WO2010143529A1 - Procédé destiné à induire la différenciation d'une cellule-souche pluripotente en une cellule épithéliale thymique - Google Patents

Procédé destiné à induire la différenciation d'une cellule-souche pluripotente en une cellule épithéliale thymique Download PDF

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WO2010143529A1
WO2010143529A1 PCT/JP2010/058781 JP2010058781W WO2010143529A1 WO 2010143529 A1 WO2010143529 A1 WO 2010143529A1 JP 2010058781 W JP2010058781 W JP 2010058781W WO 2010143529 A1 WO2010143529 A1 WO 2010143529A1
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cells
culture
cell
differentiation
thymic
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健一 磯部
雄太 稲見
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国立大学法人名古屋大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/065—Thymocytes
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor

Definitions

  • the present invention relates to a method for inducing differentiation of pluripotent stem cells into a specific cell lineage. Specifically, the present invention relates to a method for inducing differentiation of pluripotent stem cells into thymic epithelial progenitor cells or medullary thymic epithelial cells.
  • Immunity is a mechanism that protects individuals from external attack by microorganisms and destruction from inside by cancer.
  • Representative of such cells responsible for immunity are phagocytic cells and lymphocytes, and these cells migrate throughout the body via blood vessels and lymphatic vessels.
  • Phagocytes are composed of neutrophils and macrophages, and these are mobilized to the invasion site of antigen.
  • one lymphocyte forms an antibody or a receptor that binds to only one antigen, one individual has more than 10 6 types of lymphocytes corresponding to the number of antigens.
  • the immune system consists of central lymphoid tissues such as the thymus and bone marrow, where lymphocytes and phagocytic cells are generated and differentiated, and peripheral lymphoid tissues such as the spleen and lymph nodes where the cells produced there move and gather. Lymphocytes that gather in peripheral tissues include thymus-derived T cells and bone marrow-derived B cells.
  • the immune system is triggered by signal exchange between cells triggered by antigen stimulation, and these signal exchanges between cells are macrophages and T cells that have incorporated the antigen as a foreign substance, resulting in activated T and B cells, activity It occurs between activated lymphocytes and phagocytes (references 34, 37).
  • T cells are involved in both cellular immunity that destroys virus-infected cells and humoral immunity that helps B cells that produce specific antibodies, which are the main role in bacterial defense. T cells also circulate throughout the body through blood vessels and lymphatic vessels. T cells have TCR, which is a receptor that recognizes antigens and MHC molecules. There is only one type of TCR per T cell, and T cells can handle myriad antigen presentations. Must have. There are roughly two types of T cells: cells with CD4 protein on the cell surface and cells with CD8 protein. Helper T cells composed of naive Th, Th1, Th2, etc. with CD4 on the cell surface recognize antigen-presenting cells and regulate the functions of other immune cells by producing and releasing cytokines.
  • Th2 controls humoral immunity through B cell-mediated antibody production, and Th1 has a killing effect by releasing cytokines against macrophage-engaged bacteria.
  • Killer T cells with CD8 protein on the cell surface are responsible for cell-mediated immunity that prevents further infection by destroying whole cells infected with the virus.
  • T cells are differentiated on thymic stromal cells, which are composed of various cells such as thymic epithelial cells, fibroblasts, macrophages, and dendritic cells.
  • thymic stromal cells which are composed of various cells such as thymic epithelial cells, fibroblasts, macrophages, and dendritic cells.
  • thymic epithelium the thymic epithelium occupies most of the matrix.
  • the stroma itself can be divided histologically into two areas, cortex and medulla.
  • T cells differentiate through several different phenotypes. It can be divided into DN (Double Negative), DP (Double Positive), and SP (Single Positive) based on the difference in the expression of CD4 and CD8. Furthermore, at the DN stage, there are four further stages due to the difference in the expression of CD44 and CD25. It can be divided into DNs 1-3 and 4 (pre-DP). Thymic T cells at different stages of differentiation are located in spatially different regions of the mature thymus, and thymic T cells and thymic epithelial cells are closely related to T cell differentiation from thymic transfer It has been known.
  • thymic T cells of DP In the cortical region of the thymus, positive differentiation is performed in which cells that recognize their own MHC survive after differentiation from thymic progenitor cells to thymic T cells of DP. In the medulla region, differentiation of thymic T cells from DP to SP and subsequent negative selection in which only thymic cells that do not recognize self-antigens survive (Negative Selection), and immune tolerance to self cells is established. And messy gene expression in medullary thymic epithelial cells (mTEC) suggests that it plays an important role in the induction of immune tolerance.
