Construction of three-dimensional organs from pluripotent stem cells

Abstract

To provide solutions to the problems of conventional methods for constructing a three-dimensional structure (organ primordium) by co-culturing functional cells with endothelial cells derived from umbilical codes and mesenchymal cells derived from bone marrow, the problems being: [1] donor dependent quality diversity; [2] limited proliferation capability of cell sources; and [3] difficulty in guaranteeing immunocompatibility because of different cell sources.SOLUTION: Disclosed is an organ bud prepared from vascular cells, mesenchymal cells and tissue or organ cells, where the vascular cells, mesenchymal cells and tissue or organ cells have been induced from pluripotent stem cells respectively. Also disclosed is a method for preparing an organ bud, the method comprising culturing the vascular cells, mesenchymal cells and tissue or organ cells in vitro, where the vascular cells, mesenchymal cells and tissue or organ cells have been induced from pluripotent stem cells respectively.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This invention relates to the construction of three-dimensional organs from pluripotent stem cells. [Background technology]

[0002] The inventors have previously developed a method for constructing three-dimensional tissue (organ primordia) by mixing functional cells used in regenerative medicine (such as organ cells derived from pluripotent stem cells) with vascular endothelial cells derived from umbilical cord and mesenchymal cells derived from bone marrow. They have reported that these organ primordia are superior to cells differentiated by planar culture in terms of in vitro function and therapeutic effect on disease model animals (Non-patent Literature 1: Nature 2013, Non-patent Literature 2: Cell Stem Cell 2015, Patent Literature 1, 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Takebe T et al., Nature 499, pp481-484, 2013 [Non-Patent Document 2] Takebe T et al., Cell Stem Cell 16, pp556-565, 2015 [Patent Documents]

[0004] [Patent Document 1] WO2013 / 047639 [Patent Document 2] WO2015 / 012158 [Overview of the project] [Problems that the invention aims to solve]

[0005] Conventional methods using two types of umbilical cord and bone marrow-derived cells have faced several challenges in practical application, including: [1] significant variability in quality depending on the donor; [2] finite proliferative capacity of the cell source; and [3] difficulty in ensuring immunocompatibility due to different origins. Furthermore, because umbilical cord and bone marrow-derived cells are highly mature cells, their differentiation stage differs significantly from the immature cells required for organoid creation, resulting in a large discrepancy with in vivo development. [Means for solving the problem]

[0006] The inventors succeeded in creating organ primordia from immature cells at synchronized differentiation stages by generating all three cell types used to create three-dimensional organ primordia from induced pluripotent stem cells (iPS cells). Specifically, human liver buds were created using a combination of iPS cell-derived hepatic endodermal cells, iPS cell-derived vascular endothelial cells, and iPS cell-derived mesenchymal cells, and compared in vitro albumin secretion capacity and gene expression of differentiation markers with human liver buds created using the conventional method of combining iPS cell-derived hepatic endodermal cells, umbilical cord-derived vascular endothelial cells, and bone marrow-derived mesenchymal cells. As a result, a significant improvement in function was observed compared to the conventional method. Furthermore, when liver buds equivalent to 6 x 10^6 iPS cell-derived hepatic endodermal cells were transplanted into immunodeficient animals (NOD / scid mice), similar results were obtained from the amount of human albumin secreted into the serum. This invention significantly improves function while reducing the cost and effort involved in quality evaluation and manufacturing. Similar effects are highly likely to be obtained when using other pluripotent stem cells (e.g., embryonic stem cells (ES cells)) instead of iPS cells.

[0007] The gist of this invention is as follows: (1) An organ bud prepared from vascular cells, mesenchymal cells, and tissue or organ cells, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells. (2) An organ bud is a structure that can differentiate into an organ upon maturation, as described in (1). (3) The organobud described in (1) or (2), wherein the pluripotent stem cells are of human origin. (4) An organobud as described in any of (1) to (3), wherein the pluripotent stem cell is at least one cell selected from the group consisting of induced pluripotent stem cells and embryonic stem cells. (5) An organ bud as described in any of (1) to (4), wherein the organ cell is a liver cell and the organ bud is a hepatocyte. (6) Organ buds as described in (5), in which hepatocytes are TBX3-positive and ADRA1B-positive. (7) An organ bud as described in any of (1) to (6), wherein the mesenchymal cells are CD166 positive and CD31 negative. (8) An organ bud as described in any of (1) to (7), wherein the mesenchymal cells are LHX2-positive and WT1-positive. (9) The organ bud described in (8) in which the transcription of FOXF1, HLX1, COL4A, and ALCAM is activated in mesenchymal cells, and the mesenchymal cells are LHX2-positive, WT1-positive, and MIIA-positive. (10) An organ bud as described in any of (1) to (9), wherein the vascular cells are CD31-positive and CD144-positive. (11) The organobud described in (10) in which the expression of at least one gene selected from the group consisting of PECAM1, CDH5, KDR, and CD34 in vascular cells is elevated compared to pluripotent stem cells before differentiation induction. (12) An organ bud according to any one of (1) to (11), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a factor belonging to the transforming growth factor β family, a factor belonging to the Wnt family, and a class I histone deacetylase (HDAC) inhibitor, and further cultured in the presence of a factor belonging to the transforming growth factor β family and a factor belonging to the Wnt family, and the cells obtained are cultured in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation, and the TBX3-positive and ADRA1B-positive cells are used as tissue or organ cells. (13) An organ bud according to any one of (1) to (11), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a β-catenin activator, a PI3K inhibitor, and a factor belonging to the transforming growth factor β family, and further cultured in the presence of a factor belonging to the transforming growth factor β family and a BMP inhibitor, and the cells obtained are differentiated by culturing them in the presence of FGF and a factor belonging to the TGFβ superfamily, and the cells obtained are TBX3-positive and ADRA1B-positive, and these cells are used as tissue or organ cells. (14) An organ bud according to any one of (1) to (11), in which TBX3-positive and ADRA1B-positive cells are obtained by culturing pluripotent stem cells in the presence of a ROCK inhibitor, followed by culturing the cells in the presence of a factor belonging to the transforming growth factor β family, and then culturing the cells in the presence of a factor belonging to the FGF and TGFβ superfamily to induce differentiation, and these cells are used as tissue or organ cells. (15) An organ bud according to any one of (1) to (12), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a factor belonging to the transforming growth factor β family and a β-catenin activator, and further cultured in the presence of a factor belonging to the transforming growth factor β family, and the cells obtained are cultured in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation, and the cells obtained are TBX3-positive and ADRA1B-positive, and are used as tissue or organ cells. (16) An organ bud according to any one of (1) to (15), wherein pluripotent stem cells are cultured in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, then cultured in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family, and further cultured in the presence of FGF, and the resulting LHX2-positive and WT1-positive cells are used as mesenchymal cells. (17) An organ bud according to any one of (1) to (15), wherein pluripotent stem cells are cultured in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, then cultured in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family, further cultured in the presence of FGF, and then maintained in a mesenchymal cell medium, and the CD166-positive and CD31-negative cells obtained by this process are used as mesenchymal cells. (18) An organ bud according to any one of (1) to (17), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, and further cultured in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an adenylyl cyclase activator, and the resulting CD31-positive and CD144-positive cells are used as vascular cells. (19) An organ bud according to any one of (1) to (17), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a factor belonging to the TGFβ superfamily, and further cultured in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an inhibitor of the TGF-β type I receptor, wherein CD31-positive and CD144-positive cells are used as vascular cells. (20) An organobud according to any one of (1) to (17), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then in the presence of a factor belonging to the transforming growth factor β family, then in the presence of factors belonging to the FGF and TGFβ superfamily, and subsequently in the presence of a vascular endothelial growth factor receptor (VEGFR) activator, and the resulting CD31-positive and CD144-positive cells are used as vascular cells. (21) An organ bud according to any one of (1) to (17), wherein pluripotent stem cells are cultured in the presence of a ROCK inhibitor, then cultured in the presence of a factor belonging to the TGFβ superfamily, and further cultured in the presence of a factor belonging to the TGFβ superfamily, a vascular endothelial growth factor receptor (VEGFR) activator, and FGF, and the resulting CD31-positive and CD144-positive cells are used as vascular cells. (22) A method for producing an organ bud, comprising culturing vascular cells, mesenchymal cells, and tissue or organ cells in vitro, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells. (23) The method described in (22) wherein cells are cultured without using scaffolding material. (24) A method for producing tissue or organs, comprising transplanting an organ bud described in any of (1) to (21) into a non-human animal and differentiating it into tissue or organ. (25) A method for transplanting an organ bud, comprising transplanting an organ bud described in any of (1) to (21) into a human or non-human animal. (26) A method for regenerating or restoring the function of a tissue or organ, comprising transplanting an organ bud described in any of (1) to (21) into a human or non-human animal and differentiating it into a tissue or organ. (27) A method for producing a non-human chimeric animal, comprising transplanting an organ bud described in any of (1) to (21) into a non-human animal and differentiating it into a tissue or organ. A method for evaluating drugs using at least one selected from the group consisting of organ buds described in any of (1) to (21), tissues and organs prepared by the method described in (24), and non-human chimeric animals prepared by the method described in (27). (29) A method for producing cells that are positive for TBX3 and ADRA1B, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a factor belonging to the transforming growth factor-β family, a factor belonging to the Wnt family, and a class I histone deacetylase (HDAC) inhibitor, and further culturing in the presence of a factor belonging to the transforming growth factor-β family and a factor belonging to the Wnt family, and then culturing the obtained cells in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation into cells that are positive for TBX3 and ADRA1B. (30) A method for producing cells that are positive for TBX3 and ADRA1B, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a β-catenin activator, a PI3K inhibitor, and a factor belonging to the transforming growth factor-β family, and further culturing in the presence of a factor belonging to the transforming growth factor-β family and a BMP inhibitor, and then culturing the obtained cells in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation into cells that are positive for TBX3 and ADRA1B. (31) A method for producing cells that are positive for TBX3 and ADRA1B, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing the obtained cells in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation into cells that are positive for TBX3 and ADRA1B. (32) A method for producing cells that are positive for TBX3 and ADRA1B, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a factor belonging to the transforming growth factor-β family and a β-catenin activator, and further culturing in the presence of a factor belonging to the transforming growth factor-β family, and then culturing the obtained cells in the presence of FGF and a factor belonging to the TGFβ superfamily to induce differentiation into cells that are positive for TBX3 and ADRA1B. (33) A method for producing LHX2-positive and WT1-positive cells, comprising culturing pluripotent stem cells in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, then culturing in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family, and further culturing in the presence of FGF. (34) A method for producing CD166-positive and CD31-negative cells, comprising culturing pluripotent stem cells in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, then culturing in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family, further culturing in the presence of FGF, and then maintaining the culture in a medium for mesenchymal cells. (35) A method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily, and further culturing in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an adenylate cyclase activator. (36) A method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator and a factor belonging to the TGFβ superfamily, and further culturing in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an inhibitor of the type I receptor of TGF-β. (37) A method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, then culturing in the presence of a factor belonging to the transforming growth factor β family, further culturing in the presence of FGF and a factor belonging to the TGFβ superfamily, and then culturing in the presence of a vascular endothelial growth factor receptor (VEGFR) activator. (38) A method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, culturing them in the presence of a factor belonging to the TGFβ superfamily, and further culturing them in the presence of a factor belonging to the TGFβ superfamily, a vascular endothelial growth factor receptor (VEGFR) activator, and FGF. [Effects of the Invention]

[0008] Compared to conventional methods using two types of umbilical cord and bone marrow-derived cells, this invention offers several advantages, including: [1] it is donor-independent, leading to stable quality; [2] organoids using all three types of iPS cell-derived cells exhibit dramatically superior functionality; and [3] it facilitates ensuring immunocompatibility. This specification includes the content described in the specification and / or drawings of the Japanese Patent Application No. 2017-230647, which forms the basis of the priority of this application. [Brief explanation of the drawing]

