WO2013047639A1 - 組織及び臓器の作製方法 - Google Patents
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Definitions
- the present invention relates to a method for producing organ buds, tissues, and organs from undifferentiated cells such as induced pluripotent stem cells (iPS cells).
- undifferentiated cells such as induced pluripotent stem cells (iPS cells).
- the differentiation induction method using pluripotent stem cells attempts to induce cell differentiation by combining various differentiation factors such as addition of humoral factors and gene transfer.
- these conventional methods have not been able to induce terminally differentiated functional cells, and have not sufficiently achieved induction of early differentiation into tissue stem cells, which are the precursor populations.
- the cells that make up tissues and organs are not only functional cells but also multiple cell types such as vascular cells and mesenchymal cells, and they must have an ordered spatial arrangement. Therefore, an organizational structure is established by a cooperative interaction.
- a carrier such as a scaffold material as a reconstruction technique for human tissues / organs
- the engraftment rate of the seeded functional cells is extremely low, and long-term culture is difficult.
- the function of the tissue and organs made is extremely immature.
- the present invention aims to solve the above problems and provide means for reconstructing tissues and organs having a mature function.
- organ cells In the process of physiologic organ development, organ cells have a close cell-cell interaction with vascular endothelial cells and undifferentiated mesenchymal cells, so that organ formation with autonomous tissue structure and cell differentiation proceeds. To do.
- the early process of organ development is artificially reproduced to induce early differentiation through the interaction of multiple cell lineages, thereby inducing the tissue formation ability of the organ cells that have undergone initial differentiation.
- organ bud organ bud
- it is intended to artificially produce an organ bud (organ bud) that is the source of a tissue / organ in a test tube.
- the organ buds induced in the culture system are transplanted into the living body to start blood flow, thereby producing tissues / organs composed of terminally differentiated functional cells and vascular systems.
- organ cells at the optimal differentiation stage obtained from pluripotent stem cells such as iPS cells are co-cultured with vascular endothelial cells and mesenchymal cells. These three different cell components should be cultured at an optimal mixing ratio, and in a special environment supported by extracellular matrix components, a short period of time in a differentiation-inducing medium containing specific trophic and humoral factors
- a three-dimensional organ bud having a microvascular structure can be induced in a test tube.
- transplanting organ buds induced in the culture system into the living body promotes vascularization and initiates blood flow, thereby creating tissues and organs with a highly ordered tissue structure equivalent to adult tissues. It becomes possible to do.
- vascular endothelial cells and mesenchymal cells are caused by the presence of factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, and both vascular endothelial cells and mesenchymal cells. It can be replaced with secreted factors and other substances.
- the gist of the present invention is as follows.
- Vascular endothelial cells, mesenchymal cells, factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, factors secreted by the presence of both vascular endothelial cells and mesenchymal cells A method for producing an organ bud, comprising culturing an organ cell together with at least one cell and / or factor selected from the group consisting of: (2) The method for producing an organ bud according to (1), wherein the organ cell is a differentiated cell. (3) The method for producing an organ bud according to (1), wherein the organ cell is an undifferentiated cell.
- Organ cells can be differentiated into endoderm organ cells or endoderm organ cells, mesodermal organ cells or mesodermal organ cells, or ectodermal organ cells or ectoderm
- the method for producing an organ bud according to (4), wherein the organ cells are cells that can differentiate into endoderm organ cells or endoderm organ cells.
- Vascular endothelial cells, mesenchymal cells, factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, factors secreted by the presence of both vascular endothelial cells and mesenchymal cells The method for producing an organ bud according to any one of (1) to (8), wherein an organ cell is cultured in a vascular endothelial cell culture medium together with at least one cell and / or factor selected from the group consisting of: (10) Vascular endothelial cells, mesenchymal cells, factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, factors secreted by the presence of both vascular endothelial cells and mesenchymal cells.
- a method for transplanting an organ bud comprising transplanting an organ bud produced by the method according to any one of (1) to (14) to a human or non-human animal.
- Regeneration or function of tissue or organ including transplanting an organ bud produced by the method according to any one of (1) to (14) to a human or non-human animal and differentiating it into a tissue or organ Recovery method.
- a method for producing a non-human chimeric animal comprising transplanting an organ bud produced by the method according to any one of (1) to (14) into a non-human animal and allowing it to differentiate into a tissue or organ.
- An organ bud produced by the method according to any one of (1) to (14), a tissue and an organ produced by the method according to (15), and a non-organ produced by the method according to (19) A method for evaluating a drug using at least one selected from the group consisting of human chimeric animals.
- tissue stem cells cannot be obtained at all.
- tissue stem cells in conjunction with human hepatic stem cell manipulation technology developed in the past by the present inventors (International Publication Number: WO / 2009/139419), It is expected to become a cell manipulation technique useful for mass production of human hepatocytes.
- a three-dimensional human tissue structure having a vascular system can be reconstructed by artificially reproducing a plurality of cell-cell interactions occurring in organ development. Therefore, it is expected to become a basic technology for producing a human tissue / organ having a blood flow in which a vascular system is appropriately arranged, which has never been achieved by the conventional techniques.
- the figure which shows the autonomous organization of the liver endoderm cell derived from a human iPS cell The lower left figure shows human liver endoderm cells.
- the upper left figure shows a three-dimensional structure (hepatic bud) formed by the co-culture of liver endoderm cells, vascular endothelial cells and undifferentiated mesenchymal cells (4 days after the start of culture).
- the upper right figure is a fluorescence micrograph of the three-dimensional structure.
- Vascular endothelial cells (HUVEC) are labeled with EGFP
- undifferentiated mesenchymal cells hMSC
- iPS cells are not labeled.
- Hep.End. HUVEC MSC represents cells constituting the organ bud
- “undiff iPS” and “iPS” represent iPS cells
- “Def End” represents endoderm cells induced by activin.
- Hep End represents liver endoderm cells induced by BMP4 and FGF2.
- ⁇ IH-like '' represents immature hepatocyte-like cells described in K.Si-Taiyeb, et al. Hepatology, 51 (1): 297- 305 (2010)
- ⁇ MH-like '' represents K.Si -Taiyeb, et al. Hepatology, 51 (1): Represents mature hepatocyte-like cells described in 297- 305 (2010).
- Hep.End. HUVEC ⁇ MSC represents cells constituting the organ bud
- iPS represents iPS cells
- Def End represents endoderm cells induced by activin
- Hep End "Represents liver endoderm cells induced by BMP4 and FGF2.
- ⁇ IH-like '' represents immature hepatocyte-like cells described in K.Si-Taiyeb, et al. Hepatology, 51 (1): 297- 305 (2010)
- ⁇ MH-like '' represents K.Si -Taiyeb, et al.
- the left figure shows the co-cultured pancreatic ⁇ cells, and the right figure shows the pancreatic ⁇ cells cultured alone.
- FIG. 1 The figure which shows the spheroid formation process of a pancreatic beta cell.
- Vascular endothelial cells (HUVEC) are labeled with GFP, and pancreatic ⁇ -cell fistula (MIN6) is labeled with KO.
- Vascular endothelial cells (HUVEC) are labeled with GFP, and pancreatic ⁇ -cell fistula (MIN6) is labeled with KO.
- hiPSC-LBs human liver buds
- A Schematic representation of the method.
- C Three-dimensional self-assembly of hiPSC-LBs when hiPSC-Heps is co-cultured with HUVECs and hMSCs. Endothelial germination was observed in hiPSC-LBs. Green, EGFP-labeled HUVEC; red, KOFP (Kusabira Orange Fluorescent Protein) labeled hMSC.
- the length of the bar is 100 ⁇ m.
- D Formation of hiPSC-LBs was not observed in the culture system not containing hMSCs.
- the length of the bar is 1mm.
- E By time-lapse observation using a confocal microscope, autonomous organization of hiPSC-Heps labeled with Cell Tracker Red CMTMR (Molecular Probes) was confirmed. The image is a projection of the image with the maximum intensity in the confocal Z direction.
- the ratio of each cell trait is as follows: hepatoblasts, AFP positive / CK8.18 positive; proliferating cells, (PCNA positive or BrdU positive) /CK8.18 positive; endothelium Cells, (CD31 positive or Flk1 positive) / DAPI positive; mesenchymal cells, Desmin positive / DAPI positive.
- the ratio of each cell type in hiPSC-LBs is almost similar to that of E10.5 mouse LBs (mLBs).
- mLBs E10.5 mouse LBs
- (A) hiPSC-LBs were transplanted into the skull window of NOD / SCID mice. As a result of observation of the transplanted hiPSC-LBs with the naked eye, blood inflow into human blood vessels was observed about 48 hours after the transplantation. The dot area indicates transplanted hiPSC-LBs. The length of the bar is 1mm.
- the dotted line indicates the end of the graft.
- the bar length is 500 ⁇ m.
- D The connection between HUVECs (green, GFP) and host blood vessels (blue, Alexa647-labeled mouse-specific CD31, intravenous administration) was directly visualized. The bar length is 250 ⁇ m.
- E, f The localization of hMSCs or hiPSC-derived cells in the formed liver tissue was observed on the 15th day of transplantation. The bar length is 100 (e) and 250 (f) ⁇ m.