  • mTEC medullary thymic epithelial cells
  • bone marrow stromal cells transfected with delta 1 (Dll1) which is a Notch-ligand
  • Dll1 delta 1
  • OP9 OP9
  • thymic epithelial cells which are important cells in T cell differentiation, leads to elucidation of immune functions related to the thymus.
  • immune function declines with age, but this is due to changes in T cell composition and ability, and due to changes in sex hormones androgens and estrogens, the thymus itself becomes adolescent. It is known that when it passes, it begins to atrophy rapidly. It is expected to provide important findings for the elucidation of the causal relationship between such atrophy (aging) of the thymus and the decline in immune function including T cell composition, and the mechanism of thymus development and immune tolerance.
  • thymic epithelial cells can be regenerated using iPS cells (Induced pluripotent stem ⁇ ⁇ cells) created from the self, treatment of DiGeorge syndrome and normal T cells that cannot produce normal thymus due to genetic disease
  • iPS cells Induced pluripotent stem ⁇ ⁇ cells
  • Clinical applications such as cell differentiation and transplantation can also be expected.
  • Non-Patent Document 1 reports on factors involved in thymus development (Non-Patent Documents 1 to 5) are listed.
  • an object of the present invention is to provide means for inducing differentiation of pluripotent stem cells into thymic epithelial progenitor cells or medullary thymic epithelial cells aiming at thymic regeneration.
  • thymus was derived from two germ layers (cortex derived from ectoderm, medulla derived from endoderm), but it was reported that no clear relationship was found between differentiation of ectoderm and thymus The thymus was proven to be derived from the endoderm. Subsequent studies have also shown that thymic early organogenesis is closely linked to the parathyroid glands, both of which develop from organ primordia originating from the third pharyngeal sac, the endoderm, around mouse E11 I understood it.
  • the primordium at this stage contains progenitor cells to the thymus and parathyroid gland and was found to be divided into thymic and parathyroid regions from the point of gene expression.
  • the fact that the thymus originated from the endoderm revealed that cortical thymic epithelial cells (cTEC) and medullary thymic epithelial cells (mTEC) in the thymus are derived from the same endoderm.
  • cTEC cortical thymic epithelial cells
  • mTEC medullary thymic epithelial cells
  • the endoderm consists of the visceral endoderm, which forms the outer and inner layers of the anterior gastrulation, and the upper blastoderm, which is derived from the inner cell mass of the blastocyst.
  • the endoderm replaces the yolk sac near the extraembryonic body. Also, during gastrulation, blastocyst cells enter through the section behind the primitive streak and form definitive endoderm.
  • the definitive endoderm differentiates into gut formation and internal organs associated with the intestine such as the liver and pancreas.
  • the thymus was thought to originate from the definitive endoderm and this differentiation method was investigated.
  • Differentiation methods into these two endoderm using ES cells are already known, and specific factors are not required for differentiation into the proximal endoderm, and they can be induced by increasing the cell concentration.
  • an induction method using activin A (AA) which is known as a mesoderm inducing factor in the early stage of development, is known. It is also known that activin A can be induced in all three germ layers at different concentrations. Induction of differentiation into definitive endoderm using activin A is mediated through endomesoderm, and it has also been demonstrated that it is similar to differentiation into definitive endoderm via embryonic primitive streak ( Non-patent documents 1, 2).
  • FGF Fibroblast Growth Factor
  • FGF10 Fibroblast Growth Factor 7 and FGF10 are released from the mesenchymal tissue surrounding the thymic primordia and are deeply related to thymic development (Non-patent literature) 4
  • BMP4 is expressed in the thymic primordium, and it has been confirmed that the thymus is dysplastic in BMP (bone morphogenesis protein) 4 deficient mice (Non-patent literature) 5)
  • Induction of differentiation into thymic epithelial progenitor cells based on a unique viewpoint, referring to the fact that thymic dysplasia has been confirmed in FGF8-deficient mice (Non-patent Document 6). Tried.
  • Non-patent Document 7 RANK ligand and CD40 were suggested as important factors for development of medullary thymus region and self-tolerance, and it was reported that RANK ligand is important for the construction of early medullary thymic epithelial cells.
  • Non-patent Document 7 In order to promote differentiation into medullary thymic epithelial cells, it was decided to add RANK ligand. As a result, differentiation into medullary thymic epithelial cells was confirmed.
