Method for producing primitive nodule cells

Abstract

To provide a method for efficiently inducing differentiation in vitro of spherical human cell aggregates containing axial mesodermal cells from human pluripotent stem cells, in order to realize the transplant application to patients with degenerative disc disease; and to provide the spherical human cell aggregates containing axial mesodermal cells induced to differentiate in vitro.SOLUTION: A spherical human cell aggregate has the following characteristics: (1) it has two distinct regions, a primitive node area and a non-node area; (2) the primitive node area and the non-node area have a structure consisting of at least two layers; (3) axial mesoderm cells are present in the first layer on the surface of the primitive node area, and endoderm cells are substantially absent in the primitive node area; and (4) endoderm cells are present in the first layer on the surface of the non-node area.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently inducing differentiation in vitro of human pluripotent stem cells into spherical human cell aggregates containing axial mesoderm cells, and to spherical human cell aggregates containing axial mesoderm cells that have been induced to differentiate in vitro. [Background technology]

[0002] Approximately 13 million people in Japan suffer from lower back pain, and 20-40% of cases are thought to be caused by intervertebral disc degeneration. Intervertebral disc degeneration is thought to begin in many cases with the degeneration or loss of the nucleus pulposus. The nucleus pulposus is the tissue that makes up the central part of the intervertebral disc, providing cushioning and mobility to the spine, but once degenerated, the nucleus pulposus does not revert. There was hope for the development of regenerative therapy using nucleus pulposus transplantation as a means of treating intervertebral disc degeneration, and it has recently been reported that the function of the nucleus pulposus can be complemented by creating a chondroid nucleus pulposus from human iPS cells and replacing the nucleus pulposus in vivo (Non-Patent Document 1).

[0003] In models of nucleus pulposus differentiation, it is believed that in vivo, cells differentiate from the epiblast through gastrulation into axial mesoderm, which then differentiates into the primitive node and notochord, before finally developing into the nucleus pulposus. However, there have only been a few reports of inducing differentiation of mouse or human ES / iPS cells into node cells or notochord cells, which are precursor cells of nucleus pulposus cells (Non-Patent Documents 2 and 3), and the efficiency of these efforts has been low. There have been no reports of efficient differentiation into node cells and notochord cells, or their precursor cells, the axial mesoderm. Therefore, it has not yet been possible to produce nucleus pulposus from the cells of a patient with intervertebral disc degeneration and transplant it into the patient. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Takahashi et al., Biomaterials, 2022, May;284:121491 [Non-patent document 2] Loh et al., Cell, 2016, July;14:451-467 [Non-patent document 3] Rito et al., bioRxiv, 2023, Feb, 28 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a method for efficiently inducing differentiation in vitro of spherical human cell aggregates containing axial mesoderm cells from human pluripotent stem cells, and spherical human cell aggregates containing axial mesoderm cells that have been induced to differentiate in vitro, in order to realize transplant application to patients with intervertebral disc degeneration. [Means for solving the problem]

[0006] The present inventors have demonstrated that the combination of 3D culture technology and an optimal culture medium efficiently induces differentiation of hESCs / iPSCs into aggregates containing axial mesoderm cells, and further confirmed that the induced axial mesoderm possesses structural and functional properties similar to those seen in vivo. Furthermore, the present inventors confirmed through single-cell RNA sequencing (scRNA-seq) analysis that various cell types derived from ectoderm, endoderm, and mesoderm are present in the aggregates containing axial mesoderm cells. They also found that the aggregates containing axial mesoderm cells contain not only axial mesoderm but also its surrounding regions, recapitulating the development of the corresponding parts of the embryo. Based on these findings, the present inventors conducted further research and completed the present invention.

[0007] That is, the present invention is as follows. [1] Spherical human cell aggregates with the following characteristics: (1) It has two distinct regions: the primitive node area and the non-node area. (2) The primitive nodule region and the non-primitive nodule region have at least two or more layer structures, (3) primitive node cells are present in the first layer present on the surface of the primitive node region, and endoderm cells are substantially absent in the primitive node region; (4) Endodermal cells are present in the first layer, which is present on the surface of the non-primitive nodal region. [2] The human cell aggregate described in [1], wherein the primitive nodule cells are FOXJ1-positive cells. [3] A human cell aggregate according to [1] or [2], wherein FOXJ1-positive cells are present at a rate of approximately 10% to 20% of the total cell number in the cell aggregate. [4] A human cell aggregate according to any one of [1] to [3], wherein the primitive nodule cells are cells that express FOXJ1, BRA, and FOXA2. [5] A human cell aggregate described in any one of [1] to [4], wherein primary cilia extend from the primitive nodule cells toward the outside of the cell aggregate. [6] The human cell aggregate according to any one of [1] to [5], wherein the endoderm cells are SOX17-positive cells. [7] A human cell aggregate described in any one of [1] to [6], wherein the first layer present on the surface is composed of a cell layer having a thickness equivalent to 1 to 10 cells. [8] A human cell aggregate described in any one of [1] to [7], in which axial mesoderm cells are present in the second layer inside the non-primitive node region, epiblast cells are present in the third layer further inside, and epiblast cells are present in the second layer inside the primitive node region. [9] The human cell aggregate according to [8], wherein the epiblast cells are OCT4-positive cells.

[10] A human cell aggregate described in any one of [1] to

[19] , in which a basement membrane is present between the first layer present on the surface and the second layer present inside in the primitive nodule region and / or non-primitive nodule region.

[11] The human cell aggregate described in

[10] , in which the basement membrane is continuously present in the non-primitive nodule region.

[12] The human cell aggregate according to

[10] or

[11] , wherein the basement membrane is laminin-positive.

[13] A human cell aggregate described in any one of [1] to

[12] , wherein the primitive nodule cells express one or more genes selected from the group consisting of CHORDIN, NOGGIN, and SHH. [13-2] A human cell aggregate according to any one of [1] to

[13] , which is derived from pluripotent stem cells.

[14] (1) a first step of rapidly aggregating dispersed human pluripotent stem cells to form a single cell aggregate in a single culture compartment; (2) a second step of culturing the formed cell aggregates of human pluripotent stem cells in suspension in a medium containing a basement membrane preparation; (3) a third step of further suspension-culturing the cell aggregates obtained in the second step in a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator; and (4) a fourth step of further suspension-culturing the cell aggregates obtained in the third step in a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor; A method for producing human cell aggregates comprising axial mesoderm cells.

[15] The method of producing a basement membrane preparation according to

[14] , wherein the basement membrane preparation is Matrigel.

[16] The method of manufacturing according to

[14] or

[15] , wherein in the first step, cell aggregates are formed by the SFEBq method.

[17] The method according to any one of

[14] to

[16] , wherein in the first step, cell aggregates are formed in the presence of a Rock inhibitor.

[18] The method according to any one of

[14] to

[17] , wherein the culture medium in the first step and the second step contains a factor for maintaining undifferentiation.

[19] The method of producing according to

[18] , wherein the culture medium in the third and fourth steps contains a factor for maintaining undifferentiation.

[20] The method according to any one of

[14] to

[19] , wherein the culture period in the second step is 2 to 4 days.

[21] The method according to any one of

[14] to

[20] , wherein the Wnt signaling pathway activator is a GSK3 inhibitor.

[22] The method of producing according to

[21] , wherein the GSK3 inhibitor is CHIR99021.

[23] The method according to any one of

[14] to

[22] , wherein the TGFβ signaling pathway activator is Activin A.

[24] The method for production according to any one of

[14] to

[22] , wherein the TGFβ signaling pathway inhibitor is an ALK5 inhibitor, and the BMP signaling pathway inhibitor is an ALK2 / 3 inhibitor.

[25] The manufacturing method described in

[24] , wherein the ALK5 inhibitor is SB431542 and the ALK2 / 3 inhibitor is LDN-193189.

[26] Spherical human cell aggregates with the following characteristics: (1) FoxA1-positive endodermal cells are present in the outermost layer of the cell aggregates. (2) Multiple axial mesoderm cells are present inside the cell aggregate, and the floor plate of the neural tube and ventral somites are present around them. (3) Two or more of the structures described in (2) are present within a single cell aggregate.

[27] A human cell aggregate according to

[26] , wherein the floor plate of the neural tube is composed of cells expressing NKX6.1 and / or ARX, and the ventral somites are composed of cells expressing Nkx3.2 and / or PAX9.

[28] A human cell aggregate described in

[26] or

[27] , wherein the outermost layer is a cell layer having a thickness corresponding to 1 to 10 cells.

[29] A human cell aggregate described in any one of

[26] to

[28] , wherein the inside of the cell aggregate is approximately 200 μm to 300 μm from the outermost layer.

[30] The human cell aggregate according to any one of

[26] to

[29] , wherein the axial mesoderm cells are BRACHYRY-positive cells.

[31] A human cell aggregate described in any one of

[26] to

[30] , in which axial mesoderm cells are present in a group of 5 to 50 cells inside the cell aggregate.

[32] A human cell aggregate described in any one of

[26] to

[31] , in which axial mesoderm cells are gathered within a range of approximately 30 to 40 μm in length, width, and depth.

[33] A human cell aggregate according to any one of

[26] to

[32] , wherein the number of neural tubes and somites is 1 or more and 3 or less, respectively. [Effects of the Invention]