- E Venn diagram showing metabolic features of hiPSC-LB grafts measured by CE-TOFMS (FIG. 31). Of the metabolites found in hiPSC-LB grafts, 78% matched that of adult liver. The right panel shows the major metabolites detected in both hiPSC-LB grafts and healthy human adult liver but not in the original hiPSCs.
- Identification of humoral factors derived from co-cultures with stromal cells (a) Gene ontology (GO) analysis of 5000 genes up-regulated in co-cultures with endothelial and mesenchymal cells compared to undifferentiated hiPS cells. Bars represent the significance (P value) of a specific GO category within GO: 0008083 (growth factor activity) for biological processes.
- the FGF signal pathway red
- the BMP signal pathway blue
- TAT Tyrosine aminotransferase
- G6PC glucose-6-phosphatase
- TDO 2 tryptophan 2,3-dioxygenase
- GLUT2 glucose transporter 2
- GYS2 glycogen synthase 2
- APOL6 apolipoprotein L
- KNG1 kininogen 1
- CFB complement factor B
- CFI complement factor I
- PCK1 phosphoenolpyruvate carboxykinase
- LDHD lactate dehydrogenase D
- CP ceruloplasmin
- ACTB actin beta. Production of mouse liver tissue from mouse liver bud-derived cells.
- Species-specific CD31 immunostaining of explanted hiPSC-LB grafts shows a direct link between human and mouse blood vessels in hiPSC-derived liver tissue. The length of the bar is 100 ⁇ m.
- the length of the bar is 100 ⁇ m.
- the bar length is 50 ⁇ m.
- Liver tissue derived from hFLC-LB grafts express hepatocyte specific antigen (HSA) but not AFP (left panel). Immunostaining for human CD31 and ⁇ -smooth muscle actin (SMA) shows the formation of major blood vessels inside liver tissue (right panel). The bar length is 100 ⁇ m ⁇ .
- HSA hepatocyte specific antigen
- SMA ⁇ -smooth muscle actin
- organ cells are divided into vascular endothelial cells, mesenchymal cells, factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, vascular endothelial cells and mesenchymal cells.
- the organ cells are cultured with at least one cell and / or factor selected from the group consisting of factors secreted by the presence of both.
- an “organ bud” is a structure that can be differentiated into an organ upon maturation, and includes three types of organ cells, vascular endothelial cells, and undifferentiated mesenchymal cells or cells differentiated therefrom.
- a structure containing cells Whether a structure is an organ bud is examined, for example, by examining whether the structure can be transplanted into a living body and differentiated into the target organ (if it has differentiated into the target organ, it is determined to be an organ bud) And / or whether or not the structure contains all the three types of cells described above (if all three types of cells are included, it can be determined that the bud is an organ bud).
- Organ buds may be organ buds that differentiate into organs such as kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, spinal cord, etc., but differentiate into liver Organ buds that differentiate into endoderm organs such as organ buds (liver buds), organ buds that differentiate into pancreas (pancreatic buds), and organ buds that differentiate into the intestinal tract are preferred. Whether or not a certain structure is an organ bud that differentiates into an endoderm organ can be confirmed by examining the expression of a marker protein (if one or more of the marker proteins described below are expressed, the organ bud Can be determined.) For example, in liver buds, HHEX, SOX2, HNF4A, AFP, ALB, etc.
- liverlivebud liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, intestinal organoid (K. Matsumoto, et al .Science.19; 294 (5542): 559-63. (2001)) and the like are included in the organ buds of the present invention.
- organ cell refers to a functional cell constituting an organ or an undifferentiated cell that differentiates into a functional cell.
- the “undifferentiated organ cell” may be, for example, a cell that can differentiate into organs such as kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, and spinal cord.
- Brain spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, cells that can differentiate into ectodermal organs such as lens, kidney, ureter, heart, blood, gonad, adrenal cortex, muscle
- mesodermal organs such as skeleton, dermis, connective tissue, mesothelium, cells that can differentiate into endoderm organs such as liver, pancreas, intestine, lung, thyroid, parathyroid, urinary tract, etc.
- endoderm organs such as liver, pancreas, intestine, lung, thyroid, parathyroid, urinary tract, etc.
- a cell is a cell that can differentiate into an ectodermal organ, mesodermal organ, or endodermal organ can be confirmed by examining the expression of a marker protein (either one or more of the marker proteins are expressed). If so, it can be determined that the cell can differentiate into an endoderm organ.
- a marker protein either one or more of the marker proteins are expressed.
- HHEX, SOX2, HNF4A, AFP, ALB, etc. are markers
- cells that can differentiate into the pancreas cells that can differentiate into the intestinal tract, such as PDX1, SOX17, SOX9, etc. Then, CDX2, SOX9, etc.
- markers are markers, and in cells that can differentiate into kidney, SIX2, SALL1, in cells that can differentiate into heart, NKX2-5 MYH6, ACTN2, MYL7, HPPA, in cells that can differentiate into blood, For C-KIT, SCA1, TER119, HOXB4, and cells that can differentiate into the brain and spinal cord, HNK1, AP2, NESTIN, etc. are markers.
- hepatoblast hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal s , 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), , Et al. EMBO J .; 25 (21): 5214-5228. (2006), J. Zhang, et al.
- Undifferentiated organ cells can be prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to a known method.
- pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)
- iPS cells induced pluripotent stem cells
- ES cells embryonic stem cells
- organ cells that can differentiate into the liver are K. Si-Taiyeb, et al. Hepatology, 51 (1): 297- 305 (2010), T. Touboul, et al. Hepatology. 51 (5): 1754- 65. (2010), organ cells that can differentiate into pancreas can be made according to D.
- Organ cells that can differentiate into the intestinal tract are J.JCai, et al. J Mol Cell Biol.; 2 (1): 50-60 (2010), R. Spence, et al. Nature.; 470 (7332): 105-9. (2011), organ cells that can be differentiated into the heart can be prepared according to J. Zhang, et al. Circulation Research .; 104: e30-e41 (2009) In addition, cells that can differentiate into the spinal cord can be prepared according to G. Lee, et al.
- “Different organ cells” include pancreatic endocrine cells, pancreatic pancreatic duct epithelial cells, liver hepatocytes, intestinal epithelial cells, kidney tubular epithelial cells, kidney glomerular epithelial cells, heart cardiomyocytes, blood Lymphocytes and granulocytes, red blood cells, brain neurons and glial cells, spinal cord neurons and Schwann cells.
- Organ cells are mainly derived from humans, but organ cells derived from animals other than humans, such as mice, rats, dogs, pigs, monkeys, etc. may be used.
- vascular endothelial cell refers to a cell constituting the vascular endothelium or a cell that can differentiate into such a cell. Whether a cell is a vascular endothelial cell can be confirmed by examining whether a marker protein, for example, TIE2, VEGFR-1, VEGFR-2, VEGFR-3, or CD41 is expressed (any of the above-mentioned marker proteins). If one or more of them are expressed, it can be judged as a vascular endothelial cell).
- vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144.
- endothelial cells As used by those skilled in the art, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, hemangioblast (HJ. Joo, et al. Blood. 25; 4 18: 8: 8; -104. (2011)) is included in the vascular endothelial cells in the present invention.
- vascular endothelial cells are mainly derived from humans, vascular endothelial cells derived from animals other than humans such as mice, rats, dogs, pigs, monkeys, etc. may be used.
- the term “mesenchymal cell” refers to a connective tissue cell that is present in connective tissue mainly derived from mesoderm and forms a support structure for cells that function in the tissue. Is a concept that includes cells that have not yet differentiated into mesenchymal cells.
- the mesenchymal cells used in the present invention may be differentiated or undifferentiated. Whether a cell is an undifferentiated mesenchymal cell is confirmed by examining whether a marker protein such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, or Nestin is expressed (If any one or more of the marker proteins are expressed, it can be determined that they are undifferentiated mesenchymal cells).
- mesenchymal cell that does not express any of the markers in the preceding paragraph can be determined as a differentiated mesenchymal cell.
- mesenchymal stem cells mesenchymal progenitor cells, mesenchymal cells (R. Peters, et al. PLoS One. 30; 5 (12): e15689. (2010)) Included in mesenchymal cells.
- mesenchymal cells are mainly derived from humans, undifferentiated mesenchymal cells derived from animals other than humans such as mice, rats, dogs, pigs, monkeys and the like may be used.
- vascular endothelial cells and mesenchymal cells include factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, and both vascular endothelial cells and mesenchymal cells. It can be replaced by a substance such as a secreted factor.
- substances such as factors secreted from vascular endothelial cells, factors secreted from mesenchymal cells, and factors secreted by the presence of both vascular endothelial cells and mesenchymal cells include FGF2, FGF5 , BMF4, BMP6, CTGF and the like can be exemplified, but are not limited thereto.
- 10 to 100 ng / ml per 1 ⁇ 10 ⁇ 6 cells is appropriate for FGF2, and preferably about 20 ng / ml, and 10 to 10 ng / ml for BMF4. 100 ng / ml is appropriate, and about 20 ng / ml is preferable.