[0009] [Figure 1](Figure 1) Scalable hepatomegaly production and differentiation through the development of the Omniwell array platform. (A) Schematic design of an Omniwell array plate for mass production (center). Fluorescence image analysis on the right confirms large-scale (>20,000) production of microhepatomegaly (LB) from entirely human iPSCs. (B) Appearance of a 1(omni)well array plate (left) and plate flatness by a 3D profiler (right). (C) SEM image of iPSC-LB generated from co-culture of three types of progenitor cells. (D) Confocal image of the generated iPSC-LB showing endothelial sprouting (white arrow) inside. Green indicates iPSC-HE and red indicates HUVEC. (E) Macroscopic morphology of hepatomegaly generated from different endodermal stages (ranging from 0 to 20 days). The X-axis represents 3D culture time (cumulative), and the Y-axis represents the stage of source cells from 2D culture. The two columns on the right show low and high magnification images after collection on day 22. (F) Violin plot analysis of size uniformity in liver buds of various endoderm origins. Data are collected by quantifying the morphology of over 600 liver buds at cumulative day 22 compared to day 1 of 3D culture. [Figure 2](Figure 2) De-optimization of hepatocytes derived from human iPSCs that form multiple progenitor cells. (A) Quantification of time-dependent AFP production and ALB production per 2 hours / 2 × 10⁵ cells at day 20 based on ELISA. Data represent mean ± sd. n = 4, *: P < 0.05 (B) PCA analysis of hepatocytes generated from endodermal cells at multiple stages revealed important involvement in liver fate. The X axis is shown by maturation order based on in vitro experiments. (C) Characterization of markers reported specifically for each differentiation stage by qRT-PCR analysis of pluripotency, embryonic endoderm, and hepatic endodermal cell markers. Microscopic morphology of cells at various stages is shown in the right panel. The lower panel shows immunostaining for HNF4A at stages 8 and 10. (D) Volcano plot gene expression data. Genes that are expressed differently at a log2 change level between stages 6 and 8, with a p value < 0.05, are shown as red circles. Cell surface proteins or nucleoproteins are shown in bold with the gene name in the red circle. (E) Transitional endoderm cells on day 8 are labeled by both TBX3 and ADRA1B genes by qRT-PCR, and proteins are labeled by immunostaining (see Figure 3A). Data represent mean ± sd. n = 3, *: P < 0.05 (F) Screening based on qRT-PCR for the early mesoderm gene T (BRACHURY), and the STM genes HLX1, GATA4, FOXF1, COL4A1, and ALCAM. Meso: iPSC-derived lateral plate mesoderm on day 3, STM: cells on day 10 after simultaneous exposure to FGF2 and PDGFB, MSC: cells on day 20 maintained in MSC medium after STM passage. Error bars represent sd from values ​​of three independent experiments (n = 4). (G) Hierarchical clustering of adult human liver mesenchymal cells in iPSC-STM based on their characteristics (Asahina et al., 2009, Asahina et al., 2011, El Taghdouini et al., 2015). (H) Time-course image analysis of self-agglomeration from iPSC-STM using phase-contrast microscopy. The rate of aggregated cell movement is shown as the average of three independent experiments. Cultured tissues using BMSCs and iPSC-MSCs for two days are shown as controls.(I, J) Microscopic characterization of iPSC-ECs and time-course quantification of CD31 and CD144 based on FACS (I), followed by endothelial sprouting assay on Matrigel (J). (K) In vivo generation of human vascular networks by simultaneous transplantation of iPSC-STM and AAVS1::mCherry iPSC-ECs. Dextran is shown in green, and mouse-specific CD31 is shown in blue. Bars are 500 μm. [Figure 3](Figure 3) Complete hepatocyte self-organization from multiple iPSC-derived progenitor cells. (A) Outline of the protocol for generating all iPSC-hepatocytes from human iPSCs without feeder. Process validation was routinely performed by immunostaining. Immunostaining was performed on TBX3 (red) and ADRA1B (red) on tHE, WT1 (red), LHX2 (red) and myosin IIA (green) on STM, and CD144 (red) and CD31 (green) on EC. (B) Self-organization into hepatocytes with endothelial sprouting after 72 hours of culture was confirmed by bright-field (top) and light-sheet (bottom) 4D time-lapse imaging, respectively. Green represents iPSC-tHE and red represents iPSC-EC. iPSC-STM is unlabeled. Bars are 500 μm. (C) Confocal imaging of all iPSC buds confirmed the presence of the endothelial network. (D) Macroscopic lateral view of all developed iPSC buds. The panel shows a top view. Bars are 1,000 μm. (E) Macroscopic observation of all transplanted iPSC-LBs showing human vascular perfusion on day 2. The area enclosed by the dotted line shows all transplanted iPSC-LBs. (F) In vivo imaging of all transplanted iPSC-LBs demonstrating human vascular formation from iPSC-ECs. Red represents AAVS1 :: mCherry iPSC-ECs. Bars are 500 μm. (G) Presence of human iPSC-derived blood vessels and aligned iPSC-hepatocytes within all iPSC-LB transplants on day 28. Green represents iPSC-tHEs and red represents iPSC-ECs. (H) Studies co-injecting dextran and fluorescent mouse CD31 antibody reveal connections (white dotted lines or arrows) between iPSC-ECs and host mouse blood vessels. Green represents dextran, red represents iPSC-EC, and blue represents mouse CD31. (I) Close relationship between iPSC-STM and iPSC-EC. Green represents iPSC-STM, and red represents iPSC-EC. [Figure 4](Figure 4) Functional validation of mass-produced miniaturized human liver buds. (A) Strategic batch validation scheme in each 10⁸ cell scale production cycle by evaluating in vitro and in vivo function. The central panel shows the homogeneity of the collected LB morphology. Green represents iPSC-tHE. (B) Albumin production in all differentiated iPSC-LBs. For controls, human adult hepatocytes in 2D (AdHep 2D), AdHep co-culture (AdHep LB), and control LB (made from iPSC-tHE, HUVEC, and BMSC) are shown. Data are mean ± sd. n = 6. (C) Multiple hepatocyte-derived protein production, and (D) ammonia metabolism in iPSC-LB cultured in vitro at 21 days of culture. Data are mean ± sd. n = 6 in (C), n = 3 in (D). (E) Visualization of liver maturation status of liver buds from iPSCs (all iPSC-LBs) based on tSNE. The approach was compared with conventional approaches and primary human samples. (F) APRES profiles between human adult hepatocytes (F) and all differentiated iPSC-LBs (G) shown in the aster plot. (G) Kaplan-Meier survival curves of the transplant and sham groups after a subacute liver failure model using alb-Tk-NOG mice. n = 114 for LBs and n = 39 for the sham transplant group. **: P = 0.0013. The X axis represents days after transplant. (H) Time-dependent human serum albumin production in all iPSC-LB transplants (n = 48) or human adult hepatocyte transplants (n = 12). Data represent mean ± sem. (I) Detection of human-specific diclofenac metabolites in iPSC LB transplanted mice. 3'-Hydroxy-4'-methoxydifluorofenac (VI) was quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). 3'-Hydroxy-4'-methoxydifluorofenac(VI) is a human-specific metabolite known to accumulate in plasma. [Figure 5](Fig. S1) Optimization and recovery of coatings for mass-produced LBs. (A) Coating with pHEMA and MPC polymers before cell seeding. Green represents HUVECs and red represents MSCs. (B) Recovery of LBs by pipetting. Microscopic images obtained before (left) and after (right) LB recovery are shown. No remaining LBs were detected after recovery, suggesting successful recovery of LBs from the plate. [Figure 6] (Fig. S2) Cell dose and mixing ratio-dependent studies for LB generation. (A) Various proportions of mesenchymal cells (MCs) were tested on the Elplasia platform for LB generation. Microscope and electron microscope images are shown. Numerous LBs were generated at the indicated MC ratios. (B) qRT-PCR analysis of HNF4A and ALB revealed that 1 / 20 of the total cells were most efficient for hepatocyte generation. Data represent mean ± sd. n = 3 (C, D) Total cell-dependent increase in LB diameter up to 4000 cells per spot. 6000 cells did not form tissue. Green represents HUVEC and red represents MSC. Quantification of LB size is shown in D. Gene expression (E) and protein production capacity (F) of LB at each indicated cell dose. Data represent mean ± sd. n = 3 [Figure 7] (Fig. S3) Establishment of omniwell plates for large-scale production. (A) Elplasia 24-well, 6-well, and 1(omni)well plates. All plates meet the SBS footprint dimensions. By minimizing dead space, the number of microwells per plate was increased to 14,400 (600 × 24 wells), 18,000 (3,000 × 6 wells), and 20,000 (1 well). (B) qRT-PCR analysis of liver marker genes HNF4A, RBP4, AFP, ALB, TTR, TAT, TDO2, and GLUT2 in LB from 24-well, 6-well, and omni(1)well plates. Data represent mean ± sd. n = 3 [Figure 8](Fig. S4) Optimization of iPSC-derived endodermal cell induction protocols. (A) Three representative protocols for generating embryonic endodermal cells. (B, C) qRT-PCR analysis of pluripotency markers at the DE stage and hepatic endodermal markers at the HE stage following their respective indicated protocols. Data represent mean ± sd. n = 12. (D) Microscopic morphology of mature hepatocyte-like cells from different protocols. (E) qRT-PCR analysis of hepatocyte markers at the iPSC-MH stage. Data represent mean ± sd. n = 6. (F) Reproducible generation of iPSC-MH from multiple donor-derived iPSC clones. Data are shown as ng / ml / 24 hours / 2 × 10⁵ cells. n = 10. (G) ELISA quantification of the DE secretion marker cerberus1 during the transition from day 4 to day 10. [Figure 9] (Fig. S5) Optimization of iPSC-derived hepatocyte formation protocol. (A) Hepatocytes from iPSCs (day 0), DE (day 6 or 7), HE (day 10), and MH (day 14) cells. Gene expression analysis revealed that only endodermal cells from day 6 and day 10 showed the highest liver function after extended culture. (B) Hierarchical clustering of 2-D cells and 3-D tissues. 2-D cells are iPSCs, DE, HE, IH, and MH. 3-D tissues are human iPSC-hepatocytes, human fetal, and adult liver tissue using a signature developed by Si-Tayeb et al. (2010). FLT is 10gwk or 22-40gwk pooled fetal liver tissue, ILT is 0-year infant liver tissue, ALT is 5-year, 30-year, 44-year, or 55-year old liver tissue, AHEP is human primary hepatocytes, and AHEP-3D is 3D cultured human primary hepatocytes. (C) GSEA analysis and heatmap visualization of angiogenesis characteristic genes between buds on day 6 and day 8. [Figure 10] (Fig. S6) Identification of TBX3 and ADRA1B as markers for iPSC-tHE. (A, B) Time-dependent expression of TBX3(a) and ADRA1B(b). Data represent mean ± sd. n = 3 [Figure 11](Fig. S7) Highly efficient differentiation of feeder-free human iPSCs into primitive endothelial progenitor cells. (A) Four independent, stepwise, specific protocols for EC differentiation. (B) Microscopic characterization of each differentiated iPSC-EC, followed by FACS-based initial screening with CD31 and CD144. (C) Analysis of EC markers based on qRT-PCR. (D) Growth curve of iPSC-EC after 4 passages. [Figure 12] (Fig. S8) In vivo functionalization of human iPSC liver buds. (A) Whisker plot comparison of ALB levels (5th to 95th percentile) in all iPSC-LB transplantation groups at day 40. Each graph represents raw values ​​taken from all 10 different mice. (B) Time-dependent human serum albumin production in the presence or absence of iPSC-EC and STM. Data represent mean ± sem. n = 6. *: P = 0.0356. The number of transplanted cells was LB equivalent to 3 × 10⁶ cells in panels A and B. [Figure 13] In the differentiation induction of iPSCs into DEs, Wnt3a can be replaced by CHIR99021. Figure 13A: Schematic diagram of the protocol using CHIR99021 as a substitute for Wnt3a. After conditional analysis, a condition in which CHIR99021, 2 μM was added for 3 days out of 6 days of DE differentiation induction was selected. Figure 13B: Cell morphology at each differentiation stage using the conventional method with Wnt3a and under the condition of adding CHIR99021, 2 μM for 3 days. No morphological differences were observed compared to when Wnt3a was used. Figure 13C: Analysis of the CXCR4 positivity rate, a DE marker, at the DE stage by flow cytometry using the conventional method with Wnt3a and under the condition of adding CHIR99021, 2 μM for 3 days. The CXCR4 positivity rate was equivalent to when Wnt3a was used. Figure 13D: Expression analysis of each differentiation marker by quantitative PCR (qPCR) was equivalent to when Wnt3a was used. Figure 13E: ELISA analysis of secretory albumin levels at the MH stage. Similar or higher levels were observed in multiple iPSC clones compared to those using Wnt3a. [Figure 14]Comparison of Wnt3a and CHIR99021 (in vitro MH, LB). Figures 14A and B: Cell morphology in MH (Figure 14A) and LB (Figure 14B). No morphological differences were observed compared to when Wnt3a was used. Figure 14C: Marker expression analysis in MH and LB by quantitative PCR (qPCR). The results were comparable to when Wnt3a was used, and no increase in the expression of marker genes from other cell lineages, such as intestinal markers, was observed. [Figure 15] EC induction. A modified protocol for inducing differentiation of iPSC-derived vascular endothelial cells (iPSC-ECs). [Figure 16] Evaluation of differentiation induction media for clinical application. Figure 16A: Schematic diagram of the differentiation induction protocol. Figure 16B: Cell morphology of the conventional and revised methods. No morphological differences were observed. Figure 16C: Analysis of EC marker positivity rates by flow cytometry for the conventional and revised methods. Figure 16D: Summary of the flow cytometry analysis in Figure 16C. The revised method shows a more stable and higher CD31 / CD144 positivity rate compared to the conventional method. Figure 16E: Expression analysis of each differentiation marker by quantitative PCR (qPCR). EC marker expression levels remain stable even after passage. Figure 16F: Cell proliferation after each passage. The revised method shows higher proliferative capacity after passage compared to the conventional method. [Modes for carrying out the invention]

[0010] The present invention will be described in detail below.

