Apochromat Lambda 40x objective (NA 1.3, oil). Filter cubes for wavelength separation were as follows: Chroma 89402X (Hoechst, Cy5), Chroma 89403X (AlexaFluor750), Semrock GFP-A-Basic (AlexaFluor488), Semrock Cy3-4040C (Cy3), and Semrock CFP-2432C (Atto425). For multiplex analysis, each gene was assigned a unique four-digit code, with each digit corresponding to one of four fluorescent dye-conjugated detection probes (1: AlexaFluor750, 2: AlexaFluor488, 3: Cy3, 4: Cy5). All fingerprint codes were reconstructed by four rounds of hybridization and imaging in situ. Optical crowding was avoided by imaging multiple genes individually: ACTB, HOXB9, HOXA3, HOXB3, and HOXD4 for 96 h for tissues 1, 2, and 3, and ACTB, HOXB9, HOXA3, HOXB3, and HOXD4 for 120 h for tissue 4; ACTB and HOXB9 for 120 h for tissues 1 and 3. Multispectral images were acquired in multiple cycles. Each image consisted of multiple tiles (10% overlap) covering the entire tissue section, with each field of view consisting of a Z-stack in 0.8 μm increments through the entire thickness of the tissue. Tiles were stitched together and Z-sections were merged into maximum intensity projections (NIS-Elements). 8-bit TIF images of each channel from each round were exported for data analysis.

[0192] Analysis of HybISS data HybISS data analysis and subsequent quantification were performed using a home-written Python code and Fiji. To decode gene spots, first, images were roughly aligned to the first-round images using Hoechst staining. Then, images were top-hat filtered and divided into several smaller images (hereafter, these images are called tiles). For each tile in each round, a composite image of the four detection probe channels was created, aligned to the first-round composite image, and again divided into each channel. Then, each tile was stitched together to form the whole image. Gene spots were detected using a Laplacian of Gaussian filter in the first-round images, and the signal intensity of each channel of the spot was calculated in the remaining rounds. Spot intensities were normalized by dividing by the 99th percentile value in that channel. The following spots were excluded from the analysis due to poor quality: maximum intensity in the channel set is less than 0.15; maximum intensity of the channel set divided by the sum of all channels is less than 0.5. Spots that passed the quality control were assigned to genes according to their reconstructed d4-digit code. The density of gene spots along the anterior-posterior axis was quantified as follows: First, the anterior-posterior axis was manually drawn and the tissue was divided so that each segment had the same area. Then, the density of each gene spot was calculated as the number of spots / 1,000 μm 2 was calculated for each gene and for each segment, and the centroid of the segment with the highest MESP2 density was taken as the reference position. The distance between the centroids of adjacent segments was calculated, and the sum of the distance from the reference position is shown on the x-axis (where posterior to anterior is negative to positive). To create line graphs, gene densities were normalized so that the maximum density was 1. To create heat maps, gene densities plus 1 were log-transformed (base e). All Python codes used in the above analyses are available on GitHub (xxxxx) or can be requested from the authors.

[0193] Live cell imaging with Ti2 Human axioloids were cultured in 1% BSA (Nacalai)-treated 96-well flat-bottom low-attachment plates (Watson) with 80 μl per well of AK02-NC-based embedding medium (+ / - MG, + / - RA, RAL or ROL, + / - small molecule modulators of signaling pathways) in a stage-top incubator (Tokai Hit) set at 37°C and 5% CO2. Bright-field live-cell imaging was performed on an inverted Ti2 microscope system (Nikon) equipped with a PlanApo λ 10x objective, with the microscope operating in autofocus mode and focusing at 10 μm intervals over a total range of 190 μm. Images were taken every 3 min and processed in Fiji.

[0194] Bioluminescence live cell imaging Bioluminescence live-cell imaging of human axioloids derived from HES7-reporter (201B7Luc) iPS cells was performed, and signals were analyzed as previously reported. 55,56The axioloids were quantified according to the method described in the previous section. Briefly, the glass-bottom 96-well plates (Iwaki) were coated with 50 μl of 1.5% PVA (Nacalai) the day before imaging, the PVA solution was aspirated off, and the plates were dried on a clean bench at room temperature overnight. Before imaging, the axioloids were placed in the coated glass-bottom 96-well plates with 80 μl of AK02N-C medium containing 100 mM D-luciferin and 10% Matrigel. Small molecule agonists and antagonists of signaling pathways were added according to the experimental setup. Bioluminescence signals of axioloids induced from HES7 reporter cell line were recorded with an IX83 (Olympus) equipped with an iXon EMCCD camera (and / or) cultured in a stage-top incubator (Tokai Hit). Signals were acquired with 2x2 binning and 1-min exposure. A spike noise filter was applied to remove cosmic rays from the raw images, which were then smoothed with a median filter. Temporal variations in the signal baseline were corrected by background subtraction. Kymographs were generated by averaging the emission intensity values ​​along the lateral axis of the axioloid, and the resulting values ​​were aligned in time order.

[0195] Imaging of fixed samples For confocal imaging of stained axioloids, samples were placed in glass-bottom 35 mm dishes or 8-well chamber covers (Matsunami) in PBS containing BSA and imaged. Confocal microscopy was performed on an LSM980 (Carl Zeiss Microscopy), a Ti2 fluorescence microscope (Nikon) equipped with Dragonfly (and / or), or a Nikon A1R MP (multiphoton + N-STORM) fluorescence microscope. For HCR-stained samples, images were acquired as tiled z-stacks with a z-spacing of 10 μm. For immunostained samples, the central z-plane of each axioloid was observed. For 3D reconstruction of immunostained axioloids, samples were cleared in ScaleS4 clearing solution. 53The sections were cleared and mounted at 4°C for 24 h. Tiled multistack images were acquired with z-spacing of 1.4–5 μm. 3D reconstructions of the immunostained samples were generated using NIS-Elements and 3D Slicer software.

[0196] Somite volume measurement Measurements of somite volume were performed using 3D Slicer software (https: / / discourse.slicer.org / ) based on z-stack images of immunostained axioloid and CS11 human embryo data previously acquired using confocal microscopy and OPT techniques. Using the segment editor, each somite was individually highlighted based on manual selections made on multiple images of each stack, and the overall structure of each somite across each image was automatically estimated by the software using the fill between slices function. This updated selection was then used to recreate a 3D view of the highlighted structures, after manual preservation processing. The number of voxels and volume of each segment were then automatically extracted using the segment statistics function.

[0197] Measurement period (vibration) The periodicity of HES7 oscillations was quantified from time series luminescence intensity data obtained from live imaging or Kronos HT (Atto) measurements using axioloids derived from the HES7 reporter. For Kronos HT-based oscillation measurements, axioloids were transferred at 72 h (48 h after aggregation) into 24-well film-bottom plates (Eppendorf) containing 400 μl of embedding solution (+ / - MG) supplemented with D-luciferin (100 μM) (bioWORLD). Axioloid-containing culture plates were incubated at 37 °C and 5% CO2. Oscillations were measured for 48 h, with each well measured for 8 min at 10 s intervals. Intensity values ​​were processed in Matlab. Time series trends were obtained by subtracting a moving average (window size 10 h), and detrended signals were smoothed with a Savitsky-Gorhai filter (window size 3 h). The instantaneous vibration phase was calculated by applying the Hilbert transform, followed by peak detection on the cosine of the instantaneous vibration phase, after which the peak-to-peak period was quantified for each n-th vibration.

[0198] Measurement period (Ti2 bright field live imaging) Periodic cell division was quantified from brightfield live-cell imaging data acquired using an Inverted Ti2 microscope system (Nikon) and a PlanApo λ 10x objective. All images were processed in Fiji to document the time course of cell division over a 24- and 48-h period after axioloid embedding in MG.

[0199] Quantification of imaging data Quantification of length or intensity based on image data is performed using Fiji 57This was performed using a 3D model. The rostrocaudal length of the formed segments was quantified from the central plane of z-stack images of phalloidon stained axons. The long axis of the axoid was measured on brightfield images using the segmentation line tool. To quantify the fluorescence intensity of HCR or IHC staining, we first created maximum intensity z-projection images from the multichannel z-stack confocal images, then quantified the intensity of each channel using the segmentation line tool along the long axis of the axoid, with a line width corresponding to approximately 80% of the lateral length of the axoid. Intensity values ​​were further processed and plotted using custom Python code. A Savitzky-Golay filter was applied to the raw intensity values, which were then normalized. For HCR datasets containing MESP2 staining, position values ​​were normalized using the MESP2 peak before calculating the average intensity across multiple samples.

[0200] pA-Tn5 transposome preparation Recombinant protein A-linked Tn5 transposase (pA-Tn5) was synthesized by solubilizing T7 Express lysY / Iq competent E. coli (NEB) harboring the 3XFlag-pA-Tn5-Fl plasmid (Addgene, #124601, a gift from Prof. Steven Hennicho) with sonication and then lysing the resulting plasmid in a column packed with chitin slurry resin (NEB) as previously described. 58 The transposase was assembled with equimolar amounts of two oligo DNA adapters (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1) and 5'-GTCTCGT GGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2)), each of which was pre-annealed with 5'-[PHO]CTGTCTCTTATACACATCT-3' (SEQ ID NO: 3).