[0008] The present invention provides a method for efficiently inducing differentiation in vitro of human pluripotent stem cells into spherical human cell aggregates containing axial mesoderm cells. The present invention also makes it possible to realize transplantation therapy for patients with intervertebral disc degeneration by using spherical human cell aggregates containing axial mesoderm cells. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 illustrates the differentiation of hESCs into axial mesoderm. (A) Predictive model for lineage separation of paraxial mesoderm (PM) and axial mesoderm (AM) arising from the anterior primitive streak (APS). Whether axial mesoderm arises from the APS was previously unknown. UD: Undifferentiated state. Representative marker genes for each lineage are also shown. (B) Schematic diagram of the differentiation procedure using CDM2 medium for inducing cell differentiation under 2D or 3D culture conditions. See Figure 2B for detailed procedures. SF: StemFit medium, CDM2: CDM2 medium, AC: Activin A + CHIR, CLS: CHIR + LDN + SB, MG: Matrigel. (C) RT-qPCR analysis of various lineage marker genes in hESCs undergoing second differentiation. Increased expression of the axial mesoderm marker gene FOXJ1 was observed only under 3D culture conditions. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of None in 2D culture was set to 1.0. UD: undifferentiated state, APS: anterior primitive streak, PM: paraxial mesoderm, AM: axial mesoderm, DE: definitive endoderm. (D) Schematic diagram of the differentiation procedure during cell differentiation induction under 3D culture conditions using CDM2, E6, or SF medium. (E) RT-qPCR analysis of the PM marker gene TBX6 and the AM marker genes FOXJ1 and NOTO in hESCs undergoing secondary differentiation under 3D culture conditions. SF medium showed the highest expression of AM marker genes. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of None treated with CDM2 medium is set to 1.0. [Figure 2]Figure 2 shows that induction of axial mesoderm differentiation is accompanied by definitive endoderm differentiation. (A) Definitive endoderm (DE) can also be induced from the anterior primitive streak (APS). UD: undifferentiated state. PM: axial mesoderm. AM: axial mesoderm. Representative marker genes for each lineage are shown. (B) Detailed schematic diagram of Figure 1B showing the culture procedures, media, and components used to obtain none and secondary differentiation products. SF: StemFit medium, CDM2: CDM2 medium, Y: Y-27632, MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (C) The same schematic diagram as Figure 1B, along with phase-contrast images of hESCs cultured under these conditions. The number of days in culture is indicated in the upper right corner of each image. (D) RT-qPCR analysis of the DE marker gene SOX17 in hESCs cultured according to the procedures in Figures 1B and 2C. SOX17 expression was elevated only in the secondary differentiation products of 3D culture. ΔCt values ​​were calculated by subtracting the Ct values ​​of SOX17 from the Ct values ​​of β-ACTIN and are shown as the mean ± standard deviation of triplicate experiments (n = 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of None in 2D culture is set to 1.0. (E) The same schematic diagram as in Figure 1D, with phase-contrast images of hESCs cultured under these conditions. The number of days of culture is indicated in the upper right corner of each image. (F) RT-qPCR analysis of lineage marker genes in hESCs cultured according to the procedures in Figures 1D and 2E. High expression of DE marker genes was observed when SF medium was used. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of triplicate experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of None in CDM2 medium was set to 1.0. ND: Not detected. [Figure 3]Figure 3 shows the axial mesoderm differentiation of other pluripotent cell lines. (A) Schematic diagram of the differentiation procedure during cell differentiation induction under 3D culture conditions using Essential 6 (E6) medium or StemFit (SF) medium. MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (B) Phase-contrast images of three hESC / iPSC lines (hESC line: KhES-1, hiPSC line: 253G1, hiPSC line: 201B7) cultured under the conditions in (A). The number of days in culture is indicated in the upper right corner of each image. (C) RT-qPCR analysis of the paraxial mesoderm (PM) marker gene TBX6 and the axial mesoderm (AM) marker genes FOXJ1 and NOTO in the three hESC / iPSC lines. Similar gene expression patterns were observed in all three cell lines. The delta Ct values ​​were calculated by subtracting the Ct values ​​of each gene from the Ct value of β-actin, and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of None in the KhES-1 cell line treated with E6 medium was set to 1. [Figure 4]Figure 4 shows that axial mesoderm differentiation products rarely differentiate into early somites. (A) Lineage stage model for early somites (ES). ES is induced from paraxial mesoderm (PM) by tertiary differentiation (3rd differentiation) using LSIP (LDN, SB, IWR, PD). However, the differentiation products induced from axial mesoderm (AM) by tertiary differentiation are unknown and are indicated by "?". UD: undifferentiated state; APS: anterior primitive streak. (B) Schematic diagram of the differentiation procedure for tertiary differentiation using Essential 6 (E6) medium or StemFit (SF) medium under 3D culture conditions. When E6 medium is used, PM is effectively induced during secondary differentiation (2nd differentiation), but axial mesoderm (AM) is hardly induced. On the other hand, the opposite is true when SF medium is used. MG: Matrigel; AC: Activin A + CHIR; CLS: chir + ldn + sb; LSIP: LDN + SB + IER + PD. (C) Phase-contrast images of hESCs cultured under the conditions described in (B). The number of days of culture is indicated in the upper right corner of each image. (D) RT-qPCR analysis of the ES marker genes FOXC2 and MEOX1, as well as other lineage marker genes, in hESCs cultured under the same conditions as in (B). When cells were differentiated into axial mesoderm using SF medium, the expression of ES marker genes was significantly lower than when cells were cultured in E6 medium after the third spreading. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN, and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change), with the expression level of None treated with E6 medium set at 1.0. ND: Not detected. [Figure 5]Figure 5 shows the evaluation of the necessity of each component used in cell differentiation. (A) Schematic diagram of the axial mesoderm differentiation procedure. StemFit (SF) medium was used throughout the culture period, with the following components: Y-27632 (Y) on days 0-1, Matrigel (MG) on days 1-4, Activin A + CHIR (AC) on days 4-5, and CHIR + LDN + SB (CLS) on days 5-6. (B) Evaluation of the necessity of Y-27632 treatment on days 0-1. Phase-contrast images on day 1 of hESCs cultured without Y treatment (Y(-)) and with Y treatment (Y(+)). Y(-) induces cell death and does not form cell aggregates. The number of days of culture is indicated in the upper right corner of each image. (C) Evaluation of the necessity of Matrigel (MG) treatment on days 1-4. Phase contrast images of hESCs on day 4 (before differentiation induction [Before diff.]), day 5 (after primary differentiation [1st diff.]), and day 6 (after secondary differentiation [2nd diff.]). MG(-) indicates cultured without MG treatment, and MG(+) indicates cultured with MG treatment. The number of days of culture is indicated in the upper right corner of each image. (D) RT-qPCR analysis of lineage marker genes expressed in hESCs cultured under the same conditions as in (C). Expression of the axial mesoderm (AM) marker genes FOXJ1 and NOTO was higher in MG(+) than in MG(-) on days 4-5 (before differentiation induction [Before diff.]), 5 (after primary differentiation [1st diff.]), and 6 (after primary differentiation [1st diff.]). ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of the product before differentiation induction was set to 1.0 after MG(+) treatment. UD: undifferentiated, APS: anterior primitive streak, PM: paraxial mesoderm, DE: definitive endoderm. (E) Activin expression during primary differentiation on days 4-5. The necessity of A and CHIR, and the necessity of CHIR, LDN, IWRI, and SB during secondary differentiation on days 5-6 were examined. RT-qPCR analysis of lineage marker genes expressed in hESCs induced to differentiate with each compound combination. The highest expression of the AM marker gene FOXJ1 was observed when cells were treated with all components. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN, and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3).The numbers above each bar indicate the relative expression level (fold change) when the expression level of the product before differentiation induction in MG(+) was set to 1.0. ND: Not detected. [Figure 6] Figure 6 shows that axial mesoderm differentiation can be induced more effectively in 3D culture than in 2D culture. (A) Schematic diagram of the axial mesoderm differentiation procedure using StemFit (SF) medium, showing the culture period under 2D or 3D culture conditions. AC: Activin A + CHIR, CLS: CHIR + LDN + SB, MG: Matrigel. (B) Phase-contrast images of hESCs (none), primary differentiated (1st differentiation), and secondary differentiated (2nd differentiation) following the procedure in (A). The number of days in culture is indicated in the upper right corner of each image. (C) RT-qPCR analysis of axial mesoderm (AM) marker genes FOXJ1 and NOTO, as well as other lineage marker genes, in 2D or 3D cultured hESCs. Expression of axial mesoderm marker genes was observed to be higher in 3D culture than in 2D culture. The ΔCt value was calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN, and is shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The number above each bar indicates the relative expression level (fold change) when the expression level of None in 2D culture is set to 1.0. (D) Immunostained images of the AM marker FOXJ1 in 2D or 3D culture secondary differentiation products on days 3 or 6. (E) Quantification of the percentage of FOXJ1-positive areas in hESCs cultured under the same conditions as in (D). The ratio of the FOXJ1-positive area to the DAPI-positive area was calculated. Values ​​are shown as the mean ± standard deviation of four experiments (n = 4). [Figure 7-1] Figure 7 shows immunostaining of hESCs differentiated into axial mesoderm in 2D or 3D culture. (A) Immunostaining images of FOXJ1 (axial mesoderm marker), BRA (axial mesoderm, primitive streak, and pan-mesoderm marker), and FOXA2 (axial mesoderm, primitive streak, and mesoderm marker) in undifferentiated (non-differentiated) and secondarily differentiated (secondary differentiated) hESCs clustered using the procedure shown in Figure 6A. The 2D culture products are from day 3 of differentiation, and the 3D culture products are from day 6 of differentiation. (B) Immunostaining images of OCT4 (undifferentiated epidermal marker), SOX17 (definitive endoderm marker), and FOXA2. [Figure 7-2] Figure 7 shows immunostaining of hESCs differentiated into axial mesoderm in 2D or 3D culture. (C) Immunostaining images of FOXJ1, TBX6 (a paraxial mesoderm marker), and CDX2, which is expressed in the extraembryonic mesoderm, posterior epiblast, and node of mouse embryos. [Figure 8] Figure 8 shows single-cell RNA-seq analysis of the products from the first (day 5) and second (day 6) rounds. (A) UMAP analysis of cells positive for the stem cell marker UTF1, the primitive streak and pan-mesoderm marker TBXT (same as BRA), and the mesoderm marker LHX1 at primary differentiation (day 5). (B) Cluster analysis of the products from the first (day 5) and second (day 6) rounds combined. Left panel: s1: primary differentiation (day 5), s2: secondary differentiation. Right panel: 11 clusters classified by gene expression. (C) Heatmap of lineage marker gene expression in each cluster shown in the right panel of (B). (D) Location of each cell in the lineage groups, including the primitive streak (PS), anterior primitive streak (APS), axial mesoderm (AxM), endoderm (Endo), and early paraxial somitic mesoderm (same as paraxial mesoderm) (PSM). (E) The location of individual cells expressing lineage marker genes, such as UTF1, the endoderm marker SOX17, the early somitic marker FOXC2, and the axial mesoderm (primitive node) marker NOTO. (F) Predicted differentiation pathways derived from pseudo-time series analysis. Undifferentiated epiblast cells differentiate not only into APS cells but also into axial mesoderm progenitor cells (AMP). Endo and PSM cells are formed from APS, and AxM cells are formed from AMP. (G) Heatmap showing the expression levels of each gene over time. Cluster 1: XXX, Cluster 2: YYY, Cluster 3: ZZZ. (H) Comparison with single-cell RNA-seq analysis of early human embryos (Tyser et al., Nature, 2021, Dec;600(7888):285-289). Left panel: Dots from Tyser's study overlap with dots from a clustering analysis combining the results of Tyser's study and this example. Right panel: Clustering dots from a combination of Tyser and this example overlaid with dots from this example: Endoderm, Epi: epiblast, AxM: axial mesoderm, NE: neuroectoderm, PS: primitive streak, SM: somitic mesoderm. [Figure 9] Figure 9 shows the characteristics of primitive nodes observed in cell aggregates differentiated into axial mesoderm. (A) Immunostained images of hESC aggregates on day 6 after secondary differentiation (2nd differentiation) under 3D culture conditions. These gene expression patterns recapitulate those observed in primitive nodes in vivo. Top panel: Immunostained images for FOXJ1 (axial mesoderm marker), BRA (axial mesoderm, primitive ganglion, and pan-mesoderm marker), and FOXA2 (axial mesoderm and mesoderm marker). Bottom panel: Immunostained images for OCT4 (undifferentiated epiblast marker), SOX17 (definitive endoderm marker), and FOXA2. (B) Immunostained images for cilia markers Ac-α-Tubulin, FOXJ1, and SOX17 on the surface of cell aggregates induced by axial mesoderm differentiation. Gray arrows indicate primary cilia. (C) Scanning electron microscopy images of cell aggregates induced by axial mesoderm differentiation. Gray arrows indicate primary cilium. (D) RT-qPCR analysis of the BMP antagonist secreted factor genes CHIRDIN and NOGGIN, and the ventralization factor Sonic hedgehog (SHH), in hESCs differentiated into various lineages. These genes were most highly expressed in axial mesoderm (AM) cells. ΔCt values ​​were calculated by subtracting the Ct value of each gene from that of β-ACTIN. The values ​​are expressed as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of the UD product is set to 1.0. UD: undifferentiated state; AM: axial mesoderm; PM: paraxial mesoderm; DE: definitive endoderm. [Figure 10] Figure 10 shows immunostaining analysis of lineage markers, apical markers, and basal markers in cell aggregates differentiated into axial mesoderm. (A) Immunostaining images of day 6 hESC aggregates differentiated into non-differentiated (Non-Diff.) and secondarily differentiated (2nd Diff.) under 3D culture conditions. FOXJ1: axial mesoderm marker; BRA: axial mesoderm, primitive streak, and pan-mesoderm marker; FOXA2: axial mesoderm, primitive streak, and mesoderm marker; SOX17: definitive endoderm marker; OCT4: undifferentiated epiblast marker; LAMININ: basal marker; EZRIN: apical marker. (B) Enlarged image of the non-nodal area enclosed in the gray box in (A). (C) Enlarged image of the nodal area enclosed in the gray box in (A). [Figure 11] Figure 11 shows the expression of cilia marker proteins on the surface of cell aggregates differentiated into axial mesoderm. (A) Low-magnification image of Figure 12B, (B) Low-magnification image of Figure 12C, (C) Low-magnification image of Figure 8C. [Figure 12] Figure 12 shows the expression of other secretory factor genes. (A) RT-qPCR analysis of each lineage marker gene in hESCs differentiated into each lineage. Cells differentiated into each lineage specifically express the lineage marker gene of interest. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of the UD product is set to 1.0. UD: undifferentiated state, AM: axial mesoderm, PM: paraxial mesoderm, DE: definitive endoderm. (B) RT-qPCR analysis of secretory factor genes known to be expressed during early development. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of the UD product is set to 1.0. [Figure 13]Figure 13 shows the confirmation of the ability of axial mesoderm to induce neural differentiation and ventralization. (A) Schematic diagram of the long-term axial mesoderm differentiation procedure. Days 6, 8, 11, and 14 are sample collection days. SF: StemFit medium, MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (B) RT-qPCR analysis of the axial mesoderm (AM) marker genes FOXJ1 and NOTO in hESCs undergoing long-term axial mesoderm differentiation. Expression of these genes peaked on day 6 and decreased over time. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change), with the expression level of undifferentiated products at day 6 set at 1.0. (C) RT-qPCR analysis of the BMP antagonist genes CHIRDIN and NOGGIN. The ventralization factor gene Sonic hedgehog (SHH) in hESCs undergoing long-term axial mesoderm differentiation. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN. The values ​​are expressed as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of undifferentiated products on day 6 is set to 1.0. (D) Schematic diagram of the experiment confirming the neuronal differentiation-inducing ability and ventralization activity using culture supernatants. CM_Non and CM_AM represent culture supernatants from cell aggregates that had not differentiated into axial mesoderm and differentiated cell aggregates, respectively. (E) RT-qPCR analysis of the early neuroectoderm marker genes PAX6, SIX2, and RX in hESCs cultured in the supernatants. The delta Ct values ​​were calculated by subtracting the Ct values ​​of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of hESCs maintained in an undifferentiated state in fresh StemFit medium is set to 1.0. SF+LDN refers to SF medium supplemented with LDN, and hESCs treated with this medium were used as a positive control for neural differentiation induction by BMP signaling inhibition. (F) RT-qPCR analysis of the SHH target genes HHIP and PTCH1 in hESCs cultured in the supernatant.The delta Ct values ​​were calculated by subtracting the Ct values ​​of each gene from the Ct value of β-ACTIN, and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of hESCs maintained in an undifferentiated state in fresh StemFit medium is set to 1.0. [Figure 14] Figure 14 shows changes in morphology and gene expression in cell aggregates undergoing long-term axial mesoderm differentiation. (A) The same schematic diagram as in Figure 13A, along with phase-contrast images of hESCs cultured according to this procedure. The number of days of culture is indicated in the upper right corner of each image. Days 6, 8, 11, and 14 are days for observation and sample collection. SF: StemFit medium; MG: Matrigel; AC: Activin A + CHIR; CLS: CHIR + LDN + SB. (B) RT-qPCR analysis of lineage marker genes in hESCs undergoing long-term axial mesoderm differentiation. Expression peaked on day 6 and then decreased for all genes. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change), with the expression level of undifferentiated products on day 6 set at 1.0. APS: anterior primitive streak, PM: paraxial mesoderm, AM: axial mesoderm, DE: definitive endoderm. [Figure 15] Figure 15 shows the expression of other early neuroectoderm marker genes in hESCs cultured in the supernatant of cell aggregates differentiated into axial mesoderm. RT-qPCR analysis of the early neuroectoderm marker genes OTX2, ZNF521, and OCT6. hESCs cultured in CM_non and CM_AM represent the supernatants of undifferentiated and differentiated cell aggregates, respectively. ΔCt values ​​were calculated by subtracting the Ct value of each gene from the Ct value of β-ACTIN and are shown as the mean ± standard deviation of three or more experiments (n ≥ 3). The numbers above each bar indicate the relative expression level (fold change) when the expression level of hESCs maintained in an undifferentiated state in fresh StemFit medium is set to 1.0. SF+LDN refers to SF medium supplemented with LDN. hESCs treated with this medium were used as a positive control for neural differentiation induction via BMP signaling inhibition. [Figure 16]Figure 16 shows single-cell RNA-seq analysis of long-term axial mesoderm differentiation products (day 11). (A) Cluster analysis of long-term cultured cell aggregates at day 11. Gene expression patterns were classified into nine clusters. (B) The location of cells expressing lineage marker genes such as TBXT is the same as BRA (early and late axial mesoderm, primitive streak, and pan-mesoderm), POU5F1 (same as OCT4) (undifferentiated epiblast), FOXJ1 (early axial mesoderm), FOXA1 (pan-endoderm), GSC (anterior axial mesoderm called the prechordal plate), ARX (floor plate of the neural tube), LBH1 (mesoderm), and secreted factors expressed in the node, CHRD (BMP antagonist) and SHH (ventralizing factor). (C) The location of individual cells expressing somite-related genes such as FOXC2 (early somites), NKX3-2, SOX9, and SCX (ventral somites), and the SHH target genes HHIP and PTCH1. [Figure 17]Figure 17 shows that long-term cultured cell aggregates differentiated into axial mesoderm contain cells contained in the ventral neural tube and ventral somites. (A) Schematic diagram of the long-term axial mesoderm differentiation procedure. Days 6, 8, 11, 14, and 18 are sample collection days. SF: StemFit medium, MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (B) RT-qPCR analysis of the late axial mesoderm marker gene BRA. Values ​​are shown as means ± standard deviations of triplicate experiments (n = 3). (C) RT-qPCR analysis of the ventralizing factor gene SHH. Values ​​are shown as means ± standard deviations of triplicate experiments (n = 3). (D) Immunostaining images of BRA and SHH (day 11). The lower panel is a partial enlargement of the upper panel. (E) RT-qPCR analysis of the neural tube floor plate markers NKX6.1 and ARX. Values ​​are shown as means ± standard deviations of triplicate experiments (n = 3). (F) RT-qPCR analysis of ventral somite markers NKX3.2 and PAX9. Values ​​are shown as means ± standard deviations of triplicate experiments (n = 3). (G) Top and middle rows: Immunostained images of NKX6.1, BRA, and SHH (day 18). The middle row is a magnified view of a portion of the top row. Bottom row: Immunostained images of both floorplate markers NKX6.1 and ARX (day 18). (H) Immunostained images of NKX3.2 and NKX6.1 (day 18). (I) RT-qPCR analysis of SHH and SHH target genes HHI and PTCH1 in long-term cultured hESCs (day 14) treated with the SHH signaling pathway inhibitor cyclopamine (Cyc) from day 8. Values ​​are shown as means ± standard deviations of triplicate experiments (n = 3). (J) RT-qPCR analysis of floor plate marker NKX6.1 and roof plate marker PAX6 in long-term cultured hESCs (day 14) treated with cyclopamine from day 8. Values ​​are shown as mean ± standard deviation of triplicate experiments (n = 3). (K) RT-qPCR analysis of ventral somite marker NKX3.2 and dorsal somite marker MYOD in long-term cultured hESCs (day 14) treated with cyclopamine from day 8. Values ​​are shown as mean ± standard deviation of triplicate experiments (n = 3). (L) Comparison of short-term (day 6) and long-term (days 11-18) cultured cell aggregates generated in this example with corresponding human embryos. [Figure 18]Figure 18 shows differentiation into neuroectoderm, somites, and intestine observed after long-term induction of metabolic mesoderm. (A) Schematic diagram of the differentiation induction procedure and phase-contrast images of cell-length aggregates from uninduced (Non-diff.) and differentiated axial mesoderm (AM diff.). Days 6, 8, 11, 14, and 18 are sample collection days. SF: StemFit medium, MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (B) RT-qPCR analysis of midbrain, mid-hindbrain, and hindbrain marker genes. Values ​​are shown as means ± standard deviation. From three or more experiments (n ≥ 3). (C) RT-qPCR analysis of somite marker genes. Values ​​are shown as means ± standard deviation. From three or more experiments (n ≥ 3). (D) RT-qPCR analysis of pan-endodermal marker genes. Values ​​are shown as means ± standard deviation. From three or more experiments (n ≥ 3). (E) RT-qPCR analysis of foregut, hindgut, and midgut / hindgut marker genes. Values ​​are shown as mean ± standard deviation. n≥3 experiments. (F) Immunostaining for the general endoderm marker FOXA1 (pan-endodermal marker, Foxa1) (day 14). FOXA1-positive cells are present in the cell layer on the surface of the aggregates. (G) Immunostaining for the basement membrane marker LAMININ (day 14). Gray arrows indicate the basement membrane surrounding the BRA-positive notochord-like cell population. [Figure 19] Figure 19 shows that administration of the SHH signaling pathway inhibitor cyclopamine inhibits somite ventralization. (A) Schematic diagram of differentiation induction and cyclopamine (Cyc) treatment procedures, and phase-contrast images of elongated cell aggregates untreated (-Cyc) and treated (+Cyc) with cyclopamine. Days 6, 8, 11, 14, and 18 are sample collection days. SF: StemFit medium, MG: Matrigel, AC: Activin A + CHIR, CLS: CHIR + LDN + SB. (B) RT-qPCR analysis of the floor plate marker gene ARX. (C) RT-qPCR analysis of the ventral somite brain marker gene PAX9. [Figure 20]Figure 20 shows a lineage separation model of axial mesoderm. This example suggests a lineage separation model of axial mesoderm (AM) differentiation. Undifferentiated stem cells differentiate into axial mesoderm via precursor cells (here, axial mesoderm precursor cells (AMP)) that arise from a pathway other than anterior gastrulation (APS). UD: undifferentiated state, APS: anterior gastrulation, PM: paraxial mesoderm, DE: definitive endoderm, AMP: axial mesoderm precursor cells, AM: axial mesoderm. DETAILED DESCRIPTION OF THE INVENTION

[0010] 1. Cell aggregates of the present invention The present invention provides spherical cell aggregates (hereinafter referred to as the cell aggregates of the present invention). In this specification, the origin of the cell aggregates is not particularly limited, but mammals are preferred, and include rodents, ungulates, cats, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Carnivores include dogs, cats, etc. Lagomorphs include rabbits, etc. Primates refer to mammals belonging to the order Primates, and include prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans. In one embodiment, the cell aggregates of the present invention are spherical human cell aggregates.

[0011] As used herein, the term "cell aggregate" refers to a mass formed by the assembly of cells, in which the cells are adhered to one another. Embryoid bodies, spheres, spheroids, and organoids are also included in cell aggregates. In cell aggregates, cells are preferably adhered to one another at their surfaces. In one embodiment, cells are adhered to one another in part or all of the cell aggregate, forming, for example, adherens junctions. In another embodiment, two or more cell aggregates can be further artificially adhered or aggregated together. Masses in which cell populations are further adhered or aggregated together, and assembloids are also included in cell aggregates. The shape of the cell aggregate is not limited to a spherical shape, and may be, for example, a bisphere, a beaded shape, a spherical aggregate, a string-like or branched shape (the shapes described in Scientific Reports, 11:21421 (2021) and Patent Application No. 2021-078154), etc.