[0011] The present invention provides an organobud prepared from vascular cells, mesenchymal cells, and tissue or organ cells, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells.

[0012] In the present invention, "organ bud" refers to a structure that can differentiate into an organ upon maturation, and which contains three types of cells: vascular cells, mesenchymal cells, and tissue or organ cells. Whether a structure is an organ bud can be confirmed, for example, by transplanting the structure into a living organism and examining whether it can differentiate into the target organ (if it differentiates into the target organ, it can be determined to be an organ bud), and / or by examining whether the structure contains all three types of cells mentioned above (if it contains all three types of cells, it can be determined to be an organ bud). Organ buds may be organ buds that differentiate into organs such as the brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, and lens; ectodermal organs such as the spleen, kidneys, ureters, heart, blood, gonads, adrenal cortex, muscles, skeleton, dermis, connective tissue, and mesothelium; and endodermal organs such as the liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestines), lungs, thyroid gland, parathyroid gland, urinary tract, and thymus. However, organ buds that differentiate into endodermal organs, such as organ buds that differentiate into the liver (hepatomegaly), organ buds that differentiate into the pancreas (pancreaticomegaly), and organ buds that differentiate into the intestines, are preferred. Whether a structure is an organ bud that differentiates into an endodermal organ can be confirmed by examining the expression of marker proteins (if one or more of the marker proteins described later are expressed, it can be determined to be an organ bud). For example, in liver buds, HHEX, SOX2, HNF4A, AFP, and ALB serve as markers; in pancreatic buds, PDX1, SOX17, and SOX9 serve as markers; and in organ buds differentiating into the intestinal tract, CDX2 and SOX9 serve as markers. Among the terms used by those skilled in the art, liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, and intestinal organoid (K. Matsumoto, et al. Science.19;294(5542):559-63.(2001)) are included in organ buds in this invention.

[0013] The organ buds of the present invention are produced from three types of cells—vascular cells, mesenchymal cells, and tissue or organ cells—all derived from pluripotent stem cells.

[0014] Examples of pluripotent cells include pluripotent cells obtained from living organisms (e.g., ES cells), pluripotent cells induced from reprogramming (e.g., iPS cells, MUSE cells (Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. PNAS, 2011), iMPC cells (induced multipotent progenitor cells; Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature, 2014)), and combinations thereof.

[0015] Pluripotent stem cells are preferably of human origin, but they may also be derived from animals other than humans (for example, animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0016] The organobuds of the present invention can be produced by co-culturing three types of cells (vascular cells, mesenchymal cells, and tissue or organ cells) derived from pluripotent stem cells in vitro. The present invention provides a method for producing organobuds, comprising culturing vascular cells, mesenchymal cells, and tissue or organ cells in vitro, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells.

[0017] In the present invention, "tissue or organ cells" is a concept that includes cells that have differentiated into functional cells constituting a tissue or organ, or undifferentiated cells that can differentiate into functional cells. Undifferentiated cells include stem cells, progenitor cells, endodermal cells, organoblasts, etc. Preferably, undifferentiated cells are cells whose differentiation fate into functional cells has been determined, but which have not yet differentiated into functional cells. "Undifferentiated tissue or organ cells" may include, for example, cells that can differentiate into organs such as the kidneys, heart, lungs, spleen, esophagus, stomach, thyroid gland, parathyroid gland, thymus, gonads, brain, and spinal cord. Examples include cells that can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, and lens; cells that can differentiate into mesodermal organs such as the spleen, kidneys, ureters, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesothelium; and cells that can differentiate into endodermal organs such as the liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestines), lungs, thyroid gland, parathyroid gland, urinary tract, and thymus. Whether a cell is capable of differentiating into an ectodermal, mesodermal, or endodermal organ can be determined by examining the expression of marker proteins (if one or more of these marker proteins are expressed, the cell can be determined to be capable of differentiating into an ectodermal, mesodermal, or endodermal organ). For example, markers for cells capable of differentiating into the liver include HHEX, SOX2, HNF4A, AFP, and ALB; for cells capable of differentiating into the pancreas, PDX1, SOX17, and SOX9; for cells capable of differentiating into the intestinal tract, CDX2 and SOX9; for cells capable of differentiating into the kidney, SIX2 and SALL1; for cells capable of differentiating into the heart, NKX2-5, MYH6, ACTN2, MYL7, and HPPA; for cells capable of differentiating into blood, C-KIT, SCA1, TER119, and HOXB4; and for cells capable of differentiating into the brain or spinal cord, HNK1, AP2, and NESTIN are among the markers.Among the terms used in the art are hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, metanephric mesenchymal precursor cells, multipotent nephron progenitor, renal progenitor cell, cardiac mesoderm, cardiovascular progenitor cells, cardiac progenitor cells, (JR. Spence, et al. Nature.;470(7332):105-9.(2011), Self, et al. EMBO J.; 25(21): 5214-5228.(2006), J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009), G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007), etc., are included in the undifferentiated tissue or organ cells of the present invention. Undifferentiated cells can be produced from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.For example, cells that can differentiate into liver cells can be prepared according to K. Si-Taiyeb, et al. Hepatology, 51 (1): 297-305 (2010) and T. Touboul, et al. Hepatology. 51(5): 1754-65 (2010); cells that can differentiate into pancreatic cells can be prepared according to D. Zhang, et al. Cell Res.; 19(4): 429-38 (2009); cells that can differentiate into intestinal cells can be prepared according to J. Cai, et al. J Mol Cell Biol.; 2(1): 50-60 (2010) and R. Spence, et al. Nature.; 470(7332): 105-9 (2011); and cells that can differentiate into heart cells can be prepared according to J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009). These cells can be produced according to the method described above, and cells that can differentiate into brain and spinal cord cells can be produced according to G. Lee, et al. Nature Biotechnology 25, 1468 - 1475 (2007). Examples of functional cells that make up organs and tissues include endocrine cells of the pancreas, pancreatic ductal epithelial cells of the pancreas, hepatocytes of the liver, epithelial cells of the intestinal tract, renal tubular epithelial cells of the kidney, renal glomerular epithelial cells, cardiac cardiomyocytes, lymphocytes and granulocytes of the blood, red blood cells, nerve cells and glial cells of the brain, and nerve cells and Schwann cells of the spinal cord.

[0018] In the preparation of organ buds according to the present invention, the tissue or organ cells used are those produced (differentiated) from pluripotent stem cells.

[0019] Regarding the induction of differentiation from pluripotent stem cells, such as iPS cells, into hepatic endodermal cells (iPSC-HE), by comparing the differentiation stages, liver function was significantly improved by using TBX3 ADR1AB co-positive cells on Day 7 (see the example below). In this specification, expressions such as "positive" or "+" for cell surface markers mean that the cell surface marker can be confirmed to be expressed in the cell by methods such as immunohistochemistry, and expressions such as "negative" or "-" mean that the expression cannot be confirmed by methods such as immunohistochemistry. TBX3 ADR1AB co-positive hepatic endoderm cells (iPSC-HE) can be produced by the method described in the examples below (Protocols 1-3 in Figure 8A and Figure 13A). According to Protocol 1 in Figure 8A, pluripotent stem cells are cultured in the presence of a ROCK inhibitor, a factor belonging to the transforming growth factor β family, a factor belonging to the Wnt family, and a class I histone deacetylase (HDAC) inhibitor (optional) for (e.g., 1-2 days). Then, the cells obtained through the process of culturing in the presence of a factor belonging to the transforming growth factor β family and a factor belonging to the Wnt family (e.g., 0-3 days) can be differentiated into TBX3-positive and ADRA1B-positive cells by culturing in the presence of FGF and a factor belonging to the TGFβ superfamily (e.g., 1-4 days). According to Protocol 2 in Figure 8A, pluripotent stem cells are cultured in the presence of a ROCK inhibitor (e.g., for 1-2 days), followed by culture in the presence of a β-catenin activator, a PI3K inhibitor, and a factor belonging to the transforming growth factor β family (e.g., for 1-2 days). Furthermore, cells obtained through the process of culturing in the presence of a factor belonging to the transforming growth factor β family and a BMP inhibitor (e.g., for 2-4 days) can be differentiated into TBX3-positive and ADRA1B-positive cells by culturing them in the presence of FGF and a factor belonging to the TGFβ superfamily (e.g., for 1-4 days). According to Protocol 3 in Figure 8A, pluripotent stem cells can be cultured in the presence of a ROCK inhibitor (e.g., for 1-2 days), followed by culturing them in the presence of a factor belonging to the transforming growth factor β family (e.g., for 2-5 days). The resulting cells (Definitive Endoderms) can then be cultured in the presence of factors belonging to the FGF and TGFβ superfamily (e.g., for 1-4 days) to induce differentiation into TBX3-positive and ADRA1B-positive cells. According to the protocol in Figure 13A, pluripotent stem cells are cultured in the presence of a ROCK inhibitor, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a class I histone deacetylase (HDAC) inhibitor (optional) for (e.g., 1-2 days), followed by further culture in the presence of a factor belonging to the transforming growth factor β family, a β-catenin activator, and a class I histone deacetylase (HDAC) inhibitor (optional) for (e.g., 1-2 days). The cells obtained through this process (Definitive Endoderm) can then be cultured in the presence of FGF and a factor belonging to the TGFβ superfamily (e.g., 1-4 days) to induce differentiation into TBX3-positive and ADRA1B-positive cells. Additional culture steps may be included before and after each of the culture steps described above, from the differentiation of pluripotent stem cells to TBX3-positive and ADRA1B-positive cells.

[0020] In the present invention, "vascular cells" is a concept that includes cells that have differentiated into cells that constitute blood vessels, or undifferentiated cells that can differentiate into such cells. Undifferentiated cells include stem cells, progenitor cells, mesoderm cells, etc. Preferably, undifferentiated cells are cells whose differentiation fate into vascular cells has been determined, but which have not yet differentiated into vascular cells. Examples of vascular cells include vascular endothelial cells, vascular endothelial progenitor cells, endocardial progenitor cells, and angioblasts, but vascular endothelial cells are preferred. Whether a cell is a vascular endothelial cell can be confirmed by examining whether it expresses marker proteins, such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD41 (if any one or more of the above marker proteins are expressed, it can be determined to be a vascular endothelial cell). The vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether a vascular endothelial cell is a differentiated cell can be confirmed by CD31 and CD144. Among the terms used by those skilled in the art, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, and hemangioblast (HJ. joo, et al. Blood. 25;118(8):2094-104.(2011)) are included in vascular endothelial cells in the present invention.

[0021] In the preparation of organ buds according to the present invention, vascular cells are produced (differentiated) from pluripotent stem cells.