[0201] CUT&Tag library preparation Axioloids were pooled and dissociated into single cell suspensions, as in the case of single cell RNA-seq library preparation. The cells were then centrifuged and flash frozen in liquid nitrogen. The cells were then analyzed as previously described in the literature. 58The procedure was carried out as described with minor modifications. Briefly, 50,000 cells per experiment were first washed with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1X protease inhibitor (Roche)) and then resuspended in wash buffer again. The cell suspension was mixed with concanavalin A-coated beads (Bangs Laboratories) resuspended in binding buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). The cells were then resuspended in 100 μL of ice-cold wash buffer and supplemented with 0.05% digitonin, 0.1 mM EDTA, 0.1% BSA, and 0.5 μl of each primary antibody. The antibodies used were mouse monoclonal IgG1 antibody against H3K4me3 (MAB Laboratories, #MA304B) and mouse monoclonal IgG1 antibody against H3K27me3 (MAB Laboratories, #MA323B). After 2 hours of incubation at room temperature, the buffer containing the primary antibody was replaced with 100 μl of ice-cold washing buffer containing 0.05% digitonin, and incubated overnight at 4°C with 1 μl of the secondary antibody rabbit anti-mouse IgG antibody (Abcam, ab46540). After washing the beads twice with Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 1X protease inhibitor (Roche)), 100 μl of Dig-300 buffer containing 0.5 femtomoles of pA-Tn5 transposomes prepared above was added and incubated at room temperature for 1 h. After washing the beads twice with Dig-300 buffer, they were suspended in 50 μl of Dig-300 buffer supplemented with 10 mM MgCl2 and incubated at 37 °C for 1 h. The supernatant was then removed and the beads were resuspended in 20 μl of 10 mM TAPS buffer (pH 8.5). Heat-labile protease K (NEB) was then added and the mixture was incubated at 37 °C for 30 min, followed by incubation at 55 °C for 20 min to inactivate the protease.PCR amplification of the library was performed directly from the tube after adding the reaction mixture (KAPA BIOSYSTEMS, #KK2502) using the primers listed in Supplementary Table 7.

[0202] The amplified libraries were purified with 1.3 volumes of AMPure XP beads, and the libraries were sequenced in paired-end mode on a NovaSeq 6000 Sequencing System (Illumina) using NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina, #20028401).

[0203] CUT and Tag data processing Cutadapt (version 3.4) 59 After removing tagmentation adapter sequences with Bowtie2 (version 2.2.4) 60 Paired-end reads were aligned to the human genome reference hg38 using the option --very-sensitive -X 2000. From the mapped reads, properly paired (Samtools flag 0×2), MAPQ ≥ 20, and non-mitochondrial chromosomal reads were selected using Samtools (version 1.12) 61 PCR duplicate reads were removed with Picard (version 2.25.2) (http: / / broadinstitute.github.io / picard / ), and reads were then mapped to a blacklist (http: / / mitra.stanford.edu / kundaje / akundaje / release / blacklists / hg38-human / hg38.blacklist.bed.gz) using bedtools (version 2.30). 62 The BAM file was removed by deepTools (version 3.5) 63 The sequences were converted to bigwig format and visualized with the Integrative Genome Viewer (IGV) (version 2.12.2).

[0204] RNA library preparation for scRNA-seq analysis The plastic materials used in the dissociation process of human axioloids (e.g. round-bottom 96-well cell suspension plates, tubes, tips, cell strainers) were pre-coated with 0.1% BSA (SIGMA) in HBSS(-) (Wako). Axioloids were collected in wells of a 96-well plate, transferred to another well filled with HBSS(-) and washed three times with 0.1% BSA (SIGMA). Then, the desired stage and number of axioloids were transferred to another well containing 100 μl of enzyme P solution from the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) and incubated at 37°C for 10 min. After adding 100 μl of 0.1% BSA in HBSS(-), the axioloids were dispersed by pipetting 20 times with a P200 uncut tip. Dispersed cells were filtered through Flowmi Cell Strainers (Merck) with a pore size of 40 μm and placed into a 2 ml tube (Eppendorf) coated with BSA. Cells were washed twice with HBSS(-) 1% BSA with a centrifugation step at 400 x g for 6 min at room temperature. Cells were then resuspended in HBSS(-) 1% BSA using an uncut P200 tip coated with BSA, and the concentration of the cell suspension was measured with a hemocytometer. The cell suspension was subjected to library preparation using Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics), aiming for the recovery of 5000–10000 target cells. Library preparation was performed according to the manufacturer's instructions, 10X Genomics (CG000315 Rev B).

[0205] scRNA-seq library sequencing Libraries were sequenced in paired-end mode using the NovaSeq 6000 Sequencing System (Illumina) using NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina, #20028401) and NovaSeq 6000 S1 Reagent Kit v1.5 (100 cycles) (Illumina, #20028319) as described in the 10X Genomics (CG000315 Rev B) instructions.

[0206] scRNA-seq data processing The scRNA-seq data were first mapped to the human reference genome (hg38) and a matrix of UMI counts for each gene and each cell was generated using Cell Ranger (10X Genomics, version 6.0.1). Presumed doublet clusters were removed using Scrublet (version 0.2.3) with expected_doublet_rate set to 0.0664. The count data were then compiled using the Suerat package (version 4.0.3). 65 The raw counts were then imported into and used for downstream analysis. Only cells with nfeature > 1.500, nCount between 2,500 and 50,000, and a percentage of mitochondrial gene counts between 2% and 12% were included in further analysis. Raw counts were normalized using the log-normalization method. Cell cycle scores and cell cycle phases were then determined as previously described. 66 .

[0207] UMAP analysis, clustering, and marker identification Count matrices from different samples were first combined and simultaneously projected onto a UMAP plot to normalize for differences in total UMI counts and variations due to cell cycle phase. The SCTransform function was used with the vars.to.regress option. 67Next, principal component analysis (PCA) using the RunPCA function in the Seurat package was performed. The RunUMAP function was then performed with default parameters except dim = 1:30, at a resolution of 0.5 for Fig. 2b and 0.15 for Fig. 3k in the Seurat package. To identify marker genes for each cluster, the FindAllMarkers function in Seurat was used with the Wilcoxon rank sum test, with the min.pct and logfc.threshold parameters set to 0.15 and 0.3, respectively.

[0208] Batch correction and integrated analysis Two runs of single-cell transcriptome data after 96 h of MG-embedded axioloids were subjected to inverse principal component analysis using the Seurat package to correct for batch effects, followed by integration with a mutual nearest neighbor algorithm. 36 For this, we first performed SCTransform normalization as described above, and 3,000 integration features were selected with the SelectIntegrationFeatures function. Then, the RunPCA function was run using the selected features. Then, the integration anchor set was obtained by FindIntegrationAnchors with the normalization and reduction methods set to “SCT” and “rpca”, respectively. The k.anchor parameter was set to 5. The obtained anchor set was then used to integrate the two trials. After performing PCA using the integrated data, UMAP and clustering analysis were performed as described above with dim = 1:20 and resolution 0.25 for Fig. 2g and extended Fig. 5a. The integration of the two trial sets of MG embedded and non-embedded axioloids after 96 h was performed in a similar manner as above. UMAP and clustering were performed with resolution 0.1 and dim = 1:20 for Fig. 3e. Similarly, the 96-h and 120-h data sets for unembedded, MG-embedded, retinal and MG-embedded, and retinoic acid and MG-embedded were merged in a similar manner as above. UMAP and clustering were performed at dim = 1:30 and resolution 0.04 in extended Fig. 8a.

[0209] Differentially expressed gene analysis To call differentially expressed genes (DEGs) between different axioloid culture conditions, we first performed batch correction and cluster annotation as described above to define cell sets of the same cell type in the conditions to be compared. In each defined cell cluster, we compared gene expression levels based on normalized count data using the FindMarkers function in Seurat. For the comparison between MG-plus and MG-minus, we first called DEGs for each of the two replicates with a log2 fold change threshold of 0.25. We then listed genes that were commonly up- or down-regulated by MG in both replicates. We also calculated the expression changes in the combined two-trial dataset using the Wilcoxon rank sum test and the FindMarkers function to generate Volvox plots in Fig. 3f and extended Fig. 5g. For the effects of retinal and retinoic acid addition, the gene expression changes between the different conditions at each time point in each annotated cluster defined after the above integration were similarly calculated. To obtain DEGs with the addition of retinal, genes whose expression levels were increased or decreased at 96 and 120 hours and whose log2 fold change threshold was 0.25 were listed. The KEGG_2021_Human and MSigDB_Hallmark_2020 databases were used, and the Enrichr package (version 3.0) was used. 68 Enrichment analysis was performed using commonly up- or down-regulated genes as queries.

[0210] RNA kinetics and pseudotime analysis To perform kinetic analysis, fastq sequence data were analyzed using Kallisto (version 0.46.0). 69 and loompy (version 3.0.6) to obtain count matrices for both spliced ​​and unspliced ​​transcripts, and then filtered out cells that were not included in the UMAP and clustering analyses described above. Then, we used scVelo (version 0.2.3) and a probabilistic model70 RNA velocities were analyzed using scv.pp.filter_and_normalize. The parameters used were min_shared_counts=20 and n_top_genes=2000 for the scv.pp.filter_and_normalize function, and n_pcs=30 and n_neighbors=30 for the scv.pp.moments function. The velocities are projected onto the UMAP plot generated above. For the integrated dataset consisting of two trials of 96h_MG, pseudotime analysis was then performed based on the velocity graphs using the scv.tl.velocity_pseudotime function in the scVelo package. Cells, excluding those annotated as IM-like or EC-like cells according to the rank order of pseudotime, were ordered in the heatmap plots in Fig. 2i, Fig. 4n, Extended Fig. 4a-c, and 11a-c.