[0012] In one embodiment, the cell aggregate of the present invention may have a major axis (or equivalent circle diameter) of, for example, 0.5 to 7 mm, and preferably 1 to 5 mm. The method for measuring the major axis (or equivalent circle diameter) of the cell aggregate is not particularly limited, and for example, it can be measured from an image captured under a microscope. For example, cell aggregates cultured in a 96-well culture plate can be imaged using a 10x lens on a Keyence inverted microscope, and the major axis can be measured from the image. Here, the major axis refers to the longest line segment connecting the two endpoints of the cell aggregate in the captured image, and its length. The equivalent circle diameter refers to the diameter of a perfect circle, which corresponds to the area of ​​the figure (circle or ellipse) obtained when projected onto a two-dimensional surface. The cell aggregates of the present invention are spherical aggregates, and the major axis and equivalent circle diameter are similar, and the difference between the major axis and equivalent circle diameter becomes smaller as the aggregate approaches a spherical sphere.

[0013] The structure of the cell aggregates of the present invention changes depending on the differentiation stage. They can be broadly divided into two stages: cell aggregates in the early differentiation stage (approximately 6 days after the start of the first step, described below) and cell aggregates in the late differentiation stage (approximately 11 to 18 days after the start of the first step, described below). First, the characteristics of cell aggregates in the early differentiation stage will be described.

[0014] 1-1. Cell aggregates in the early stages of differentiation The cell aggregate of the present invention has the above-mentioned characteristics and is constituted by stacking two or more different cell layers.

[0015] The cell aggregate of the present invention has the following characteristics. (1) It has two distinct regions: the primitive node area and the non-node area. (2) The primitive nodal region and non-primitive nodal region have a layer structure of at least two layers. (3) Primitive node cells are present in the first layer present on the surface of the primitive node region, and endodermal cells are substantially absent in the primitive node region. (4) Endodermal cells are present in the first layer, which is present on the surface of the non-primitive nodal region.

[0016] The cell aggregates of the present invention have two distinct regions: (1) a primitive nodal region and a non-primitive nodal region. As used herein, a cell layer structure region refers to a region in which specific cell layers (cell layers with the same number of cell layers and the same types of cells present in each cell layer) exist continuously.

[0017] As used herein, the "primitive node" is one of the earliest structures to appear in vertebrate embryos. In mouse embryos, the node begins to appear on the ventral surface of the distal end at approximately embryonic day (E) 7.0 and is recognized as a morphologically distinct structure, a teardrop-shaped, concave structure composed of contracted epithelial cells, at the rostral end of the primitive streak. Furthermore, the node expresses BMP inhibitors and plays an important role in determining left-right asymmetry.

[0018] The ratio of the primitive nodule region and the non-primitive nodule region constituting the cell aggregate of the present invention is not particularly limited as long as both are present simultaneously, but may be 1:100 to 1:10000 in volume ratio and 1:10 to 1:1000 in aggregate surface area ratio.

[0019] In the cell aggregate of the present invention, (2) the primitive nodule region and the non-primitive nodule region have a layer structure of at least two layers. Each layer structure of the primitive nodule region and non-primitive nodule region is a cell layer composed of one or more types of cells. Each cell layer has a thickness equivalent to at least one (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) cell constituting the cell layer. For example, of each layer structure of the primitive nodule region and non-primitive nodule region, the first layer present on the surface may be a cell layer having a thickness equivalent to 1 to 10 cells (e.g., 2 to 5 cells). Here, the first layer present on the surface corresponds to the outermost cell layer of the cell aggregate of the present invention in which the primitive nodule region or non-primitive nodule region is present. Different cell populations exist in different cell layers, and the layer structure may be identified based on the differences in the cell populations present.

[0020] In the cell aggregate of the present invention, (3) axial mesoderm cells are present in the first layer present on the surface of the primitive node region, and endoderm cells are substantially absent in the primitive node region. As used herein, the term "axial mesoderm" refers to one of the tissues that differentiate from the epiblast in vivo. Axial mesoderm cells are cells present in the axial mesoderm, and are, for example, cells that express markers for axial mesoderm cells, such as BRA-positive cells, which will be described later.

[0021] Those skilled in the art can determine whether axial mesoderm cells are present in the first layer present on the surface of the primitive node region using conventional methods. For example, this can be confirmed by detecting the expression of a marker gene by RT-PCR or immunohistochemistry using an antibody specific for an axial mesoderm cell marker. Examples of markers for axial mesoderm cells include BRA, FOXA2, FOXJ1, and NOTO. Therefore, cells positive for at least one marker selected from the group consisting of BRA, FOXA2, FOXJ1, and NOTO can be determined to be axial mesoderm cells. In one embodiment, axial mesoderm in the early differentiation stage (approximately days 4 to 8 of the culture method of the present application) expresses all of BRA, FOXA2, and FOXJ1. In another embodiment, in the late differentiation stage (approximately days 10 to 15 of the culture method of the present application), axial mesoderm cells lose FOXJ1 expression and express BRA, or BRA and FOXA2.

[0022] Axial mesoderm further differentiates into the primitive node in vivo, and the primitive node can differentiate into the notochord, and the notochord can differentiate into the nucleus pulposus. The axial mesoderm cells present in the primitive node are defined as primitive node cells. Therefore, in the cell aggregate of the present invention, all of the axial mesoderm cells contained in the first layer present on the surface of the primitive node region are primitive node cells. Mesoderm other than axial mesoderm may be present in the primitive node region. The proportion of axial mesoderm cells (primitive node cells) present in the primitive node region may be approximately 70% to approximately 90% (e.g., approximately 80%) of all cells present in the first layer of the primitive node region.

[0023] In the cell aggregate of the present invention, when the axial mesoderm cells present in the first layer of the primitive node region are primitive node cells and the primitive node cells are FOXJ1-positive cells, the FOXJ1-positive cells are present at a ratio of approximately 10% to 20% of the total cell number of the cell aggregate of the present invention.

[0024] Because axial mesoderm cells present in the first layer of the primitive node region are primitive node cells, axial mesoderm cells and primitive node cells are the same cells at least temporarily and share many gene expression patterns. Therefore, for example, marker gene expression can be detected by RT-PCR or immunohistochemistry using a primitive node cell marker-specific antibody. Primitive node cell markers overlap with the axial mesoderm cell markers described above, specifically, BRA, FOXA2, FOXJ1, NOTO, etc. Therefore, cells positive for at least one marker selected from the group consisting of BRA, FOXA2, FOXJ1, and NOTO, preferably FOXJ1-positive cells, and more preferably FOXJ1-positive, BRA-positive, and FOXA2-positive cells, can be determined to be primitive node cells. Meanwhile, primitive node cells present at the outermost surface of the primitive node region may have primary cilia. The definition of primary cilia and methods for identifying primary cilia are described below.

[0025] In the cell aggregates of the present invention, primitive node cells also express secreted proteins of BMP signaling pathway inhibitors and ventralizing factors. Therefore, whether all or part of the axial mesoderm cells are primitive node cells can be determined, for example, by detecting the expression of BMP signaling pathway inhibitors or ventralizing factors by RT-PCR or immunohistochemistry using antibodies specific for BMP signaling pathway inhibitors or ventralizing factors. Proteins known to inhibit the BMP signaling pathway include, for example, secreted proteins belonging to the noggin, chordin, follistatin, gremlin, inhibitor, twisted gastration, coco, and dan families. Proteins known to ventralize factors include, for example, proteins belonging to the hedgehog family (e.g., shh, ihh) and shh receptors. Therefore, cells that express at least one secretory protein selected from the group consisting of NOGGIN, CHORDIN, FOLLISTATIN, GREMLIN, INHIBIN, TWISTED GASTRULATION, COCO, secretory proteins belonging to the DAN family, SHH and IHH, preferably at least one secretory protein selected from the group consisting of CHORDIN, NOGGIN and SHH, more preferably CHORDIN, NOGGIN and SHH, can be determined to be primitive nodule cells.

[0026] The cell aggregates of the present invention also have primary cilia extending from the primitive nodal cells toward the outside of the cell aggregate. Here, "primary cilia extending from the primitive nodal cells toward the outside of the cell aggregate" refers to primary cilia extending from the primitive nodal cells present on the surface of the primitive nodal region of the cell aggregates of the present invention. Furthermore, the term "primary cilia" refers to cilia that protrude from the cell membrane and whose rotation creates a fluid flow, forms a concentration gradient of growth factors, and destroys left-right symmetry.

[0027] Those skilled in the art can determine whether a primary cilium has extended from a primitive nodal cell using standard methods. For example, this can be confirmed by detecting the expression of a marker gene by RT-PCR or immunohistochemistry using antibodies specific to markers of axial mesoderm cells or primitive nodal cells. Examples of primary cilium markers include Ac-α-Tubulin. Therefore, Ac-α-Tubulin-positive cells can be determined to be primitive nodal cells from which a primary cilium has extended.

[0028] Furthermore, in the cell aggregate of the present invention, "substantially no endoderm cells are present in the primitive node region" means that the cell layer structure of the primitive node region is substantially free of cell layers containing endoderm cells. "Substantially no" means, for example, that the proportion of endoderm cells to the total number of cells in the first layer present on the surface of the primitive node region is less than 5%, preferably 0%.

[0029] As used herein, "endoderm" refers to the innermost of the three germ layers formed after fertilization of an egg during the early development of an organism. For example, the digestive system, urinary tract, pharynx, trachea, bronchi, and lungs are formed from the endoderm. During differentiation into endoderm, pluripotent stem cells express endodermal markers such as SOX17, HNF-3β / FoxA2, ​​Klf5, GATA4, GATA6, and PDX-1, preferably SOX17. Endodermal cells are cells present in the endoderm, and are, for example, cells that express endodermal cell markers, such as SOX17-positive cells, as described below.

[0030] Those skilled in the art can determine whether endoderm cells are present in the first layer present on the surface of the primitive node region using routine methods. For example, this can be confirmed by detecting the expression of marker genes by RT-PCR or immunohistochemistry using an antibody specific for an endoderm cell marker. Endoderm cell markers include, as described above, SOX17, HNF-3β / FoxA2, ​​Klf5, GATA4, GATA6, PDX-1, and the like. Therefore, if no cells expressing at least one marker selected from the group consisting of SOX17, HNF-3β / FoxA2, ​​Klf5, GATA4, GATA6, and PDX-1 (e.g., SOX17) are detected or the expression rate is less than 5% of the total cells, it can be determined that endoderm cells are substantially absent.

[0031] In the cell aggregate of the present invention, (4) endoderm cells are present in the first layer present on the surface of the non-primitive node region. Here, the first layer present on the surface of the non-primitive node region corresponds to the outermost cell layer of the cell aggregate of the present invention in which the non-primitive node region is present, just like the first layer present on the surface of the primitive node region. Furthermore, "present" means, for example, that the proportion of endoderm cells to the total number of cells contained in the first layer present on the surface of the non-primitive node region is 50% or more (60% or more, 70% or more, 80% or more, and 90% or less, 95% or less).

[0032] Those skilled in the art can determine whether endoderm cells are present in the first layer, which is present on the surface of the non-primitive node region, using standard methods. For example, this can be confirmed by detecting the expression of a marker gene by RT-PCR or immunohistochemistry using an antibody specific for an endoderm cell marker. Endoderm cell markers include, as described above, SOX17, HNF-3β / FoxA2, ​​Klf5, GATA4, GATA6, PDX-1, and the like. Therefore, the presence of endoderm cells can be determined when cells expressing at least one marker selected from the group consisting of SOX17, HNF-3β / FoxA2, ​​Klf5, GATA4, GATA6, and PDX-1, preferably SOX17, account for 50% or more (60% or more, 70% or more, 80% or more, and 90% or less, 95% or less) of the total cell population.

[0033] The cell aggregates of the present invention also have axial mesoderm cells in a second layer inside the non-primitive node region, epiblast cells in a third layer further inside that, and epiblast cells in a second layer inside the primitive node region. Here, the second layer inside the non-primitive node region (or primitive node region) corresponds to another cell layer adjacent to the inner side of the outermost cell layer of the cell aggregate of the present invention in which the non-primitive node region (or primitive node region) is present. As mentioned above, the types of cells present in the first, second, and third layers are different, so each layer can be identified. Furthermore, "present" means, for example, that the proportion of axial mesoderm cells to the total number of cells contained in the second layer inside the non-primitive node region is 5% or more. Note that in addition to axial mesoderm cells, other mesoderm cells are present in the second layer of the non-primitive node region. Furthermore, the third layer inside the non-primitive nodal region corresponds to another cell layer adjacent to the inside of the second cell layer of the cell aggregate of the present invention in which the non-primitive nodal region is present. Furthermore, "present" means, for example, that the proportion of epiblast cells to the total number of cells contained in the third layer inside the non-primitive nodal region is 20% or more (30% or more, 40% or more). Here, the second layer inside the primitive nodal region corresponds to another cell layer adjacent to the inner side of the outermost cell layer (first layer) of the cell aggregate of the present invention in which the primitive nodal region is present. Furthermore, "present" means, for example, that the ratio of epiblast cells to the number of pre-cells contained in the second layer inside the non-primitive nodal region is 20% or more (30% or more, 40% or more). As used herein, the term "epiblast" refers to an undifferentiated cell layer that proliferates within the blastocyst formed within the blastocoel after a fertilized egg develops into a morula. Epiblast cells are cells present in the epiblast, and are, for example, cells that express epiblast cell markers, such as OCT4-positive cells, which will be described later.

[0034] Those skilled in the art can determine whether axial mesoderm cells are present in the second layer of the non-primitive node region using conventional methods. For example, this can be confirmed by detecting the expression of marker genes using RT-PCR or immunohistochemistry using antibodies specific to axial mesoderm cell markers. Markers for axial mesoderm cells include BRA, FOXA2, FOXJ1, and NOTO. Therefore, cells positive for at least one marker selected from the group consisting of BRA, FOXA2, FOXJ1, and NOTO can be determined to be axial mesoderm cells. As mentioned above, markers can change depending on the differentiation stage, so those skilled in the art can select the optimal combination of markers depending on the differentiation stage.

[0035] Those skilled in the art can determine whether epiblast cells are present in the third layer of the non-primitive node region or the second layer of the primitive node region using conventional methods. For example, this can be confirmed by detecting the expression of a marker gene by RT-PCR or immunohistochemistry using an antibody specific for an epiblast cell marker. Examples of epiblast cell markers include OCT4, NANOG, SOX2, and FGF5. Therefore, OCT4-positive cells can be determined to be epiblast cells.

[0036] The cell aggregate of the present invention also has a basement membrane between the first layer present on the surface and the second layer present on the inside in the primitive nodal region and / or non-primitive nodal region.

[0037] As used herein, the term "basement membrane" refers to a thin, membranous structure composed of extracellular matrix. In living organisms, basement membranes are formed on the basal side of epithelial cells. Examples of basement membrane components include type IV collagen, laminin, heparan sulfate proteoglycan (perlecan), entactin / nidogen, cytokines, and growth factors, with laminin being preferred. Whether a basement membrane is present between the first layer present on the surface and the second layer present inside in the primitive nodule region and / or non-primitive nodule region can be detected by, for example, tissue staining such as PAM staining, or immunohistochemistry using antibodies against basement membrane components (e.g., anti-laminin antibody, anti-type IV collagen antibody). Therefore, for example, if laminin is positive, it can be determined that a basement membrane is present between the first layer present on the surface and the second layer present inside in the primitive nodule region and / or non-primitive nodule region.

[0038] In addition, in the non-primitive nodule region, the basement membrane exists continuously between the first layer present on the surface and the second layer present inside. Here, "continuously present" means that the basement membrane appears to be stained as a continuous line in immunostaining using an anti-laminin antibody. The first and second layers of the non-primitive nodule region do not necessarily need to be completely continuous throughout, and this also includes cases where the line appears to be interrupted and separated. Note that "continuously stained" means that the basement membrane is stained continuously (without interruptions) over a length of at least 4-5 cells.

[0039] 1-2. Cell aggregates at the late stage of differentiation The cell aggregate of the present invention has the following characteristics. (1) FoxA1-positive endodermal cells are present in the outermost layer of the cell aggregates. (2) Inside the cell aggregate, multiple axial mesoderm cells are present in a cluster (sometimes referred to as a cluster of axial mesoderm cells), and around it are the neural tube and somites. (3) Two or more of the structures described in (2) are present within a single cell aggregate.

[0040] The cell aggregate of the present invention (1) has FoxA1-positive endodermal cells present in the outermost layer of the cell aggregate. The outermost layer is the layer present at the outermost part of the cell aggregate, and has a thickness equivalent to the number of cells constituting at least one (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of said cell layer. Whether FoxA1-positive endodermal cells are present in the outermost layer of a cell aggregate can be determined by those skilled in the art using standard methods in this field, such as the methods described above.

[0041] The cell aggregate of the present invention comprises (1) a cell aggregate in which a plurality of axial mesodermal cells are present inside the cell aggregate, with the neural tube and somites present around the cell aggregate. The inside of the cell aggregate refers to the inside of the outermost layer described above. It can also be defined as a region of the cell aggregate in which FoxA1-positive endodermal cells are not present. In one embodiment, the inside of the cell aggregate refers to a region approximately 200 μm to 300 μm inside from the outermost layer.