[0022] For differentiation induction of pluripotent stem cells, such as iPS cells, into vascular endothelial cells (iPSC-ECs), it is recommended to use cells cultured in medium 1 (DMEM / F12 medium + 1-2% B27 + 1% Glutamax, 25 ng / mL BMP4, 8 μM CHIR 99021) for 3 days after dispersed seeding in the presence of Rho kinase, followed by culture in medium 2 (StemPro34-SFM + 200 ng / mL VEGF + 2 μM Forskolin) for 3-4 days. Alternatively, the cells may be cultured in medium 3 (StemPro34-SFM + 50 ng / mL VEGF) for up to 7 days. The resulting vascular endothelial cells (iPSC-ECs) should be those in which more than 90% express CD31 (PECAM1), a marker for vascular endothelium, as determined by immunohistochemistry and FACS. Gene expression analysis revealed high expression of vascular endothelial markers such as PECAM1, CDH5, KDR, and CD34, with expression levels 10 to 100 times higher than in iPS cells before differentiation induction (see example below). In addition to CD31, a marker for vascular endothelial cells, the expression of CD144 and CD309 proteins was confirmed by immunohistochemistry (see example below). The present invention provides a method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), culturing them in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 2-3 days), and further culturing them in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an adenylyl cyclase activator (e.g., for 4-8 days) (Pr1 in Figure 11A). The CD31-positive and CD144-positive cells may be iPSC-ECs. The CD31-positive and CD144-positive cells can be used to produce organobuds. The present invention also provides a method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), culturing them in the presence of a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 2-4 days), and further culturing them in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an inhibitor of the TGF-β type I receptor (e.g., for 4-7 days) (Pr2 in Figure 11A). CD31-positive and CD144-positive cells may be iPSC-ECs. CD31-positive and CD144-positive cells can be used to produce organobuds. The present invention also provides a method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor and a factor belonging to the transforming growth factor β family (e.g., for 1-2 days), then culturing them in the presence of a factor belonging to the transforming growth factor β family (e.g., for 1-3 days), further culturing them in the presence of a factor belonging to the FGF and TGFβ superfamily (e.g., for 1-3 days), and then culturing them in the presence of a vascular endothelial growth factor receptor (VEGFR) activator (e.g., for 2-7 days) (Pr3 in Figure 11A). CD31-positive and CD144-positive cells may be iPSC-ECs. CD31-positive and CD144-positive cells can be used to produce organobuds. The present invention also provides a method for producing CD31-positive and CD144-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), culturing them in the presence of a factor belonging to the TGFβ superfamily (e.g., for 2-4 days), and further culturing them in the presence of a factor belonging to the TGFβ superfamily, a vascular endothelial growth factor receptor (VEGFR) activator, and FGF (e.g., for 2-7 days) (Pr4 in Figure 11A). CD31-positive and CD144-positive cells may be iPSC-ECs. CD31-positive and CD144-positive cells can be used to produce organobuds. Additional culture steps may be included before and after each of the culture steps described above, from the differentiation of pluripotent stem cells to CD31-positive and CD144-positive cells. Furthermore, the positivity rate of CD31 and / or CD144 can be increased by reseeding CD31-positive and CD144-positive cells and culturing them in expanded medium (see Example 3 below). The type of expanded medium may be changed when it is replaced after a certain period of time. StemPro-34SFM supplemented with VEGF-A is preferred as the expanded medium for reseeding, and Miracell (registered trademark) EC (Takara Bio) is preferred as the medium to replace it with, but the medium is not limited to these. It is advisable to add a ROCK inhibitor for one day during reseeding. In the preparation of organ buds according to the present invention, vascular cells are preferably CD31-positive and CD144-positive. Furthermore, vascular cells are preferably more expressive than pluripotent stem cells before differentiation induction, with at least one gene selected from the group consisting of PECAM1, CDH5, KDR, and CD34 being expressed.

[0023] In the present invention, "mesenchymal cells" is a concept that includes cells that have differentiated into connective tissue cells, which are mainly found in the connective tissue derived from the mesoderm and form the supporting structure of cells that function in tissues, or undifferentiated cells that can differentiate into such cells. Undifferentiated cells include stem cells, progenitor cells, mesoderm cells, etc. It is preferable that undifferentiated cells are cells whose differentiation fate into mesenchymal cells has been determined, but which have not yet differentiated into mesenchymal cells. Whether a cell is an undifferentiated mesenchymal cell can be confirmed by examining whether it expresses marker proteins, such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin (if any one or more of the above marker proteins are expressed, it can be determined to be an undifferentiated mesenchymal cell). Furthermore, mesenchymal cells that do not express any of the above markers can be determined to be differentiated mesenchymal cells. Among the terms used by those skilled in the art, Septum Mesenchyme, Septum Transversum Mesenchyme, mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal cells (R. Peters, et al. PLoS One. 30;5(12):e15689.(2010)) are included in the mesenchymal cells of the present invention.

[0024] In the preparation of organ buds according to the present invention, mesenchymal cells are used that have been prepared (differentiated) from pluripotent stem cells.

[0025] To induce differentiation of pluripotent stem cells, such as iPS cells, into mesenchymal cells (iPSC-MCs), it is recommended to disperse seeding in the presence of Rho kinase, culture in medium 1 (DMEM / F12 medium + 1-2% B27 + 1% Glutamax + 8uM CHIR 99021 + BMP4 25ng / ml) for 3 days, and then culture in medium 2 (DMEM / F12 medium + 1-2% B27 + 1% Glutamax + 10ng / ml PDGFBB + 2ng / ml Activin A) for 2 days. It is also recommended to use cells cultured for 2 days in medium 3 (DMEM / F12 medium + 1-2% B27 + 1% Glutamax + 10ng / ml bFGF + 12ng / ml BMP4). Alternatively, cells maintained in a mesenchymal cell medium such as MSCGM may be used. The generated mesenchymal cells (iPSC-MCs) may be CD166-positive and do not express CD31 (PECAM1), a marker for vascular endothelium. The present invention also provides a method for producing CD166-positive and CD31-negative cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), culturing them in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 3-5 days), culturing them in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family (e.g., for 1-4 days), culturing them in the presence of FGF and a factor belonging to the transforming growth factor β family (e.g., for 2-6 days), and then maintaining culture in mesenchymal cell medium (e.g., for 3-20 days). Additional culture steps may be included before and after each culture step from pluripotent stem cells to the differentiation induction of CD166-positive and CD31-negative cells. The CD166-positive and CD31-negative cells may be mesenchymal cells (iPSC-MCs). The CD166-positive and CD31-negative cells can be used to produce organobuds. Examples of culture media for mesenchymal cells include MSCGM, but are not limited to this. In the preparation of organ buds according to the present invention, the mesenchymal cells may be septomesenchymal (STM) cells. STM cells may be LHX2-positive and WT1-positive. STM cells may have activated transcription of FOXF1, HLX1, COL4A, and ALCAM, and may be LHX2-positive, WT1-positive, and MIIA-positive. The present invention also provides a method for producing LHX2-positive and WT1-positive cells, comprising culturing pluripotent stem cells in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), culturing them in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 3-5 days), culturing them in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family (e.g., for 1-4 days), and further culturing them in the presence of FGF and a factor belonging to the transforming growth factor β family (e.g., for 2-6 days). Additional culture steps may be included before and after each culture step from pluripotent stem cells to the differentiation induction of LHX2-positive and WT1-positive cells. The LHX2-positive and WT1-positive cells may be septomesenchymal (STM) cells. The LHX2-positive and WT1-positive cells can be used to produce organobuds.

[0026] This section describes the factors that can be used in the differentiation induction method from pluripotent stem cells to various cell types (organ cells, vascular cells, and mesenchymal cells) as described above. Examples of ROCK inhibitors include Y-27632, GSK429286A, SR3677, Ripasudil (K-115), Fasudil, and Thiazovivin, of which Y-27632 is preferred. Examples of factors belonging to the transforming growth factor β family include ActivinA and Nodal, of which ActivinA is preferred. Examples of factors belonging to the Wnt family include Wnt3a, Wnt3a-AFM, CHIR99021, R-Spondin-1, and BIO(6-bromoindirubin-3′-oxime), of which Wnt3a is preferred. Factors belonging to the Wnt family should be capable of activating β-catenin. Class I histone deacetylase (HDAC) inhibitors include butyrate (e.g., sodium butyrate), Valproic acid, Panobinostat (LBH589), Apicidin, BML-210, Depudecin, HC Toxin, M344, Oxamflatin, Scriptaid, Splitomicin, Suberoyl bis-hydroxamic acid, Trichostatin A, Vorinostat (SAHA, MK0683), Entinostat (MS-275), Panobinostat (LBH589), Mocetinostat (MGCD0103), Biphenyl-4-sulfonyl chloride, ACY-738, Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), MC1568, Tubastatin A, Givinostat (ITF2357), and Dacinostat. (LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat, Abexinostat (PCI-24781), RGFP966, AR-42, Rocilinostat (ACY-1215), Tacedinaline (CI994), CUDC-907, Curcumin, Tubacin, RG2833 (RGFP109), Resminostat, Divalproex Sodium, Sodium Phenylbutyrate, Tubastatin A, TMP269, Santacruzamate A (CAY10683), TMP195, Tasquinimod, BRD73954, Citarinostat (ACY-241), HPOB, LMK-235, Nexturastat Examples include A, Tucidinostat (Chidamide), (-)-Parthenolide, CAY10603, 4SC-202, BG45, ITSA-1 (ITSA1), among which sodium butyrate is preferred. Examples of FGF (fibroblast growth factor) include basic FGF (bFGF, sometimes written as FGF2), FGF4, and FGFC (Chimeric Fibroblast Growth Factor), of which basic FGF is preferred. Examples of factors belonging to the TGFβ superfamily include BMP4, BMP2, and BMP, of which BMP4 is preferred. Examples of β-catenin activators include CHIR99021, Wnt3a, Wnt3a-AFM, R-Spondin-1, and BIO(6-bromoindirubin-3′-oxime), of which CHIR99021 is preferred. CHIR99021 can serve as a substitute for Wnt3a in the differentiation induction of iPSCs into DEs (CHIR d3 in Example 2 and Figure 13A, described later). Examples of PI3K inhibitors include PI-103, ZSTK474, NVP-BEZ235, LY294002, and Wortmannin, of which PI-103 is preferred. The PI3K inhibitor should preferably be a PI3K, Akt, or mTOR inhibitor. Examples of BMP inhibitors include LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, Pirfenidone, GW788388, LY364947, RepSox, K02288, SD-208, LDN-214117, SIS3 HCl, Vactosertib (TEW-7197), DMH1, LDN-212854, ML347, Kartogenin, Hesperetin, and Alantolactone, among which LDN-193189 is preferred. The BMP inhibitor should preferably be an ALK2 and ALK3 inhibitor. Examples of vascular endothelial growth factor receptor (VEGFR) activators include VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PLGF, of which VEGF is preferred. Examples of adenylate cyclase activators include Forskolin, HJC0350, 8-Br-cAMP, Adenosine 3′,5′-cyclic monophosphate (cAMP), and Dibutyryl-cAMP (Bucladesine), of which Forskolin is preferred. The adenylate cyclase activator should increase the intracellular cAMP concentration. Examples of TGF-β type I receptor inhibitors include SB431542, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, Vactosertib (TEW-7197), ML347, LDN-212854, DMH1, Pirfenidone, Alantolactone, SIS3, and Hesperetin, among which SB431542 is preferred. Inhibitors of the TGF-β type I receptor include ALK4, ALK5, and ALK7 inhibitors, and preferably activin inhibitors. Examples of PDGF receptor activators include PDGFBB, PDGF-AA, PDGF-AB, PDGF-CC, and PDGF-DD, of which PDGFBB is preferred.

[0027] The culture ratio of the three types of cells in co-culture is not particularly limited as long as it is within the range in which organ buds can be formed, but a suitable cell ratio is tissue or organ cells:vascular endothelial cells:mesenchymal cells = 10:10 to 5:2 to 1.

[0028] Any culture medium that can form organ buds may be used during culture, but it is preferable to use a culture medium for vascular cells (e.g., vascular endothelial cells), a culture medium for tissue or organ cells, or a mixture of the two aforementioned media. Any culture medium for vascular endothelial cells may be used, but it is preferable to use one that contains at least one of the following: hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, Antibiotics (e.g., gentamicin, amphotericin B, etc.), heparin, L-Glutamine, Phenolred, or BBE. For vascular endothelial cell culture, EGM-2 BulletKit (Lonza), EGM BulletKit (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), and Human Microvascular Endothelial Cell Growth Medium (TOYOBO) can be used. Any medium can be used for tissue or organ cell culture, but if the organ cells are hepatocytes, it is preferable to use a medium containing at least one of ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000. For hepatocyte culture, HCM BulletKit (Lonza) without hEGF (recombinant human epidermal growth factor), RPMI1640 (Sigma-Aldrich) with 1% B27 Supplements (GIBCO) and 10 ng / mL hHGF (Sigma-Aldrich) can be used. Regarding the formation of human liver buds, it has been found that adding Dexamethasone, Oncostatin M, and HGF to a 1:1 mixture of GM BulletKit (Lonza) and HCM BulletKit (Lonza), with hEGF (recombinant human epidermal growth factor) removed, is effective in maturing liver buds.

[0029] While scaffolding materials are not required for cell culture, it is preferable to culture a mixture of three types of cells on a gel-like support that allows mesenchymal cells to contract.

[0030] The contraction of mesenchymal cells can be confirmed by morphological observation (microscope or with the naked eye) of three-dimensional tissue formation, or by demonstrating that the tissue has sufficient strength to maintain its shape when retrieved with a spatula or similar tool (Takebe et al. Nature 499 (7459), 481-484, 2013).