[0211] Comparison with human embryo scRNA-seq data To compare axioloids with human embryos, single-cell RNAseq data from CS12 human embryos was used 35 From this dataset, a Seurat object for the embryo was created using Seurat (version 4.0.6) and an SCTransform was performed with options vars.to.regress = c('S.Score', 'G2M.Score'), variable.features.n = 5000. UMAP analysis and clustering were performed for Fig. 4i using the RunUMAP function with option dims = 1:60 and the FindClusters function with a resolution of 0.65. Cell clusters were named essentially based on the annotated cell types by Xu et al. 35The cluster called "limb" is composed mainly of cells previously annotated as "limb", but also contains cells annotated as mesoderm cells. The same SCTransform function was used for the datasets sampled at 96 and 120 hours from MG plus axioloid and retinal additives. Cell type annotation for axioloid is from the extended Fig. 8a. The integration of axioloid and embryo data was performed similarly to the method described above, except that the number of integration features was 5000 and k.anchor was 10. UMAP analysis and clustering were also performed similarly, with dimensionality set to 40 and resolution 1.0. Calculation of Pearson's correlation coefficients for axioloid cell groups and embryos was performed based on the number of cells in each cell group assigned to the clusters defined after integration in Fig. 15k.

[0212] Display of gene expression levels Log-normalized UMI counts were used to represent the expression level of each gene in the UMAP plots in Fig. 2e and Extended Fig. 5d, the scatter plots in Extended Fig. 3f, and the violin plots in Fig. 3h and Extended Fig. 8f-h. Gene expression levels were also scaled using the Seurat ScaleData function to have a mean expression level of zero and a variance of one across cells. The scale values ​​were used in the heatmaps in Figs. 2f and i, 4n, and Extended Figs. 4a-c and 11a-c. In Extended Figs. 3d and e, 5c, 10h and i, the mean values ​​of the scaled data were calculated and plotted across cells in each cluster.

[0213] Data Acquisition All single-cell RNA-seq and CUT&Tag data used in this study have been deposited in the NCBI Gene Expression Omnibus under the accession number GSE199576. To view the data, please visit https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE199576 and enter the reviewer token ktunygmklvclpwp in the box.

[0214] Code Availability The computational codes and scripts used in this study are available on GitHub (https: / / github.com / Alev-Lab) or upon request from the authors.

[0215] Supplementary Video Video 1: Effect of Matrigel on axioloid morphology. Live cell imaging of 409B2-derived axoloids (top) and 201B7 Luc-derived axoloids (bottom) embedded in Matrigel (MG) and cultured in +MG (right) or -MG (left) after 72 and 120 hours in culture. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0216] Video 2: The effect of Matrigel embedding on the kinetics of HES7 gene expression. Live imaging of spatiotemporal morphogenetic expression of the HES7 gene from 72 to 120 hours after incubation of 201B7 Luc-derived axioloids embedded in MG. BF video (left) and HES7:luciferase signal (right). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0217] Video 3: Effects of RA signaling on axioloid growth and morphology. Live imaging of 409B2-derived axioloids (top row) and 201B7 Luc-derived axioloids (bottom row) after 72 to 120 h of culture in MG alone (left), or in MG plus retinal (RAL) (middle), or retinoic acid (RA) (right). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0218] Video 4: 3D visualization of the axioloid structure. 3D reconstruction of an axioloid embedded in MG+RA for 120 h. F-actin (phalloidin) is stained in grey, TBXT (BRA) in blue, fibronectin (FN) in green, and MEOX1 in red.

[0219] Video 5: Formation of the midline and bipartites in the axilloid. Visualization of midline formation in axioloids derived from 409B2 embedded in MG+RAL (top), and formation of single bilateral somites in axioloids derived from 409B2 embedded in MG+ROL (bottom). Live imaging was performed between 72 and 120 hours of culture. Scale bar, 200 μm.

[0220] Video 6: 3D reconstruction of axioloid and segments in human CS11 embryo. 3D reconstructions of an axioloid somite from 409B2 (top right) and 201B7 Luc (bottom right), and a reconstruction of the posterior 8 somites of a CS11 human embryo (right). Each somite-like structure is highlighted in a different color depending on its position along the anterior-posterior axis.

[0221] Video 7: The effect of RAL, RA or BMS493 supplementation on the kinetics of HES7 gene expression. Live imaging of spatiotemporal morphogenetic expression of the HES7 gene from 72 to 120 h of culture of 201B7 Luc-derived axioloids embedded in MG and supplemented with RAL (top left) or RA (top right), or with RAL+DMSO (bottom left) or RAL+BMS493 (bottom right). BF videos (left) and HES7:luciferase signal in each condition (right). Data are representative of at least three independent experiments. Scale bar 200 μm.

[0222] Video 8: Effects of RA, NOTCH, FGF and WNT pathway inhibition on axioloid growth and morphology (From left to right) Live imaging of 201B7 Luc-derived axioloids at 72 to 120 hours of culture after embedding in MG+RAL supplemented with DMSO, BMS493, DAPT, PD173074, or XAV939. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0223] Video 9: Effects of HES7 gene KO on axioloid growth and morphology. Top, Live imaging of axioloids embedded in MG+RAL from 201B7 Luc (top left), HES7 KO1 (top middle) and HES7 KO2 (top right) cell lines. Data are representative of at least three independent experiments. Scale bar 200 μm. Bottom, 3D reconstructions of axioloids embedded in MG+RAL from HES7 KO1 (middle) and HES7 KO2 (bottom). F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green and MEOX1 in red.

[0224] Video 10: Effects of HES7 gene KO on HES7 gene expression dynamics. Live imaging of spatiotemporal expression of the HES7 gene in axioloids from 201B7 Luc (top), HES7 KO1 (middle), and HES7 KO2 (bottom) embedded in MG+RAL. BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0225] Video 11: Effect of HES7 point mutation (rs113994160: c.73C>T; HES7 R25W ) influence. Top row: 201B7 Luc (left) embedded in MG+RAL, HES7 R25W MT1 (center), HES7 R25W Live imaging of axioloids from the MT2 (right) cell line. Data are representative of at least three independent experiments. Scale bar, 200 μm. Bottom row, HES7 R25W MT1-derived (top) and HES7 R25W 3D reconstruction of axioloids embedded in MG+RAL from MT2 (bottom), stained for F-actin (phalloidin) in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0226] Video 12: Effect of HES7 point mutation (rs113994160: c.73C>T; HES7 R25W ) influence. 201B7 Luc (top), HES7 R25W MT1 (medium), HES7 R25WLive imaging of spatiotemporal expression of the HES7 gene in axioloids derived from MT2 (bottom). BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0227] Video 13: Effects of MESP2 gene KO on axioloid growth and morphology. Top, Live imaging of axioloids from 201B7 Luc (top left), MESP2 KO1 (top middle), and MESP2 KO2 (top right) cell lines embedded in MG+RAL. Data are representative of at least three independent experiments. Scale bars are 200 μm. Bottom panels, 3D reconstructions of axioloids from MESP2 KO1 (middle) and MESP2 KO2 (bottom) embedded in MG+RAL. F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0228] Video 14: Effect of MESP2 gene KO on HES7 gene expression dynamics. Live imaging of spatiotemporal expression of HES7 gene in axioloids from 201B7 Luc (top), MESP2 KO1 (middle), and MESP2 KO2 (bottom) embedded in MG+RAL. BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bar 200 μm.

[0229] Supplementary considerations Formation of the midline and somites in axioloids In MG- and RA-treated axioloids, a single axis was typically observed in which epithelial somites formed sequentially with a central single spinal foramen. Interestingly, axioloids frequently displayed a superficial sulcus or midline-like structure that started from the PSM and ran through most of the forming segment (Supplementary Fig. 22a, b), but it usually remained superficial and did not separate the segment into two completely separated somites (Supplementary Fig. 22a, b, Supplementary Video 8). In retinoid-treated axioloids, fully segmented true somites, each with a central somite cavity in the middle, were still occasionally present, either as an isolated pair of fully segmented somites or, rarely, as a series of two or more somite pairs along the AP axis of the axioloid (Supplementary Fig. 22c-e, Supplementary Video 8). These bipartites usually exhibited normal protein and gene expression patterns, including proper anterior-posterior polarity (Supplementary Fig. 22f-k). The exact molecular nature and possible mechanisms underlying the formation of unilateral and bilateral somites in axioloids remain to be elucidated.

[0230] Supplementary Table S Supplementary Table 1 (Tables 1-A to 1I): Genes expressed in identified clusters from scRNA-seq data.

[0231] [Table 1-A]

[0232] [Table 1-B]

[0233] [Table 1-C]

[0234] [Table 1-D]

[0235] [Table 1-E]

[0236] [Table 1-F]

[0237] [Table 1-G]

[0238] [Table 1-H]

[0239] [Table 1-I]

[0240] Supplementary Table 2 (Tables 2-A to 2-B): NMP and NE module score genes. List of neuromesodermal progenitor (NMP) and neuroectoderm (NE)-related genes used to calculate the module scores in Figure 8g.

[0241] [Table 2-A]

[0242] [Table 2-B]

[0243] Supplementary Table 3 (Tables 3-A to 3-D): Recombinant proteins, small molecules, reagents and consumables / equipment used in this study. List of recombinant proteins and small molecules (3.1), signaling pathway regulators (3.2), reagents (3.3) and consumables / equipment (3.4) used.