[0042] The axial mesoderm cells may be cells that express the above-mentioned markers. In one embodiment, the axial mesoderm cells may be BRACHYRY (BRA) positive. The axial mesoderm cells may also be cells that express SHH. The axial mesoderm cells at this differentiation stage are sometimes called notochord cells. The axial mesoderm cells may be present in a group of 5 or more cells (10 or more cells, 15 or more cells, 20 or more cells). There is no particular upper limit, but the number is usually around 50 cells or less.

[0043] Axial mesoderm cells (or notochord cells) aggregates do not have the long tubular structures seen in living organisms. They are amorphous, with cells densely packed within an area of ​​approximately 30-40 μm in length, width, and depth.

[0044] The cell aggregate of the present invention (2) has a plurality of axial mesoderm cells gathered inside the cell aggregate, with neural tubes and somites present around it.

[0045] The term "neural tube" refers to the fetal precursor of the central nervous system, consisting of the brain and spinal cord, in developing chordates. The dorsal wall of the neural tube is called the roof plate, and the ventral wall is called the floor plate. The presence or absence of a neural tube or neural tube cells can be determined by those skilled in the art using standard methods. For example, confirmation can be achieved by detecting the expression of marker genes using RT-PCR or immunohistochemistry using antibodies specific to neural tube cell markers. Examples of neural tube cell markers include ARX and Nkx6.1. Therefore, cells positive for at least one marker selected from the group consisting of ARX and Nkx6.1 can be determined to be neural tube cells (particularly neural floor plate cells (also called neural tube floor plate cells)). In one embodiment, the neural tube floor plate, i.e., neural floor plate cells expressing ARX and Nkx6.1, are present around a collection of axial mesoderm cells.

[0046] "Somites" refer to structures that are part of the mesoderm and develop along the sides of the neural tube to form various tissues and structures, such as the dermis, skeletal muscle, vertebrae, tendons, and cartilage. The presence or absence of somites or somite cells can be determined by those skilled in the art using routine methods. For example, confirmation can be achieved by detecting the expression of marker genes using RT-PCR or immunohistochemistry using a somite cell marker-specific antibody. Somites can be classified into mature somites (sometimes referred to as somite 1) and early somites before bud formation (sometimes referred to as somite 2) based on their degree of maturation. They can also be classified as ventral or dorsal. Somite 1 markers include Nkx3.2 and PAX9 (ventral somite markers), MYOD (dorsal somite marker), etc. Somite 1 may be negative or weakly positive for SOX9. Examples of somite 1 markers include SOX9. The somite cells present around the aggregate of axial mesoderm cells may be any of the above, but are preferably ventral somite cells that express Nkx3.2.

[0047] The surroundings of the axial mesoderm cell aggregate refers to the inner region of the cell aggregate other than the outermost layer, and is close to the axial mesoderm cell aggregate. Close to the axial mesoderm cell aggregate means, for example, a range of about 100 to 200 μm.

[0048] The number of neural tubes and somites present around the aggregate of axial mesoderm cells is not particularly limited, but is usually about 1, 2, and 3, respectively. The number of neural tubes and somites does not need to be the same.

[0049] If a collection of axial mesoderm cells and the surrounding neural tube and somites are considered to be one structure, then two or more (three or more, four or more, five or more) such structures are present in one cell aggregate. There is no upper limit to the number of such structures, but it is usually five or less.

[0050] 2. Method for producing cell aggregates of the present invention The present invention provides a method for producing a cell aggregate containing axial mesoderm cells (hereinafter referred to as a method for producing the cell aggregate of the present invention). The axial mesoderm cells and the cell aggregate may be as described for the cell aggregate of the present invention.

[0051] The method for producing a cell aggregate of the present invention includes the following steps. (1) A first step in which dispersed pluripotent stem cells are rapidly aggregated to form a single cell aggregate in one culture compartment. (2) A second step of culturing the formed cell aggregates of pluripotent stem cells in suspension in a medium containing an extracellular matrix. (3) A third step in which the cell aggregates obtained in the second step are further cultured in suspension in a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator. (4) A fourth step in which the cell aggregates obtained in the third step are further cultured in suspension in a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor.

[0052] In one aspect, the method for producing a cell aggregate of the present invention is a method for producing a human cell aggregate, and in this case, the pluripotent stem cells used are human pluripotent stem cells.

[0053] As used herein, "stem cells" refer to undifferentiated cells that have the ability to differentiate and proliferate (particularly the ability to self-renew). Stem cells include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation ability. "Pluripotent stem cells" refer to stem cells that can be cultured in vitro and have the ability to differentiate into all cells that make up a living organism (pluripotency). "All cells" refer to cells derived from the three germ layers: ectoderm, mesoderm, and endoderm. "Multipotent stem cells" refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. "Unipotent stem cells" refer to stem cells that have the ability to differentiate into specific tissues or cells.

[0054] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Pluripotent stem cells also include Muse cells (Multi-lineage differentiating Stress Enduring cells) obtained from mesenchymal stem cells (MSCs), and GS cells prepared from germ cells (e.g., testes). Human embryonic stem cells are established from human embryos within 14 days of fertilization.

[0055] Embryonic stem cells were first established in 1981 and have been used to generate knockout mice since 1989. Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. ES cells can be produced by culturing internal cell populations on feeder cells or in medium containing leukemia inhibitory factor (LIF). Methods for producing ES cells are described in, for example, International Publication No. 96 / 22362, International Publication No. 02 / 101057, U.S. Patent No. 5,843,780, U.S. Patent No. 6,200,806, and U.S. Patent No. 6,280,718. Embryonic stem cells are available from designated institutions or commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. EB5 cells, which are mouse embryonic stem cells, are available from RIKEN, and the D3 strain is available from the American Type Culture Collection (ATCC). Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg cell from which the nucleus has been removed.

[0056] EG cells can be produced by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF, and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).

[0057] "Induced pluripotent stem cells" are cells induced to pluripotency by reprogramming somatic cells using known methods. Specific examples of induced pluripotent stem cells include cells induced to pluripotency by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4)pp. 663-676). In 2007, induced pluripotent stem cells were established from human fibroblasts and possess the same pluripotency and self-renewal capabilities as embryonic stem cells (Cell, 2007, 131(5)pp. 861-872; Science, 2007, 318(5858)pp. 1917-1920; Nat. Biotechnol., 2008, 26(1)pp. 101-106). In addition to direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by the addition of chemical compounds (Science, 2013, 341, pp. 651-654).

[0058] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0059] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes (e.g., four factors: Oct3 / 4, Sox2, Klf4, and Myc), the means for expressing the genes is not particularly limited. Examples of means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated viral vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors and episomal vectors) (e.g., calcium phosphate transfer, lipofection, retronectin transfer, and electroporation), gene transfer methods using RNA vectors (e.g., calcium phosphate transfer, lipofection, and electroporation), and direct protein injection.

[0060] It is also possible to obtain established induced pluripotent stem cell lines, for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University, which are available from Kyoto University and iPS Academia Japan, Inc. Examples of established induced pluripotent stem cell lines available from Kyoto University include Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University.

[0061] Pluripotent stem cells may be genetically modified. Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques. Examples of genes on chromosomes that can be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes due to disorders of nervous system cells. Target genes on chromosomes can be modified using methods described in "Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)," "Gene Targeting, A Practical Approach," IRL Press at Oxford University Press (1993)," and "Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells," Yodosha (1995).

[0062] Specifically, for example, the genomic gene of the target gene to be modified (e.g., a cell marker gene, a gene for a histocompatibility antigen, or a disease-related gene) is isolated, and a target vector for homologous recombination of the target gene is prepared using the isolated genomic gene. The prepared target vector is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the target vector are selected, thereby producing stem cells in which a gene on the chromosome has been modified.

[0063] Methods for isolating the genomic gene of the target gene include known methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. Genomic DNA Library Screening System (Genome Systems) and Universal GenomeWalker Kits (Clontech) can also be used.

[0064] Construction of a target vector for homologous recombination of a target gene and efficient selection of homologous recombinants can be performed according to methods such as those described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) and Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells, Yodosha (1995). Either replacement or insertion type target vectors can be used. Selection methods include positive selection, promoter selection, negative selection, and poly(A) selection. Methods for selecting the desired homologous recombinants from the selected cell lines include Southern hybridization of genomic DNA and PCR.

[0065] Pluripotent stem cells can also be those that have undergone genome editing. "Genome editing" is a technique for intentionally modifying target genes or genomic regions through site-specific cleavage of genomic DNA strands using nucleases or chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFN), TALEN, and CRISPR / Cas9. Genome editing technology can be used to create knockout cell lines in which specific genes are deleted, knockin cell lines in which a different sequence is artificially inserted into a specific gene locus, and so on.

[0066] Pluripotent stem cells can be, for example, those of warm-blooded animals, preferably mammals. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, livestock such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, and primates such as humans, monkeys, orangutans, and chimpanzees. Pluripotent stem cells are preferably those of rodents (such as mice and rats) or primates (such as humans), and more preferably human pluripotent stem cells.

[0067] The method for producing a cell aggregate of the present invention includes (1) a first step of rapidly aggregating dispersed pluripotent stem cells to form a single cell aggregate in one culture compartment.

[0068] Before the start of the first step, the pluripotent stem cells are dispersed into single cells. The "dispersed cells" obtained by the dispersion step are preferably single cells, but may also include cell clumps consisting of a small number of cells, for example, 2 to 100, or may include cell clumps consisting of 2 to 50 cells. The "dispersed cells" may, for example, be 70% or more single cells and 30% or less cell clumps, and preferably 80% or more single cells and 20% or less cell clumps.

[0069] Methods for dispersing pluripotent stem cells include mechanical dispersion treatment, cell dispersion treatment, and cell protective agent addition treatment, and these treatments may be performed in combination. Preferably, the cell dispersion method involves simultaneously performing cell protective agent addition treatment and cell dispersion treatment, followed by mechanical dispersion treatment.

[0070] Examples of the cytoprotective agent used in the cytoprotective agent addition treatment include substances acting on the FGF signaling pathway, heparin, Rho-associated protein kinase (ROCK) inhibitors, myosin inhibitors, polyamines, integrated stress response (ISR) inhibitors, caspase inhibitors, serum, serum substitutes, etc. Preferred cytoprotective agents include ROCK inhibitors.

[0071] ROCK inhibitors include Y-27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide, dihydrochloride), Fasudil (HA1077) (1-(5-Isoquinolinylsulfonyl)homopiperazine, hydrochloride), H-1152 (5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]-4-methyl-isoquinoline, dihydrochloride), HA-1100 (Hydroxyfasudil) ([1-(1-Hydroxy-5-isoquinolinesulfonyl)homopiperazine, hydrochloride), Chroman 1 ((3S)-N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-6-methoxy-3,4-dihydro-2H-chromene-3-carboxamide), Belumosudil (KD025, 2-[3-[4-[(1H-Indazol-5-yl)amino]quinazolin-2-yl]phenoxy]-N-isopropylacetamide), HSD1590 ([2-Methoxy-3-(4,5,10-triazatetracyclo[7.7.0.02,6.012,16]hexadeca-1(9),2(6),3,7,10,12(16)-hexaen-11-yl)phenyl]boronic acid), CRT0066854 ((S)-3-phenyl-N1-(2-pyridin-4-yl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)propane-1,2-diamine), RKI1447 (1-(3-hydroxybenzyl)-3-(4-(pyridin-4-yl)thiazol-2-yl)urea), Ripasudil (4-Fluoro-5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]isoquinoline)、GSK269962A(N-[3-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethylimidazo[4,5-c]pyridin-6-yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide)、GSK429286A(N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide)、Y-33075((R)-4-(1-Aminoethyl)-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide)、LX7101(N,N-Dimethylcarbamic acid 3-[[[4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinyl]carbonyl]amino]phenyl ester)、AT13148((alphaS)-alpha-(Aminomethyl)-alpha-(4-chlorophenyl)-4-(1H-pyrazol-4-yl)benzenemethanol)、SAR407899(6-(piperidin-4-yloxy)isoquinolin-1(2H)-one hydrochloride)、GSK180736A(4-(4-fluorophenyl)-N-(1H-indazol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide)、Hydroxyfasudil(1-(1-hydroxy-5-isoquinolinesulfonyl)homopiperazine,HCl)、bdp5290(4-Chloro-1-(4-piperidinyl)-N-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-1H-pyrazole-3-carboxamide)、sr-3677(N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidehydrochloride)、CCG-222740(N-(4-Chlorophenyl)-5,5-difluoro-1-(3-(furan-2-yl)benzoyl)piperidine-3-carboxamide)、ROCK inhibitor-2(N-[(1R)-1-(3-methoxyphenyl)ethyl]-4-pyridin-4-ylbenzamide)、Rho-Kinase-IN-1(N-[1-[(4-methylsulfanylphenyl)methyl]piperidin-3-yl]-1H-indazol-5-amine)、ZINC00881524(N-(4,5-dihydronaphtho[1,2-d]thiazol-2-yl)-2-(3,4-dimethoxyphenyl)acetamide)、SB772077B((3S)-1-[[2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride)、Verosudil(N-(1,Examples of suitable cytoprotective agents include 2-Dihydro-1-oxo-6-isoquinolinyl)-alpha-(dimethylamino)-3-thiopheneacetamide), GSK-25 (4-(4-chloro-2-fluorophenyl)-2-(2-chloropyridin-4-yl)-1-(6-fluoro-1H-indazol-5-yl)-6-methyl-4H-pyrimidine-5-carboxamide), and derivatives thereof, with Y-27632 being preferred. Pre-prepared cytoprotective agents can also be used. Examples of pre-prepared cytoprotective agents include RevitaCell Supplement (Thermo Fisher Scientific) and CloneR (Stemcell Technologies). These substances may be used alone or in combination.

[0072] Examples of cell dispersion solutions used in cell dispersion treatment include solutions containing at least one enzyme, such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain, and a chelating agent, such as ethylenediaminetetraacetic acid. Commercially available cell dispersion solutions, such as TripLE Select (Thermo Fisher Scientific), TripLE Express (Thermo Fisher Scientific), and Accumax (Innovative Cell Technologies), can also be used. Preferred cell dispersion solutions for treating pluripotent stem cells include, but are not limited to, TrypLE Select or phosphate buffered saline (PBS) supplemented with 5 mM EDTA.

[0073] Methods for mechanical dispersion include pipetting or scraping with a scraper. The dispersed cells are suspended in a medium.

[0074] Therefore, methods for dispersing pluripotent stem cells include, for example, treating pluripotent stem cell colonies with TrypLE Select, ethylenediaminetetraacetic acid, or Accumax in the presence of a ROCK inhibitor, and then dispersing the colonies in serum-free medium by pipetting.

[0075] Examples of serum-free media that can be used include serum-free media for culturing pluripotent stem cells based on basal media (e.g., Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow Minimum Essential Medium (Glasgow MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle Minimum Essential Medium (Eagle MEM), Alpha Modified Eagle Minimum Essential Medium (αMEM), Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, DMEM / F12, IMDM / F12, Ham's medium, RPMI 1640, Fischer's medium, and mixtures thereof), preferably known serum-free media for embryonic stem cells or induced pluripotent stem cells, and serum-free media for culturing pluripotent stem cells in a feeder-free environment (feeder-free medium).Many synthetic feeder-free media have been developed and are commercially available, including StemFit (Ajinomoto Co.), Essential 8 (Thermo Fisher Scientific), S-medium (DS Pharma Biomedical), StemPro (Thermo Fisher Scientific), hESF9, mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), TeSR-E8 (STEMCELL Technologies), mTeSR Plus (STEMCELL Technologies), ReproMed iPSC Medium (ReproCELL), NutriStem XF (Biological Industries), NutriStem V9 (Biological Industries), Cellartis DEF-CS Xeno-Free Culture Medium (Takara Bio), Stem-Partner SF (Kyokuto Pharmaceuticals), and PluriSTEM Human ES / iPS Cell Examples of such a medium include StemSure hPSC Medium (manufactured by Merck) and StemSure hPSC MediumΔ (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and preferably StemFit.

[0076] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO 98 / 30679. Commercially available serum substitutes may also be used. Examples of commercially available serum substitutes include Knockout Serum Replacement (Thermo Fisher Scientific) (hereinafter sometimes referred to as "KSR"), Chemically-defined Lipid Concentrated (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), B27 Supplement (Thermo Fisher Scientific), and N2 Supplement (Thermo Fisher Scientific).

[0077] The serum-free medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like, as appropriate.

[0078] The serum-free medium used in the present invention is preferably a medium containing chemically defined components (CDM) from the viewpoint of avoiding contamination with chemically undefined components.

[0079] The method for aggregating pluripotent stem cells in the first step includes a method of suspension culture of dispersed pluripotent stem cells in one culture compartment. When performing suspension culture, a suspension of pluripotent stem cells dispersed in a serum-free medium is seeded into one non-adherent culture compartment. When the culture compartment is non-adherent, the cells are cultured in suspension, and multiple pluripotent stem cells aggregate to form cell aggregates.

[0080] As used herein, "suspension culture" refers to culturing cells while maintaining a state in which they exist suspended in a culture medium. In other words, suspension culture is performed under conditions that do not allow cells to adhere to the cultureware or feeder cells, etc., on the cultureware (hereinafter referred to as "cultureware, etc."), and is distinguished from culture performed under conditions that allow cells to adhere to the cultureware, etc. (adherent culture). More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substrate bonds to form between the cells and the cultureware, etc. Those skilled in the art can easily distinguish whether cultured cells are in suspension culture or adherent culture by, for example, rocking the cultureware during microscopic observation.