  • the present invention is mainly based on the above results.
  • a method for inducing differentiation of pluripotent stem cells into thymic epithelial progenitor cells or medullary thymic epithelial cells A first induction step for differentiating pluripotent stem cells into definitive endoderm cells; A second induction step of differentiating from definitive endoderm cells to thymic epithelial progenitor cells or medullary thymic epithelial cells by using FGF8, FGF7, FGF10, BMP4 and lithium chloride in combination, In the second induction step, following the first culture under the condition where FGF8 is added to the medium and FGF7 and FGF10 are not added, the second culture is performed under the condition where FGF7 and FGF10 are added to the medium and FGF8 is not added.
  • a method of culturing [2] The method according to [1], wherein the first induction step is a step of culturing cells under conditions in which activin and lithium chloride are added to the medium. [3] The method according to [2], wherein the concentration of lithium chloride in the medium is about 5 mM. [4] The method according to any one of [1] to [3], wherein the first culture is a step of culturing cells under conditions in which FGF8 and lithium chloride are added to the medium. [5] The method according to any one of [1] to [3], wherein the first culture is a step of culturing cells under conditions where FGF8, lithium chloride and activin are added to the medium.
  • the second induction step includes a third culture under a condition in which RANK ligand or CD40 ligand is added to the medium, and medullary thymic epithelial cells are induced by the culture, any of [1] to [7] The method according to claim 1.
  • ES cells were cultured for 4 days in serum-free medium SF-O3 supplemented with activin A (10 ng / ml) and analyzed by flow cytometry. There were 13.2% of cells (endomesoderm) positive for both E-cadherin and PDGFR- ⁇ (left). On the other hand, 25.3% of the cells (definitive endoderm) were positive for both E-cadherin and CXCR4 (right). Examination of conditions for inducing differentiation into definitive endoderm (using ES cells). The effect of combining activin A and LiCl was analyzed by flow cytometry. The induction efficiency to the endomesoderm changes depending on the concentration of LiCl.
  • D7 After 7 days of culture (D7), the cells are cultured in a serum-free medium supplemented with FGF7, FGF10, BMP10 and LiCl. Changes in gene expression by RT-PCR. Cells were collected on day 0 (D0), day 4 (D4), day 7 (D7), day 9 (D9), day 12 (D12) and day 15 (D15), and RT- The expression state of various genes was analyzed by PCR. Flow cytometry results using cells on day 14 of culture. Two cell populations (top, 12% and 58%) were observed. EpCAM1 and MTS24 are expressed in a small cell population (12%). Protocol for inducing differentiation into medullary thymic epithelial cells.
  • Cells were collected on the 0th day (D0), 4th day (D4), 12th day (D12) and 16th day (D16) of culture, and the expression state of various genes was analyzed by RT-PCR. Gene expression analysis by real-time PCR. Cells were collected on the 0th day (D0), 12th day (D12) and 16th day (D16) of culture, and the expression states of Pax1, FoxN1 and K5 were examined. Protocol of differentiation induction method to thymic epithelial progenitor cells (improved method). Incubate in serum-free medium supplemented with activin A and LiCl until day 4 of culture (D4).
  • the cells are cultured in a serum-free medium supplemented with FGF8, LiCl and activin A.
  • the cells are cultured in a serum-free medium supplemented with FGF7, FGF10, BMP10 and LiCl.
  • H.E stained image showing changes in cell groups induced to differentiate by the improved method.
  • the scale bar is 200 ⁇ m. Changes in gene expression of cells induced to differentiate by an improved method (RT-PCR results).
  • AA Activin A
  • Bmp4 bone morphogenetic protein 4
  • Bmps bone morphogenetic proteins
  • cad11 cadherin 11
  • cTEC cortical thymic epithelial cell
  • Dll1 delta-like 1
  • Dll4 delta-like 4
  • DN double negative
  • DP double positive
  • EB Embryoid Body
  • E-cad (ECD) E-cadherin
  • ES cell embryonic stem cell
  • Eya1 eyes absent 1 homologue
  • FCM Flow cytometry
  • Fgf10 fibroblast growth factor 10
  • Fgf7 fibroblast growth factor 7
  • Fgf8 fibroblast growth factor 8
  • Fgfr2-IIIb Fgf receptor 2 isoform IIIb
  • Fgfs fibroblast growth factors
  • FoxN1 forkhead box N1
  • FTOC fetal thymic organ culture
  • Gcm2 gliotanib
  • the present invention provides a method for inducing differentiation of pluripotent stem cells into thymic epithelial progenitor cells or medullary thymic epithelial cells (hereinafter also referred to as “differentiation induction method of the present invention”).