- the medium used for the culture may be any medium as long as an organ bud is formed, but a medium for culturing vascular endothelial cells, a medium for organ cell culture, and a mixture of the two media. Etc. are preferably used. Any medium can be used for vascular endothelial cell culture, but hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS Antibiotics (for example, gentamicin, amphotericin B, etc.), Heparin, L-Glutamine, Phenolred, and BBE are preferably used.
- EGM-2 BulletKit (Lonza), EGM BulletKit (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen)
- TOYOBO Human microvascular endothelial cell growth medium
- Any medium for organ cell culture may be used.
- the organ cells are hepatocytes, a medium containing at least one of ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000 is used. It is preferred to use.
- HCM BulletKit (Lonza) minus hEGF (recombinant human epidermal growth factor), RPMI1640 (Sigma-Aldrich) 1% B27 Supplements (GIBCO) And 10 ng / mL HHGF (manufactured by Sigma-Aldrich)
- a mixture of GM BulletKit (Lonza) and HCM BulletKit (Lonza) excluding hEGF (recombinant human epidermal growth factor) in a 1: 1 mixture Addition of Oncostatin M and HGF has been found to be effective in liver bud maturation.
- Organ cells are preferably seeded on a gel and cultured.
- the gel to be used is not particularly limited, but BD-Matrigel (manufactured by BD Pharmingen) or the like can be used.
- the temperature during the cultivation is not particularly limited, but is preferably 30 to 40 ° C, more preferably 37 ° C.
- the culture period is not particularly limited, but is preferably 3 to 10 days, and more preferably 6 days.
- a tissue or organ can be produced by transplanting the organ bud produced as described above to a non-human animal and allowing it to mature in the non-human animal.
- non-human animals to be used include mice, rabbits, pigs, dogs and monkeys.
- the non-human animal used is preferably an immunodeficient animal in order to avoid immune rejection.
- the present invention also provides a method for transplanting organ buds, including transplanting the organ buds produced by the above method to a human or non-human animal.
- the transplantation site of the organ bud may be any site as long as transplantation is possible, but examples thereof include intracranial, mesentery, liver, spleen, kidney, subrenal capsule, and portal vein.
- 5mm large organ buds may be transplanted, and when transplanted on the portal vein, about 1 to 20 5mm large organ buds prepared in vitro may be transplanted.
- the tissues and organs produced as described above can be used for drug discovery screening and regenerative medicine.
- the present invention provides a tissue or organ regeneration or function recovery method comprising transplanting an organ bud produced by the above method into a human or non-human animal and differentiating it into a tissue or organ.
- non-human animals include mice, rabbits, pigs, dogs and monkeys.
- the present invention also provides a method for producing a non-human chimeric animal, which comprises transplanting an organ bud produced by the above method into a non-human animal and allowing it to differentiate into a tissue or organ.
- a non-human animal for example, a mouse
- transplanted with an organ bud can mimic the physiological function of an organ cell-derived species (for example, a human) used for producing the organ bud.
- organ cell-derived species for example, a human
- the present invention also provides a method for evaluating a drug using at least one selected from the group consisting of organ buds, tissues and organs produced by the above method, and non-human chimeric animals.
- drug evaluation include prediction of drug metabolism profile of drug candidate compounds, drug efficacy evaluation, toxicity evaluation, and drug interaction evaluation.
- tissue stem cells from tissues and organs prepared by the method of the present invention, and the present invention can be applied to cell manipulation techniques for mass production of human tissue cells and organ cells.
- ⁇ experimental method ⁇ (1) Preparation of human liver endoderm cells Human iPS cells (human skin-derived TkDA3 hiPSC clone (assigned from Mr. Koji Eto and Mr. Hiromitsu Nakauchi)) were cultured in serum-free medium with CXCR4 and E -Cadherin-positive endoderm cells were induced. The obtained endoderm cells were added with BMP4 and FGF2 and cultured for 2 days to obtain a CXCR4-negative HNF4 ⁇ -positive liver endoderm population. The expression of CXCR4 and HNF4 ⁇ was confirmed by immunostaining and gene expression analysis as described in Hepatology, 51 (1), 297-305, 2010.
- vascular endothelial cells human umbilical cord blood-derived venous endothelial cells
- undifferentiated mesenchymal cells human mesenchymal stem cells
- a human three-dimensional structure (hepatic bud).
- the formation process and the formed structure were observed with a confocal microscope, and kinetic / static analysis such as cell morphology and localization was performed.
- gene expression analysis of the formed human three-dimensional structure was performed.
- (3) Preparation of human liver tissue The formed human three-dimensional structure was transplanted into an immunodeficient mouse (NOD / SCID mouse (Sankyo Lab. Co., Tsukuba, Japan)) in vivo. Live observations with the naked eye and confocal microscope were performed to confirm the engraftment and proliferation of human cells and to analyze the vascular maturation process after transplantation. A sample (2 weeks) at an early stage of transplantation was collected and analyzed histologically.
- Example 2 Preparation of organs using differentiated organ cells
- Pancreatic ⁇ cell line (MIN6), vascular endothelial cells (human umbilical cord blood-derived venous endothelial cells) and undifferentiated mesenchymal cells (human mesenchymal stem cells) were mixed at a ratio of 5: 5-10: 2, respectively.
- Co-culture was performed.
- the pancreatic ⁇ cell line (KO) and vascular endothelial cell (EGFP) were each fluorescently labeled.
- the cell suspension was seeded on a culture dish on which a stock solution or a 2-fold diluted Matrigel (BD pharmingen) had been immobilized.
- BD pharmingen 2-fold diluted Matrigel
- endothelial cell culture medium kit-2 EGM-2 BulletKit (product code CC-3162: Lonza) was used.
- Short-term culture (3-10 days) was performed to produce a three-dimensional structure.
- the formation process and the formed structure were observed with a confocal microscope, and dynamic / static analysis such as cell morphology and localization was performed.
- the formed three-dimensional structure was transplanted into an immunodeficient mouse (NOD / SCID mouse (Sankyo Lab. Co., Tsukuba, Japan)) in vivo. Live observations with the naked eye and confocal microscope were performed to confirm cell engraftment and proliferation, and to analyze the maturation process after transplantation. Transplanted samples (4 weeks) were collected and histologically analyzed.
- FIG. 11 Histological analysis of the transplanted sample confirmed the formation of insulin-positive island structures.
- the formed structure had a complex blood vessel structure inside, and had a structure similar to that of normal mouse islets (FIG. 12).
- Example 3 Creation of functional vascularized human liver by transplanting organ buds derived from induced pluripotent stem cells Donor organs for treating end-stage organ failure are remarkably insufficient, and patient-derived induced pluripotent stem cells The need to create organs using (hiPSCs) 1, 2 is increasing. Although many papers have reported functional cell differentiation 3-7 , there are no successful examples of organs with a three-dimensional vascular network such as the liver. We succeeded in creating a functional human liver tissue with vascular structure by transplanting in vitro prepared hiPSC-derived liver buds (hiPSC-LBs).
- hiPSC-LBs When endothelial cells and mesenchymal cells 8 were added to promote organogenesis, iPS cell-derived hepatic endoderm cells self-assembled into three-dimensional hiPSC-LBs. As a result of immunostaining and gene expression analysis, similarity was observed between hiPSC-LBs grown in vitro and liver buds in vivo. Within 48 hours, the human vascular network in the hiPSC-LBs graft connected with the host vessel and blood perfusion started. The formation of functional vasculature revealed that hiPSC-LBs matured into tissues similar to adult liver. Additionally, transplanted tissue from hiPSC with high metabolic capacity, without requiring replacement 9, 10 of the recipient liver was exhibited liver-specific functions such as human protein production and human-specific drug metabolism.
- transplantation of hiPSC-LBs into the mesentery improved the survival rate of the drug-induced lethal liver failure model.
- this is the first example of creating a functional human organ from pluripotent stem cells.
- organs with complex structures such as livers from pluripotent stem cells, but there has been no successful example. This is because it has been considered to be difficult to reproduce the complex interactions among cells and tissues that occurs organ formation process by in vitro 2, 11. We addressed this issue by focusing on the first process in organogenesis, namely the cell-cell interaction during organ bud development.
- liver bud LB
- hepatic endoderm cells with endothelial and mesenchymal cells ( Figure 13a).
- hiPSC-Hep human iPSCs were first induced to differentiate into hepatic endoderm cells (hiPSC-Hep) by the stepwise addition of inducers.
- HNF4A hepatic marker
- hiPSC-Hep cells were co-cultured with stromal cell populations to reproduce early liver formation. Endothelial and mesenchymal cells were used unless otherwise specified human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Notably, despite being cultured in 2D culture conditions, hiPSC-Hep cells grown on specific matrix proteins are 3D cells that are large enough to be confirmed by the naked eye due to their intrinsic organization ability. Clusters formed within 24 hours after culture (FIGS. 13c, d, e). The clusters presumed to be hiPSC-Hep-derived liver buds (hiPSC-LBs) had moderate mechanical strength and could withstand subsequent transplantation operations.