[0081] In cell aggregates during suspension culture, cells adhere to each other through a plane. In cell aggregates during suspension culture, strong cell-substrate bonds are not formed between the cells and the cultureware, and cell-substrate bonds are barely formed, or even if they are formed, their contribution is small. Internal cell-substrate bonds may exist within cell aggregates during suspension culture. "Plane attachment between cells" refers to cell-to-cell adhesion through a plane. More specifically, "plane attachment between cells" refers to the proportion of the surface area of ​​a cell that is attached to the surface of another cell, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. Cell surfaces can be observed by staining with membrane-staining reagents (e.g., DiI) or immunostaining for cell adhesion factors (e.g., E-cadherin, N-cadherin, etc.).

[0082] The cultureware used for suspension culture is not particularly limited as long as it is capable of supporting suspension culture, and those skilled in the art can appropriately select the appropriate cultureware. Examples of such cultureware include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, spinner flasks, and roller bottles. These cultureware are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive cultureware include those whose surfaces are not artificially treated to improve cell adhesion. Examples of non-cell-adhesive cultureware include those whose surfaces are artificially treated to reduce cell adhesion. The culture surface of the cultureware may be flat, U-, or V-bottom, or may be uneven. Examples of treatments that reduce adhesion to cells include ultrahydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), and polyethylene glycol (PEG), as well as treatments to reduce protein adsorption.

[0083] To protect cell aggregates from physical stresses such as shear forces that occur during suspension culture, and to increase the local concentrations of growth factors and cytokines secreted by the cells and promote tissue development, cell aggregates can be embedded in gels or encapsulated in permeable capsules before suspension culture (Nature, 2013, 501. 7467:373). The encapsulated cell aggregates can also be cultured with shaking. As used herein, "shaking culture" refers to a culture method in which the culture medium is agitated by shaking the culture vessel, promoting oxygen supply to the medium and material exchange with the surrounding cells. Agitation culture, flow channel culture, and other methods can also be used. The gel or capsule used for embedding can be made of either a biologically derived or synthetic polymer. Gels or capsules used for such purposes include, for example, Matrigel (manufactured by Corning), PuraMatrix (manufactured by 3D Matrix), VitroGel 3D (manufactured by The Well Bioscience), collagen gel (manufactured by Nitta Gelatin), alginate gel (manufactured by PG Research), and Cell-in-a-Box (manufactured by Austrianova).

[0084] More specifically, in the method for producing cell aggregates of the present invention, dispersed pluripotent stem cells may be seeded in a relatively large culture compartment, such as a 10-cm dish, during suspension culture, allowing multiple cell aggregates to form simultaneously in one culture compartment. However, from the viewpoint of minimizing variations in size among cell aggregates, it is preferable to seed a fixed number of dispersed pluripotent stem cells in each well of a multi-well plate (U-bottom, V-bottom) such as a non-cell-adhesive 96-well microplate. When this is subjected to static culture, the cells rapidly aggregate, forming one cell aggregate in each culture compartment (Serum-free culture of Embryoid Body-like aggregates with quick reaggregation; SFEBq method). Therefore, the first step involves forming cell aggregates using the SFEBq method. One embodiment is as follows: hESCs / iPSCs cultured to maintain an undifferentiated state were dissociated into single cells using 0.5x TrypLE select and cultured at 3.0 x 10 in SF medium containing 10 μM Y-27632. 3 The cells are seeded onto a low-cell-adhesion 96-well plate, PrimeSurface (Sumitomo Bakelite), at a density of 100 cells / well. The day the cells are seeded onto the 96-well plate is counted as day 0, and after one day of culture, cell aggregates are allowed to form in each well.

[0085] To prepare non-adhesive culture compartments, the surface of the culture compartment can be coated with a superhydrophilic polymer, or other processing to make the culture compartment non-adhesive. Examples of non-adhesive multi-well plates include the PrimeSurface 96V bottom plate (MS-9096V, manufactured by Sumitomo Bakelite Co., Ltd.). Centrifugation may be performed to more rapidly form cell aggregates. Cell aggregates formed in each culture compartment can be collected from multiple culture compartments to obtain a uniform population of cell aggregates. Uniform cell aggregates can provide more stable production efficiency for each culture compartment and each replicate experiment in subsequent processes, allowing for more reproducible production of cell aggregates containing axial mesoderm cells.

[0086] Other examples of cultureware for forming cell aggregates from dispersed pluripotent stem cells include mortars with a bottom surface that allows cells to settle in one place and promotes aggregate formation, downward-facing pyramids, concave shapes, grids, protrusions, etc., or cultureware with a bottom surface that is treated to allow cells to adhere to only a portion of the surface to facilitate aggregate formation. Examples of such cultureware include, but are not limited to, the AggreWell embryoid body formation plate (manufactured by StemCell Technologies), PAMCELL (manufactured by ANK), spheroid microplates (manufactured by Corning), NanoCulture Plate / Dish (manufactured by Organogenix), Cell-able (manufactured by Toyo Gosei), EZSPHERE (manufactured by AGC Technoglass), SPHERICALPLATE 5D (manufactured by Mito Kogyo Co., Ltd.), and TASCL (manufactured by Sims Bio).

[0087] It is also preferable to use a three-dimensional cell culture vessel as the cultureware, which allows for simultaneous replacement of the culture medium in all culture compartments while the cell aggregates remain in each culture compartment. An example of such a three-dimensional cell culture vessel is the PrimeSurface 96-slit well plate (manufactured by Sumitomo Bakelite Co., Ltd.). This plate has narrow openings (slits) at the top of each of the 96 wells, allowing for the entry and exit of culture medium. The slits are designed to be narrow enough to prevent cell aggregates from passing through, allowing for simultaneous replacement of the culture medium in the entire plate while preventing adhesion between cell aggregates, improving operational efficiency and the quality of the cell aggregates.

[0088] The concentration of pluripotent stem cells used in the first step can be appropriately set so as to form cell aggregates more uniformly and efficiently. For example, when pluripotent stem cells are cultured in suspension in a 96-well microwell plate, the concentration is usually about 0.5 × 10 per well. 3 to approximately 1 x 10 4 cells, preferably about 1 x 10 3 From about 8 x 10 3 cells, more preferably about 2 x 10 3 from about 4 × 10 3 A solution prepared to form cells is added to each well, and the plate is left to stand to form cell aggregates. The number of cells can be determined by counting using a hemocytometer.

[0089] The suspension culture time required to form cell aggregates can be determined appropriately depending on the pluripotent stem cells used, but it is desirable to keep the time as short as possible to form uniform cell aggregates. The process by which dispersed cells form cell aggregates can be divided into a cell aggregation process and an aggregate formation process. The time from the time of seeding dispersed cells (i.e., the start of suspension culture) to cell aggregation is preferably within about 24 hours, more preferably within about 12 hours, in the case of, for example, human pluripotent stem cells (human iPS cells, etc.). The time from the time of seeding dispersed cells (i.e., the start of suspension culture) to the time of cell aggregate formation is preferably within about 36 hours, more preferably within about 24 hours, in the case of, for example, human pluripotent stem cells (human iPS cells, etc.). The time until cell aggregate formation can be adjusted appropriately by adjusting the cell aggregation tool, centrifugation conditions, etc.

[0090] The suspension culture of the present invention is preferably carried out under xeno-free conditions. "Xeno-free" refers to conditions in which components derived from organisms other than the organism species of the cells to be cultured are excluded.

[0091] The serum-free medium used in the first step preferably contains a cytoprotective agent to suppress cell death of pluripotent stem cells. The cytoprotective agent may be the same as the cytoprotective agent used in the cytoprotective agent addition treatment, and a ROCK inhibitor is preferred. In one embodiment, when Y-27632 is added as the ROCK inhibitor, it is added to the culture environment at a concentration of typically about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, and more preferably about 1 μM to about 100 μM. In another embodiment, when Chroman 1 is added as the ROCK inhibitor, it is added to the culture environment at a concentration of typically about 10 pM to about 1 mM, preferably about 100 pM to about 100 μM, and more preferably about 1 nM to about 10 μM.

[0092] Therefore, in one embodiment of the first step, hESCs / iPSCs cultured to maintain an undifferentiated state were dissociated into single cells using 0.5x TrypLE select, and then cultured at a concentration of 3.0 x 10 in SF medium containing 10 μM Y-27632. 3 Cells can be seeded at a density of 1000 cells / well onto a low-cell-attachment 96-well plate, PrimeSurface (Sumitomo Bakelite). The day the cells are seeded onto the 96-well plate is considered day 0, and after one day of culture, cell aggregates can be formed in each well.

[0093] The serum-free medium used in the first step may also contain factors for maintaining undifferentiated states to enable culturing to maintain the undifferentiated state. The factors for maintaining undifferentiated states are not particularly limited, as long as they have the effect of suppressing the differentiation of pluripotent stem cells. Factors for maintaining undifferentiated states commonly used by those skilled in the art include, in the case of primed pluripotent stem cells (e.g., human ES cells and human iPS cells), FGF signaling pathway activators, TGFβ family signaling pathway activators, insulin, etc. Specific examples of FGF signaling pathway activators include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Furthermore, examples of TGFβ family signaling pathway activators include TGFβ signaling pathway activators and Nodal / Activin signaling pathway activators. Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling pathway activators include Nodal, Activin A, and Activin B. These substances may be used alone or in combination.

[0094] The factor for maintaining the undifferentiated state may be one produced by any host or one artificially synthesized, as long as it has the ability to maintain the undifferentiated state of the pluripotent stem cells to be cultured. The factor for maintaining the undifferentiated state used in the present invention is preferably one that has been modified in the same way as that occurring in vivo, and more preferably one that has been produced in cells of the same type as the pluripotent stem cells to be cultured under conditions that do not contain any xenogeneic components.

[0095] The concentration of the undifferentiation maintenance factor in the medium used in the first step is a concentration that allows the cultured pluripotent stem cells to be maintained in an undifferentiated state, and can be appropriately determined by one skilled in the art. For example, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually about 4 ng / mL to about 500 ng / mL, preferably about 10 ng / mL to about 200 ng / mL, and more preferably about 30 ng / mL to about 150 ng / mL.

[0096] The method for producing cell aggregates of the present invention includes (2) a second step of suspension-culturing the formed cell aggregates of pluripotent stem cells in a medium containing an extracellular matrix. Examples of the extracellular matrix include basement membrane preparations, laminin or fragments thereof, entactin, collagen, and gelatin. In one embodiment, the extracellular matrix used in the second step of the present invention is a basement membrane preparation such as Matrigel.

[0097] As used herein, the term "basement membrane preparation" refers to a preparation containing basement membrane components that provide a medium with sufficient hardness to support 3D culture of cell aggregates and efficiently induce the differentiation of pluripotent stem cells into axial mesoderm cells. Here, "basement membrane components" refers to thin membrane-like extracellular matrix molecules present between epithelial cell layers and interstitial cell layers in animal tissues. Basement membrane preparations can be prepared, for example, by removing cells with basement membrane-forming ability that are attached to a support via a basement membrane from the support using a solution capable of dissolving lipids in the cells or an alkaline solution. Examples of basement membrane preparations include commercially available basement membrane preparations, such as Matrigel (Corning) and Geltrex (Thermo Fisher Scientific), and preparations containing extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.), with Matrigel being preferred.

[0098] As used herein, the term "laminin or a fragment thereof" is not particularly limited as long as it has high affinity for at least the integrin α3β1 complex, and examples thereof include laminin-111 or a fragment comprising its E8 region, laminin-211 or a fragment comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment comprising its E8 region, laminin-221 or a fragment comprising its E8 region, laminin-332 or a fragment comprising its E8 region, laminin-3A11 or a fragment comprising its E8 region, laminin-411 or a fragment comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment comprising its E8 region, and laminin-511 or a fragment comprising its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment comprising its E8 region, laminin-213 or a fragment comprising its E8 region, laminin-423 or a fragment comprising its E8 region, laminin-523 or a fragment comprising its E8 region, laminin-212 / 222 or a fragment comprising its E8 region, and laminin-522 or a fragment comprising its E8 region.

[0099] In the second step, the concentration of the extracellular matrix contained in the medium can be appropriately set so that the cell aggregates of pluripotent stem cells formed in the first step can be cultured in three dimensions, the cell aggregates can be proliferated while maintaining their pluripotency, and the cell aggregates of the present invention can be formed through the third and fourth steps described below. In one embodiment, when Matrigel, a basement membrane preparation, is used as the extracellular matrix, it can be added to the medium at a concentration of, for example, 0.05% (v / v) to 20% (v / v), 0.1% (v / v) to 5% (v / v), or 0.5% (v / v) to 2% (v / v).

[0100] The suspension culture of the pluripotent stem cell aggregates formed in the first step may be carried out using the same serum-free medium and culture compartment as those used in the first step. In one embodiment, the second step may be carried out by replacing the serum-free medium contained in the culture compartment used in the first step with a medium containing an extracellular matrix (e.g., a basement membrane preparation). In another embodiment, the pluripotent stem cell aggregates formed in the first step may be collected and suspended in a medium containing an extracellular matrix (e.g., a basement membrane preparation), and the second step may be carried out in a new culture compartment.

[0101] The time required for suspension culture in the second step can be determined appropriately, for example, about 1 to 10 days, preferably about 3 to 5 days, and more preferably about 2 to 4 days.

[0102] Therefore, in one embodiment of the second step, the serum-free medium on day 1 after the start of the first step is switched to a serum-free medium containing 1% v / v Matrigel, and the cells are cultured in that medium for 3 days.

[0103] The serum-free medium used in the second step may also contain a factor for maintaining undifferentiation, as in the first step, to enable culture to maintain the undifferentiated state. The factor for maintaining undifferentiation and its concentration contained in the serum-free medium in the second step may be the same as the factor for maintaining undifferentiation and its concentration used in the first step.

[0104] The method for producing cell aggregates of the present invention includes (3) a third step of further suspension-culturing the cell aggregates obtained in the second step in a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator.

[0105] As used herein, the term "TGFβ signaling pathway" refers to a signaling pathway transduced intracellularly by the Smad family, with transforming growth factor β (TGFβ), Nodal / Activin, or BMP as the ligand. Furthermore, a TGFβ signaling pathway activator refers to a substance that enhances the TGFβ signaling pathway, i.e., the signaling pathway transduced by the Smad family, and includes not only TGFβ signaling pathway activators but also Nodal / Activin signaling pathway activators. Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling pathway activators include Nodal, Activin A, and Activin B. These substances may be used alone or in combination, with Activin A being preferred.

[0106] The concentration of the TGFβ signaling pathway activator in the medium can be appropriately set depending on the substance used, as long as the above-mentioned enhancing effect can be achieved. When Activin A is used as the TGFβ signaling pathway activator, it is usually used at a concentration of about 1 ng / ml to about 1000 ng / ml, preferably about 5 ng / ml to about 200 ng / ml, and more preferably about 10 ng / ml to about 100 ng / ml. When a TGFβ signaling pathway activator other than Activin A is used, it is desirably used at a concentration that exhibits the same TGFβ signaling pathway enhancing activity as Activin A at the above-mentioned concentration.

[0107] As used herein, the term "Wnt signaling pathway" refers to a signaling pathway that utilizes Wnt family proteins as ligands and primarily Frizzled as a receptor. Examples of such pathways include the canonical Wnt pathway and the non-canonical Wnt pathway. The canonical Wnt pathway is transduced by β-catenin. Non-canonical Wnt pathways include the planar cell polarity (PCP) pathway, Wnt / JNK pathway, Wnt / Calcium pathway, Wnt-RAP1 pathway, Wnt-Ror2 pathway, Wnt-PKA pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, and Wnt-mTOR pathway. In the non-canonical Wnt pathway, there are signaling factors that are common to signaling pathways other than Wnt. However, activators of these factors are also included in the Wnt signaling pathway inhibitors. Furthermore, the Wnt signaling pathway activator refers to a substance that activates the Wnt signaling pathway, that is, the signaling pathway transmitted in the Wnt pathway, and examples thereof include GSK3 inhibitors.Examples of GSK3 inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), 1-Azakenpaullone (9-bromo-7,12-dihydro-pyrido[3',2':2,3]azepino[4,5-b]indol-6(5H)-one), and AZD 2858 (3-amino-6-[4-[(4-methyl-1- piperazinyl)sulfonyl]phenyl]-N-3-pyridinyl-2-pyrazinecarboxamide), BIO(6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3- dihydro-(3Z)-2H-indol-2-one), CP21R7 (3-(3-aminophenyl)-4-(1-methyl-1H-indol3-yl)-1H-pyrrole-2,5-dione), Sotrastaurin (3-(1H-indol-3-yl)-4-[2-(4-methyl1-piperazinyl)-4-quinazolinyl]-1Hpyrrole-2,5-dione), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol), Valproic Acid (2-propyl-pentanoic acid), and the like are included, and preferably CHIR99021 is used.