[0031] The support is preferably a gel-like substrate having appropriate hardness (for example, a Young's modulus of 200 kPa or less (such as in the case of a flat gel coated with Matrigel), although the appropriate hardness of the support may vary depending on the coating and shape). Examples of such substrates include hydrogels (e.g., acrylamide gel, gelatin, Matrigel, etc.), but it is not limited to these. Depending on the shape, size, and quantity of the target aggregate, the hardness of the support does not necessarily have to be uniform, and it is possible to set spatial and temporal gradients or patterns in the hardness. If the hardness of the support is uniform, the hardness of the support is preferably 100 kPa or less, more preferably 1 to 50 kPa. The gel-like support may be flat, or the cross-section of the culture side of the gel-like support may be U or V-shaped. Having a U or V-shaped cross-section on the culture side of the gel-like support is advantageous because it allows cells to accumulate on the culture surface of the support, and a cell aggregate can be formed with a smaller number of cells and / or tissues. Furthermore, the support material may be subjected to chemical or physical modifications. Examples of modifying substances include Matrigel, laminin, entactin, collagen, fibronectin, and vitronectin.

[0032] One example of setting a spatial gradient in the stiffness of a gel-like culture support is a gel-like culture support in which the stiffness of the center is greater than that of the periphery. The appropriate stiffness of the center is 200 kPa or less, and the stiffness of the periphery should be softer than that of the center, however, the appropriate stiffness of the center and periphery of the support can vary depending on the coating and shape. Another example of setting a spatial gradient in the stiffness of a gel-like culture support is a gel-like culture support in which the stiffness of the periphery is greater than that of the center.

[0033] One example of a patterned gel culture support is a gel culture support having one or more patterns in which the hardness of the center is harder than the hardness of the periphery. The hardness of the center is appropriate to be 200 kPa or less, and the hardness of the periphery should be softer than the center, but the appropriate hardness of the center and periphery of the support may vary depending on the coating and shape. Another example of a patterned gel culture support is a gel culture support having one or more patterns in which the hardness of the periphery is harder than the hardness of the center. The hardness of the periphery is appropriate to be 200 kPa or less, and the hardness of the center should be softer than the periphery, but the appropriate hardness of the center and periphery of the support may vary depending on the coating and shape.

[0034] The temperature during cultivation is not particularly limited, but it is preferably 30-40°C, and more preferably 37°C.

[0035] The culture period is not particularly limited, but it is preferably 3 to 10 days, and more preferably 6 days.

[0036] In this invention, by producing all three types of cell species used to create organobuds from pluripotent stem cells, the differentiation stages of the three cell species can be synchronized, and by producing organobuds from these cells, the function of the organobuds can be improved. Furthermore, the cost and effort required for quality evaluation and manufacturing of the three cell species can be reduced.

[0037] The organ buds of the present invention can be transplanted into non-human animals and allowed to mature within those non-human animals to produce tissues or organs. In other words, the present invention also provides a method for producing tissues or organs, which includes transplanting the above-mentioned organ buds into non-human animals and allowing them to differentiate into tissues or organs. Examples of non-human animals used include those used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc. Specifically, these include mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, and the like. Furthermore, it is preferable that the non-human animals used are immunodeficient animals in order to avoid immune rejection.

[0038] Accordingly, the present invention also provides a method for transplanting organ buds, which includes transplanting the above-mentioned organ buds into humans or non-human animals. The transplantation site for organ buds may be any site where transplantation is possible, but examples include the intracranial region, mesentery, liver, spleen, kidney, sub-renal capsule, and portal vein. When transplanting into the skull, it is good to transplant about 1 to 3 organ buds about 5 mm in size that have been prepared in vitro. When transplanting into the mesentery, it is good to transplant about 1 to 6 organ buds about 5 mm in size that have been prepared in vitro. When transplanting onto the portal vein, it is good to transplant about 1 to 20 organ buds about 5 mm in size that have been prepared in vitro. When transplanting into the renal capsule, it is good to transplant about 1 to 5 organ buds about 5 mm in size that have been prepared in vitro. When transplanting into the liver, spleen, or kidney, it is good to transplant about 100 to 200 organ buds about 100 μm in size that have been prepared in vitro.

[0039] The tissues and organs produced in the manner described above can be used for drug discovery screening, regenerative medicine, and other applications.

[0040] Accordingly, the present invention provides a method for regenerating or restoring the function of tissue or organs, comprising transplanting the above-mentioned organ buds into humans or non-human animals and differentiating them into tissue or organs. Non-human animals include animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc. Specifically, examples include mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, and the like.

[0041] The organ buds of the present invention can be formulated and used in the form of a regenerative medicine composition. The composition of the present invention can be implanted in a living body to produce tissue or organs. Furthermore, the composition of the present invention can be implanted in a living body to regenerate or restore the function of tissue or organs.

[0042] After the composition of the present invention is transplanted into a living organism, organ buds can differentiate into tissues or organs having a vascular network. Vascular perfusion can occur in this vascular network. It is believed that the occurrence of vascular perfusion in the vascular network makes it possible to create tissues and organs with a highly ordered tissue structure equivalent to or close to that of adult tissue.

[0043] The composition of the present invention may also contain tissue angiogenesis promoters such as FGF2, HGF, and VEGF, a gelatin sponge for hemostasis during transplantation (product name: Spongel, Astellas Inc.), and tissue adhesives such as Volheal (Teijin Pharma Limited), Beriplast (CSL Behring Co., Ltd.), and Tacocomb (CSL Behring Co., Ltd.) used for fixing transplanted tissue.

[0044] Furthermore, the present invention also provides a method for producing non-human chimeric animals, which includes transplanting the above-mentioned organ buds into non-human animals and differentiating them into tissues or organs. Non-human animals (e.g., mice) to which organ buds have been transplanted can mimic the physiological functions of the species from which the tissues or organ cells used to produce the organ buds originated (e.g., humans).

[0045] Furthermore, the present invention also provides a method for evaluating drugs using at least one selected from the group consisting of organ buds, tissues and organs, and non-human chimeric animals. Examples of drug evaluation include evaluation of drug metabolism (e.g., prediction of drug metabolism profiles), evaluation of pharmacokinetics (e.g., screening for drugs that are effective as pharmaceuticals), toxicity evaluation, and drug interaction evaluation.

[0046] The evaluation of drug metabolism can be performed by collecting and analyzing biological samples after administering a candidate drug compound to at least one organism selected from the group consisting of organ buds, tissues, organs, and non-human chimeric animals created by the above method, thereby obtaining a human-type drug metabolism profile. This makes it possible to predict the distribution, metabolism, and excretion processes of drugs in humans, which was extremely difficult to achieve with conventional techniques, and is expected to dramatically accelerate the development of safe and effective drugs.

[0047] The screening of effective pharmaceuticals can be performed by administering a novel drug candidate compound to at least one selected from a group consisting of organ buds, tissues, and organs created from cells established from diseased patients using the method described above, and non-human chimeric animals. This is expected to significantly improve the accuracy of predicting drug efficacy when actually administered to humans, which was insufficient in conventional in vitro tests.

[0048] Toxicity assessment can be improved by administering the test substance to at least one selected from a group consisting of organ buds, tissues, and organs prepared by the above method, and non-human chimeric animals, and then measuring tissue damage markers, thereby improving the accuracy of damage prediction.

[0049] Drug interaction evaluation can be performed by administering multiple drugs to at least one of the groups consisting of organ buds, tissues and organs, and non-human chimeric animals prepared by the above method, and then evaluating the pharmacokinetics, toxicity, and efficacy of each drug, including their distribution, metabolism, and excretion processes.

[0050] Furthermore, it is possible to create tissue stem cells from tissues and organs produced by the method of the present invention, and the present invention can be applied to cell manipulation technology for the mass production of human tissue cells and organ cells. [Examples]

[0051] The present invention will be described in more detail below with reference to examples. [Example 1] Establishment of a method for mass production of liver buds from human pluripotent stem cells.

[0052] summary Organoid transplantation therapy has the potential to become a revolutionary paradigm for disease treatment, but ensuring reproducibility and scalability has been the most critical challenge. In this study, we attempted to address these challenges by constructing a platform for mass production of organobuds from human induced pluripotent stem cells (iPSCs). First, we identified effective progenitor cell populations (hepatic endoderm, endothelium, and transverse septal mesenchyme) for generating hepatobuds with high reproducibility through large-scale "reverse" screening experiments. Furthermore, we found that >10 8 By developing an Omniwell array culture platform for mass-producing homogeneous and miniaturized liver buds on a scale, we achieved the volume levels required for transplant therapy. The liver tissue, entirely generated from iPSCs, was vascularized and functional, significantly improving subsequent liver function enhanced by stepwise developmental progenitor cell interactions. Furthermore, this liver tissue enabled functional relief from acute liver failure through transplantation. This research realizes a manufacturing platform for supplying multicellular liver bud organoids, which is expected to significantly accelerate clinical and drug discovery applications for the treatment of liver diseases in the future.

[0053] Introduction Organoid technology is a recently evolving approach to the treatment of intractable diseases and to human development and disease models (Huch and Koo, 2015; Lancaster and Knoblich, 2014; Sasai, 2013). Based on the principle of self-condensation, we have recently succeeded in constructing further complexity in organoids by developing multicellular organ buds such as liver buds, pancreatic buds, and kidney buds that have therapeutic potential for various mouse disease models (Takebe et al., 2015; Takebe et al., 2013; Takebe et al., 2014). Nevertheless, broader applications of organoid-based approaches face challenges in scalability and reproducibility in order to generate sufficiently large and stable organoids for transplantation and drug testing in humans (Ding and Cowan, 2013). To promote the future therapeutic applications of organ bud-based approaches, we aimed to establish a comprehensive, scalable, and reproducible method for completely generating angiogenic human liver buds (LBs) from feeder-free human iPSCs and to validate their functional capabilities for transplantation.

[0054] result Initially, we aimed to establish a scalable three-dimensional organoid culture platform using liver buds (LBs) as an example. The overall strategy is summarized in Figures 1A and B. Briefly, we developed a U-shaped-bottom microwell plate by combinatorial chemistry techniques to develop a microstructured membrane by precisely transferring a template structure onto a resin membrane. Our microwell array has a very high aspect ratio (as shown in Figure 1C, the opening diameter of each well is approximately 500 μm, the depth is 400 μm, and the wells are closely spaced 30 μm apart and arranged in a triangular pattern). Such an aspect ratio is difficult to transfer widely with conventional molding machines. Therefore, we improved the transfer accuracy by using an internally optimized molding machine (see Methods). As a result, prototyping was carried out in 24-well and 6-well formats, i.e., Elplasia TM Success was demonstrated with RB (round-bottom) plates (600 spots / well in 24 wells; 3,000 spots / well in 6 wells). Subsequently, we adapted this plate for hepatomegaly culture by mixing human iPSC-derived hepatic endoderm (iPSCHE), human umbilical vein endothelial cells (HUVEC), and bone marrow mesenchymal stem cells (BMSC) as previously described (Takebe et al., 2013). By optimizing the coating material (Fig. 5 (Fig. S1)), depression shape (data not shown), mixing ratio (Fig. 6A-C (Fig. S2 AC)), and cell number (Fig. 6D-F (Fig. S2 DF)), small hepatomegaly can be effectively formed using this microwell array-based approach (supplementary text).

[0055] To intensively scale up this strategy, we devised an omni(1) well array plate containing more than 20,000 microspots per well (Fig. 7A (Fig. S3A)). The external dimensions (length, width, height) of the omniwell array plate frame meet the SBS standard, similar to general microplates. The plate consists of two modules: a bottomless plate frame and a microwell transfer film. These were assembled by laser welding (Fig. 1A (left)). The application of the microwell transfer film used in the 24 / 6 well format was not effective for the omniwell format because a significant depression in the central portion caused abnormal organoid displacement and subsequent fusion (Fig. 1B (center)). Therefore, we attempted to improve flatness by following two strategies: 1. reducing residual stress in the film, and 2. increasing the stiffness of the film. First, we minimized the molding pressure required to transfer the pattern and reduced the limitations of template transfer by optimizing molding conditions such as molding temperature and press time, and improving film deflection during laser welding. Next, we increased the rigidity by increasing the film thickness (Figure 1B, caption). We found that a film thickness of 1.1 mm satisfied acceptable rigidity without interfering with microscopic observation under various thickness conditions ranging from 0.8 mm to 1.4 mm. The 3D surface profile analyzer showed minimal height variation under the improved conditions, enabling subsequent LB cultures on a larger scale (Figure 1B, right). Importantly, more than 99% of the generated LBs were successfully collected by simple manual pipetting (Fig. 5B). Once seeded with three types of progenitor cells in an Omniwell plate, more than 20,000 endothelialized LBs self-organized, whereas single iPSC-HE cultures did not (Figure 1C, D). Quality confirmation analysis using qRT-PCR showed that LB in Omniwell had equivalent characteristics to LB in a 24-well / 6-well format (Figure 7B (Fig. S3B)).In summary, we have developed a prototype design for an omniwell array plate for large-scale organoid production.