  • pluripotent stem cell refers to a cell having the ability to differentiate into various tissues and organs. Examples of pluripotent stem cells are ES cells (embryonic stem cells), EG cells (embryonic germ cells) and iPS cells (artificial pluripotent stem cells).
  • the pluripotent stem cell is preferably a human cell.
  • Pluripotent stem cells can be prepared according to a known method. For example, for the production of ES cells, Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al Stem Cells 24: 2669-2676, 2006; Wassarman, PM et al. Methods in Enzymology, Vol.365, 2003, etc. Regarding the production of iPS cells, Takahashi K., et al.
  • ES cells can be obtained from Center for Stem Cell Medicine, Institute for Regenerative Medicine, Kyoto University, WiCell Research Institute (Madison, USA), ES Cell International Pte Ltd (Singapore), and the like.
  • iPS cells can be provided from Kyoto University or RIKEN BioResource Center.
  • iPS cells produced by Dr. Yamanaka et al. Have excellent characteristics such as being able to solve ethical problems associated with conventional pluripotent stem cells (ES cells, etc.). Therefore, iPS cells are preferably employed as pluripotent stem cells.
  • Thymic epithelial progenitor cells refer to proliferating cells that are destined to differentiate into thymic epithelial cells (ie, differentiate into thymic epithelial cells when placed in an appropriate environment).
  • the “medullary thymic epithelial cell” is an epithelial cell constituting the medulla of the thymus.
  • the cells that make up the cortical region of the thymus are called cortical thymic epithelial cells (cTEC).
  • “Inducing differentiation” means acting to differentiate along a specific cell lineage. In the present invention, differentiation is induced into thymic epithelial progenitor cells or further differentiated medullary thymic epithelial cells.
  • the differentiation induction method of the present invention comprises two stages of induction, ie, a step of differentiating pluripotent stem cells into definitive endoderm cells (first induction step), FGF8, FGF7, FGF10, BMP4 and lithium chloride (LiCl). ) In combination with a thymic epithelial progenitor cell or a further differentiated medullary thymic epithelial cell (second induction step).
  • first induction step a step of differentiating pluripotent stem cells into definitive endoderm cells
  • FGF8 FGF7 FGF10
  • BMP4 lithium chloride
  • LiCl lithium chloride
  • pluripotent stem cells are cultured and differentiated into definitive endoderm cells.
  • pluripotent stem cells are cultured under conditions that induce differentiation into definitive endoderm cells.
  • iPS cells with few ethical problems are preferably used as pluripotent stem cells, other pluripotent stem cells such as ES cells can also be used.
  • the definitive endoderm is formed via the mesendoderm.
  • the aim is to differentiate the definitive endoderm via the endomesoderm into thymic epithelial progenitor cells.
  • pluripotent stem cells are cultured in a medium to which, for example, activin (preferably activin A) and lithium chloride (LiCl) are added so as to differentiate into endomesoderm.
  • activin concentration is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 30 ng / ml.
  • concentration of LiCl is, for example, 0.5 to 30 mM.
  • the concentration of LiCl is preferably 2 to 10 mM, more preferably about 5 mM.
  • the period of the first induction step is, for example, 2 to 7 days, preferably 3 to 5 days, and more preferably 4 days.
  • the culture conditions are not particularly limited except that the culture conditions described above are satisfied.
  • a culture condition of “use a medium not containing serum” is employed as an additional culture condition.
  • a serum-free medium supplemented with activin and LiCl is preferably used. Use of a serum-free medium increases the efficiency of differentiation induction.
  • FGF8, FGF7, FGF10, BMP4 and LiCl are used together to differentiate from definitive endoderm cells to thymic epithelial progenitor cells or medullary thymic epithelial cells.
  • this process is characterized in that five types of factors (FGF8, FGF7, FGF10, BMP4, and LiCl) are used in combination.
  • FGF8 is added to the medium and FGF7 and FGF10 are not added (first culture)
  • FGF7 and FGF10 are added to the medium and FGF8 is not added.
  • the lower culture (second culture) is performed.
  • differentiation into the pharyngeal endoderm proceeds by the action of FGF8, and in the second culture, differentiation further proceeds by the action of FGF7 and FGF10, resulting in thymic epithelial progenitor cells.