[0108] The concentration of the Wnt signaling pathway activator in the medium can be appropriately set depending on the substance used, within a range that allows the above-mentioned effects to be achieved. When CHIR99021, a type of GSK3 inhibitor, is used as the Wnt signaling pathway activator, its concentration is generally about 0.1 μM to about 100 μM, preferably about 0.5 μM to about 20 μM, and more preferably about 1 μM to about 10 μM. When a Wnt signaling pathway activator other than those mentioned above is used, it is desirably used at a concentration that exhibits Wnt signaling pathway enhancing activity equivalent to the above-mentioned concentrations.

[0109] In the third step, the cell aggregates obtained in the second step may be cultured in suspension in the same serum-free medium and culture compartment as those used in the second step. In one embodiment, the third step may be carried out by replacing the serum-free medium contained in the culture compartment used in the second step with a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator. In another embodiment, the cell aggregates of pluripotent stem cells formed in the second step may be collected and suspended in a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator, and the third step may be carried out in a new culture compartment.

[0110] The suspension culture time required in the third step can be determined appropriately, for example, from about 6 hours to about 42 hours, preferably from about 12 hours to about 36 hours, and more preferably from about 18 hours to about 30 hours.

[0111] Therefore, in one embodiment of the third step, on the fourth day after the start of the first step, the cell aggregates are washed with PBS and treated with 30 ng / ml Activin A and 3 μM CHIR99021 in serum-free medium for 24 hours to achieve primary differentiation.

[0112] The serum-free medium used in the third step may also contain a factor for maintaining undifferentiation in order to enable culture for maintaining undifferentiation, as in the first or second step. The factor for maintaining undifferentiation and its concentration contained in the serum-free medium in the third step may be the same as the factor for maintaining undifferentiation and its concentration used in the first or second step.

[0113] The method for producing cell aggregates of the present invention includes a fourth step of further suspension-culturing the cell aggregates obtained in the third step in a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor.

[0114] The Wnt signaling pathway activator and its concentration used in the fourth step may be the same as the Wnt signaling pathway activator and its concentration used in the third step.

[0115] As used herein, the term "BMP signaling pathway" refers to the pathway in which binding of BMP ligands to type I BMP receptors ALK-1, ALK-2, ALK-3, and ALK-6 induces phosphorylation of intracellular molecules Smad-1, Smad-5, Smad-8, and Smad-9, resulting in activation or repression of target gene transcription. BMP signaling pathway inhibitors are not limited to those capable of inhibiting signal transduction induced by BMP family proteins. They may be nucleic acids, proteins, or small organic compounds. Examples of such inhibitors include substances that inhibit BMP processing and extracellular secretion, substances that act directly on BMP (e.g., proteins, antibodies, aptamers), substances that suppress the expression of BMP-encoding genes (e.g., antisense oligonucleotides, siRNAs), substances that inhibit the binding of BMP receptors to BMPs, and substances that inhibit the physiological activity resulting from signal transduction mediated by BMP receptors. There are two types of BMP receptors: type I BMP receptors and type II BMP receptors. Type I BMP receptors include ALK-1, ALK-2, ALK-3, and ALK-6, while type II BMP receptors include TGF-beta R-II, ActR-II, ActR-IIB, BMPR2, and MISR-II.

[0116] Proteins known to inhibit the BMP signaling pathway include, for example, secreted proteins belonging to the NOGGIN, CHORDIN, FOLLISTATIN, GREMLIN, INHIBIN, TWISTED GASTRULATION, COCO, and DAN families.

[0117] Compounds well known to those skilled in the art can also be used as BMP signaling pathway inhibitors. Examples of BMP signaling pathway inhibitors include Alk2 / 3 inhibitors. Examples of compounds having the above-mentioned activity include LDN-193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride), K02288(3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol,3-[6-Amino-5-(3,4,5 -trimethoxyphenyl)-3-pyridinyl]-phenol), Dorsomorphin(6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyrid inyl)pyrazolo[1,5-a]pyrimidine), LDN-212854(5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyriMidin-3-y l]quinoline), LDN-214117(1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine), ML347 (5-[6-(4-Methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline)), DMH1(4-(6-(4-Isopropoxyphenyl)pyrazo lo[1,5-a]pyrimidin-3-yl)quinoline), DMH2(4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimid in-3-yl]-quinoline), VU0465350(7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine), VU046 9381(5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone), OD36(4-chloro-7,10-dioxa-13,17,18,Examples of Alk2 / 3 inhibitors include 21-tetrazatetracyclo[12.5.2.12,6.017,20]docosa-1(20),2(22),3,5,14(21),15,18-heptaene) and OD52. These substances may be used alone or in combination.

[0118] The BMP signaling pathway inhibitor is preferably an Alk2 / 3 inhibitor, more preferably selected from the group consisting of LDN-193189, K02288, Dorsomorphin, LDN-212854, LDN-214117, ML347, DMH1 and DMH2, and even more preferably LDN-193189.

[0119] The concentration of the BMP signaling pathway inhibitor in the medium can be appropriately set depending on the substance used, as long as the above-mentioned effects can be achieved. When LDN-193189 is used as the BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 1 μM, and even more preferably about 100 nM to about 500 nM. When K02288 is used, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 25 μM. When LDN-212854 is used, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When BML-347 is used, it is generally used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 1 μM to about 25 μM. When DMH2 is used, it is generally used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When a BMP signaling pathway inhibitor other than those mentioned above is used, it is desirably used at a concentration that exhibits BMP signaling pathway inhibitory activity equivalent to the above concentrations.

[0120] The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ receptors to TGFβ, and substances that inhibit physiological activities caused by signal transduction by TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). An example of a protein known as an inhibitor of the TGFβ signaling pathway is Lefty.

[0121] As the TGFβ signaling pathway inhibitor, compounds well known to those skilled in the art can be used. Specifically, SB431542 (sometimes abbreviated as "SB431") (4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide), SB505124 (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methylpyridine), SB525334 (6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY2157299 (4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyr azol-3-yl]-6-quinolinecarboxamide), LY2109761(4-[5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2 -b]pyrazol-3-yl]-7-[2-(4-morpholinyl)ethoxy]-quinoline), GW788388(4-{4-[3-(Pyridin-2-yl)-1H- pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), LY364947(4-[3-(2-Pyridin yl)-1H-pyrazol-4-yl]quinoline), SD-208(2-(5-Chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl amine), EW-7197(N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-Imidazole-2-methanamine)、A83-01(3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole)、RepSox(2-[5-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine)、SM16(4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]bicyclo[2.2.2]octane-1-carboxamide)、R268712(4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol)、IN1130(3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide)、Galunisertib(4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide)、AZ12799734(4-({4-[(2,6-dimethylpyridin-3-yl)oxy]pyridin-2-yl}amino)benzenesulfonamide)、A77-01(4-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline)、KRCA 0008(1,1-[(5-Chloro-2,4-pyrimidinediyl)bis[imino(3-methoxy-4,1-phenylene)-4,1-piperazinediyl]]bisethanone)、GSK 1838705(2-[[2-[[1-[(Dimethylamino)ethanoyl]-5-(methyloxy)-2,3-dihydro-1H-indol-6-yl]amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino]-6-fluoro-N-methylbenzamide)、Crizotinib(3-[(1R)-1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-2-aMinopyridine)、Ceritinib(5-Chloro-N2-[2-isopropoxy-5-Methyl-4-(4-piperidyl)phenyl]-N4-(2-isopropylsulfonylphenyl)pyriMidine-2,4-diaMine)、ASP 3026(N2-[2-Methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfon)、TAE684(5-Chloro-N2-[2-methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-2,4-pyrimidinediamine)、AZD3463(N-[4-(4-Amino-1-piperidinyl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)-2-pyrimidinamine)、TP0427736(6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole)、TGFBR1-IN-1(5-(1,3-benzothiazol-6-yl)-N-(4-hydroxyphenyl)-1-(6-methylpyridin-2-yl)pyrazole-3-carboxamide)、TEW-7197(2-fluoro-N-[[5-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,LY3200882(2-[4-[[4-[1-cyclopropyl-3-(oxan-4-yl)pyrazol-4-yl]oxypyridin-2-yl]amino ]pyridin-2-yl]propan-2-ol), BIBF-0775((3Z)-N-Ethyl-2,3-dihydro-N-methyl-2-oxo-3-[phenyl[[4-(1-piperidinylmethyl)phenyl]amino]methylen e]-1H-indole-6-carboxamide and other Alk5 inhibitors, SIS3 (1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-propen-1-one) and other SMAD3 inhibitors, and ITD-1 (4-[1,1'-Biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid). Examples of such inhibitors include receptor degradation promoters such as ALK5 (alcohol-containing methyl ester) and derivatives of these compounds. These substances may be used alone or in combination. SB431542 is a compound known as an inhibitor of the TGFβ receptor (ALK5) and Activin receptor (ALK4 / 7) (i.e., a TGFβR inhibitor). SIS3 is a TGFβ signaling pathway inhibitor that inhibits the phosphorylation of SMAD3, an intracellular signaling factor under the control of the TGFβ receptor. ITD-1 is a substance that promotes the proteasomal degradation of the TGF-β type II receptor.

[0122] The TGFβ signaling pathway inhibitor is preferably an Alk5 inhibitor, and the Alk5 inhibitor is preferably selected from the group consisting of SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, RepSox, SM16, R268712, IN1130, Galunisertib, AZ12799734, A77-01, KRCA 0008, GSK 1838705, Crizotinib, Ceritinib, ASP 3026, TAE684, AZD3463, and TP0427736, and more preferably SB431542.

[0123] The concentration of the TGFβ signaling pathway inhibitor in the medium can be appropriately set depending on the substance used, as long as the above-mentioned effects can be achieved. When SB431542 is used as the TGFβ signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 1 μM to about 10 μM. When a TGFβ signaling pathway inhibitor other than SB431542 is used, it is desirably used at a concentration that exhibits TGFβ signaling pathway inhibitory activity equivalent to that of SB431542 at the above-mentioned concentrations.

[0124] In the fourth step, the cell aggregates obtained in the third step may be cultured in suspension in the same serum-free medium and culture compartment as those used in the third step. In one embodiment, the third step may be carried out by replacing the serum-free medium contained in the culture compartment used in the third step with a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor. In another embodiment, the cell aggregates obtained in the third step may be collected and suspended in a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor, and the fourth step may be carried out in a new culture compartment.

[0125] The suspension culture time required in the fourth step can be determined appropriately, for example, from about 6 hours to about 30 days, preferably from about 12 hours to about 16 days, and more preferably from about 24 hours to about 12 days.

[0126] Therefore, in one embodiment of the fourth step, the cell aggregates are washed with PBS on the fifth day after the start of the first step, and then cultured in serum-free medium with 3 μM CHIR99021, 100 nM LDN-193189, and 2 μM SB431542 for 24 hours to achieve secondary differentiation.

[0127] The serum-free medium used in the fourth step may also contain a factor for maintaining undifferentiation in order to enable culture for maintaining undifferentiation, as in the first, second, or third step. The factor for maintaining undifferentiation and its concentration contained in the serum-free medium in the fourth step may be the same as the factor for maintaining undifferentiation and its concentration used in the first, second, or third step.

[0128] The fourth step of the method for producing cell aggregates of the present invention may include two steps ((4-1) and (4-2)). Step (4-1) is a step in which the cell aggregates obtained in the third step are cultured in suspension as described above to obtain the above-mentioned "1-1. Cell aggregates in the early stage of differentiation."

[0129] In addition, the above-mentioned (4-2) step is a step in which the cell aggregates obtained in step (4-1) are cultured in suspension in a manner similar to the fourth step of the above-mentioned method for producing cell aggregates of the present invention, thereby obtaining the above-mentioned "1-2. Cell aggregates in the late stage of differentiation." Step (4-2) may be performed by further continuing step (4-1). In addition, the cell aggregates to be subjected to suspension culture in step (4-2) may be obtained by other methods as long as they have the same characteristics as those of the above-mentioned "1-1. Cell aggregates at the early stage of differentiation."

[0130] 3. Uses of the Cell Aggregates of the Present Invention The cell aggregate of the present invention can be suitably used in regenerative medicine. Therefore, in another aspect, a transplantation therapy agent (hereinafter, sometimes referred to as "transplantation therapy agent of the present invention") containing the cell aggregate of the present invention is provided. The present invention also encompasses a method for treating or preventing spine-related diseases, in which an effective amount of the cell aggregate of the present invention is administered or transplanted into a subject (human) to be treated. Examples of spine-related diseases include diseases manifested as symptoms of damage, degeneration (degenerative disc disease), or herniation of the intervertebral disc (nucleus pulposus), such as lumbar or cervical disc disease, herniated disc, cervical spondylotic myelopathy, radiculopathy, spondylolysis / spondylolisthesis, lumbar spinal stenosis, degenerative lumbar spondylolisthesis, and degenerative lumbar scoliosis. Treatment of spine-related diseases also encompasses the regeneration of damaged tissues that cause the disease. Regeneration of damaged tissue includes not only complete tissue regeneration but also cases in which a tissue-like structure is formed.

[0131] The transplantation therapeutic agent of the present invention can be administered or transplanted into the body of a subject (human) in need thereof. The transplantation is preferably carried out into an area of ​​the body where the cell aggregate of the present invention can be fixed at a fixed position, for example, into an intervertebral disc, more specifically, into the nucleus pulposus. The cell aggregate to be transplanted only needs to be administered in a therapeutically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the recipient, and is not particularly limited, but for example, the number of cells contained in the cell aggregate to be transplanted may be 10 × 10 3 cells ~10×10 12 It can be made equivalent to a cell size.

[0132] When the cell aggregates of the present invention are used in cell transplantation therapy, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to prevent rejection. Here, "substantially the same" means that the HLA genotype matches the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C, or six loci including HLA-DP and HLA-DQ. If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0133] The transplantation therapy agent of the present invention can be prepared by, for example, mixing with a pharmaceutically acceptable aqueous liquid. Thus, in one embodiment, there is also provided a method for producing a transplantation therapy agent containing a cell aggregate, etc., which includes a step of formulating the cell aggregate, etc., of the present invention. Such a production method may include a step of preparing the cell aggregate, etc., of the present invention. Furthermore, it may also include a step of preserving the cell aggregate, etc., of the present invention.

[0134] The pharmaceutically acceptable aqueous liquid that can be contained in the transplantation therapy agent of the present invention can contain, for example, an appropriate selected buffer, isotonicity agent, pH adjuster, antioxidant, chelating agent, etc., within a range that does not affect the viability and physiological activity of the transplanted cell aggregate.

[0135] Examples of buffers include phosphate buffers, borate buffers, citrate buffers, tartrate buffers, acetate buffers, amino acids, epsilon-aminocaproic acid, etc. Examples of isotonicity agents include sugars such as D-sorbitol, D-glucose, and D-mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, and boric acid. Chelating agents include sodium edetate and citric acid. Examples of pH adjusters include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or its salts (borax), hydrochloric acid, citric acid or its salts (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or its salts (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or its salts (sodium acetate, ammonium acetate, etc.), and tartaric acid or its salts (sodium tartrate, etc.). Examples of antioxidants include ascorbic acid, glutathione, sodium hydrogen sulfite, dried sodium sulfite, sodium pyrosulfite, and tocopherol. Specific examples of "pharmaceutically acceptable aqueous liquids" include aqueous liquids such as saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The transplantation therapy agent of the present invention may be formulated with, for example, a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc.

[0136] The transplantation therapy agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be about 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0137] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0138] Materials and Methods hESC / iPSC culture The hESC line KhES-1 and the hiPSC lines 253G1 and 201B7 were cultured using a feeder-free system as described in (Kuroda et al., Stem Cell Res., 2019 Aug;101514). Undifferentiated hESCs / iPSCs were cultured in StemFit (SF) medium (Ajinomoto) at 37°C in an atmosphere containing 5% CO2, with the recombinant human laminin-511 E8 fragment, iMatrix-511 (Matrixome) (0.5 μg / cm). 2 After passage, hESCs / hiPSCs were treated with 0.5x TrypLE select (TrypLE select [Life Technologies] diluted 1:1 with 0.5 mM EDTA / PBS), dissociated into single cells by pipetting, and then cultured at 1.0 x 10 in SF medium containing 10 μM Y-27632 (FUJIFILM Wako Pure Chemical). 4 hESCs / hiPSCs were seeded onto iMatrix-511-coated 6-well culture plates at a density of 1000 cells / well. The next day, the medium was switched to SF medium without Y-27632. The medium was changed every other day. hESCs / hiPSCs were passaged once a week.