[0244] [Table 3-A]

[0245] [Table 3-B]

[0246] [Table 3-C]

[0247] [Table 3-D]

[0248] Supplementary Table 4 (Tables 4-A to 4-D): Antibodies used in this study. List of primary antibodies for immunohistochemistry (4.1), secondary antibodies for immunohistochemistry (4.2), primary antibodies for CUT&Tag library construction (4.3), and secondary antibodies for CUT&Tag library construction (4.4).

[0249] [Table 4-A]

[0250] [Table 4-B]

[0251] [Table 4-C]

[0252] [Table 4-D]

[0253] Supplementary Table 5 (Table 5): List of probes used for HCR. List of probes used for HCR-based whole-tissue in situ hybridization analysis in human axioloid specimens.

[0254] [Table 5]

[0255] Supplementary Table 6 (Tables 6-A to 6-R): List of probe sequences used for HybISS analysis. List of probe sequences used for HybISS-based spatial transcriptional analysis in human axioloid specimens.

[0256] [Table 6-A]

[0257] [Table 6-B]

[0258] [Table 6-C]

[0259] [Table 6-D]

[0260] [Table 6-E]

[0261] [Table 6-F]

[0262] [Table 6-G]

[0263]

Table 6-H

[0264]

Table 6-I

[0265]

Table 6-J

[0266]

Table 6-K

[0267]

Table 6-L

[0268]

Table 6-M

[0269]

Table 6-N

[0270]

Table 6-O

[0271]

Table 6-P

[0272]

Table 6-Q

[0273] [Table 6-R]

[0274] Supplementary Table 7 (Table 7): List of primers used in the CUT&Tag experiments. List of primers used in the CUT&Tag analysis of human axioloides.

[0275] [Table 7]

[0276] Supplementary Video S Supplementary Video 1: Symmetry breaking and initial elongation of the axioloid Live imaging of 409B2-derived xyloids (top) and 201B7 Luc-derived xyloids (bottom) from 24 to 72 hours after culture. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0277] Supplemental Video 2: Effect of Matrigel on axilloid morphology Live imaging of axioloids derived from 409B2 (top) and 201B7 Luc (bottom) embedded in Matrigel (MG) and cultured for 72 to 120 h with or without +MG (right) or -MG (left). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0278] Supplementary Video 3: HES7 gene expression dynamics in MG-embedded axioloids Live imaging of spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived axioloids embedded in MG from 72 to 120 hours in culture. BF videos (left) and HES7:luciferase signals in each condition (right). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0279] Supplemental Video 4: Effects of retinoid signaling on axioloid growth and morphology Live imaging of axioloids derived from 409B2 (top) and 201B7 Luc (bottom) at 72 to 120 hours of culture after embedding in MG alone (left) or MG supplemented with retinol (ROL), retinal (RAL), or retinoic acid (RA) (right). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0280] Supplementary Video 5: 3D reconstruction of axolotls treated with retinoids 3D reconstruction of axoloids from 409B2 embedded in MG alone (top left) or MG supplemented with retinol (ROL) (bottom left), retinal (RAL) (bottom right) or retinoic acid (RA) (top right) at 120 hours in culture. Stained for F-actin (phalloidin) in grey, TBXT (BRA) in blue, fibronectin (FN1) in green and MEOX1 in red.

[0281] Supplemental Video 6: Effects of RA pathway inhibition on axioloid growth and morphology (From left to right) Live imaging of 201B7 Luc-derived axolotls at 72 to 120 hours of culture after embedding in +MG +RAL supplemented with DMSO, BMS493, AGN193109 or ER50891. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0282] Supplementary Video 7: 3D visualization of axioloids treated with RA pathway inhibitors 3D reconstruction of axoloids from 201B7 Luc cell line embedded in MG +MG+RAL (top left) or +MG+RAL supplemented with BMS493 (top right), AGN193109 (bottom left) or ER50891 (bottom right) at 120 hours of culture. F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0283] Supplemental Video 8: Midline and somite formation in axioloids Visualization of midline formation in 409B2-derived axoloids embedded in +MG +RAL (top) and in 409B2-derived axoloids embedded in +MG +ROL (bottom). Live imaging was performed at 72 to 120 hours of culture. Scale bar, 200 μm.

[0284] Supplementary Video 9: 3D reconstruction of axioloids and somites in human CS9-11 embryos 3D reconstructions of somites formed in axioloids (top) embedded in MGs derived from 409B2 treated with retinol (ROL) (left), retinal (RAL) (middle) or retinoic acid (RA), and of human embryonic somites found in human embryos CS9 (left), CS10 (middle) and CS11 (right). Each somite-like structure is highlighted in a different color depending on its position along the anterior-posterior axis.

[0285] Supplementary Video 10: Effect of ROL, RAL or RA on HES7 gene expression dynamics Live imaging of spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived axioloids embedded in MG alone (top left) or supplemented with retinol (ROL) (bottom left), retinal (RAL) (bottom right) or retinoic acid (RA) (top right) from 72 to 120 h of culture. BF videos (left) and HES7:luciferase signal (right) in each condition. Data are representative of at least three independent experiments. Scale bar 200 μm.

[0286] Supplementary Video 11: Effect of BMS493 supplementation on HES7 gene expression dynamics Live imaging of spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived axioloids embedded in MG supplemented with RAL+DMSO (left pair) or RAL+BMS493 (right pair) from 72 to 120 h of culture. BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bars are 200 μm.

[0287] Supplemental Video 12: Effects of NOTCH, FGF and WNT pathway inhibition on axillary tract Live imaging of 201B7 Luc-derived axolotls from 72 to 120 h of culture after embedding in +MG +RAL supplemented with DMSO (top left), DAPT (top right), PD173074 (middle left), PD0325901 (middle right), XAV939 (bottom left), and IWP2 (bottom right). Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0288] Supplementary Video 13: Effects of HES7 gene KO on axioloid growth and morphology Top, live imaging of axioloids embedded in +MG+RAL from 201B7 Luc (top left), HES7 KO1 (top middle), and HES7 KO2 (top right) cell lines. Data are representative of at least three independent experiments. Scale bars are 200 μm. Bottom panels, 3D reconstructions of axioloids embedded in +MG +RAL from HES7 KO1 (middle) and HES7 KO2 (bottom). F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0289] Supplementary Video 14: Effect of HES7 gene KO on HES7 gene expression dynamics Live imaging of spatiotemporal expression of HES7 gene in axioloids from 201B7 Luc (top pair), HES7 KO1 (middle pair), and HES7 KO2 (bottom pair) embedded in +MG +RAL. BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0290] Supplementary Video 15: Effect of the HES7 point mutation (rs113994160: c.73C>T; HES7R25W) on axioloid growth and morphology Top, live imaging of axioloids embedded in +MG +RAL from 201B7 Luc (top left), HES7R25W MT1 (top middle), and HES7R25W MT2 (top right) cell lines. Data are representative of at least three independent experiments. Scale bars are 200 μm. Bottom panels, 3D reconstructions of axioloids embedded in +MG +RAL from HES7R25W MT1 (middle) and HES7R25W MT2 (bottom). F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0291] Supplementary Video 16: Effect of HES7 point mutation (rs113994160:c.73C>T;HES7R25W) on HES7 gene expression dynamics Live imaging of spatiotemporal expression of the HES7 gene in axioloids from 201B7 Luc (top pair), HES7R25W MT1 (middle pair), and HES7R25W MT2 (bottom pair). BF videos (left) and HES7:luciferase signals (right) in each condition. Data are representative of at least three independent experiments. Scale bar, 200 μm.

[0292] Supplementary Video 17: Effects of MESP2 gene KO on axioloid growth and morphology Top, Live imaging of axioloids embedded in +MG +RAL from +MG +RAL-derived 201B7 Luc (top left), MESP2 KO1 (top middle), and MESP2 KO2 (top right) cell lines. Data are representative of at least three independent experiments. Scale bars are 200 μm. Bottom panels, 3D reconstructions of axioloids embedded in +MG +RAL from MESP2 KO1 (middle) and MESP2 KO2 (bottom). F-actin (phalloidin) stained in grey, TBXT (BRA) in blue, fibronectin (FN1) in green, and MEOX1 in red.

[0293] Supplementary Video 18: Effect of MESP2 gene knockout on HES7 gene expression Live imaging of spatiotemporal expression of HES7 gene in axioloids from 201B7 Luc (top pair), MESP2 KO1 (middle pair), and MESP2 KO2 (bottom pair) embedded in +MG +RAL. BF videos (left) and HES7:luciferase signals (right) in each condition.

[0294] Addendum to the patent application: "Axioloid: A stem cell-based model of human axis development" introduction Somitogenesis is a core developmental event by which somitic structures form in vertebrates. It has been well studied in model organisms such as mouse, zebrafish, and chicken, but remains poorly understood in humans and other primates. Although in vitro models of organogenesis and embryonic development based on pluripotent stem cells (PSCs) have shown progress in recent years, experimental model systems that can recapitulate key features of somitogenesis in vitro remain elusive. Using in vitro induced presomitic mesoderm (PSM), we have previously succeeded in reconstituting and quantifying the oscillatory activity of the segmentation clock, a molecular clock thought to control somitogenesis. We further expanded on these initial findings to test whether the actual processes of somitogenesis and epithelial somitogenesis, as well as the segmentation clock, could be recapitulated in vitro.