[0056] Next, we attempted to define the phenotype of the optimal endodermal cell population for hepatocyte formation in humans. Therefore, we conducted a large-scale "reverse" screening experiment by comparing multiple endodermal stages based on the quality of the obtained organoids. After selecting the most reproducible published mature hepatocyte differentiation protocol (Figures 8A-F (Fig. S4A-F) and supplemental text) (Kajiwara et al., 2012, Lohet al., 2014, Si-Tayeb et al., 2010), we further attempted to accurately determine the best endodermal stage for iPSC-LB function by initiating hepatocyte culture from cells from day 0 to day 20 (Figure 9 (Fig. S5)) and Figures 2A, B (Figure 2A, B)). First, quantitative fluorescence-based evaluation of morphological changes in recovered LBs generated from multiple stages (0-20 days) revealed that day 8 cells were the only population capable of maintaining highly homogeneous LBs with a size of 100-200 μm (Figure 1E, F). Subsequent qRT-PCR of liver marker genes (Figure 9A (Fig. S5A)) and ELISA of protein production (Figure 2A (Fig. 2A)) showed that iPSC-LBs derived from day 8 cells were of higher quality than hepatocyte cultures derived from day 0 (iPSC), day 5, day 6, day 7, day 10, and day 20. Transcriptome analysis showed that the expression characteristics of the LBs were more similar to those of human adult liver tissue than to those of 2-D differentiated cells (Figure 9B (Fig. S5B)). Consistently, principal component analysis of overall gene expression showed that the PC1 axis profile in hepatic buds at day 8 most closely resembled that of human adult liver tissue samples compared to other stages (Figure 2B). Interestingly, significant enhancement in the gene set characteristic of angiogenesis was observed in hepatic buds at day 8, but not in buds at day 7 (Figure 9C). This may be useful for favorable angiogenesis outcomes in vivo.

[0057] To further characterize these "reverse" identified 8-day populations, we performed profiling based on SOX17 / HNF4A co-immunostaining, qRT-PCR, FACS, and Cerberus1 ELISA, identifying that the 8-day cells represent a population in the transition period from embryonic endoderm to hepatic endoderm cells (Fig. 8G (Fig. S4G) and Fig. 2C (Figure 2C)). Here, we define these cells as transitional hepatic endoderm cells (tHE). Since the 8-day cells are negative for DE markers but do not express HE markers such as HNF4A (Fig. 2C (Figure 2C)), we attempted to define the cells by identifying definable markers in comparative transcriptome analysis between the 6-day and 8-day populations. Based on transcriptome dynamics, volcano plot analysis (comparison of iPSC-DE and iPSC-tHE) identified T-box transcription factor 3 (TBX3) and adrenaline receptor alpha 1B (ADRA1B) (Figure 2D and Fig. S6). This was confirmed by immunohistochemistry and qRT-PCR (Figure 2E and Figure 3A). In summary, although the developmental association of these reverse-identified populations remains unclear, we concluded that TBX3 and ADRA1B co-positive tHE represents the most effective stage for hepatometabolism.

[0058] One major obstacle to the future application of such LB-based approaches is the use of postnatal tissue-derived stromal progenitor cells (i.e., HUVECs and BMSCs). Therefore, we aimed to induce two hepatocyte stromal progenitor cells from human iPSCs. In the early stages of hepatic organogenesis, hepatic endodermal cells migrate to LIM Homeobox 2 (LHX2) and Wilms tumor 1 (WT1)-positive septal mesenchyme (STM) cells to form hepatocytes (Delgado et al., 2014, Kolterud et al., 2004). STM cells are involved not only in hepatocyte formation but also in the proliferation and survival of hepatocytes, mediated at least by paracrine factors derived from STM cells (Zaret, 2002). However, differentiation protocols for STM cells have not been developed (Iyer et al., 2015, Witty et al., 2014). To dictate the fate of STM cells, we exposed iPSC-derived lateral plate mesoderm (iPSC-Meso) cells to potential inducers and their combinations on day 4. While the PSC marker NANOG and the early mesoderm marker T were barely detectable, transcriptional activation of STM markers FOXF1, HLX1, COL4A, and ALCAM was successfully induced on day 10 with co-exposure to FGF2 and PDGFB (Figure 2F). Immunostaining of WT1, MIIA, and LHX2 also confirmed the induction of correct cell fate in putative STM cells (Figure 3A). Global gene array analysis suggested that the characteristics of iPSC-STM cells show significant alterations to those of reported human adult liver mesenchymal cells (Asahina et al., 2009, Asahina et al., 2011, El Taghdouini et al., 2015) (Figure 2G). We previously demonstrated that one function of mesenchymal cells in the LB generation is myosin IIA-dependent autoaggregation. Consistently, we demonstrated using time-lapse imaging that the self-aggregating ability of iPSC-STMs is comparable to that of conventional BMSCs (Figure 2H).

[0059] To reproducibly generate endothelial progenitor cells (iPSC-ECs), we developed four independent protocols adapted from previous literature after adapting them to feeder-free iPSC culture (Narazaki et al., 2008, Orlova et al., 2014, Patsch et al., 2015, Samuel et al., 2013) (Fig. 11A (Fig. S7A)). iPSC-ECs subjected to a complete differentiation program under each protocol were evaluated by flow cytometry (FACS) and qRT-PCR (Fig. 11B, C (Fig. S7B, C)). The results showed that cells following protocol 1 exhibited the highest expression of EC markers and minimized pluripotency markers. The combination of VEGF and Forskolin can induce the highest levels of co-expression of CD144 (VE-Cadherin) and CD31 by regular FACS analysis (>92.8%, n = 12) without the need for sorting based on magnetic beads (Figure 2I). After continuous passage, ECs proliferated vigorously (~200-fold increase) (Fig. 11D), and this expression was maintained up to four passages. This was confirmed by immunostaining of endothelial markers; after plating on Matrigel plugs, the cells showed vigorous migration and subsequent endothelial sprouting ability (Figure 2J). More importantly, co-culture of iPSC-ECs and iPSC-STMs on a soft substrate resulted in the formation of condensed tissue (Takebe et al., 2015), and patency of blood vessels could be generated 48 hours after transplantation. This was confirmed by confocal imaging of cells using AAVS1 :: mCherry iPSC-EC after fluorescent dextran injection (Figure 2K). These results suggest that human iPSC-STM and -EC populations successfully differentiate from feeder-free iPSCs with functional capabilities for autoaggregation and angiogenesis.

[0060] The potent induction of three types of progenitor cells allowed us to investigate the potential for hepatocyte generation from iPSCs (all iPSC-LB) (Figure 3A). These three distinct progenitor cells were successfully induced from multiple human feeder-free iPSC sources (five independent donor-derived clones tested), including HLA homozygous clones such as Ff-l01 and Ff-l14. 4D bright-field and light-sheet imaging analysis revealed successful autoaggregation in the presence of STM (Figure 3B, top) and self-organizing iPSC-EC networks (Figure 3B, bottom), respectively. Confocal wide-field imaging of cultured tissue for three days confirmed sprouting iPSC-ECs aligned with iPSC-tHE (Figure 3C, D). The potential for angiogenesis was further evaluated by transplantation into the cranial windows of immunodeficient mice. In vivo imaging demonstrated the formation of functional blood vessels and the eventual engraftment of iPSC-tHE at 48 hours (Figure 3E-G). The iPSC-EC directly anastomosed mouse CD31 endothelial cells (Figure 3H) and were surrounded by iPSC-STM in a perivascular location (Figure 3I). Thus, we successfully generated fully angiogenic and functional liver tissue from human iPSCs.

[0061] To correct specific metabolic liver function, at least 10 8 Because transplantation of 10 hepatocytes is required (Martin et al., 2014), the biggest challenge is adapting all iPSC-LB strategies to a culture platform that can be implemented on an omniwell array scale. For this purpose, our strategy for mass production and batch validation of all iPSC-LBs is outlined in Figure 4A. Briefly, we aim to achieve 10 per culture. 8We prepared 5-10 Omniwell array plates to produce cell-scale hepatocytes and elucidated their in vitro function and in vivo therapeutic potential. As a result, after 10 days of culture, all mass-produced iPSC-LBs showed higher albumin production than conventional LBs (HUVEC / BMSC) and adult human hepatocytes (AdHep) (10 μg / ml / 24hr / 10). 6(More than 1 / 2 cells) (Figure 4B and Fig. S5B). In addition, these iPSC-LBs also produced several important liver serum proteins, including complement factor H, coagulation factor VIII, transferrin, and AAT (Figure 4C). Notably, the ammonium clearance capacity of all long-term cultured iPSC-LBs was equivalent to that of primary adult human hepatocytes in culture (Figure 4D). Further global transcriptome analysis showed that all differentiated iPSC-LBs were more mature than conventional LBs and equivalent to AdHep (although not as mature as 30-year-old adult liver tissue) (Figure 4E and Fig. S5B). To make a fair comparison with human AdHep, we developed a scoring method based on liver function gene characteristic data. This scoring method was named APRES (Aster Plot of Relative Enrichment Score). Using a characteristic gene set in MSigDB version 5.0 (Subramanian et al., 2005), we determined the width of each component in the aster plot using the relative magnitude of mNES scores calculated from the expression levels of progressive traits in human adult hepatocytes (AdHep 2D) related to fetal liver (see Methods). For each tissue or cell type, the relative mNES score was multiplied by the width and height of the component and then shown as the height of each component in the aster plot, and the total relative score was defined as the sum. Unbiased APRES-based scoring clearly demonstrated that the profile of differentiated whole iPSC-LBs was similar to that of AdHep and conventional iPSC-MHs, including complement, angiogenesis, and cholesterol homeostasis cascades (Figure 4F).

[0062] Finally, functional batch validation of mass-produced human liver enzymes was performed in an immunodeficient mouse model of liver failure. 10 8 Prepare liver buds equivalent to individual liver cells, and for each mass production, 10 7The LB corresponding to individual cells was divided among approximately 10 mice, and they were evaluated by determining the therapeutic potential of whole liver buds. Transplantation was performed under the renal capsule of Alb-Tk-NOG mice. Overall, the results obtained from 114 transplanted mice provided strong evidence with statistical significance for improved viability by relieving total liver dysfunction (Figure 4G). Importantly, follow-up serum analysis also supported the fact that the mass-produced liver buds were functionally stable by producing ALB in vivo and reducing AFP (undetectable), independent of the production cycle (Fig. S8A). Notably, the size and persistence of human albumin were significantly higher than those obtained from transplantation of human primary hepatocytes (N = 12, 4 donors) (Figure 4H). The drug metabolism ability of LB was also confirmed by detection of human-specific diclofenac metabolites (Figure 4I). Transplantation of iPSC-tHE cells without stromal cells hardly functioned as evaluated by human ALB in transplanted mice (Fig. S8B). This suggested the potential role of iPSC-STM and -EC in liver functionalization, as shown in recent studies of single-cell RNA sequencing (Camp et al., 2017). In summary, our organoid mass production culture platform not only solves the scaling problem but also provides a rigorous and reproducible differentiation platform for human iPSC-derived human liver tissue with functions equivalent to 10 8 or more adult hepatocytes.

[0063] Discussion In conclusion, the technology outlined in this study reveals an exciting strategy for supplying iPSC-based multicellular organoids for drug testing and regenerative applications. In particular, the production of liver buds at a cell scale of 10 8 or more is considered a reasonable scale for human transplantation applications. Because the number of cell transplantation trials is, especially for pediatric patients, the minimum dose of 10 8This is because its clinical efficacy has been demonstrated in cellular studies (Enosawa et al., 2014, Jorns et al., 2012). The standard treatment scale is 10 per patient. 9 If we are dealing with individual hepatocytes, or even more (Horslen and Fox, 2004), then ongoing scaling efforts are expected in studies of corresponding efficacy in animal models of metabolic disorders. Nevertheless, this protocol has been developed through multiple academic and industrial collaborative efforts using clinical-grade components. These clinical-grade components include genetically characterized iPSCs, defined media and substrates, microwell array plates, and microscopic examination in preparation for future clinical trials. To accelerate this, fully compliant all iPSC-LB strategies with cGMP-grade systems is crucial for future clinical applications and safety assessments through clinically appropriate transplantation routes. Ultimately, we believe that the integration of a range of manufacturing technologies makes it feasible to develop clinically effective hepatocyte transplantation therapies for treating currently challenging liver diseases.