[0139] Axial mesoderm differentiation from hESCs / iPSCs For the 3D culture method, hESCs / iPSCs cultured to maintain an undifferentiated state were dissociated into single cells using 0.5x TrypLE select, and then cultured in SF medium containing 10 μM Y-27632 until they reached 3.0 x 10 3Cells were seeded at a density of 1000 cells / well onto low-cell-attachment 96-well plates (PrimeSurface, Sumitomo Bakelite). The day of seeding was designated day 0, and after 1 day of culture, cell aggregates were allowed to form in each well. On day 1, the medium was switched to SF medium containing 1% v / v Matrigel (Corning), and the cells were cultured in this medium for 3 days (days 1 to 4). On day 4, the cells were washed with PBS and treated with 30 ng / ml Activin A (R&D Systems) and 3 μM CHIR99021 (Stemgent) in SF medium for 24 hours (days 4 to 5) to achieve primary differentiation (anterior primitive streak [APS]). The cells were then washed with PBS and cultured in SF medium with 3 μM CHIR99021, 100 nM LDN-193189 (Stemgent), and 2 μM SB431542 (FUJIFILM Wako Pure Chemical) for 24 hours (from day 5 to day 6) to achieve secondary differentiation (axial mesoderm [AM] including the primitive node). To examine the effect of medium on AM differentiation, we used CDM2 (50% IMDM [Life Technologies] and 50% F12 [Life Technologies], 1 mg / ml polyvinyl alcohol [Sigma], 1% v / v chemically modified lipid concentrate [Life Technologies], 450 μM monothioglycerol [FUJIFILM Wako Pure Chemical], 0.7 μg / ml insulin [Sigma], and 15 μg / ml transferrin [Sigma]) and Essential 6 (E6) medium (Thermo Fisher Scientific). For 2D culture, undifferentiated hESCs / iPSCs were dissociated into single cells and then cultured at 5.0 x 10 in SF medium containing 10 μM Y-27632 on an iMatrix-511-coated 24-well culture plate. 4Cells were seeded at a density of 1000 cells / well and cultured for 1 day (day 0 to day 1). They were then treated with 30 ng / ml Activin A and 3 μM CHIR99021 in SF medium for 24 hours (day 1 to day 2) to achieve primary differentiation (APS). Cells were then washed with PBS and treated with 3 μM CHIR99021, 100 nM LDN-193189, and 2 μM SB431542 in SF medium for 24 hours (day 2 to day 3) to achieve secondary differentiation (AM with primitive nodules).

[0140] Paraxial mesoderm, definitive endoderm, early somitic differentiation Paraxial mesoderm (PM) and definitive endoderm (DE) in Figures 9 and 14, and early somites (ES) in Figure 4, were derived from hESCs / iPSCs according to the method of switching from 2D culture to 3D culture (Loh et al., Cell, 2016, July; 14:451-467). Undifferentiated hESCs / iPSCs were dissociated into single cells and plated at 3.0 x 10 in SF medium containing 10 μM Y-27632 on a 96-well PrimeSurface plate. 3Cells were seeded at a density of 1000 cells / well and cultured for 1 day (day 0 to day 1). They were then treated with 1% v / v Matrigel in SF medium for 3 days (day 1 to day 4). For PM differentiation, day 4 cells were treated with 30 ng / ml Activin A and 3 μM CHIR99021 in E6 medium for 24 hours (day 4 to day 1), followed by 3 μM CHIR9902, 100 nM LDN-193189, and 2 μM SB431542 in SF medium for 24 hours (day 5 to day 6). For DE differentiation, day 4 cells were treated with 30 ng / ml Activin A and 3 μM CHIR99021 in SF medium for 24 hours (day 4 to day 5), followed by 100 ng / ml Activin A and 100 nM LDN-193189 in SF medium for 48 hours (day 5 to day 7). For ES differentiation, day 4 cells were treated with 30 ng / ml Activin A and 3 μM CHIR99021 in SF medium for 24 hours (days 4 to 5), followed by 3 μM CHIR99021, 100 nM LDN-193189, and 2 μM SB431542 in SF medium for 24 hours (days 5 to 6), followed by 100 nM LDN-193189, 2 μM SB431542, 5 μM IWR-1-endo (Merck), and 500 nM PD0325901 (Sigma).

[0141] Long-term culture and cyclopamine treatment The method for differentiating axial mesoderm under 3D culture conditions by day 6 was as described previously. The hESC / iPSC aggregates were then cultured in SF medium containing 3 μM CHIR9902, 100 nM LDN-193189, and 2 μM SB431542 until day 18. The medium was completely changed every 2–3 days. To inhibit the sonic hedgehog signaling pathway, cyclopamine (LKT Labs) was added to the medium at a concentration of 1 μM or 3 μM on day 8, and subsequent medium changes were performed using medium containing cyclopamine.

[0142] RT-qPCR Total RNA was extracted using the RNeasy Mini kit (QIAGEN) and purified with Oligo(dT). 12-18 Reverse transcription was performed using Primer (Thermo Fisher Scientific) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative PCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's instructions. Data analysis was performed using the comparative Ct method. Expression levels were normalized to those of β-actin. The primers used for RT-qPCR are listed in Table 1.

[0143] [Table 1-1]

[0144] [Table 1-2]

[0145] [Table 1-3]

[0146] immunostaining For frozen sections, cultured cell aggregates were fixed in 4% paraformaldehyde in phosphate-buffered saline (4% PFA / PBS) (FUJIFILM Wako Pure Chemical) for 15 minutes at room temperature, washed with PBS, and then equilibrated overnight at 4°C in 15% sucrose in PBS. Cell aggregates were embedded in OCT compound (Sakura Finetek), frozen, and sectioned at 10 μm thickness using a cryostat (Leica Biosystems). Sections were placed on glass slides (Matsunami glass) and air-dried at 37°C for 30 minutes. The sections were washed with PBS and incubated in blocking solution (5% BSA and 0.3% Triton X-100 in PBS) for 1 hour at room temperature. The sections were then incubated with primary antibodies in blocking solution overnight at 4°C, washed with PBS, and then incubated with secondary antibodies in blocking solution containing 1 μg / ml DAPI for 1 hour at room temperature. After washing with PBS, sections were mounted with SlowFade Gold Antifade Mountant (Thermo Fisher Scientific), and immunofluorescence images were obtained using a TSC SP-8 (Leica Camera) laser scanning confocal microscope. For whole mounts, cell aggregates cultured in 96-well low-adhesion plates were transferred to cell culture dishes (IWAKI) and incubated in culture medium for 30 minutes. Once the cell aggregates adhered to the dishes, they were fixed with 4% PFA / PBS for 10 minutes at room temperature. The fixed cell aggregates were washed with PBS, simultaneously permeabilized, and blocked with blocking solution (5% donkey serum, 0.1% Triton X, PBS) for 1 hour at room temperature. The cell aggregates were then incubated with primary antibodies in blocking solution overnight at 4°C, washed with PBS, and then incubated with secondary antibodies in blocking solution containing 1 μg / ml DAPI for 1 hour at room temperature. After washing with PBS, immunofluorescence was performed using a TSC SP-8 laser scanning confocal microscope. For 2D cultured cells, the methods of fixation, permeabilization, blocking, and antibody staining were the same as for whole-mount immunofluorescence. The antibodies used in this method are listed in Table 2.

[0147] [Table 2]

[0148] scanning electron microscope Day 5.5 axial mesoderm-differentiated cell aggregates were transferred to chamber slides (Thermo Fisher Scientific) coated with a 1:30 dilution of Matrigel and cultured in the corresponding medium for 12 hours to induce differentiation and allow them to adhere to the bottom of the culture slide. The adhered cell aggregates were fixed overnight at 4°C with 4% PFA / 2% glutaraldehyde in phosphate buffer. The fixed cell aggregates were then incubated in 1% osmium tetroxide (Nacalai Tesque), dehydrated, dried by critical point drying, and coated with a thin layer of platinum-palladium. Samples were observed under a scanning electron microscope S-4700 (Hitachi Co., Tokyo, Japan).

[0149] Single-cell RNA sequencing Cell aggregates differentiated into APS on day 5, AM on day 6, and long-term AM on day 11 were dissociated into single cells using TrypLE select for 10 minutes at 37°C. Dissociated cells were suspended in PBS containing 10 μM Y-27632 and 0.1% BSA. Immediately, single-cell targeted library preparation was performed using the Chromium Single Cell 3' Reagent Kits v3 (10x Genomics) according to the manufacturer's instructions. Approximately 10,000 to 17,000 cells were analyzed at each time point, and libraries were sequenced on a NovaSeq 6000 (Illumina) using sequencing parameters according to the manufacturer's instructions.

[0150] Single-cell RNA-sequencing data analysis The raw sequencing files were separated using unique index combinations to generate FASTQ files.

[0151] Example 1: Axial mesoderm can be induced from hESCs / iPSCs in 3D culture but not in 2D culture under CDM2 medium conditions. StemFit medium promotes differentiation into axial mesoderm while inhibiting differentiation into paraxial mesoderm. The opposite phenomenon is observed under Essential 6 medium conditions. Although several papers have reported the differentiation of mouse and human ESCs into node- and notochord-like cells, the differentiation efficiency was very low and was confirmed only by the expression of marker genes. Therefore, the inventors attempted to more efficiently differentiate hESCs / iPSCs into axial mesoderm, which has the structure and function of the node and notochord. In mice, cells destined to become the axial mesoderm (AM) reside in the anterior primitive streak (APS) during early gastrulation (Figure 2A). Paraxial mesoderm (PM) and definitive mesoderm (DE) cells also originate from the APS (Figure 2A). Therefore, we investigated whether we could modify the method for inducing AM cells from hESCs / iPSCs and their differentiation into PM (Figure 1A). According to a previous report, PM differentiation was performed in two steps under two-dimensional (2D) culture conditions (Loh et al., 2016) (Figure 1A, B; Figure 2B: 2D culture). The first differentiation step (1st differentiation) involved exposing undifferentiated hESCs (UD) to APS cells with Activin A and the WNT signaling agonist CHIR-99021 (CHIR). The second differentiation step (2nd differentiation) involved exposing APS cells to PM cells with CHIR, the BMP signaling inhibitor LDN193189 (LDN), and the TGF-β type I receptor inhibitor SB431542 (SB). On day 3, phase-contrast microscopy revealed that differentiated cells exhibited large, flat morphologies (Figure 2C: top). Consistent with previous results, expression of the UD marker OCT4 was decreased, while expression of the pan-mesodermal marker BRACHYURY (BRA) and PM marker BX6 was increased (Figure 1C: gray bars). Unfortunately, expression of the AM-specific marker FOXJ1 was not elevated under these conditions (Figure 1C: gray bars). The movement of AM cells to form the node and notochordal plate is highly complex. Presumptive AM cells in the epiblast undergo epithelial-mesenchymal transition (EMT) in the anterior part of the primitive striatum and then invade the mesoderm layer. They then migrate rostrally, undergo mesenchymal-epithelial transition (MET), and emerge along the midline at the embryonic surface. There, AM cells are surrounded by endodermal cells (proximal endoderm and / or definitive endoderm), indicating that cells of different germ layers coexist in a patterned, adjacent fashion. To differentiate hESCs / iPSCs into AM cells, we hypothesized that such complex conditions and environments must be recapitulated in cell culture. Therefore, we next converted 2D culture to 3D culture (Figure 1B, Figure 2B: 3D culture). 3D culture is suitable for recapitulating early embryonic development because it can strongly induce self-organization. In 3D culture, undifferentiated hESCs were aggregated using the SFEBq protocol from day 0 to day 1, and then treated with Matrigel from day 1 to day 4 to induce the basal side of the cell aggregates. Subsequently, AM differentiation was performed in two stages (first differentiation: day 4 to day 5, second differentiation: day 5 to day 6) as in 2D culture (Figure 1B, Figure 2C: bottom). As expected, increased expression of FOXJ1 was observed in 3D culture (Figure 1C: dark gray bar). Unexpectedly, despite mesoderm induction, the expression of the endoderm marker SOX17 was also increased (Figure 2D: dark gray bar). These results suggest that 3D culture is suitable for axial mesoderm differentiation. Next, we examined the media used during the differentiation period (days 4 to 6). We compared three media: the original CDM2 medium, Essential 6 (E6) medium, and StemFit (SF) medium, which was also used during the hESC / iPSC maintenance culture period (Figure 1D). There were no obvious differences in the appearance and size of cell aggregates (Figure 2E) or in the gene expression of OCT4 and BRA (Figure 2F). However, TBX6 expression was significantly elevated in CDM2 and E6 media, but only slightly elevated in SF medium (Figure 1E). Furthermore, expression of FOXJ1 and another AM-specific marker, NOTO, was highest in SF medium. Increased expression of FOXJ1 and NOTO was also observed in CDM2 medium, as shown in Figure 1C, but not in E6 medium (Figure 1E). SOX17 was also highly expressed in SF medium (Figure 2F). These data suggest that under 3D culture conditions, SF medium induced AM differentiation most efficiently, with little PM differentiation. On the other hand, E6 medium induced PM-specific differentiation, while CDM2 medium induced both AM and PM differentiation, although AM differentiation was limited.

[0152] Example 2: The preferential differentiation into axial mesoderm and paraxial mesoderm in SF medium and E6 medium, respectively, has been replicated in other pluripotent stem cell lines. Because there is considerable variability in the differentiation potential of hESCs / iPSCs into specific cell lineages, multiple cell lines must be used to confirm the phenomenon observed in a single cell line. The preferential differentiation into AM in SF medium and PM in E6 medium was also observed in other pluripotent stem cells, 253G1 and 201B7 (both hiPSC lines) (Figure 3). These results indicated that this phenomenon is common across cell lines.

[0153] Example 3: Axial mesoderm exhibited different differentiation characteristics than paraxial mesoderm. To confirm the differences in the properties of AMs and PMs selectively produced by the difference in culture medium, we further differentiated the 2nd differentiation products, differentiated in E6 medium and SF medium, into early somites (ES) according to the procedure shown in Figure 4B. ES were derived from PMs through the 3rd differentiation step using the LDN, SB, and WNT signaling inhibitor IWR-1e (IWR) and the MEK inhibitor PD0325901 (PD) (Figure 4A, B). After 3rd differentiation, the appearance of cell aggregates was clearly different between E6 medium and SF medium. PM derivatives in E6 medium had a smooth surface (Figure 4C, left panel), while AM ​​derivatives in SF medium had an uneven surface with multiple vesicles (Figure 4C, right panel). The 3rd differentiation products in E6 medium showed significantly increased expression of the ES markers FOXC2 and MEOX1 (Figure 4D, light gray bars), but not in SF medium (Figure 4D, dark gray bars). These results indicate that AM cells induced in SF medium have distinct properties that are distinct from PM cells induced in E6 medium, and that the AM differentiation products are largely free of PM cells.

[0154] Example 4: Examination of optimal differentiation conditions We investigated the necessity of each component (extracellular matrix, recombinant proteins, and small molecules) used to induce AMs using SF medium under 3D culture conditions. Figure 5A shows the exposure timing and treatment period for each component. Treatment with the Rho-kinase inhibitor Y-27632 (Y) from day 0 to day 1 was found to be essential for cell survival and aggregation (Figure 5B). Comparison of cell aggregates with and without Matrigel from days 1 to 4 revealed that cell aggregates without Matrigel were significantly smaller in size than those with Matrigel, and appeared to maintain an undifferentiated state, with a highly permeable, smooth surface. However, cell aggregates with Matrigel became rougher on day 5 and, although smooth, had a less transparent surface and a thinner epithelial layer on day 6 (Figure 5C). In cell aggregates without Matrigel, expression of OCT4 and another UD marker, NANOG, did not decrease even after secondary differentiation, and expression of differentiation markers BRA, FOXA2, FOXJ1, and NOTO was not elevated. These data indicate that Matrigel is also required for efficient cell differentiation (Figure 5D). Finally, we examined the effects of Activin A (A) and CHIR99021 (C) on days 4–5 of primary differentiation, and CHIR99021, LDN-193189 (L), and SB431542 (S) on days 5–6 of secondary differentiation (Figure 5E). We examined the combination of A and C when CLS exposure was fixed during secondary differentiation, and the combination of C, L, and S when AC exposure was fixed during primary differentiation. When A and C were used during primary differentiation, no addition (none) or A alone induced only weak expression of BRA, FOXA2, and FOXJ1. However, C alone and AC induced high expression of BRA, but slightly lower expression of FOXA2 and FOXJ1, and SOX17 expression was undetectable with C alone. When C, L, and S cells were examined during the second differentiation stage, high expression of BRA was observed only in CS and CLS, whereas low expression of FOXJ1 and high expression of TBX6 were observed in CS. This indicates that inhibition of the BMP signaling pathway by LDN-193189 is important for the conversion of PM to AM differentiation. Finally, the most efficient differentiation to AM was achieved when all components were exposed. These results demonstrate the role and necessity of each component in AM differentiation.

[0155] Example 5: Highly efficient and specific differentiation into axial mesoderm by combining SF medium and three-dimensional culture We found that SF medium is suitable for AM differentiation from hESCs / iPSCs (Figure 1D, E). Therefore, we compared 2D and 3D culture in detail using SF medium (Figure 6A, B). Gene expression analysis revealed that the expression of OCT4 and NANOG decreased with differentiation, while the expression of BRA, FOXA2, GSC, FOXJ1, and NOTO increased in a similar pattern in 2D and 3D cultures. However, the expression levels of the latter four AM markers were higher in 3D culture than in 2D culture (Figure 6C). Furthermore, unlike in CDM2 medium, FOXJ1 and NOTO were expressed at low levels in 2D culture, but were still sufficient after the second differentiation stage (Figure 1C, Figure 6C). Compared to 2D culture, the expression of TBX6 and MSGN1 was significantly suppressed in 3D culture, while the expression of SOX17 was significantly increased (Figure 6C). Immunostaining also confirmed higher FOXJ1 expression in 3D cultures than in 2D cultures (Figure 6D). Interestingly, while FOXJ1-positive cells were dispersed in 2D cultures, multiple FOXJ1-positive cell populations were present on or near the outer surface of cell aggregates in 3D cultures (Figure 6D, Figures 7A, 7C). The locations of BRA, FOXA2, OCT4, SOX17, TBX6, and CDX2-positive cells were also clearly evident in both 2D and 3D cultures (Figure 7). To evaluate differentiation efficacy, the percentage of FOXJ1-positive areas was quantified by image analysis. As shown in Figure 6E, the percentage in 3D cultures was more than three times higher than in 2D cultures (2D: 5.4 ± 0.9%, 3D: 17.3 ± 1.0%). These results demonstrate that the combination of SF medium and 3D culture is the optimal condition for the most efficient and specific induction of AM differentiation.