[0295] To this end, we generated the "axioloid," a self-organizing 3D in vitro model of human somitogenesis that shares morphological and molecular features of the emerging vertebrate embryonic tail and axis, including the presence of major cell populations associated with somitogenesis, opposing morphogen gradients and signaling activities, and the cyclic formation of properly patterned epithelial somites synchronized with the segmentation clock. Using this model, we uncovered a previously unknown function of retinoic acid (RA) signaling in the stabilization and epithelialization of newly formed somite-like structures within the axioloid.

[0296] Here, the effects of varying the concentration and nature of molecules and reagents used during axioloid induction on initial and final axioloid morphogenesis are evaluated and reported, including the effects of different levels of CHIR-mediated WNT activation, bFGF-mediated FGF pathway activation, and different levels of TGFβ inhibitor-mediated TGF-β pathway activation. Additionally, the effects of various extracellular matrix compounds and synthetic analogs of the retinoic acid pathway were also evaluated.

[0297] material and method Cultivation of human induced pluripotent stem cells (iPSCs) In this study, a human iPS cell line derived from a healthy donor, e.g., 409B2, was used. Human iPS cells were cultured in 50 U of penicillin and 50 μg ml -1 The cells were maintained on iMatrix-511 silk-coated plates or dishes (Nippi) using StemFit AK02N (Reprocell) medium supplemented with 100 mg of streptomycin (Gibco). StemFit AK02N (Reprocell) medium consists of three components, A, B, and C, which were mixed together and used for standard maintenance culture of human iPS cells at 37°C and 5% CO2 in a humidified incubator. The iPS cells used were regularly tested and reported to be negative for mycoplasma contamination.

[0298] Based on the axioloid protocol, we investigated whether alternative compounds, molecules, and inhibitors could be used to support axioloid generation from PSCs. We started by culturing NDiff227 (Takara, Cat: Y40002) medium or RPMI 1640 (Nacalai, Cat: 30264-85) supplemented with B27 with or without retinol (Gibco, Cat: 17504-044) (AK02N-C induction medium) to test whether they could be used as an alternative to AK02N-C induction medium. We also cultured different concentrations of bFGF (5–250 ng ml -1 The effects of CHIR99021 (2.5–10 μM), or SB431542 (1.25–15 μM) on the axioloid form were tested. Also, another member of the FGF superfamily, FGF8b (PeproTech, Cat. No. 100-25) (20–250 ng ml -1 We also tested whether bFGF or another TGFβ inhibitor, A83-01 (Selleck Chemicals, Cat: S7692) (1.25 to 15 μM), would produce results similar to those described for bFGF and SB431542. Finally, we tested whether alternative ECM-containing compounds, Geltrex (Gibco, Cat: A14132-0), Cultrex (R&D Systems, Cat: 3433-005-01), and ECMgel (Sigma-Aldrich, Cat: E6909), could be used in place of Matrigel during the embedding step and support axioloid morphogenesis. We also evaluated whether the synthetic RA selective agonist TTNPB (4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) (Selleck Chemicals, Cat: S4627) could replace vitamin A derivatives such as retinol or retinal.

[0299] result Basic strategy for axioloid induction from human pluripotent stem cells (PSCs) Recently, the generation of a mesoderm-based human iPSC (induced pluripotent stem cell)-derived human axial development 3D model has been reported, termed "axioloid". It was found that an initial induction step in 2D with both bFGF (20 ng / ml) and CHIR99421 (5 μM) could efficiently and reproducibly promote the formation of primitive streak-like cells from human PSCs. This was followed by a 3D aggregation step in the presence of bFGF and CHIR (same concentrations as in the first step) plus 10 μM SB431542 (a TGF inhibitor) and 10 μM Y-27632 (a ROCK inhibitor). This step aims to promote the commitment of early primitive streak cells to a promesoderm or paraaxial mesoderm (PSM) fate. The formed mesoderm aggregates spontaneously broke symmetry, started to elongate, and assumed an initial "bean-like" morphology. At this point, when they were embedded in medium containing 10% Matrigel (MG) supplemented with retinoid, they further elongated and somite-like epithelialized segments appeared sequentially along the anterior-posterior axis of these structures (which we call "axioloids").

[0300] Alternative media can also support axioloid induction and morphogenesis In addition to the standard AK02N medium, various media were tested. Several groups in the field have reported successful generation of mouse and human PSC-derived gastroids and similar structures using N2B27 medium (PMID: 30283134). A quality-controlled, commercially available N2B27 medium, called NDiff227, was first tested and shown to be able to support axioloid formation (Figure 25a). RPMI was then tested as an alternative medium supplemented with B27 supplement, with or without rectinol. The results showed that although it was able to support early axioloid outgrowth, the structures generated were morphologically different from normal axioloids, with no visible segments or somite-like structures formed (Figure 25b).

[0301] Effective concentration ranges of bFGF, CHIR (WNT agonist) and SB431542 (TGFβ inhibitor) It was shown in the corresponding paper (Yamanaka, Hamidi et al., Nature 2023) that bFGF, CHIR99421 (WNT agonist) and SB431542 (TGFβ inhibitor) are necessary and sufficient for proper axioloid induction. Here, different concentrations of the three components were tested to evaluate their effect on axioloid induction and morphology. The results showed that high concentrations of bFGF did not significantly affect the overall axioloid morphology, both in terms of elongation and segmentation observed after 96 and 120 h, and after 120 h the structures appeared thinner with higher bFGF concentrations. Complete deprivation of bFGF during axioloid induction resulted in loss of polarity, elongation and inability to generate axioloid structures from PSCs (Figure 26a). The effect of various concentrations of CHIR on axioloid induction and morphology was also tested, and at low concentrations, e.g., 2 μM or 2.5 μM CHIR99421, round cell aggregates were observed without signs of symmetry breaking or elongation. Intermediate levels of CHIR, e.g., 3 μM CHIR99421, formed elongated neural tube-like structures. Higher concentrations (5 μM) of CHIR, e.g., 7.5 μM or 10 μM CHIR99421, did not dramatically affect the overall axioloid morphology, although they had a slight negative effect on the overall axioloid length (Figure 26b). This result also suggests that the concentration of CHIR used for axioloid induction may show different results depending on the cell line used.

[0302] Additionally, we evaluated different concentrations of the TGFβ pathway inhibitor SB431542 used for axioloid induction. As a result, we found that SB431542 was active and effective over a wide concentration range (Figure 26c). In summary, the CHIR99421 concentration utilized appears to be the most important factor determining the overall success rate of axioloid generation, while the concentrations of bFGF and TGFβ inhibitors show a wider range of activity. For most PSC lines, CHIR concentrations around 5 μM may be effective, but need to be determined and optimized for each cell line. The overall data suggests that signaling molecules have a "range of activity" rather than a discrete single acting concentration that is sufficient to contribute to proper and efficient axioloid induction and morphogenesis. FGF8b is not a substitute for bFGF, but A-83-01 can be used as a substitute for SB431542.

[0303] Basic FGF (bFGF) is a member of the fibroblast growth factor family that contains 23 heparin-binding peptides and is widely expressed during embryonic development. A number of recombinant FGFs were tested for their putative effects on axioloid induction and morphogenesis. The effect of recombinant FGF8b, which behaved differently from bFGF, was observed. At higher concentrations of FGF8b (100 ng / ml), elongated structures were obtained, but somites could not be discerned within the structures formed (Figure 27a).

[0304] Similarly, we tested whether an alternative TGFβ pathway inhibitor, A-83-01, could be used in place of SB431542. The results showed that axioloids generated with different concentrations of A-83-01 recapitulated the characteristics of proper axioloid formation, similar to that described for SB431542, with lower concentrations of A-83-01 (e.g., 1.25 μM or 2.5 μM) producing the best results (Figure 27b).

[0305] Alternative extracellular matrix (ECM)-rich components can be used to induce axioloids. Matrigel (MG) is a solubilized basement membrane rich in extracellular matrix (ECM) secreted by EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells that supports the formation of complex tissue structures and helps mimic morphogenetic processes in vitro. In our experimental system, the addition of 5% or 10% MG to the culture medium (embedding stage after the initial symmetry breaking) is sufficient, resulting in effective axioloid induction in the presence of retinoids. Here, we evaluated the addition of ECM-rich compounds such as Cultrex, Geltrex, and ECMgel (10%) to the culture medium. All three ECM-rich compounds tested led to changes in the morphological axioloid phenotype, and axioloid elongation was observed with all three compounds, although to a lesser extent than Matrigel. It is believed that other ECM-containing compounds can be used as well and could replace MG. Furthermore, it is envisioned that well-defined mixtures of defined recombinant matrix proteins and basement membrane components could also replace MG or similar complex ECM-rich compounds to achieve reproducible and efficient axioloid induction and morphogenesis in the presence of active retinoid signaling, which appears to be essential in acting synergistically with MG.

[0306] In addition to evaluating ECM-rich compounds, we also investigated and confirmed that TTNPB, a synthetic analog of retinoic acid selective for retinoic acid receptor (RAR) subtypes, can be used as an alternative to retinoids. TTNPB showed similar overall effects on axilloid morphology and somite epithelialization compared to other retinoids, including retinoid (ROL), retinal (RAL), and retinoid acid (RA).

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[0308] Although 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 be apparent to those skilled in the art can be made in the configuration and specifications of the present disclosure without departing from the scope of the present disclosure.