- HAVECs human umbilical vein endothelial cells
- hMSCs human mesenchymal stem cells
- hiPSC-LBs vascular network development with precise endothelial cell sprouting in hiPSC-LBs was visualized using cells labeled with fluorescent proteins (FIG. 13c). Moreover, the formation of hiPSC-LB was kept uniform by using umbilical cord-derived MSCs and HUVECs obtained from the same donor (FIG. 18a, right). As a result of qPCR analysis, cells in hiPSC-LBs transcribe high levels of alpha fetoprotein (AFP), retinol-binding protein (RBP4), transthyretin (TTR) and albumin (ALB) 5 that are early differentiation markers of the liver. (Figure 18d).
- AFP alpha fetoprotein
- RBP4 retinol-binding protein
- TTR transthyretin
- ARB albumin
- stromal cell-dependent liver maturation of hiPSC-Hep cells was maintained to a certain extent even in the co-culture system using transwells (FIG. 13f).
- Microarray and qPCR analyzes were performed to investigate the mediators that induce liver maturation.
- BMP4 and FGF2 were significantly elevated in the co-culture system with endothelial and mesenchymal cells (FIG. 19).
- Noggin and SU-5402 inhibitors of BMP- and FGF-specific signaling, inhibited the differentiation promoting effect by co-culture with stromal cells.
- BMP4 and FGF2 were added to the hiPSC-Hep medium, the same liver differentiation-inducing effect was observed (FIGS. 13g and h).
- hiPSC-LB was very similar to LBs (mLBs) in E10.5 mice, unlike the developed liver as in late pregnancy and postnatal mice (FIG. 14a).
- hiPSC-LB and E10.5 mLBs like mesenchymal and endothelial progenitor cells , are expressed by proliferating hepatoblasts that express AFP 14, 15 and have the ability to differentiate.
- hiPSC-LB and E10.5 mLBs more than 90% of the estimated hepatocytes expressed AFP, but not in E15.5 and E17.5 mLBs (FIG. 14b).
- the hepatocytes in hiPSC-LB had the same growth ability as E10.5 mLBs (FIG. 14c).
- hiPSC-LB was composed of mesenchymal and endothelial progenitor cells in the same proportion as E10.5 mLBs (FIGS. 14d and e).
- E10.5 mLBs corresponds to the human fetal liver at 3-4 weeks of gestation (3-4GW) 16, 17 , to be consistent with fetuses at 22-40 GW of pregnancy.
- 83 gene expression patterns in hiPSC-LB were found to be at appropriate differentiation stages (FIGS. 14f and 20).
- LB maturation 18 In order to examine whether hiPSC-LBs reconstructs a fully functional liver tissue, we transplanted it to a cranial observation window where repeated imaging 19 can be performed over a long period of time. First, it was confirmed that this model can reproduce the maturation process of LB by using mouse liver bud-derived cells (mLB) for transplantation experiments (Supplementary discussion, FIGS. 21 and 22). Furthermore, fetal hepatocytes similarly having the ability to form LB (hFLC-LBs) were used as a control during the culture (FIG. 18a).
- mLB mouse liver bud-derived cells
- LB grafts were analyzed histologically after 60 days. Similar to hFLC-LB, the hiPSC-LB graft has a hepatocyte cord-like structure characteristic of adult liver (Fig. 16a and Fig. 28a), and this structure is tight junction protein zona occlusion 1 ( ZO-1), ALB, cells expressing cytokeratins 8 and 18 (CK8.18) (FIG. 16b), and a basement membrane containing collagen IV normally found along the entire length of the sinusoidal vessel 22 (FIG. 29a) ). Furthermore, it was shown that asialoglycoprotein receptor 1 (ASGR1), which is a mature hepatocyte marker, was expressed, and the immature hepatocyte marker AFP was not expressed (FIGS.
- ASGR1 asialoglycoprotein receptor 1
- ketoprofen 23 or debrisoquine 24, 25, which are known to be metabolized in different ways in humans and mice were administered to mice.
- human-specific metabolite formation was observed in urine and serum samples of mice transplanted with hiPSC-LB (FIGS. 16f, g, h and supplemental considerations). From this, it was shown that the human drug metabolism profile can be predicted by imitating human physiological functions in vivo using this mouse.
- the conventional method required transplantation of high-quality adult hepatocytes using mice 26 and 27 with severely damaged liver as the host. Is significant in that it can be accurately verified.
- HUVECs and hMSCs were maintained at 37 degrees in the presence of 5% CO 2 in a humidified incubator using Endothelial Growth Medium (EGM) (Lonza) or MSC Growth Medium (Lonza) .
- HCM hepatocyte culture medium
- Oncostatin M 10 ng / ml, R & D System, Minneapolis, MN
- HGF including 20 ng / ml, PromoKine
- SingleQuots SingleQuots (Lonza) with 1x10 6 hiPSC-derived hepatocytes, 0.8-1x10 6 HUVECs, and 2x10 5 hMSCs Turbid and coated on Matrigel (BD Biosciences, Bedford, MA, USA).
- the generated hiPSC-LBs were peeled off and collected, and then transplanted into the skull window 19 of an immunodeficient mouse.
- TkDA3 hiPSC clones were a kind gift from Koji Eto and Hiromitsu Nakauchi.
- Undifferentiated hiPSC cells were grown on mouse embryo fibroblasts as feeder cells.
- hiPSCs were spread on a matrigel-coated dish, transferred to RPMI1640 medium containing 1% B27 (without insulin) and (100 ng / ml), and cultured for 5-6 days .
- the endoderm cells derived from hiPSC were treated with PRMI1640 containing hbFGF (10 ng / ml), hBMP4 (20 ng / ml) and 1% B27 for further 3-4 days to specify the liver.
- HGF Human recombinant HGF (50 ng / ml) and EGF (20 ng / ml) (Sigma) were added prior to culture.
- Retroviral transduction For live imaging, 19 cells were infected with retroviruses expressing EGFP or kusabira orange (KOFP) as described in the literature. That is, retroviral vectors pGCDNsam IRES-EGFP or KOFP were transfected into 293gp and 293gpg packaging cells (provided by Masafumi Onodera) and viral particle production was induced using a tetracycline-inducible system. The culture supernatant of cells infected with retrovirus was passed through a 0.45- ⁇ m filter (Whatman, GE Healthcare, Japan) and immediately used for infection. KOFP showed a major absorption maximum wavelength at 548 nm with a small shoulder at 515 nm and a bright orange fluorescence with a peak at 561 nm 32 .
- Transplantation LB generated in vitro was peeled off and collected, and then transplanted into a cranial window applied to a severely immunodeficient NOD / SCID mouse (Sankyo Lab. Co., Tsukuba, Japan).
- the in vivo progress of the transplanted cells was observed by in vivo imaging using a fluorescence microscope (model BZ-9000; Keyence, Osaka, Japan) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany). Survival curves were analyzed using TK-NOG mice (weight ⁇ 20-30 g) (provider Central Institute for Experimental Animals, Kanagawa, Japan) 29 .
- mice After transplanting dozens of hiPSC-LBs into the mesentery, on the 7th and 10th day, mice were administered non-toxic ganciclovir (GCV, 50mg / kg, intraperitoneal injection) to human and mouse tissues. The introduced parenchymal cells of the liver were erased in a tissue-specific manner. Mice were bred and maintained in accordance with our institutional guidelines for animal experimentation.
- GCV non-toxic ganciclovir
- RNA of human fetal liver (Lot No .: A601605) and adult human liver (Lot No .: B308121) was obtained from Biochain Institute (Hayward, CA, USA).
- cRNA is amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, Calif.) and supplied to the 44K 60-mer oligo microarray (Human Gene Expression 4x44K v2 Microarray Kit; Agilent Technologies). Hybridization according to Hybridized microarray slides were scanned using an Agilent High-Resolution Microarray Scanner. Relative hybridization intensity and background hybridization values were calculated using Feature Extraction Software version 10.7.3.1 (Agilent Technologies). The raw signal intensity and the flag for each probe were calculated from the hybridization intensity and spot information using the flag criteria in GeneSpring 11.5.1 Software according to the procedure recommended by Agilent Technologies.
- the raw signal intensity of the sample was log2 transformed and normalized by a displacement algorithm.
- a heat map was created by GeneSpring. Normalized intensity values were loaded and scaled according to the distance from the median value of each probe.
- 83 genes were selected as liver characteristic genes. This is because these genes continuously increased during liver development in both humans and mice.
- the ELISA blood sample was coagulated in a centrifuge tube at room temperature (about 5 minutes), separated from the side of the tube, and kept at 4 ° C. (melted ice) for 20 minutes.
- the coagulated blood was centrifuged for 10-15 minutes at 400 g, 4 ° C, and the serum fraction was removed, taking care to exclude red blood cells or coagulated material.
- Human ALB and AAT in mouse serum samples were analyzed using Human Albumin ELISA Quantitation Kit (Bethyl Laboratories Inc., Montgomery, TX, USA) and human alpha 1-antitrypsin ELISA Quantitation Kit (GenWay Biotech, Inc., San Die, ) was measured according to the manufacturer's instructions.
- mice were perfused by cardiac puncture using 4% paraformaldehyde (PFA) dissolved in PBS.