[0156] Example 6: Tracking differentiation of the anterior primitive streak to axial mesoderm by single-cell RNA sequencing Single-cell RNA sequencing (scRNA-seq) analysis was performed on APS cell aggregates induced by primary differentiation (day 5) and AM cell aggregates induced by secondary differentiation (day 6). UMAP analysis of a mixture of APS and AM cell aggregates showed that UTF1-positive epiblast cells differentiated into BRA-positive primitive streak cells, which further differentiated into LHX1-positive endodermal and mesodermal cells (Figure 8A). Figure 8B shows the differences between APS cell aggregates and AM cell aggregates, shown in black and gray, respectively (Figure 8B left panel). Clustering identified 11 different cell populations (Figure 8B right panel). Figure 8C shows the expression of various lineage marker genes in these cell populations, and Figures 8D and 8E show the distribution of cells expressing each marker gene. When the expression of these marker genes was evaluated in conjunction with previous data, APS cell aggregates corresponded to mouse embryos at E6.5 and E7.0 and human embryos at 14 days post-fertilization (dpf), respectively, and AM cell aggregates corresponded to mouse embryos at E7.5 and human embryos at 16 dpf, respectively. Pseudo-time series analysis revealed that paraxial parietal mesoderm (PSM) and endoderm (Endo) were generated via the APS, but axial mesoderm (AxM) was induced via a pathway distinct from APS after leaving the Epi state (Figure 8F), and differentiated via a cell population distinct from AxM, Epi, and APS. This cell population was named axial mesoderm progenitor cells (AMP). This data was very intriguing, as the origin of axial mesoderm had not been clearly demonstrated in vivo, and the existence of axial mesoderm progenitor cells was revealed. Figure 8G shows the change in expression levels of each gene over time in a pseudo-time series. reported the results of scRNA-seq analysis of human fetal embryos at stage CS7. Using AM cell aggregates (day 6), we compared our data with Tyser et al.'s data and found that although AM cell aggregates lacked endoderm, mesoderm, vascular endothelium, erythroblasts, and some extraembryonic mesoderm cells, the plots of various cell types reported in their paper neatly fit into the groupings classified by the inventors. These results indicate that AM cell aggregates recapitulate, to some extent, the development of human fetal embryos.

[0157] Example 7: Axial mesoderm cell aggregates exhibit some characteristics of nodules. This nodule is an AM derivative and has several distinctive features. First, it has a unique gene expression pattern. Second, it has a unique position relative to surrounding cells and apical-basal polarity between cells. Third, it has the presence of primary cilia protruding from the apical surface of the cells. Fourth, it expresses several secreted factors that act as signaling centers and are involved in axis determination and early embryonic development. To investigate the unique gene expression patterns in AM cell aggregates, we performed immunostaining for each protein. Figure 9A shows that most FOXJ1-positive cells coexpressed BRA and FOXA2 near the outer surface of the cell aggregates (Figure 9A: upper lane). Some FOXJ1, BRA, and FOXA2 triple-positive cells formed cell clusters at the outermost part of the cell aggregates, which are thought to correspond to the ventral side of the embryo (Fig. 9A, right panel of the upper lane). These cell clusters were SOX17-negative and were surrounded by SOX17-positive DE cells, which existed continuously over the entire outer surface of the cell aggregates (Fig. 9A, bottom row). Furthermore, OCT4-positive epiblast cells were present inside the cell aggregates, which are thought to correspond to the dorsal side of the embryo, and were in contact with the triple-positive cell clusters (Fig. 9A, right panel of the lower lane). The arrangement of these cells was very similar to that observed in the chick and mouse tubercles. Next, we examined apical-basal polarity (Fig. 10A). In the primitive streak, the basement membrane of the ventral epiblast is destroyed by gastrulation. Because the node is located at the anterior end of the primitive streak, the basement membrane between the epiblast and the node is also destroyed. In the "node region" of AM cell aggregates, the basement membrane marker LAMININ was intermittently observed between the triple-positive cell group and the OCT4-positive cell layer, indicating that the basement membrane had indeed been destroyed (Fig. 10B). In contrast, in the "non-node region," LAMININ was strongly and continuously expressed between the BRA-positive mesoderm layer and the OCT4-positive epiblast (Fig. 10C). The apical marker EZRIN was expressed inside the OCT4-positive epiblast, corresponding to the dorsal side of the embryo (Fig. 10C). This EZRIN expression pattern resembled the continuous contact between the apical surface of the epiblast and the dorsal side of the amniotic cavity seen in chick and mouse embryos, and these data indicated that the apical-basal polarity of AM cell aggregates also closely recapitulates that of the embryo. Primary cilia are one of the most distinctive features of nodule cells and play an important role in disrupting left-right symmetry. Immunostaining with the cilia marker Ac-α-Tubulin revealed the presence of primary cilia protruding outward from FOXJ1-positive AM cells (Fig. 9B), as shown in Figure 9A (Fig. 11). These cells were surrounded by SOX17-positive DE cells. Primary cilia were also confirmed by scanning electron microscopy (Fig. 9C). The AM derivatives, the node, notochordal plate, and notochord, serve as embryonic signaling centers that control embryonic patterning. Finally, we examined the gene expression of signaling factors secreted from the node (Figure 9E). Expression of the BMP antagonists CHORDIN and NOGGIN, and the Sonic Hedgehog (shh) signaling factor SHH, was higher in the AM than in the PM or DE (Figure 9D). The expression of other secreted factors associated with early embryonic development is shown in Figure 12. These results strongly suggest that AM cell aggregates share many characteristics very similar to those of the in vivo node.

[0158] Example 8: Confirmation of the function of axial mesoderm derivatives in neural induction and activation of the shh signaling pathway The neural induction factors CHORDIN and NOGGIN, which are required for forebrain development in mice, are secreted by AM derivatives. SHH is also secreted by AM derivatives and ventralizes early neural plate cells, the neural tube, and somites. It has been reported that the addition of surgically isolated chick notochord to cultures of undifferentiated hESCs induces neural differentiation and ventralization. Therefore, we investigated whether AM cell aggregates have the ability to induce neural differentiation and ventralization of hESCs (Figure 13A). We used cell aggregates not only on day 6, immediately after the end of secondary differentiation, but also on days 8, 11, and 14, when additional culture was performed to promote tissue development of the cell aggregates. When culture was extended from secondary differentiation to day 14, the surface of the cell aggregates began to become uneven on day 8, and many small vesicles were observed on the surface from day 11 onwards (Figure 14A). The expression of FOXj1 and Noto gradually decreased from day 6 to day 14 (Figure 13B), which was similar to the expression of FOXj1 and Noto in mouse embryos. Foxj1 and Noto are expressed in early AM derivatives from E7.5 to E8.0, but gradually decrease after E8.5. The expression of BRA and FOXA2 also gradually decreased after day 6, but remained higher than that of undifferentiated control cells (Figure 14B). The expression patterns of BRA and FOXA2 were maintained in the mouse embryonic notochord for a long period of time, consistent with their in vivo expression patterns. These results indicated that the additional culture, which induces a decrease in AM marker gene expression, partially recapitulates AM development in vivo. Expression of TBX6 and SOX17 also gradually decreased to the same level as the control by day 14 (Fig. 14B). Expression of CHORDIN gradually decreased but maintained a relatively high expression level even at day 14. On the other hand, expression of NOGGIN and SHH increased throughout the period (Fig. 13C). Next, to confirm whether the induced AM derivatives possessed neural differentiation and ventralization potential, undifferentiated hESCs were cultured in conditioned medium from AM cell aggregates on days 6, 8, 11, and 14. Expression of neuroectoderm markers PAX6, SIX3, RX, OTX2, ZNF521, and OCT6, as well as SHH target genes HHIP and PTCH1, were significantly increased (Figures 13E and 13F, Figure 15). These results suggest that the AM derivatives possess neural differentiation and ventralization potential, at least in relation to undifferentiated hESCs.

[0159] Example 9: Single-cell RNA-Seq analysis of long-term cultured cell aggregates We performed scRNA-seq analysis on day 11 AM cell aggregates, and nine cell populations were identified by clustering, annotated as follows: undifferentiated (undifferentiated), No. 6 (AMP: axial mesoderm precursor), node, notochord (NC), No. 3 (APS), somite 1, somite 2, and foregut (Figure 16A). POU5F1-positive undifferentiated cells, FOXC2-positive somites, FOXA1- and LBH-positive foregut, strongly FOXJ1- and BRA-positive nodes, and strongly FOXJ1- and BRA-positive notochord were detected (Figure 16B, C). Somites were divided into two types: somite 1 and somite 2. Somite 2 is an early somite before bud formation, strongly SOX9-positive and Nkx3.2-negative, while somite 1 is a mature somite, weakly SOX9-positive and Nkx3.2-positive (Figure 16C). Strong expression of the neural induction factor CHORDIN and the ventralization factor SHH was observed in the notochord compared with the node cell population (Fig. ​(Fig.16B). 16B). These results indicated that long-term culture promoted the development of cells derived from each germ layer, including the axial mesoderm.

[0160] Example 10: Embryonic development around the notochord was recapitulated in long-term cultured axial mesoderm cell aggregates. AM cell aggregates were cultured for up to 18 days (Figure 17A) and contained a central aggregate surrounded by several "buds" (Figure 18A). Since Figure 16 indicates that long-term cultured cell aggregates contain more mature cells, we examined the expression of neuroectoderm-derived brain markers PAX7, EN2, PAX8, and GBX2 (Figure 18B), PM-derived somitic markers FOXC2 and MEOX1 (Figure 18C), DE-derived pan-endodermal marker FOXA1 (Figure 18D), and gut markers IRX3, TBX1, HOXB4, and CDX2 (Figure 18E). Most of these genes began to be expressed at days 8 or 11 and reached high expression levels by day 18 (Figures 18B, C, D, and E). Regarding the anterior-posterior axis, no expression of forebrain markers was observed (data not shown), but the expression of midbrain, mid-hindbrain boundary (MHB), and hindbrain markers was observed (Figure 18B). Furthermore, expression of anterior and posterior foregut markers was observed, but not midgut / hindgut markers (Figure 18E). These results suggest that the regions corresponding to these gene expression regions are induced in long-term cultured AM cell aggregates. Furthermore, immunohistochemical analysis revealed that the surface of the cell aggregates was covered with FOXA1-positive endoderm cells (Figure 18F). As shown in Figure 14B, gene expression analysis revealed that the expression of the notochord marker BRA gradually decreased from day 6 onward but remained low at day 18 (Figure 17B), whereas SHH was highly expressed at day 18 (Figure 17C). Immunostaining of BRA and SHH revealed that their expression patterns were very similar (Figure 17D). These results are consistent with the secretion of SHH from the notochord in vertebrate embryos. SHH secreted from the notochord ventralizes the neural tube and somites. We cultured AM cell aggregates containing cells expressing brain and somite markers for the long term and examined whether the expression of floor plate markers Nkx6.1 and ARX, and ventral somite markers Nkx3.2 and PAX9 increased (Figure 18B,C). As expected, the expression of floor plate and ventral somite marker genes increased from day 11, which coincided with the significant increase in SHH gene expression (Figure 17E,F). Furthermore, Nkx6.1- and Nkx3.2-positive cells were present only in the vicinity of BRA-positive cells (Fig. 17G, H). These results suggest that SHH secreted from BRA-positive cells ventralizes neural tube and somite-differentiated cells. Interestingly, BRA-positive cells in long-term cultured AM cell aggregates were localized within the aggregates, rather than on their surface as seen on day 6. Furthermore, some BRA-positive cells were clustered together surrounded by a LAMININ-positive basement membrane (Fig. 18G). The characteristics of these BRA-positive cells resembled those of the in vivo notochord. To confirm the effect of SHH on the ventralization of neural tube and somite-differentiated cells, long-term cultured AM cell aggregates were treated with the SHH antagonist cyclopamine from day 8 (Fig. 19A). The appearance of cyclopamine-treated cell aggregates was unchanged from that of untreated aggregates (Fig. 19A). However, because SHH is autoregulated, its effect was confirmed by the inhibition of gene expression of SHH and SHH target genes (Fig. 17I). In cyclopamine-treated cell aggregates, the expression of floor plate and ventral somite markers was suppressed, while the expression of tectal plate and dorsal somite markers was increased (Fig. 17J, 17K; Fig. 19B, 19C). These results demonstrate that the in vivo ventralization of neural tube and somites induced by SHH secreted from the notochord is recapitulated in our AM cell aggregates. [Industrial Applicability]

[0161] The present invention provides a method for efficiently inducing differentiation in vitro of human pluripotent stem cells into spherical human cell aggregates containing axial mesoderm cells. The present invention also makes it possible to realize transplantation therapy for patients with intervertebral disc degeneration by using spherical human cell aggregates containing axial mesoderm cells.

Claims

1. Spherical human cell aggregates with the following characteristics: (1) It has two distinct areas: a primitive node area and a non-node area. (2) The primitive nodule region and the non-primitive nodule region have at least two or more layer structures, (3) primitive node cells are present in the first layer present on the surface of the primitive node region, and endoderm cells are substantially absent in the primitive node region; (4) Endodermal cells are present in the first layer, which is present on the surface of the non-primitive node region.

2. The human cell aggregate of claim 1, wherein the primitive nodule cells are FOXJ1-positive cells.

3. The human cell aggregate according to claim 1 or 2, wherein FOXJ1-positive cells are present at a ratio of approximately 10% to 20% of the total cell number in the cell aggregate.

4. The human cell aggregate according to any one of claims 1 to 3, wherein the primitive nodule cells are cells that express FOXJ1, BRA, and FOXA2.

5. A human cell aggregate according to any one of claims 1 to 4, wherein primary cilia extend from the primitive node cells toward the outside of the cell aggregate.

6. The human cell aggregate according to any one of claims 1 to 5, wherein the endoderm cells are SOX17-positive cells.

7. A human cell aggregate according to any one of claims 1 to 6, wherein the first layer present on the surface is composed of a cell layer having a thickness equivalent to 1 to 10 cells.

8. A human cell aggregate described in any one of claims 1 to 7, wherein axial mesoderm cells are present in a second layer inside the non-primitive node region, and epiblast cells are present in a third layer further inside that, and epiblast cells are present in the second layer inside the primitive node region.

9. The human cell aggregate described in claim 8, wherein the epiblast cells are OCT4-positive cells.

10. A human cell aggregate described in any one of claims 1 to 9, wherein in the primitive nodule region and / or non-primitive nodule region, a basement membrane is present between the first layer present on the surface and the second layer present inside.

11. The human cell aggregate described in claim 10, wherein the basement membrane is continuously present in the non-primitive nodule region.

12. The human cell aggregate described in claim 10 or 11, wherein the basement membrane is LAMININ-positive.

13. The human cell aggregate according to any one of claims 1 to 12, wherein the primitive node cells or axial mesoderm cells express one or more genes selected from the group consisting of CHORDIN, NOGGIN, and SHH.

14. (1) a first step of rapidly aggregating dispersed human pluripotent stem cells to form a single cell aggregate in a single culture compartment; (2) a second step of culturing the formed cell aggregates of human pluripotent stem cells in suspension in a medium containing a basement membrane preparation; (3) a third step of further culturing the cell aggregates obtained in the second step in suspension in a medium containing a TGFβ signaling pathway activator and a Wnt signaling pathway activator; and (4) a fourth step of further suspension-culturing the cell aggregates obtained in the third step in a medium containing a Wnt signaling pathway activator, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor; A method for producing human cell aggregates comprising axial mesoderm cells.

15. The method of claim 15, wherein the basement membrane preparation is Matrigel.

16. The production method according to claim 14 or 15, wherein in the first step, cell aggregates are formed by the SFEBq method.

17. The production method according to any one of claims 14 to 16, wherein in the first step, cell aggregates are formed in the presence of a Rock inhibitor.

18. The method according to any one of claims 14 to 17, wherein the culture medium in the first step and the second step contains a factor for maintaining undifferentiation.

19. The method of claim 18, wherein the culture medium in the third and fourth steps contains a factor for maintaining undifferentiation.

20. The method according to any one of claims 14 to 19, wherein the culture period in the second step is 2 to 4 days.

21. The method according to any one of claims 14 to 20, wherein the Wnt signaling pathway activator is a GSK3 inhibitor.

22. The method of claim 21, wherein the GSK3 inhibitor is CHIR99021.

23. The method according to any one of claims 14 to 22, wherein the TGFβ signaling pathway activator is Activin A.

24. The method of any one of claims 14 to 22, wherein the TGFβ signaling pathway inhibitor is an ALK5 inhibitor, and the BMP signaling pathway inhibitor is an ALK2 / 3 inhibitor.

25. The manufacturing method according to claim 24, wherein the ALK5 inhibitor is SB431542 and the ALK2 / 3 inhibitor is LDN-193189.

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