[0309] This application claims priority from U.S. Provisional Patent Application No. 63 / 326,611, filed April 1, 2022, the entire disclosure of which is incorporated herein by reference.

[0310] All patents, patent applications, and references cited herein are hereby incorporated by reference in their entireties, as if fully and specifically set forth herein.

[0311] <Supplementary explanation> The above exemplary embodiments and disclosed examples are described in whole or in part in the following supplemental description, but are not limited thereto. (Supplementary explanation 1) A three-dimensional cell aggregate generated in vitro from pluripotent stem cells comprising mesodermal cells, the cell aggregates are polarized along an anterior-posterior or rostrocaudal axis and an apical-basolateral axis; The cell aggregates are three-dimensional cell aggregates capable of reconstituting various aspects of somitogenesis and axis development, including axial elongation, segmentation, epithelial somitogenesis and patterning (formation of one or more somite-like structures), and oscillation of the segmentation clock under somitogenic culture conditions. (Supplementary explanation 2) A three-dimensional cell aggregate generated in vitro from pluripotent stem cells comprising mesodermal cells, the cell aggregates are polarized along an anterior-posterior or rostrocaudal axis and an apical-basolateral axis; A three-dimensional cell aggregate, wherein the percentage of mesodermal cells in the cell 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 cell number. (Supplementary explanation 3) A cell aggregate described in Supplementary Explanation 1 or 2, wherein the anterior-posterior axis is defined by an anterior region and a posterior region, and the anterior region cells have higher or lower expression of one or more markers compared to the posterior region cells. (Supplementary explanation 4) The cell aggregate described in Supplementary Explanation 3, wherein the anterior region cells have lower expression of one or more markers selected from the group consisting of TBXT, MIXL1, SOX2, TBX6, HES7, MSGN1, MEOX1, TCF15, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, HOXD13, HOXB, HOXA9, HOXA10 and CDX2 compared to the posterior region cells. (Supplementary explanation 5) The cell aggregate described in Supplementary Explanation 3 or 4, wherein the anterior region cells have higher expression of one or more markers, including LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2 and RDH10, compared to the posterior region cells. (Supplementary explanation 6) The three-dimensional cell aggregate described in Supplementary Note 5, wherein the posterior region contains tailbud (TB)-like cells. (Supplementary explanation 7) the apical-basolateral axis is defined by an apical region and a basolateral region; A cell aggregate described in any one of Supplementary Explanations 1 to 6, wherein cells in the apical region have high or low expression of one or more markers compared to cells in the basolateral region. (Supplementary explanation 8) The three-dimensional cell aggregate described in Supplementary Explanation 3, wherein cells in the apical region have lower expression of one or more markers selected from the group consisting of fibronectin, collagen V and laminin compared to cells in the basolateral region. (Supplementary explanation 9) The three-dimensional cell aggregate described in Supplementary Explanation 3 or 4, wherein cells in the apical region have higher expression of one or more markers selected from the group consisting of aPKC, CDH2, Ezrin, ZO1 and F-actin compared to cells in the basolateral region. (Supplementary explanation 10) A cell aggregate described in any one of Supplementary Explanations 1 and 3 to 9, wherein the percentage of mesodermal cells in the cell 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 cell number. (Supplementary explanation 11) The cell aggregate described in any one of Supplementary Explanations 1 to 10, wherein the mesodermal cells express 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. (Supplementary explanation 12) The cell aggregate described in any one of Supplementary Explanations 2 to 11, wherein the cell aggregate is capable of reconstituting various aspects of somite formation and axis 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 somite formation culture conditions. (Supplementary explanation 13) 13. The cell aggregate of claim 1 or 12, wherein the somitogenic culture conditions are the presence of a gel or matrix and retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist; or The cell aggregate of Supplementary Explanation 1 or 12, wherein the somitogenic culture conditions are the presence of a gel or matrix and a retinoid, including retinoic acid, a retinoic acid precursor or derivative, and / or a retinoic acid receptor (RAR) agonist. (Supplementary explanation 14) The cell aggregate of Supplementary Note 13, wherein the RAR agonist comprises retinal or retinol; or The cell aggregate of claim 13, wherein the retinoid comprises retinoic acid, a retinoic acid precursor, e.g., retinal or retinol, or a RAR agonist; or The cell aggregate of claim 13, wherein the retinoid comprises retinal or retinol; or The cell aggregate described in Supplementary Explanation 13, wherein the retinoid comprises retinoic acid, a retinoic acid precursor, such as retinal or retinol, or a RAR agonist. (Supplementary explanation 15) A cell aggregate described in Supplementary Explanation 13 or 14, wherein the cell aggregate is embedded in a gel or matrix or disposed inside a gel or matrix. (Supplementary explanation 16) A cell aggregate described in any one of Supplementary Explanations 13 to 15, wherein the matrix includes an extracellular matrix. (Supplementary explanation 17) The cell aggregate described in any one of Supplementary Explanations 13 to 16, wherein the extracellular matrix comprises collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. (Supplementary explanation 18) The cell aggregate described in any one of Supplementary Explanations 13 to 17, wherein the gel includes a hydrogel. (Supplementary explanation 19) A cell aggregate described in any one of Supplementary Explanations 13 to 18, wherein the gel contains a basement membrane matrix. (Supplementary explanation 20) The cell aggregate described in Supplementary Explanation 19, wherein the basement membrane matrix comprises laminin, collagen, heparan sulfate, proteoglycan and / or entactin. (Supplementary explanation 21) A cell aggregate described in any one of Supplementary Explanations 13 to 17, wherein the gel comprises an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. (Supplementary explanation 22) The somite or somite-like structure includes anterior and posterior portions of an anterior-posterior axis, and A cell aggregate described in any one of Supplementary Explanations 1 and 12 to 21, wherein the anterior cells have higher or lower expression of one or more markers compared to the posterior cells. (Supplementary explanation 23) The cell aggregate described in Supplementary Explanation 22, wherein the anterior cells have higher expression of one or more markers selected from the group consisting of TBX18 and ALDH1A2 compared to the posterior cells. (Supplementary explanation 24) The cell aggregate described in Supplementary Explanation 22 or 23, wherein the anterior cells have lower expression of one or more markers selected from the group consisting of UNCX 8 and LNFG compared to the posterior cells. (Supplementary explanation 25) A cell aggregate according to any one of Supplementary Explanations 1 and 12 to 24, comprising an anterior splanchnic / anterior mesoderm (PSM), The anterior PSM is a cell aggregate having expression of one or more markers selected from the group consisting of MESP1, MESP2, RIPPLY1, RIPPLY2, and PCDH8, preferably MESP2. (Supplementary explanation 26) A cell aggregate described in any one of Supplementary Explanations 1 and 12 to 25, wherein the somites are 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. (Supplementary explanation 27) A cell aggregate described in any one of Supplementary Explanations 1 to 26, comprising pluripotent stem cells. (Supplementary explanation 28) The cell aggregate described in Supplementary Explanation 27, wherein the percentage of pluripotent stem cells in the cell 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 cell number. (Supplementary explanation 29) A cell aggregate described in any one of Supplementary Explanations 1 to 28, which is substantially free of endodermal cells and / or ectodermal cells. (Supplementary explanation 30) The cell aggregate described in Supplementary Explanation 29, wherein the percentage of endoderm cells in the cell 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 cell number. (Supplementary explanation 31) The cell aggregate described in Supplementary Explanation 29 or 30, wherein the endoderm cells express a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. (Supplementary explanation 32) A cell aggregate described in any one of Supplementary Explanations 29 to 31, wherein the proportion of ectodermal cells in the cell 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 cell number. (Supplementary explanation 33) The cell aggregate according to any one of Supplementary Explanations 29 to 32, wherein the ectodermal cells express a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2 and BARX1; or The cell aggregate described in any one of Supplementary Explanations 29 to 32, wherein the ectodermal cells express a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1. (Supplementary explanation 34) A cell aggregate described in any one of Supplementary Explanations 1 to 33, wherein expression of the segmentation clock gene is subject to genetic oscillation. (Supplementary explanation 35) The cell aggregate described in Supplementary Explanation 34, wherein the segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3, and HES7. (Supplementary explanation 36) The cell aggregate described in Supplementary Explanation 34 or 35, wherein the cycle of the gene oscillation is a 3 to 7 hour cycle, a 3.5 to 6.6 hour cycle, or a 4 to 6 hour cycle. (Supplementary explanation 37) The cell aggregate described in any one of Supplementary Explanations 1 to 36, wherein the pluripotent stem cells are human pluripotent stem cells. (Supplementary explanation 38) The cell aggregate described in any one of Supplementary Explanations 1 to 37, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. (Supplementary explanation 39) A cell aggregate described in any one of supplementary explanations 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 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. (Supplementary explanation 40) A cell aggregate described in any one of Supplementary Explanations 1 to 39, having 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. (Supplementary explanation 41) 1. A method for producing three-dimensional cell aggregates generated in vitro from pluripotent stem cells, comprising the steps of: (a) culturing pluripotent stem cells to induce three-dimensional cell aggregates comprising mesodermal cells; and (b) culturing the cell aggregates comprising the mesodermal cells to induce three-dimensional cell aggregates; Including, A method, wherein the three-dimensional cell aggregate is a cell aggregate described in any one of Supplementary Explanations 1 to 40. (Supplementary explanation 42) The process is as follows: (a1) culturing the pluripotent stem cells in a medium comprising a GSK3β inhibitor and FGF to initiate commitment to primitive streak and mesoderm fates and / or induce mesoderm cells; (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium comprising a WNT agonist (GSK3β inhibitor), an FGF agonist, a TGFβ inhibitor and a ROCK inhibitor to induce three-dimensional cell aggregates comprising said mesodermal cells; and optionally, (b2) culturing said three-dimensional cell aggregates comprising mesodermal cells in a medium comprising retinoic acid, a retinoic acid derivative, and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or matrix to induce morphogenesis and / or self-organization of the three-dimensional cell aggregates, and / or the three-dimensional cell aggregates; The method according to Supplementary Explanation 41, comprising: (Supplementary explanation 43) The process is as follows: (a1) culturing the pluripotent stem cells in a medium comprising a GSK3β inhibitor and FGF to initiate commitment to primitive streak and mesoderm fates and / or induce mesoderm cells; (a2) culturing the cells derived, obtained or obtainable from step (a1) in a medium comprising a GSK3β inhibitor, an FGF, a TGFβ inhibitor and a ROCK inhibitor to induce three-dimensional cell aggregates comprising said mesodermal cells; (b1) culturing the three-dimensional cell aggregates comprising the mesodermal cells in a medium that does not contain a GSK3β inhibitor, an FGF, a TGFβ inhibitor, and a ROCK inhibitor; and optionally, (b2) culturing said three-dimensional cell aggregates comprising mesodermal cells in a medium comprising retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or matrix to induce morphogenesis and / or self-organization of the three-dimensional cell aggregates, and / or the three-dimensional cell aggregates; The method according to Supplementary Explanation 41 or 42, comprising: (Supplementary explanation 44) or the method of any one of claims 42 to 43, comprising culturing a three-dimensional cell aggregate comprising said mesodermal cells embedded in or disposed inside a gel or matrix in a medium comprising retinoic acid, a retinoic acid derivative, and / or a retinoic acid receptor (RAR) agonist to induce a three-dimensional cell aggregate; or The method described in Supplementary Description 42 or 43, comprising culturing three-dimensional cell aggregates comprising the mesodermal cells embedded in a gel or matrix or disposed inside a gel or matrix in a medium containing retinoic acid, a retinoic acid derivative, and / or a retinoid, to induce three-dimensional cell aggregates. (Supplementary explanation 45) The method of claim 43 or 44, wherein the RAR agonist is retinal or retinol; or The method of claim 43 or 44, wherein the retinoid comprises retinoic acid, a retinoic acid precursor, e.g., retinal or retinol, or a RAR agonist; or The method of claim 43 or 44, wherein the retinoid is retinal or retinol; or The method of any one of Supplementary Explanations 43 to 44, wherein the retinoid comprises retinoic acid, a retinoic acid precursor, such as retinal or retinol, or an RAR agonist. (Supplementary explanation 46) the GSK3β inhibitor is CHIR99021; The FGF is bFGF, the TGFβ inhibitor is SB431542, and / or The method according to any one of Supplementary Explanations 42 to 45, wherein the ROCK inhibitor is Y-27632. (Supplementary explanation 47) The method according to any one of Supplementary Explanations 42 to 46, wherein the matrix comprises an extracellular matrix. (Supplementary explanation 48) A method described in any one of Supplementary Explanations 42 to 47, wherein the extracellular matrix comprises collagen, laminin, fibronectin, vitronectin, gelatin, and / or entactin. (Supplementary explanation 49) A method according to any one of Supplementary Explanations 42 to 48, wherein the gel comprises a hydrogel. (Supplementary explanation 50) The method of any one of Supplementary Explanations 42 to 49, wherein the gel comprises a basement membrane matrix. (Supplementary explanation 51) The method of Supplementary Explanation 50, wherein the basement membrane matrix comprises laminin, collagen, heparan sulfate, proteoglycan, and / or entactin. (Supplementary explanation 52) The method described in any one of Supplementary Explanations 42 to 51, wherein the gel comprises an acrylamide gel, an arginine gel, an agarose gel, and / or a polyethylene glycol hydrogel. (Supplementary explanation 53) A method according to any one of Supplementary Explanations 41 to 52, wherein the percentage of mesodermal cells in the cell aggregates containing mesodermal cells 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 cell number. (Supplementary explanation 54) or the method of any one of Supplementary Explanations 41-53, wherein the mesodermal cells express a marker selected from the group consisting of TBXT, MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WNT5a, DLL1, MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, HES7, MSGN1, TCF15, MEOX1, and FLK1 / KDR; or The method described in any one of Supplementary Explanations 41 to 53, wherein the mesodermal cells express a marker selected from the group consisting of TBXT, SOX2, MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WNT5a, DLL1, MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, HES7, MSGN1, TCF15, MEOX1, and FLK1 / KDR. (Supplementary explanation 55) A method according to any one of Supplementary Explanations 41 to 54, wherein the cell aggregates containing mesodermal cells are substantially free of endodermal cells and / or ectodermal cells. (Supplementary explanation 56) The method described in Supplementary Explanation 55, wherein the percentage of endoderm cells in the cell aggregate containing the mesoderm cells 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 cell number. (Supplementary explanation 57) The method described in Supplementary Explanation 55 or 56, wherein the endoderm cells express a marker selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. (Supplementary explanation 58) A method according to any one of supplementary explanations 55 to 57, wherein the proportion of ectodermal cells in the cell aggregates containing mesodermal cells 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 cell number. (Supplementary explanation 59) Supplementary Explanation 55-58: The method according to any one of Supplementary Explanations 55 to 58, wherein the ectodermal cells express a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2 and BARX1. ;or A method described in any one of Supplementary Explanations 55 to 58, wherein the ectodermal cells express a marker selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1. (Supplementary explanation 60) A method according to any one of Supplementary Explanations 41 to 59, wherein the pluripotent stem cells are dissociated pluripotent stem cells or a cell suspension containing pluripotent stem cells. (Supplementary explanation 61) A method according to any one of Supplementary Explanations 41 to 60, wherein the pluripotent stem cells are human pluripotent stem cells. (Supplementary explanation 62) The method according to any one of Supplementary Explanations 41 to 61, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. (Supplementary explanation 63) The method according to any one of Supplementary Explanations 41 to 62, wherein the cell aggregate comprising mesodermal cells 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 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. (Supplementary explanation 64) The method described in any one of Supplementary Explanations 41 to 63, wherein the cell aggregates containing mesodermal cells have 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. (Supplementary explanation 65) A cell obtained from a cell aggregate described in any one of supplementary explanations 1 to 40. (Supplementary explanation 66) A method for producing a precursor cell or a differentiated cell, comprising the steps of culturing the cell aggregate according to any one of Supplementary Explanations 1 to 40, and inducing a precursor cell or a differentiated cell selected from the group consisting of the following (a) to (f): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) endothelial or hematopoietic cells or their precursor cells; or A method for producing a precursor cell or a differentiated cell, comprising the steps of culturing the cell aggregate according to any one of Supplementary Explanations 1 to 40, and inducing a precursor cell or a differentiated cell selected from the group consisting of the following (a) to (i): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) an endotome or endothelial or hematopoietic cell or a precursor cell thereof; (g) adipocytes or their precursor cells, including white, beige, and brown cells; (h) dermal cells or their precursor cells; and (i) Neural tube cells or their precursor cells. (Supplementary explanation 67) Prior to induction, the method includes inducing three-dimensional cell aggregates from pluripotent stem cells, The method described in Supplementary Explanation 66, wherein the three-dimensional cell aggregate induction is performed by a method described in any one of Supplementary Explanations 41 to 64. (Supplementary explanation 68) 1. A method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of the three-dimensional cell aggregates; and evaluating the three-dimensional cell aggregates after culturing; A method, wherein the three-dimensional cell aggregate is a cell aggregate described in any one of Supplementary Explanations 1 to 40. (Supplementary explanation 69) During evaluation, a test substance that alters the polarity, morphology and / or size of the cell aggregates is selected as a candidate substance that modifies, promotes or inhibits the polarity, morphology and / or size of the cell aggregates, as described in Supplementary Explanation 68. (Supplementary explanation 70) The method described in Supplementary Explanation 68, wherein the culture is under segmental culture conditions. (Supplementary explanation 71) The method described in Supplementary Explanation 70, wherein during evaluation, a test substance that alters the somitogenesis of the cell aggregate is selected as a candidate substance that modifies, promotes, or inhibits somitogenesis of the cell aggregate. (Supplementary explanation 72) The method according to Supplementary Explanation 68, wherein the culture induces a progenitor cell or a differentiated cell selected from the group consisting of the following (a) to (f): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) endothelial or hematopoietic cells or their precursor cells; or The method according to Supplementary Explanation 68, wherein the culture induces a progenitor cell or a differentiated cell selected from the group consisting of the following (a) to (i): (a) Neuro-mesodermal cells or their precursors; (b) muscle cells or their precursor cells; (c) bone cells or their precursor cells; (d) chondrocytes or their precursor cells; (e) tenocytes or their progenitor cells; and (f) an endotome or endothelial or hematopoietic cell or a precursor cell thereof; (g) adipocytes or their precursor cells, including white, beige, and brown cells; (h) dermal cells or their precursor cells; and (i) Neural tube cells or their precursor cells. (Supplementary explanation 73) During the evaluation, a test substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a) to (f) is selected as a candidate substance that promotes or inhibits the induction of a precursor cell or a differentiated cell selected from the group consisting of (a) to (f); or The method described in Supplementary Explanation 72, wherein during the evaluation, a test substance that promotes or inhibits the induction of a precursor cell or differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or inhibits the induction of a precursor cell or differentiated cell selected from the group consisting of (a) to (i). (Supplementary explanation 74) A method according to any one of Supplementary Explanations 68 to 73, wherein the evaluation is an evaluation using a control in which the test substance is not present as a reference. (Supplementary explanation 75) The culturing is the generation of three-dimensional cell aggregates from pluripotent stem cells; The method according to Supplementary Explanation 68, wherein the three-dimensional cell aggregate is produced by a method according to any one of Supplementary Explanations 41 to 64. (Supplementary explanation 76) 1. A method for assessing gene or genome function comprising the steps of: preparing pluripotent stem cells having a modified test gene or genome; generating three-dimensional cell aggregates from the pluripotent stem cells; and evaluating the three-dimensional cell aggregates after culturing; The method for producing the three-dimensional cell aggregate is carried out by the method according to any one of Supplementary Explanations 41 to 64; or 1. A method for assessing gene function or genomic sequence function comprising the steps of: preparing pluripotent stem cells modified with a test gene or test genomic sequence; generating three-dimensional cell aggregates from the pluripotent stem cells; and evaluating the three-dimensional cell aggregates after culturing; A method in which the three-dimensional cell aggregate is produced by a method described in any one of Supplementary Explanations 41 to 64. (Supplementary explanation 77) or the method of Supplementary Note 75, wherein during the evaluation, the test gene or test genome that alters the polarity of the cellular aggregates, the morphology of the cellular aggregates, and / or the size of the cellular aggregates is evaluated as a candidate gene or candidate genome that modifies, promotes, or inhibits the polarity of the cellular aggregates, the morphology of the cellular aggregates, and / or the size of the cellular aggregates; During evaluation, test genes or test genomic sequences that alter cell aggregate polarity, cell aggregate morphology and / or cell aggregate size are evaluated as candidate genes or candidate genomic sequences that modify, promote or inhibit cell aggregate polarity, cell aggregate morphology and / or cell aggregate size, as described in Supplementary Explanation 75. (Supplementary explanation 78) 76. The method of claim 75, comprising culturing in the presence of three-dimensional cell aggregates under somitogenic culture conditions, During the evaluation, the test gene or test genome that alters the somitogenesis of the cell aggregate is evaluated as a candidate gene or genome that modifies, promotes, or inhibits somitogenesis of the cell aggregate; or 76. The method of claim 75, comprising culturing in the presence of three-dimensional cell aggregates under somitogenic culture conditions, During evaluation, test genes or test genomic sequences that alter somitogenesis of said cell aggregates are evaluated as candidate genes or candidate genomic sequences that modify, promote, or inhibit somitogenesis of cell aggregates. (Supplementary explanation 79) The method of any one of Supplementary Explanations 75-78, 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 method described in any one of Supplementary Explanations 75 to 78, wherein 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.