- PFA paraformaldehyde
- the cover glass forming the cranial window was removed, and the graft (thickness: about 300 ⁇ m) was removed and placed in 4% PFA dissolved in PBS for 1.5 hours on ice.
- PFA paraformaldehyde
- For immunostaining fixed collagen gels were washed 3 times with PBS (10 min each), blocked with 3% BSA / 0.1% Triton X-100 for 1 hour, and incubated with primary antibody at 4 ° C. overnight. Next, washing was performed 3 times for 10 minutes using PBS / 0.1% -100Triton X-100. Samples were incubated with secondary antibody overnight at 4 ° C.
- TAPI samples were counterstained with DAPI, mounted on glass slides in mounting medium (Vector Laboratories, USA) and covered with coverslips.
- the following primary antibodies were used: mouse anti-human ZO1, mouse anti-human CD31, and rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse collagen IV (Millipore, USA) and desmin (Dako Corporation, Carpinteria, CA). Immunostaining was analyzed using a Leica TCS SP5 confocal microscope.
- Tissue processing and immunostaining Tissues were fixed in 4% PFA overnight at 4 ° C., processed, and embedded in paraffin. Transverse sections (4 ⁇ m) were mounted on MAS-coated slides (Matsunami, Osaka, ⁇ Japan) for immunostaining with hematoxylin / eosin (HE) or standard histological staining. Prior to immunostaining, autoclave antigen recovery using citrate buffer (pH 6.0) was performed. The primary antibodies used were anti-human: CD31; smooth muscle actin; AFP; CK8.18 (all Dako Corporation) and ALB (BD Biosciences). Tissue sections were incubated with secondary antibody Alexa Fluor (Life Technologies) for 1 hour at room temperature, followed by DAPI (Sigma) nuclear staining. Images were obtained using an LSM510 laser scanning microscope (Carl Zeiss Co., Germany).
- HUVEC MSC isolation We obtained umbilical cord samples according to guidelines established and approved by the ethics committee of Yokohama City University (approval number: 13120510008). HUVECs and MSCs were simultaneously isolated from the umbilical cord 2 as described in the literature.
- E13.5 mFLCs isolated from C57BL / 6-Tg CAG : EGFP (SLC, Japan) in Dulbecco's modified Eagle's medium (DMEM; containing 10% fetal bovine serum (FBS)) (JRH Bioscience, USA) And mechanically separated by pipetting. Hepatocytes were separated from non-parenchymal cells by performing low speed centrifugation (690 rpm / 4 ° C for 1 minute) several times. The separated cells were passed twice through a 70 ⁇ m cell strainer (Falcon, USA) to obtain single cells.
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- Agilent CE Capillary Electrophoresis System with Agilent 6210 Time-of-Flight Mass Spectrometer, Agilent 1100 Uniform Concentration HPLC Pump, Agilent G1603A CE-MS Adapter Kit and Agilent G1607A CE-ESI-MS Sprayer Kit (Agilent Technologies, Waldbronn, Germany ) was used for CE-TOFMS. The system was controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany).
- Cationic metabolites were analyzed using fused silica capillaries (inner diameter 50 ⁇ m ⁇ total length 80 cm) using Cation Buffer Solution (Human Metabolome Technologies) as electrolyte. Samples were injected for 10 seconds at a pressure of 50 mbar (about 10 nl). The applied voltage was set to 27 kV. Electrospray ionization mass spectrometry (ESI-MS) was performed in positive ion mode and the capillary voltage was set to 4,000 V. The mass spectrometer was scanned from m / z 50 to 1,000. Other conditions were the same as those in cation analysis 3 .
- ESI-MS Electrospray ionization mass spectrometry
- Anionic metabolites were analyzed using a fused silica capillary (an inner diameter of 50 ⁇ m ⁇ total length of 80 cm) using Anion Buffer Solution (Human Metabolome Technologies) as an electrolyte. Samples were injected for 25 seconds at a pressure of 50 mbar (about 25 nl). The applied voltage was set to 30 kV. ESI-MS was performed in negative ion mode and the capillary voltage was set to 3,500 V. The mass spectrometer was scanned from m / z 50 to 1,000. Other conditions were the same as those in anion analysis 4 .
- Raw data obtained by CE-TOFMS was processed by MasterHands 5 , an automatic integration software. Peak information including m / z, migration time (MT) and area was obtained. The peak area was converted into a relative peak area according to the following equation. Each peak was ordered according to similar migration times in CE and m / z values determined by TOFMS.
- Relative peak area metabolite peak area / (internal standard peak area x sample amount)
- Metabolic pathway maps were provided using VANTED: Visualization and Analysis of Networks containing Experimental Data 6 which is a public software.
- NOD / SCID mice 15 mg / kg
- Urine (0-2 hours) was collected in 0.5 M acetate buffer (pH 5.0). The urine sample was added with 1 N KOH, incubated at 80 ° C. for 3 hours, and then neutralized with an equal volume of 1 N HCL.
- Acetonitrile containing 1% acetic acid was added and centrifuged (15000 rpm, 4 ° C, 5 minutes).
- the supernatant was subjected to liquid chromatography-tandem mass spectrometry (LC / MS / MS).
- LC / MS / MS liquid chromatography-tandem mass spectrometry
- the LC-20A series Shiadzu, Kyoto, Japan
- an Intersil® ODS-3 column GL Science Inc., Tokyo, Japan
- Chromatographic separation was achieved using an Intersil® ODS-3 column (5 ⁇ m, 4.6 ⁇ x150 mm ⁇ I.D .; GL Sciences Inc., Tokyo, Japan).
- the column temperature was maintained at 40 ° C.
- a mobile phase consisting of 0.1% acetic acid (solvent A) and 0.1% acetic acid-containing acetonitrile (solvent B) was injected at a flow rate of 0.5 mL / min according to the following gradient schedule: 25-80% solvent B linear gradient (0- 15 min), 80% solvent B (15-25 min), linear gradient of 80-25% solvent B (25-26 min), and 25% solvent B (26-35 min).
- Liquid chromatography was coupled to a 4000 Q Trap system (AB SCIEX, Foster City, CA) and operated in negative electrospray ionization mode.
- the turbo gas was maintained at 600 ° C. Parent and / or fragment ions were filtered through the first quadrupole and dissociated in the collision cell using nitrogen as the collision gas.
- the ion spray voltage was -4500 V and the analyzed m / z transitions (Q1 / Q3) for ketoprofen and 1-hydroxyketoprofen were 253.1 / 209.3 and 269.1 /
- sham operated NOD / SCID mice were used as a control.
- Blood samples were collected 0.5, 1, 2 and 8 hours after dosing and sodium heparin was added.
- Plasma was separated from blood by centrifugation. Internal standard (1 ⁇ m niflumic acid) and methanol solution (100 ⁇ l) were added to 5 ⁇ l of plasma and centrifuged (15000 rpm, 4 ° C., 5 minutes). The supernatant was subjected to LC / MS / MS.
- a mobile phase consisting of 10 mM ammonium acetate (solvent A) and acetonitrile (solvent B) was injected at a flow rate of 0.8 mL / min according to the following gradient schedule: 0% solvent B (0-0.2 min), 0-30 Linear gradient of% solvent B (0.2-0.3 min), linear gradient of 30-60% solvent B (0.3-0.85 min), 60% solvent B (0.85-1.15 min), linear gradient of 60-100% solvent B ( 1.15-1.16 min), and 100% solvent B (1.16-1.5 min).
- Liquid chromatography was coupled to the API4000 system (AB SCIEX, Foster City, CA) and operated in positive electrospray ionization mode. The turbo gas was maintained at 450 ° C.
- Parent and / or fragment ions were filtered through the first quadrupole and dissociated in the collision cell using nitrogen as the collision gas.
- the ion spray voltage was 5000 V and the analyzed m / z transition (Q1 / Q3) of 4-hydroxydebrisoquin and internal standard was 192.6 / 132.1 and 283.2 / 245.4, respectively.
- Alb-TRECK / SCID mice were provided by Hiromichi Yonekawa and Kunie Matsuoka (Tokyo Metropolitan Institute of Clinical Medicine). This transgenic line expresses HBEGF from the ALB enhancer / promoter and develops fulminant hepatitis when administered a small amount of diphtheria toxin (DT) 7 .
- DT diphtheria toxin
- HFLCs-LBs were transplanted into the mesentery covered with fibrin glue.
- 1.5 ⁇ g / kg DT was injected from the tail vein, causing severe liver damage. Survival was compared between mice that received the transplant and mice that did not.
- transplanted mFLCs had successfully engrafted and that a functional vascular network was formed in the graft (FIG. 21a).
- the transplanted tissues were extensively differentiated into tissues similar to hepatic cords, sinusoids and bile ducts. These tissues are characteristic of adult liver, not donor E13.5 LBs (FIG. 21b).
- Liver tissue reconstitution with mFLCs was enhanced by the addition of HGF and EGF, which are known to stimulate hepatic stem / progenitor cell proliferation (FIG. 22) 9,10 . This suggests that this transplantation approach provides a useful in vivo monitoring system for assessing LB cell maturation and differentiation.