[0064] Experimental Procedure Culture of human liver buds in microwells All iPSC lines were maintained in StemFit® (Ajinomoto Co., Inc.) on dishes coated with the Laminin 511 E8 fragment (iMatrix-511®, kindly provided by Nippi, Inc.). Methods for specific differentiation into each line are described in the Supplementary Methods. To generate human LB in vitro, a total of 1140–10,140 cells per microwell were resuspended in a mixture of EGM and hepatocyte culture medium (HCM) (Cambrex, Baltimore, MD) in a ratio of 10:7:2 (= human iPSC-tHE:iPSC-EC:iPSC-STM). This mixed medium contains dexamethasone (0.1 μM, Sigma-Aldrich, St. Louis, MO), oncostatin M (10 ng / ml, R&D System, Minneapolis, MN), HGF (20 ng / ml, PromoKine), and SingleQuots (Lonza). The resuspended cells were seeded on either a 24-well plate or an Elplasia® platform (co-developed by Kuraray, Inc.) in a 24-well, 6-well, or 1-well plate. Phase-contrast colony images, shown above in Figure 3B, were obtained using a BioStation CT culture incubator, microscope, and digital imaging system (Nikon, Tokyo, Japan). Fluorescence time-course imaging, shown below in Figure 3B, was imaged using a Lightsheet Z.1 microscope (Zeiss, Germany). The generated human iPSC-LBs were collected by gentle pipetting and used for in vitro maturation evaluation and in vivo transplantation experiments. As controls, we used five different human primary adult hepatocytes (lot numbers: H768, H737, HC2-8, H4.1, and M00995, purchased from XenoTech, KS, USA, and Veritas, Tokyo, Japan).

[0065] Quantification and statistical analysis Data are expressed as mean ± sem or mean ± sd of the independent experiments identified in the description. This study did not employ randomization or blinding. Statistical significance of albumin production was assessed by the non-parametric Mann-Whitney U test. Two-sided p-values ​​less than 0.05 were considered significant. For survival analysis, GraphPad Prism Software version 6.0 was used for statistical analysis.

[0066] Supplementary Information The supplementary information includes supplementary experimental procedures and eight figures (Figures 5-12 (Figs. S1-S8)).

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Takebe, T., Zhang, RR, Koike, H., Kimura, M., Yoshizawa, E., Enomura, M., Koike, N., Sekine, K., and Taniguchi, H. (2014). Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 9, 396-409. Witty, AD, Mihic, A., Tam, RY, Fisher, SA, Mikryukov, A., Shoichet, MS, Li, RK, Kattman, SJ, and Keller, G. (2014). Generation of the epicardial lineage from human pluripotent stem cells. Nat Biotechnol 32, 1026-1035. Zaret, KS (2002). Regulatory phases of early liver development: paradigms of organogenesis. Nat Rev Genet 3, 499-512.

[0068] Experimental model and subject details mouse LBs generated in vitro were harvested and transplanted into pre-formed cranial fenestrae or other indicated sites in non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice (Sankyo Lab. Co., Tsukuba, Japan). The in vivo fate of the transplanted cells was monitored by in vivo imaging using a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany). For in vivo functionalization studies, 8-week-old male NOG mice (approximately 20-30g body weight) were used (supplied by the Central Institute for Experimental Animals (CIEA), Kanagawa, Japan) (Hasegawa et al., 2011). For survival curves, albumin-TK-NOG mice (approximately 20-30g body weight) were used in this study (supplied by CIEA, Kanagawa, Japan). Prior to transplantation, ganciclovir (GCV, 50 mg / kg, ip), a drug non-toxic to human or mouse tissues, was administered to induce tissue-specific ablation of transgenic liver parenchymal cells, resulting in 1 × 10⁶ mice per unit. 7 iPSC-LB cells were transplanted into the subscapular region of the kidney. Sample size was determined by the minimum size required to obtain a significant difference (P < 0.05) at 80% output when there was a 30% change in secreted albumin protein. Euthanasia timing was randomly assigned. Regarding the exclusion criteria for transplantation experiments, it was predetermined that data from mice euthanized due to disease would be excluded. Such data were not related to transplantation. Mice were housed and maintained in accordance with Yokohama City University's facility guidelines for the use of experimental animals.

[0069] Method details Human iPSC culture and tHE differentiation Human iPSC lineages (TkDA3-4, 1231A3, 1383D2, 1383D6, and Ff01) were provided by Kyoto University and the University of Tokyo. 1231A3, 1383D2, and 1383D6 are lineages established from ePBMC® (Cellular Technology Limited, OH) at Kyoto University CiRA. All iPSC lineages were maintained in StemFit® (Ajinomoto Co., Inc.) on dishes coated with the Laminin 511 E8 fragment (iMatrix-511®, kindly provided by Nippi, Inc.). For in vitro and in vivo live imaging analysis, fluorescent protein knock-in reporters were used under the expression of adeno-associated virus integration site 1 (AAVS1 :: EGFP or mCherry). To derive iPSC-tHE, we developed a two-step differentiation method (see supplemental text). In the first stage, human iPSCs were seeded on iMatrix-511 coated dishes with 10 μM ROCK inhibitor Y-27632 (Wako, catalog number 253-00513), 1% B27 was added, and RPMI-1640 containing human 100 ng / ml activin A (provided by Ajinomoto Co., Inc.) and 50 ng / ml Wnt3a (R&D Systems) was used as culture medium for 6 days. On the first day of iPSC plating, 1 mM sodium butyrate (Sigma) was added. Successful endoderm identification was quantitatively evaluated using the Cerberus 1 ELISA kit (Dojin Kagaku, Kumamoto, Japan). Next, human iPSC-derived endodermal cells were further treated for two days with RPMI-1640 containing 1% B27, 10 ng / ml human basic FGF, and 20 ng / ml human BMP4 to induce TBX3 and ADRA1B-positive transitional hepatic endodermal populations. Before adapting the protocol described for each iPSC clone, we strongly recommend examining the specificity markers of each human iPSC at each stage of differentiation by immunohistochemistry and gene expression studies, in addition to careful microscopic observation, because successful hepatome formation is crucial for functional maturation in vivo.The use of human iPSCs in this case study was approved by the Ethics Committee of Yokohama City University.

[0070] Human iPSC-EC and STM differentiation For EC differentiation, human iPSCs were dissociated using Accutase and plated onto Laminin 511 E8 fragments (iMatrix-511®, provided by Nippi, Inc.). The human iPSCs were incubated in StemFit® (Ajinomoto Co., Inc.) containing 10 μM ROCK inhibitor Y-27632 to various optimal densities (dependent on cell line). The following day, the medium was replaced with priming medium. The priming medium consisted of B27 medium (a 1:1 mixture of DMEM and F12 containing 1% Glutamax and 1% B27 (all Life Technologies)) containing 8 μM CHIR99021 (Tocris Bioscience) and 25 ng / ml BMP4 (R&D Systems). Three days later, the priming medium was replaced with EC induction medium. The EC induction medium consisted of StemPro-34 SFM medium (Life Technologies) supplemented with 200 ng / ml VEGF (Life Technologies) and 2 μM forskolin (Sigma-Aldrich). The induction medium was refreshed daily. On day 7 of differentiation, ECs were dissociated with 0.05% trypsin and subjected to FACS analysis. iPSC-derived ECs should exhibit typical endothelial morphology with junctional localization of CD144 and CD31. ECs were measured at 1 μg / cm³. 2 50,000 cells per cm² in EC expansion medium on a dish coated with fibronectin (Sigma-Aldrich). -2 Replating was performed at the following density. The EC expansion medium consisted of StemPro-34SFM supplemented with 50 ng / ml VEGF-A. The EC expansion medium was changed every other day. For STM differentiation, human iPSCs were dissociated using Accutase and placed on Laminin 511 E8 fragments in StemFit® containing 10 μM ROCK inhibitor Y-27632 at a rate of 2000-8000 cells / cm³.2 The cells were plated and cultured for 4–6 days prior to induction. During the mesoderm induction stage, the maintenance medium was replaced with mesoderm induction medium, followed by exposure to 2 ng / ml activin A and 10 ng / ml PDGFBB (R&D Systems) for 3 days. The mesoderm induction medium was a 1:1 mixture of DMEM and F12 containing 1% Glutamax and 1% B27, where B27 contained 8 μM CHIR99021 and 25 ng / ml BMP4. After 3 days, the mesoderm induction medium was replaced with STM induction medium and cultured for 3 days. The STM induction medium consisted of StmePro-34 SFM medium supplemented with 10 ng / ml FGF2 and 10 ng / ml PDGFBB.

[0071] Manufacturing process of Elplasia® microwell plates Elplasia™ microwell plates were industrially manufactured using a combination of two technologies: one was the creation of a finely structured mold, and the other was the precise transfer of the structure from the mold to the resin film. The creation of the finely structured mold began with the preparation of the master block. We precisely cut microwells into a metal plate at equal intervals using precision metal cutting. Each microwell had a U-shaped bottom, an opening diameter of approximately 500 μm, and a depth of 400 μm. To ensure that all seeded cells were separated into each microwell, they were arranged closely together in a triangular pattern at 30 μm intervals. After metal cutting, the plates were cut into squares, the back surface was polished, and a release treatment was applied to improve the release of the resin from the mold. Through these processes, a nickel plate was created as a mold with fine columnar structures on its surface, representing the inverted shape of the microwells. The transfer of the microwell shape from the mold to the resin film was performed using an internally developed transfer molding machine. Compared to conventional injection molding machines, our molding machine significantly improves transfer accuracy, especially in high aspect ratio areas such as plate shapes ("aspect ratio" is the ratio of height to width of microstructures). In fact, our microwells (depth: 400 μm, spacing: 30 μm) have a very high aspect ratio that is difficult to transfer with conventional molding machines. We mounted the mold to the molding machine and applied molten polystyrene resin to the mold. Through optimization of several conditions, including temperature, press weight, and holding time, we finally obtained a resin film with perfectly transferred microwells. The trimmed film was bonded to a pre-fabricated well plate frame without a bottom, thereby completing the Elplasia® microwell plate with a microwell array at the bottom of the plate. 1 cm 2 Since there are approximately 320 microwells per plate, a 24-well format has approximately 600 microwells per well, a 6-well format has approximately 3,000 microwells per well, and an omniwell format has approximately 20,000 microwells per plate.

[0072] In vitro imaging The phase-contrast colony images shown in the upper part of Figure 3B were obtained using a BioStation CT culture incubator, microscope, and a digital imaging system (Nikon, Tokyo, Japan) that was adjusted to optimize autofocus and cell image tiling acquisition functions according to the provided instructions. The fluorescence time-course imaging shown in the lower part of Figure 3B was imaged using a Lightsheet Z.1 microscope (Zeiss, Germany).

[0073] In vivo imaging Vascular lumens (all from Invitrogen, Carlsbad, CA, USA) were identified using tail vein injections of 1% tetramethylrhodamine-conjugated dextran (MW 2,000,000), fluorescein isothiocyanate-conjugated dextran (MW 2,000,000), and Texas Red-conjugated dextran (70,000 MW, neutral). Host endothelial cells were visualized using intravenously injected Alexa647-conjugated mouse-specific CD31 (BD). Confocal image stacks were acquired for the transplanted vessels and dextran.

[0074] Gene expression analysis qRT-PCR analysis was performed as previously described (Takebe et al., 2013). For microarrays, total RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA for gene expression profiling was hybridized on a Whole Human Genome Agilent 4x44K v2 oligonucleotide microarray or Whole Human Genome Agilent 8x60K v2 oligonucleotide (Agilent Technologies, Palo Alto, CA) according to the manufacturer's instructions. As control samples, in addition to the human primary hepatocyte samples mentioned above, human FLT (10gwk or 22-40gwk pool Fetal Liver Tissue), ILT (0yr Infant Liver Tissue), and ALT (5yrs, 30yrs, 44yrs, or 55yrs old Adult Liver Tissues) RNA samples were obtained from the Biochain Institute (Hayward, CA, USA).

[0075] Hierarchical clustering analysis Microarray data were processed using the GeneSpring standard protocol. Briefly, signal intensities less than 1 were corrected to 1 (not detected), followed by 75 percent shift normalization. After averaging signal intensities within replicated spots, batch effects observed in different 8x60k v2 array chips (data not shown) were removed by Combat (Leek et al., 2012) with the same differentiation stage covariates. When comparing with 4x44k v2 array data, we performed quantile standardization to ensure the removal of inter-chip differences and batch effects. To analyze genome-wide differentiation status, we performed principal component analysis (PCA) using scaled expression levels of protein-coding genes (Le et al., 2008). Since the upper layer principal component (PC) explained approximately 40% of the variance across all samples from 2-D cultured iPSCs to clinical specimens, we primarily used PC1 (Figure 2D). Unsupervised hierarchical clustering was performed using Pearson correlation-based distance and mean join methods. To highlight the characteristics of the development, we used Discovery LifeMap signatures from http: / / discovery.lifemapsc.com / in-vivo-development / , 2015 / 4 / 8, and p-values ​​from Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005) are shown by a heatmap (Figure 2H, comparisons are shown in this figure). Unless otherwise noted, data processing and analysis were performed using statistical software R version 3.0.1.