Claims

1. A three-dimensional cell aggregate produced in vitro from pluripotent stem cells, including mesodermal cells, The cell aggregates have polarity along the anterior-posterior axis or the rostral-tail axis and the apical-basal axis. The cell aggregate is a three-dimensional cell aggregate that can reconstruct various aspects of somite formation 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 somite-forming culture conditions.

2. A three-dimensional cell aggregate produced in vitro from pluripotent stem cells, including mesodermal cells, The cell aggregates have polarity along the anterior-posterior axis or the rostral-tail axis and the apical-basal axis. A three-dimensional cell aggregate in which the proportion of mesoderm cells in the cell aggregate is, based on the number of cells, 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%.

3. The aforementioned front-rear axis is defined by a front region and a rear region. i) The anterior region cells have lower expression of one or more markers selected from the group consisting of TBXT, SOX2, MIXL1, TBX6, HES7, MSGN1, MEOX1, TCF15, CYP26A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, HOXD13, HOXB, HOXA9, HOXA10, and CDX2, compared to the posterior region cells. ii) The cell aggregate according to claim 1 or 2, wherein the anterior region cells have high expression of one or more markers, including LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1A2, and RDH10, compared to the posterior region cells.

4. The cell aggregate according to claim 3, wherein the posterior region includes tail bud (TB)-like cells.

5. The aforementioned apex-bottom axis is defined by the apex region and the bottom region, i) The apical region of the cell has lower expression of one or more markers selected from the group consisting of fibronectin, collagen V, and laminin, compared to the basolateral region of the cell. i) The cell aggregate according to claim 1 or 2, wherein the apical region of the cell has high expression of one or more markers selected from the group consisting of aPKC, CDH2, Ezrin, ZO1, and F-actin, compared to the basolateral region of the cell.

6. The cell aggregate according to claim 1 or 2, wherein the mesodermal cells express a marker selected from the group consisting of BXT, SOX2, NODAL, WNT3a, WNT5a, DLL1, TCF15, MEOX1, TBX18, UNCX, ALDH1A2, RDH10, RIPPLY1, RIPPLY2, MESP1, MESP2, HES7, TBX6, MSGN1, and FLK1 / KDR.

7. The cell aggregate according to claim 2, wherein the cell aggregate can reconstruct various aspects of somite formation and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite-like structures), and oscillation of the segmental clock, under somite-forming culture conditions.

8. The cell aggregate according to claim 1 or 7, wherein the somite-forming culture conditions include a gel or matrix and a retinoid, retinoic acid, a retinoic acid precursor or derivative thereof, and / or a retinoic acid receptor (RAR) agonist.

9. The cell aggregate according to claim 8, wherein the cell aggregate is embedded in a gel or matrix, or is located inside a gel or matrix.

10. The cell aggregate according to claim 1 or 2, which is substantially free of endodermal cells and / or ectoderm cells.

11. The cell aggregate according to claim 1 or 2, wherein the expression of the segmental clock gene is subject to gene oscillation.

12. The cell aggregate according to claim 11, wherein the segmental clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3, and HES7.

13. A method for producing three-dimensional cell aggregates in vitro from pluripotent stem cells, comprising the following steps: (a) A step of culturing pluripotent stem cells to induce a three-dimensional cell aggregate containing mesodermal cells; and (b) A step of culturing the cell aggregate containing the mesodermal cells to induce a three-dimensional cell aggregate, Includes, The method wherein the three-dimensional cell aggregate is the cell aggregate according to claim 1 or 2.

14. The following steps: (a1) A step of culturing the pluripotent stem cells in a medium containing a GSK3β inhibitor and FGF to initiate commitment to primitive streaks and mesoderm fate, and / or induce mesoderm cells; (a2) A step of culturing cells derived from, obtained, or obtainable from step (a1) in a medium containing a WNT agonist (GSK3β inhibitor), an FGF agonist, a TGFβ inhibitor, and a ROCK inhibitor, to induce a three-dimensional cell aggregate containing the mesodermal cells; and optionally, (b) The method according to claim 13, comprising the step of culturing the three-dimensional cell aggregates, including mesodermal cells, in a medium comprising a retinoid, retinoic acid, a retinoic acid derivative and / or a retinoic acid receptor (RAR) agonist in the presence of a gel or matrix, thereby inducing morphogenesis and / or self-organization of the three-dimensional cell aggregates.

15. The method according to claim 13, wherein the proportion of mesodermal cells in the cell aggregate containing the mesodermal cells is, based on the number of cells, 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%.

16. The method according to claim 13, wherein the mesoderm cells express a marker selected from the group consisting of TBXT, SOX2, MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WNT5a, DLL1, MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, HES7, MSGN1, TCF15, MEOX1, and FLK1 / KDR.

17. Cells obtained from the cell aggregate described in claim 1 or 2.

18. A method for producing progenitor cells or differentiated cells, comprising the step of culturing the cell aggregate described in claim 1 or 2 and inducing progenitor cells or differentiated cells selected from the group consisting of (a) to (i) below: (a) Neuronal mesodermal cells or their precursor cells; (b) Myocytes or their precursor cells; (c) Osteocytes or their precursor cells; (d) Chondrocytes or their precursor cells; (e) Tendon cells or their precursor cells; and (f) Endotome or endothelium or hematopoietic cells or their progenitor cells; (g) Adipocytes or their precursor cells, including white, beige, and brown cells; (h) Dermal cells or their precursor cells; and (i) Neural tube cells or their progenitor cells.

19. A method for evaluating a test substance, comprising the following steps: A step of culturing a test substance in the presence of a three-dimensional cell aggregate; and The process includes evaluating the three-dimensional cell aggregates after the culture, The three-dimensional cell aggregate is the cell aggregate described in claim 1 or 2. Herein, during evaluation, test substances that alter the polarity of the cell aggregates, the morphology of the cell aggregates, and / or the size of the cell aggregates are selected as candidate substances that modify, promote, or inhibit the polarity of the cell aggregates, the morphology of the cell aggregates, and / or the size of the cell aggregates, in a method.

20. A method for evaluating gene function or genome sequence function, comprising the following steps: A step of preparing pluripotent stem cells modified with a test gene or test genome sequence; A step of producing a three-dimensional cell aggregate from the aforementioned pluripotent stem cells; and This includes a step of evaluating the three-dimensional cell aggregates after culturing, The three-dimensional cell aggregate is prepared by the method described in claim 13. Herein, during evaluation, test genes or test genome sequences that alter the polarity, morphology, and / or size of the cell aggregates are evaluated as candidate genes or candidate genome sequences that modify, promote, or suppress the polarity, morphology, and / or size of the cell aggregates, in a method.

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