- enzyme linked immunoassay showed the appearance of human albumin production 30 days after transplantation (FIGS. 16c and d). Therefore, in vivo monitoring of cell morphology can be an indicator for predicting the state of hepatocyte differentiation in vivo.
- KTP ketoprofen
- Liver humanized mice are a useful tool for studying human-specific drug metabolism. Human specific drug metabolism function in liver humanized mice has been previously reported using immunodeficient mice with high quality adult hepatocytes and severely damaged liver. After administration of KTP, it was observed that UGT facilitates KTP glucuronidation 14 and hydrolysis metabolizes KTP to KTP-G. The KTP / OH-KTP peak area ratio was calculated and this area ratio was compared between the hydrolyzed and non-hydrolyzed samples. A doubling of the KTP / OH-KTP peak area ratio suggests the formation of KTP-G in the sample.
- Debrisoquin which acts as a general phenotypic test for human CYP2D6, is metabolized in humans to 4-hydroxydebrisoquin (4-OHDB), but can be ignored in mice.
- human CYP2D6 is involved in 25% metabolism of known drugs and contributes to significant individual differences due to the presence of many polymorphisms. After oral administration of debrisoquin, the plasma concentration of 4-OHDB in the mesenteric or skull transplanted group was higher than that in the sham-operated group, reflecting the production of human-specific drug metabolites .
- hFLC-LB mesenteric transplant ⁇ br/>
- the intestinal tract was sealed with fibrin glue and hFLC-LBs grown in vitro Implanted on membrane.
- transgenic immunodeficient mice expressing human HB-EGF precursor under the control of hepatocyte-specific albumin promoter were used. These mice, referred to as toxin receptor-mediated cell knockout / severe combined immunodeficiency (TRECK / SCID) mice, develop fulminant hepatitis when administered small amounts of diphtheria toxin (DT) 7 .
- TRECK / SCID severe combined immunodeficiency
- the DT agent was injected from the tail vein at a dose of 1.5 ⁇ g / kg on the second day after transplantation. Survival curves revealed that all TRECK / SCID mice that did not receive the transplant died within 10 days. In contrast, 28% of TRECK / SCID mice transplanted with hFLC-LB survived for over 40 days, demonstrating the therapeutic potential of our proof of concept (Figure 32d). However, this transplant model works to some extent, but the rescue rate is not high. This is because DT is also toxic to human cells. Therefore, we adopted TK-NOG mice as an immunodeficient liver injury model. This is because administration of GCV (which is non-toxic to human tissue) induces tissue-specific removal of transgenic hepatocytes at the appropriate time. Using this model, we removed host hepatocytes at 7 and 10 days post-transplant, where grafts are expected to successfully engraft through the formation of functional human vascular networks.
- GCV which is non-toxic to human tissue
- VANTED a system for advanced data analysis and visualization in the context of biological networks.BMC Bioinformatics 7, 109, (2006). 7 Saito, M. et al. Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol 19, 746-750, (2001). 8 Yuan, F. et al. Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows.Cancer Res 54, 4564-4568, (1994). 9 Suzuki, A., Iwama, A., Miyashita, H., Nakauchi, H. & Taniguchi, H.
- the present invention can be used in the pharmaceutical industry and the like.
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Abstract
Description
(1)血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞を培養することを含む、器官芽の作製方法。
(2)臓器細胞が分化した細胞である(1)記載の器官芽の作製方法。
(3)臓器細胞が未分化な細胞である(1)記載の器官芽の作製方法。
(4)臓器細胞が、内胚葉性器官の細胞若しくは内胚葉性器官の細胞に分化可能な細胞、中胚葉性器官の細胞若しくは中胚葉性器官の細胞に分化可能な細胞、又は外胚葉性器官の細胞若しくは外胚葉性器官の細胞に分化可能な細胞である(1)~(3)のいずれかに記載の器官芽の作製方法。
(5)臓器細胞が、内胚葉性器官の細胞若しくは内胚葉性器官の細胞に分化可能な細胞である(4)記載の器官芽の作製方法。
(6)内胚葉性器官が、肝臓又は膵臓である(5)記載の器官芽の作製方法。
(7)臓器細胞が、人工多能性幹細胞由来の細胞である(1)~(6)のいずれかに記載の器官芽の作製方法。
(8)人工多能性幹細胞がヒト由来である(7)記載の器官芽の作製方法。
(9)血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞を血管内皮細胞培養用培地中で培養する(1)~(8)のいずれかに記載の器官芽の作製方法。
(10)血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞をゲル上に播種して培養する(1)~(9)のいずれかに記載の器官芽の作製方法。
(11)血管内皮細胞が分化した細胞である(1)~(10)のいずれかに記載の器官芽の作製方法。
(12)血管内皮細胞が未分化な細胞である(1)~(10)のいずれかに記載の器官芽の作製方法。
(13)間葉系細胞が分化した細胞である(1)~(12)のいずれかに記載の器官芽の作製方法。
(14)間葉系細胞が未分化な細胞である(1)~(12)のいずれかに記載の器官芽の作製方法。
(15)(1)~(14)のいずれかに記載の方法によって作製された器官芽を非ヒト動物に移植し、組織又は臓器に分化させることを含む、組織又は臓器の作製方法。
(16)(1)~(14)のいずれかに記載の方法によって作製された器官芽をヒト又は非ヒト動物に移植することを含む、器官芽の移植方法。
(17)器官芽の移植部位が、頭蓋内、腸間膜、肝臓、脾臓、腎臓、腎被膜下、門脈上からなる群より選択される(16)記載の器官芽の移植方法。
(18)(1)~(14)のいずれかに記載の方法によって作製された器官芽をヒト又は非ヒト動物に移植し、組織又は臓器に分化させることを含む、組織又は臓器の再生又は機能回復方法。
(19)(1)~(14)のいずれかに記載の方法によって作製された器官芽を非ヒト動物に移植し、組織又は臓器に分化させることを含む、非ヒトキメラ動物の作製方法。
(20)(1)~(14)のいずれかに記載の方法によって作製された器官芽、(15)記載の方法によって作製された組織及び臓器、並びに(19)記載の方法によって作製された非ヒトキメラ動物からなる群より選択される少なくとも一つを用いて、薬剤を評価する方法。