[0076] APRES (Aster plot of relative enrichment score) algorithm First, using gene sets characteristic of MSigDB version 5.0 (Subramanian et al., 2005), we performed enrichment analysis comparing human fetal liver tissue (FLT, 10w) with tissues / cells at several different differentiation stages. The normalized enrichment score (NES) calculated by GSEA software was converted to modified NES (mNES) as follows: when NES > 1, mNES is NES; when -1 < NES < 1, mNES is 1; otherwise, mNES is 1 / |NES|. This conversion enables comparison of NES regardless of whether the target gene set is enriched in the comparison of samples or FLT. Next, we determined the width of each component of the aster plot using the relative size of mNES calculated from the comparison of human adult hepatocytes (AHEP 2D) and FLT. For each tissue or cell type, the relative mNES score was calculated by dividing by the mNES of AHEP 2D vs FLT, and this score was shown as the height of each component of the aster plot. Finally, we multiplied the width and height of the components and added them to define the total relative score. The total relative score was shown at the center of the aster plot (Figure 4F).

[0077] ELISA Blood samples were allowed to coagulate in a centrifuge tube at room temperature (approximately 5 minutes), loosened from the side of the tube, and incubated at 4°C (thawed ice) for 20 minutes. The coagulated blood was centrifuged at 400 g at 4°C for 10–15 minutes, and the serum fraction was removed carefully, taking care to remove red blood cells or coagulated material. Human CER1, ALB, and AAT were measured in mouse serum samples using the Human Cerberus 1 Quantification Kit (Dojin Kagaku, Kumamoto, Japan), Human Albumin ELISA Quantitation Kit (Bethyl Laboratories Inc., Montgomery, TX, USA), and human alpha 1-antitrypsin ELISA Quantitation Kit (GenWay Biotech, Inc., San Diego, CA, USA) according to the manufacturer's instructions. All ELISA experiments were performed by blinded researchers using in vitro culture supernatant or in vivo serum.

[0078] Data and software effectiveness The accession number for the microarray data reported in this example will be updated when the data is uploaded via NCBI GEO.

[0079] Supplemental Text Small liver bud culture Following the selection of coating materials (Fig. 5 (Fig. S1)), we first optimized cell culture conditions by conducting mixing ratio and dose-dependent studies. The endothelial cell versus whole cell mixing protocol had been previously optimized based on efficient post-transplant angiogenesis (Takebe et al., 2014). However, the mesenchymal cell protocol had not yet been determined. SEM analysis confirmed that 10% to 1.4% of the mesenchymal cell mixture (1 / 5 to 1 / 40 of endodermal cells) enabled reproducible LB production (Fig. 6A (Fig. S2A)), and subsequent gene expression analysis suggested that 2.8% (1 / 20 of endodermal cells) was the most efficient ratio for stable liver differentiation and tissue formation (Fig. 6B, C (Fig. S2B, C)). Next, we conducted dose-reduction studies to determine the minimum cell number for functional LB production (Fig. 6D-F (Fig. S2D-F)). The cell number-dependent LB size is shown in Figure 6D (Fig. S2D). LB size showed a dose-dependent increase up to 4000 cells (LBs could not be formed with more than 6000 cells per microwell). Functional screening based on ELISA of long-term differentiated LBs showed that LBs containing 600 iPSC-tHE cells per LB (1130 cells in total) produced the most human albumin compared to higher or lower amounts (ranging from 150–1200 iPSC-tHE cells per LB) (Figure 6F (Fig. S2F)). This was confirmed by gene expression analysis of additional liver differentiation markers (Figure 6D, E (Fig. S2D, E)).

[0080] Selection of highly efficient differentiation protocols for generating hepatocyte-like cells We conducted numerous "reverse" screening experiments by comparing the morphology and functionality of differentiated hepatocytes using endodermal cells at multiple differentiation stages. A vast number of publications have reported that each specific method, involving the sequential addition of genes and proteins, leads to the production of hepatocyte-like cells. However, comparative analyses have been rarely performed. First, we performed a 2D-based screening to select three promising stepwise differentiation protocols (Kajiwara et al., 2012, Loh et al., 2014, Si-Tayeb et al., 2010) (Figure 8A (Fig. S4A)). Therefore, after modifying the three main protocols to our feeder-free iPSC culture by optimizing cell density, cytokine exposure duration, and basal medium, differentiated cells in protocol (Pr)1 showed minimal expression of pluripotency markers (OCT4 and NANOG) and higher expression of hepatic markers (FOXA2, HNF4A, ALB, and AFP) in endoderm (DE) and mature hepatocyte-like (MH) cells compared to Pr 2 and Pr 3 (Fig. 8B-E). These results were confirmed by ELISA analysis of human albumin secretion, which revealed that secretion from MH cells in Pr 1 was approximately 2.5 times higher than that of Pr 2 and 3 (data not shown). Under Pr1, which amplifies the Wnt signaling pathway, four different patient-derived iPSC clones were able to differentiate into functional hepatocyte-like cells in a highly reproducible manner (Fig. 4F). In summary, Wnt3A exposure during early endoderm specificization is effective in generating functional hepatocyte-like cells in 2D culture.

[0081] Future verification through detection of CER1 secretion A transcriptome-based comparison of high (good) / low (poor) albumin MHs with original DE cells suggested that better outcomes of 2D MH function correlated with the efficiency of DE specificization, which may help identify future validation markers (data not shown). Therefore, we hypothesized that successful liver maturation, particularly at the DE stage, is highly dependent on the quality of early endoderm specificization. To identify good DE markers, by comparing the overall gene expression profiles of good and poor DEs (distinguished by final iPSC-MH function), it became clear that good DEs tended to express Cerberus 1 (CER1) at much higher levels (Iwashita et al., 2013). Cerberus 1 is a secreted protein and a known endoderm marker. Its amount can be measured by ELISA in the culture supernatant. Therefore, we found that detection of CER1 protein in the culture medium on day 6 was at least essential for final ALB detection on day 20 (Figure 4G). This suggests that the amount of secreted protein on day 6 is a potential requirement for liver function after the completion of terminal differentiation.

[0082] SUPPLEMENTAL REFERENCES Iwashita, H., Shiraki, N., Sakano, D., Ikegami, T., Shiga, M., Kume, K., and Kume, S. (2013). Secreted cerberus1 as a marker for quantification of definitive endoderm differentiation of the pluripotent stem cells. PLoS One 8, e64291. Kajiwara, M., Aoi, T., Okita, K., Takahashi, R., Inoue, H., Takayama, N., Endo, H., Eto, K., Toguchida, J., Uemoto, S., et al. (2012). Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proc Natl Acad Sci U S A 109, 12538-12543. Loh, K.M., Ang, L.T., Zhang, J., Kumar, V., Ang, J., Auyeong, J.Q., Lee, K.L., Choo, S.H., Lim, C.Y., Nichane, M., et al. (2014). Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell stem cell 14, 237-252. Si-Tayeb, K., Noto, F.K., Nagaoka, M., Li, J., Battle, M.A., Duris, C., North, P.E., Dalton, S., and Duncan, S.A. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297-305. Takebe, T., Zhang, RR, Koike, H., Kimura, M., Yoshizawa, E., Enomura, M., Koike, N., Sekine, K., and Taniguchi, H. (2014). Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 9, 396-409.

[0083] [Example 2] Regarding the induction of tHE differentiation from human iPSCs, the same differentiation method as in Example 1 (human iPSC culture and tHE differentiation) was performed, The following points I changed it. In the first stage, human iPSCs were seeded on iMatrix-511 coated dishes with 10 μM ROCK inhibitor Y-27632 (Wako, catalog number 253-00513), 1% B27 was added, and 100 ng / ml human activin A (provided by Ajinomoto Co., Inc.) and 50 ng / ml Wnt3a (R&D Systems) RPMI-1640 containing [the specified substance] was used as the culture medium for 6 days. Replacing the above 50 ng / ml Wnt3a (R&D Systems) with the addition of 2 μM CHIR99021 for 3 days reduces costs, makes the mixture xeno-free, and complies with bio-based raw material standards (Figures 13, 14). A schematic diagram of the protocol using CHIR99021 as a substitute for Wnt3a is shown in Figure 13A. After testing the conditions, we selected a condition in which CHIR99021, 2 μM was added for 3 days out of the 6 days of DE differentiation induction. Figure 13B shows the cell morphology at each differentiation stage using the conventional method with Wnt3a and under conditions of adding CHIR99021, 2 μM for 3 days. No morphological differences were observed between the two methods using Wnt3a. Figure 13C shows the analysis of the CXCR4 positivity rate, a DE marker, at the DE stage using flow cytometry with the conventional method using Wnt3a and with CHIR99021, 2 μM added for 3 days. The CXCR4 positivity rate is comparable when using Wnt3a. Figure 13D shows the expression analysis of each differentiation marker by quantitative PCR (qPCR). The expression levels of the markers at each stage in CHIR d3 are equivalent to those obtained using Wnt3a. Figure 13E shows the ELISA analysis of secreted albumin levels at the MH stage. In multiple iPSC clones, ALB secretion levels at CHIR d3 tended to be equivalent to or higher than those observed when using Wnt3a. No morphological differences were observed in the cell morphology of MH (Figure 14A) and LB (Figure 14B) compared to when Wnt3a was used. Figure 14C shows the marker expression analysis in MH and LB cells by quantitative PCR (qPCR). No significant differences were observed between Wnt3a and CHIR cells in marker expression levels and ALB secretion levels. No increased expression of marker genes from other cell lineages, such as intestinal markers, was observed.

[0084] [Example 3] For EC expansion culture, the same differentiation method as in Example 1 for the human iPSC-EC and STM differentiation regions was performed, The following points I changed it. For EC differentiation, human iPSCs were dissociated using Accutase and plated onto Laminin 511 E8 fragments (iMatrix-511®, provided by Nippi, Inc.). The culture medium was replaced with a priming medium containing a Rock inhibitor. The priming medium consists of B27 medium (a 1:1 mixture of DMEM and F12 containing 1% Glutamax and 1% B27 (all from Life Technologies)) containing 8 μM CHIR99021 (Tocris Bioscience) and 25 ng / ml BMP4 (R&D Systems). The following day, replace the priming medium with one that does not contain Rock inhibitors. Three days later, the priming medium was replaced with EC induction medium. The EC induction medium consisted of StemPro-34 SFM medium (Life Technologies) supplemented with 200 ng / ml VEGF (Life Technologies) and 2 μM forskolin (Sigma-Aldrich). The induction medium was refreshed daily. On day 7 of differentiation, the ECs were dissociated with 0.05% trypsin and subjected to FACS analysis. iPSC-derived ECs should exhibit typical endothelial morphology with junctional localization of CD144 and CD31. The ECs were then analyzed. Laminin 511 E8 fragment (iMatrix-511®, provided by Nippi, Inc.) contains a Rock inhibitor. Cells were replated in EC expansion medium at a density of 50,000 cells per cm². The EC expansion medium consisted of StemPro-34SFM supplemented with 50 ng / ml of VEGF-A. The following day, the culture medium was changed to Miracell (registered trademark) EC (Takara Bio), and this was changed every other day. This makes it possible to obtain more stable and highly CD31 / CD144 co-positive cells (Figures 15, 16). A modified differentiation induction protocol for iPSC-derived vascular endothelial cells (iPSC-ECs) is shown in Figure 15. A schematic diagram of the differentiation induction protocol is shown in Figure 16A. Figure 16B shows the cell morphology obtained using the conventional method and the revised method. No morphological differences are observed. Figure 16C shows the analysis of EC marker positivity rates using flow cytometry with the conventional and revised methods. Figure 16D summarizes the flow cytometry analysis in Figure 16C. The revised method shows a more stable and higher CD31 / CD144 positivity rate compared to the conventional method. Figure 16E shows the expression analysis of each differentiation marker by quantitative PCR (qPCR). EC marker expression levels remained stable even after passage. Figure 16F shows cell proliferation after each passage. The revised method shows higher proliferative capacity after passage compared to the conventional method. Based on the above, even if the positive rate was not stable at EC P0, stable EC could be produced by resowing. Propagation after EC completion was successful. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0085] This invention can be used in regenerative medicine and drug discovery screening.

Claims

1. A method for producing mesenchymal cells, comprising culturing pluripotent stem cells in the presence of CHIR99021 and BMP4, then culturing them in the presence of PDGFBB and activin A, and further culturing them in the presence of FGF2.

2. The method according to claim 1, wherein the mesenchymal cells are CD166 positive.

3. The method according to claim 1 or 2, wherein pluripotent stem cells are cultured in the presence of Y-27632 before being cultured in the presence of CHIR99021 and BMP4.

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