〔実施例1〕 未分化な臓器細胞を用いた臓器の作製
(1)ヒト肝臓内胚葉細胞の作製
ヒトiPS細胞(ヒト皮膚由来TkDA3 hiPSCクローン(Koji Eto氏及び Hiromitsu Nakauchi氏より譲渡))を無血清培地にアクチビンを添加して培養することで、CXCR4及びE-cadherin両陽性の内胚葉系細胞を誘導した。得られた内胚葉系細胞をBMP4、FGF2を添加して2日間培養することで、CXCR4陰性 HNF4α陽性の肝臓内胚葉集団を得た。CXCR4及びHNF4αの発現は、Hepatology, 51(1), 297-305,2010の記載に従い、免疫染色及び遺伝子発現解析により確認した。
(2)ヒト肝芽の作製
得られた肝臓内胚葉細胞を、血管内皮細胞(ヒト臍帯血由来静脈内皮細胞)(Lonza、Basel、Switzerland)及び未分化間葉系細胞(ヒト間葉系幹細胞)(Lonza、Basel、Switzerland)を各々10:5-10:2の割合で混合し、共培養を行った。血管内皮細胞および未分化間葉系細胞は、各々蛍光標識を行ったものを用いた。共培養に際しては、原液ないし2倍希釈のマトリゲル(BD pharmingen)の固相化を行った培養皿の上へ細胞懸濁液を播種した。培養液は、内皮細胞培地キット-2:EGM-2 BulletKit(製品コード CC-3162:Lonza)を用いた。
(3)ヒト肝組織の作製
形成されたヒト三次元構造体を、免疫不全マウス(NOD/SCIDマウス(Sankyo Lab. Co., Tsukuba, Japan))生体内へ移植した。肉眼および共焦点顕微鏡によるライブ観察を実施し、ヒト細胞の生着・増殖を確認するとともに、移植後の血管成熟化過程を解析した。移植早期のサンプル(2週間)を回収し、その組織学的解析を行った。
(1)ヒト細胞のみから自律的に組織化が進行し、肉眼的に観察可能な立体的な構造体が形成された(図1)。
(2)培養4日目には、血管様の管腔構造が確認された(図1右上図の拡大像)。
(3)形成された三次元構造体は、未分化細胞のマーカーであるNANOGやCXCR4などの発現を減弱した(図2)。
(4)従来技術により終末分化誘導を行った細胞と比較して、肝細胞の分化マーカーであるアルブミン(ALB)の発現を100倍以上増強し(図2)、その他の分化マーカー(FOXA2、TAT、PCK2)の発現も数十倍程度の発現増強を示した(図3)。
(5)移植により、ヒト血管がマウス血管と接続し、早期(移植2日目)から血液灌流が開始した(図4)。
(6)ヒトiPS細胞由来肝臓細胞の増殖が確認された(図5)。
(7)移植早期(2週)のサンプルの組織学的解析により、アルブミン陽性の索状構造の形成が確認された。また、類洞様構造の形成も確認された(図5)。
(8)共培養の際、細胞懸濁液をマトリゲル固相化培養皿上へ播種するのではなく、細胞懸濁液をマトリゲル中に包埋した場合、非コートの培養皿へ播種した場合、あるいは培養皿をtype 1 collagenでコートした培養皿へ播種した場合、には立体構造は形成されなかった。
(9)共培養の際、培養液として内皮細胞培地ではなく、Hepatocyte Medium (XenoTech)、あるいはBMP4及びFGF2を添加した肝細胞誘導培地(Hepatology, 51(1), 297-305,2010)を用いた場合には、肝芽に特徴的な遺伝子発現の増強(Alb、TTRなど)は認められなかった。
〔実験方法〕
膵β細胞株(MIN6)を、血管内皮細胞(ヒト臍帯血由来静脈内皮細胞)及び未分化間葉系細胞(ヒト間葉系幹細胞)を各々5:5-10:2の割合で混合し、共培養を行った。膵β細胞株(KO)および血管内皮細胞(EGFP)は、各々蛍光標識を行ったものを用いた。共培養に際しては、原液ないし2倍希釈のマトリゲル(BD pharmingen)の固相化を行った培養皿の上へ細胞懸濁液を播種した。尚、マトリゲル内部へ包埋した場合、あるいは、非コート、type 1 collagenコートの培養皿では、立体構造は形成されなかった。培養液は、内皮細胞培地キット-2:EGM-2 BulletKit(製品コード CC-3162:Lonza)を用いた。
(1)細胞のみから自律的に組織化が進行し、翌日には肉眼的に観察可能な立体的な構造体が形成された(図6)。
(2)培養2日目には、膵β細胞株が集塊を形成し、その周囲を取り囲むようにヒト血管様の管腔構造が確認された(図7)。
(3)移植により、ヒト血管がマウス血管と接続し、移植早期から血液灌流が開始した(図8)。
(4)膵β細胞株は集塊を形成するように増殖し、膵島様の構造体を形成した(図9)。
(5)形成された膵島様構造体の中にヒト細胞からなる血管構造の形成が確認された(図10)。
(6)血流の可視化により、膵島様構造体の内部では、充分に血液灌流が再開していることが確認された(図11)。
(7)移植サンプルの組織学的解析により、インスリン陽性の島状構造の形成が確認された。形成された構造体は、内部に複雑な血管構造を有しており、正常マウス膵島と類似の構造を有していた(図12)。
末期臓器不全を治療するためのドナー臓器は著しく不足しており、患者由来の人工多能性幹細胞(hiPSCs)1, 2を用いて臓器を作成する必要性が一段と高まっている。多くの論文にて機能的な細胞分化が報告されているものの3-7、肝臓といった3次元の血管網を有する臓器の作成に成功した例はない。我々は、in vitroで調製したhiPSC由来の肝芽(hiPSC-LBs)の移植により、血管構造を有する機能的なヒト肝臓組織の作成に成功した。内皮細胞と間葉細胞8を加え、器官形成を促したところ、iPS細胞由来肝内胚葉細胞は3次元的なhiPSC-LBsへと自己組織化した。免疫染色及び遺伝子発現解析の結果、in vitroで成長したhiPSC-LBsとin vivoの肝芽間で類似性が認められた。hiPSC-LBs移植片内のヒト血管網は、48時間以内に宿主血管と連結し、血液灌流が開始した。機能的な血管構造が形成されることにより、hiPSC-LBsは成体肝臓と類似する組織へと成熟することが明らかとなった。さらに、高い代謝能を有するhiPSC由来の移植組織は、 レシピエントの肝臓の置換9, 10を要することなく、ヒト型のタンパク質産生やヒト特異的な薬物代謝といった肝臓特異的な機能を発揮した。さらに、hiPSC-LBsを腸間膜へ移植したところ、薬物で誘導した致死的肝不全モデルの生存率を向上させた。我々の知る限り、本報告は多能性幹細胞から機能的なヒト臓器を作成した最初の例である。本手法を臨床に応用するには更なる検討が必要であるものの、我々が実験で実証した器官芽移植は、再生医療において可能性のある新たなアプローチを提供するものである。
hiPSCsの肝初期分化誘導は先述の方法5に従い行った。HUVECsとhMSCs(Lonza、Basel、Switzerland)は、内皮増殖培地(EGM)(Lonza)もしくはMSC増殖培地(Lonza)を用いて、加湿インキュベーター内で37度、5%のCO2存在下で維持された。ヒトLBをin vitroで作成する際は、EGMと肝細胞用培地(HCM)(Cambrex、 Baltimore、MD)(デキサメタゾン(0.1 μM、Sigma-Aldrich、St Louis、MO)、オンコスタチンM(10 ng/ml、R&D System、Minneapolis、MN)、HGF(20 ng/ml、PromoKine)及び SingleQuots(Lonza)を含む)に1x106のhiPSC由来肝細胞、0.8-1x106のHUVECs、及び2x105のhMSCsを懸濁し、マトリゲル(BD Biosciences、Bedford、MA、USA)上に塗布した。培養4-6日後、生成したhiPSC-LBsを剥がし、採取した後、免疫不全マウスの頭蓋窓19に移植した。
細胞培養及び分化 TkDA3 hiPSCクローンは、Koji Eto氏及び Hiromitsu Nakauchi氏より分譲頂いた。未分化hiPSC細胞については、マウス胚線維芽細胞をフィーダー細胞として用い、その上で生育させた。内胚葉性分化の際は、hiPSCsをマトリゲルでコートしたディッシュ上に撒いた後、1%のB27(インスリン不含)と(100ng/ml)を含むRPMI1640培地へと移し、5-6日間培養した。hiPSC由来の内胚葉細胞をhbFGF(10ng/ml)、hBMP4(20ng/ml)及び1% B27を含むPRMI1640でさらに3-4日間処理することで肝臓への特定化を行った。ヒト組換体アクチビンA/ EDFは、Yuzuru Eto氏(Ajinomoto Co. Inc.)より分譲頂いた。hFLCs(CS-ABI-3716;Applied Cell Biology Research Institute)は、コラーゲンIVでコーティングされた6-wellプレート(BD Biosciences)上に撒かれた後、当研究室の標準培地[10% FBS(Lot.7219F; ICN Biochemical、USA)、50 mmol/l HEPES(Wako Pure Chemical Industries、Japan)、2 mmol/L L-glutamine(Life Technologies Corporation、USA)、50 mmol/L 2-mercaptoethanol(Sigma)、1× penicillin/streptomycin(Life Technologies)、10 mmol/L nicotinamide(Sigma)、1 × 10-4 M Dexamethasone(Sigma)及び1 μg/ml insulin(Wako)を含むF-12(Sigma Aldrich、Japan)とDMEMの1:1混液]を用いて維持された。ヒト組換体HGF(50 ng/ml)及び EGF(20 ng/ml)(Sigma)を培養前に添加した。HUVECs及びhMSCs(Lonza)は上皮増殖培地もしくはMSC増殖培地(Lonza)を用いて、加湿インキュベーター内で37度、5%のCO2存在下で維持された。
全RNAは、RNeasy Mini Kit (Qiagen, Valencia, CA)を用いて、hiPSC由来細胞/組織(hiPSC, hiPSC-Def, hiPSC-Hep, hiPSC-IH, hiPSC-MH, hiPSC-LB, hiPSC-LB-Tx) から調製した。 ヒト胎児肝臓(Lot No.: A601605) およびヒト成体肝臓 (Lot No.: B308121)の全RNAは Biochain Institute (Hayward, CA, USA)から得た。cRNAを増幅し、Low Input Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, CA)を用いて標識し、44Kの60量体オリゴマイクロアレイ(Human Gene Expression 4x44K v2 Microarray Kit; Agilent Technologies)に製造者の指示に従ってハイブリダイズさせた。ハイブリダイズさせたマイクロアレイスライドを、Agilent High-Resolution Microarray Scannerを用いてスキャンした。Feature Extraction Software バージョン 10.7.3.1 (Agilent Technologies)を用いて、相対ハイブリダイゼーション強度およびバックグラウンドハイブリダイゼーション値を計算した。Agilent Technologiesが推奨する手順に従ってGeneSpring 11.5.1 Software中のフラグ基準を用いて、生のシグナル強度および各プローブのフラグをハイブリダイゼーション強度およびスポット情報から計算した。さらに、サンプルの生シグナル強度をlog2変換し、変位値アルゴリズムによって正規化した。我々は全てのサンプルに、「妥協した」(compromised)フラグを除きプローブを選択し、検出された遺伝子として34,183個のプローブを得た。さらに、我々は遺伝子レベルにおける26,153個の発現データに焦点をあてる。GeneSpringによってヒートマップが作製された。正規化された強度値をロードし、各プローブの中央値からの距離によりスケーリング調整した。我々はユークリッド測定基準を用いる序列的クラスタリング法によってサンプルおよび遺伝子を分類した。種々の段階のhiPSCs中における遺伝子発現パターンの相違を評価するため、我々は選択した83種の遺伝子の発現変化を分析した。これらの遺伝子は、種々の異なる発生段階にあるマウス肝細胞および2つの異なる段階にあるヒト肝組織のマイクロアレイ分析による我々の以前の研究において同定された。すべての遺伝子から83種の遺伝子を肝臓特性遺伝子として選択した。なぜなら、それらの遺伝子はヒトおよびマウス両者の肝臓発生に際し継続的に増加したからである。
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HUVEC MSC単離。我々は、横浜市立大学の倫理委員会によって定められ、承認されたガイドラインに従って臍帯サンプルを入手した(承認番号:13120510008)。HUVECsおよびMSCsは、文献に記述されているように2、臍帯から同時に単離した。
機能性肝臓組織創出における頭蓋窓モデルの可能性
頭蓋窓調製のための詳しい手順は、以前に記述されている8。我々は、EGFPを発現しているE13.5マウス胎児肝細胞(mFLCs)の移植片を用いて、肝細胞の成熟を研究する上での頭蓋窓の可能性を評価した。コラーゲン/フィブロネクチンゲルに包埋したmFLCsの一片を切り出し、頭蓋窓の中心部に置いた。次に、組織適合性のシアノアクリレート糊を用いて8mmのカバーグラスを骨に接着させ、この窓をシールした。生体内蛍光顕微鏡イメージングは、移植されたmFLCsがうまく生着したこと、および機能性血管網が移植片内で形成されたことを示した(図21a)。移植された組織は、肝細胞索、類洞および胆管に類似した組織に広範に分化した。これらの組織は成体肝臓に特徴的なものであり、ドナーE13.5 LBsに特徴的なものではない(図21b)。mFLCsによる肝組織再構成は、HGFおよびEGF(これらは肝幹細胞/前駆細胞の増殖を刺激することが知られている)の添加により増強された(図22)9,10。したがって、この移植アプローチは、LB細胞の成熟および分化を評価するために有益な生体内モニタリング系となることが示唆された。
正常肝発生過程において、肝細胞の形態は円形から敷石上の細胞へと変化する11。この変化は、サイトケラチン免疫染色によって容易に可視化することができる(図16a、左)。IN Cell Investigatorソフトウエアを用いて、我々はマウス肝細胞の真円度(形状因子)がE13.5における0.833 ± 0.18 から生後8週における 0.568 ± 0.16 まで低下することを見出した。同様に、単細胞形態の生体内イメージングは、移植されたEGFP標識hFLCsの真円度が0日目の0.93 ± 0.07 から30日後には 0.512 ± 0.13 に変化することを明らかにした(図27c、右)。これらの考察と一致して、酵素結合免疫測定法(ELISA)は移植30日後からのヒトアルブミン産生の出現を示した(図16cおよびd)。したがって、細胞形態の生体内モニタリングは、in vivoにおける肝細胞分化の状態を予測するための1つの指標となりうる。
我々は、ケトプロフェン(KTP)を用いて、ヒト特異的薬物代謝機能を評価した。KTPはマウス中でシトクロームP450によって第一に代謝され、1-ヒドロキシケトプロフェン(OH-KTP)を形成する12。他方、ヒトにおいては、KTPは主としてUDP-グルコロノシルトランスフェラーゼ(UGT)によって代謝され、ケトプロフェングルクロニド(KTP-G)を形成する13。
頭蓋窓モデルは高度に侵襲性であるため、器官芽移植にとって非常に効率的な方法とは言えない。したがって、臨床応用を想定する際には、より低侵襲的な移植法の開発が必要である。さらに、移植可能な容量が肝不全を逆転させるには不十分である。よって、我々は侵襲性が最少で、臨床的適切性を有する、腸間膜移植モデルの可能性を検討しようと試みた。なぜなら、肝機能の改善にとって門脈血流が重要であると考えられたからである。我々の期待と一致して、最近なされた報告が、恐らく腸間膜血流からの宿主血管補充によって、腹腔部位はヒト成体肝細胞の生着および肝機能の維持を支持しうることを示した15。In vitroで増殖させたhFLC-LBsまたはhiPSC-LBsを腸間膜上に移植した(図33a)。
hiPSC-LB移植片における肝細胞成熟は、HGF等の再生因子によって促進しうるかどうかを決定するため、腸間膜移植後7日目に2/3部分肝切除(PH)を実施した。2/3 PH実施後、30日目にヒトアルブミンの産生が偽手術群の82.1 ng/mlから2/3 PH群では121 ng/mlに上昇した(図33c)。これらの結果は、恐らくhiPSC-LB移植片内における広範な肝細胞増殖および成熟のゆえに、hiPSC-Hepsは2/3 PH後の再生刺激に応答しうることを示唆した。
我々の移植法を用いた治療可能性を評価するため、in vitroで増殖させたhFLC-LBsをフィブリン糊でシールした腸間膜上に移植した。肝損傷モデルとして、肝細胞特異的アルブミンプロモーターの制御下でヒトHB-EGF前駆体を発現するトランスジェニック免疫不全マウスを用いた。毒素受容体媒介細胞ノックアウト/重症複合免疫不全(TRECK/SCID)マウスと呼ばれるこれらのマウスは、少量のジフテリア毒素(DT)を投与すると劇症肝炎を発症する7。DT剤は、移植後2日目に1.5 μg/kgの用量で尾静脈より注入した。生存曲線は、移植を受けなかったTRECK/SCIDマウスの全部が10日以内に死亡したことを明らかにした。対照的に、hFLC-LBを移植されたTRECK/SCIDマウスの28%は40日以上にわたって生存し、我々の概念実証の治療上の可能性を示した(図32d)。しかし、この移植モデルはある程度までうまくいくが、救助率は高くない。なぜなら、DTはヒト細胞に対しても毒性だからである。そこで、我々は免疫不全肝損傷モデルとしてTK-NOGマウスを採用した。なぜなら、GCV(ヒト組織に対して非毒性である)の投与が、適切なタイミングでトランスジェニック肝実質細胞の組織特異的除去を誘導するからである。このモデルを用いて、我々は、機能性ヒト血管網の形成を介して移植片がうまく生着することが予測される移植後7日目および10日目に、宿主肝細胞を除去した。
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Claims (20)
- 血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞を培養することを含む、器官芽の作製方法。
- 臓器細胞が分化した細胞である請求項1記載の器官芽の作製方法。
- 臓器細胞が未分化な細胞である請求項1記載の器官芽の作製方法。
- 臓器細胞が、内胚葉性器官の細胞若しくは内胚葉性器官の細胞に分化可能な細胞、中胚葉性器官の細胞若しくは中胚葉性器官の細胞に分化可能な細胞、又は外胚葉性器官の細胞若しくは外胚葉性器官の細胞に分化可能な細胞である請求項1~3のいずれかに記載の器官芽の作製方法。
- 臓器細胞が、内胚葉性器官の細胞若しくは内胚葉性器官の細胞に分化可能な細胞である請求項4記載の器官芽の作製方法。
- 内胚葉性器官が、肝臓又は膵臓である請求項5記載の器官芽の作製方法。
- 臓器細胞が、人工多能性幹細胞由来の細胞である請求項1~6のいずれかに記載の器官芽の作製方法。
- 人工多能性幹細胞がヒト由来である請求項7記載の器官芽の作製方法。
- 血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞を血管内皮細胞培養用培地中で培養する請求項1~8のいずれかに記載の器官芽の作製方法。
- 血管内皮細胞、間葉系細胞、血管内皮細胞から分泌される因子、間葉系細胞から分泌される因子、血管内皮細胞と間葉系細胞の両方が存在することによって分泌される因子からなる群より選択される少なくとも1つの細胞及び/又は因子とともに、臓器細胞をゲル上に播種して培養する請求項1~9のいずれかに記載の器官芽の作製方法。
- 血管内皮細胞が分化した細胞である請求項1~10のいずれかに記載の器官芽の作製方法。
- 血管内皮細胞が未分化な細胞である請求項1~10のいずれかに記載の器官芽の作製方法。
- 間葉系細胞が分化した細胞である請求項1~12のいずれかに記載の器官芽の作製方法。
- 間葉系細胞が未分化な細胞である請求項1~12のいずれかに記載の器官芽の作製方法。
- 請求項1~14のいずれかに記載の方法によって作製された器官芽を非ヒト動物に移植し、組織又は臓器に分化させることを含む、組織又は臓器の作製方法。
- 請求項1~14のいずれかに記載の方法によって作製された器官芽をヒト又は非ヒト動物に移植することを含む、器官芽の移植方法。
- 器官芽の移植部位が、頭蓋内、腸間膜、肝臓、脾臓、腎臓、腎被膜下、門脈上からなる群より選択される請求項16記載の器官芽の移植方法。
- 請求項1~14のいずれかに記載の方法によって作製された器官芽をヒト又は非ヒト動物に移植し、組織又は臓器に分化させることを含む、組織又は臓器の再生又は機能回復方法。
- 請求項1~14のいずれかに記載の方法によって作製された器官芽を非ヒト動物に移植し、組織又は臓器に分化させることを含む、非ヒトキメラ動物の作製方法。
- 請求項1~14のいずれかに記載の方法によって作製された器官芽、請求項15記載の方法によって作製された組織及び臓器、並びに請求項19記載の方法によって作製された非ヒトキメラ動物からなる群より選択される少なくとも一つを用いて、薬剤を評価する方法。
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