  • LiCl is preferably added to the medium throughout the entire culture period of the second induction step. This can promote differentiation.
  • BMP4 is preferably added to the medium during the second culture. Thus, BMP4 should be added when differentiation has progressed to some extent. Thereby, differentiation into thymic epithelium can be determined and differentiation can be promoted.
  • LiCl has the same action as Wnt, which is known to increase the expression of FoxN1, an important gene in thymic development.
  • FGF7 and FGF10 are factors released from the mesenchymal tissue around the thymic primordia and are closely related to thymic development.
  • BMP4 which is also a bone morphogenetic factor, is expressed in the thymic primordium, and thymic dysplasia has been reported in BMP4-deficient mice.
  • thymic hypoplasia has been reported in FGF8-deficient mice.
  • these factors are used in combination according to the unique combination and use period as defined above, and pluripotent stem cells differentiated into definitive endoderm cells in the first induction step are induced to differentiate into thymic epithelium.
  • FGF8 0.4 ng / ml to 40 ng / ml (2 ng / ml to 20 ng / ml)
  • FGF7 2 ng / ml to 200 ng / ml (10 ng / ml to 100 ng / ml)
  • FGF10 0.5 ng / ml to 50 ng / ml (2.5 ng / ml to 25 ng / ml)
  • BMP4 1 ng / ml to 100 ng / ml (5 ng / ml to 50 ng / ml)
  • LiCl 0.5 mM to 30 mM (2 mM to 10 mM)
  • the culture period of the first culture is, for example, 2 to 7 days, preferably 2 to 4 days, and more preferably 3 days.
  • the culture period of the second culture is, for example, 4 to 20 days, preferably 6 to 15 days, and more preferably 7 to 10 days.
  • the culture conditions are not particularly limited except that the culture conditions described above are satisfied.
  • the second induction step preferably employs a culture condition of “use a medium not containing serum” as an additional culture condition. Use of a serum-free medium increases the efficiency of differentiation induction.
  • the culture is performed under the condition where RANK ligand or CD40 ligand is added to the medium (third culture).
  • This third culture is performed to induce differentiation and induce medullary thymic epithelial cells. It is more preferable to use a medium supplemented with LiCl in addition to RANK ligand or CD40 ligand. Therefore, in a preferred embodiment, the cells are cultured using a serum-free medium supplemented with RANK ligand or CD40 ligand and LiCl.
  • One or more factors FGF7, FGF10, BMP4 added to the medium during the second culture may be used in combination.
  • RANK ligand and CD40 ligand are not exclusive and may be used in combination.
  • the concentration of RANK ligand or CD40 ligand added is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 15 ng / ml.
  • the culture period of the third culture is, for example, 2 to 20 days, preferably 3 to 15 days, and more preferably 4 to 10 days.
  • RANK and CD40 ligands are important factors for the development of the medullary region and the construction of self-tolerance in the thymus (Ref. 18). By using these, medullary thymic epithelial cells are selectively generated from thymic epithelial progenitor cells. Can be made.
  • the RANK ligand is preferably used in the present invention.
  • the first culture is performed under the condition of adding FGF8, LiCl and activin.
  • FGF8 LiCl and activin.
  • target cells can be obtained efficiently.
  • the use of this condition and the use of the third culture are not mutually exclusive. That is, also in this embodiment, the third culture may be performed.
  • the cell population obtained through the first induction step or a part thereof is subjected to the second induction step without being selected.
  • the second induction step may be performed.
  • the selection of definitive endoderm cells may be performed, for example, with a flow cytometer (cell sorter) using a cell surface marker as an index.
  • Thymic epithelial progenitor cells are obtained by the second induction step. That is, thymic epithelial progenitor cells are recognized as at least part of the cell population.
  • a thymic epithelial progenitor cell can be confirmed by the expression of Pax1, Pax9, Eya1, Six1, etc., for example.
  • the third culture is also carried out following the first culture and the second culture, further differentiated medullary thymic epithelial cells are obtained. That is, medullary thymic epithelial cells are recognized as at least a part of the cell population.
  • the cell population is a state in which thymic epithelial progenitor cells and medullary thymic epithelial cells are mixed.
  • a medullary thymic epithelial cell can be confirmed, for example, by the fact that keratin 5 is expressed but keratin 8 is not expressed (K5 +, K8-) and FoxN1 is expressed.
  • a cell population consisting only of the target cells (thymic epithelial progenitor cells and / or medullary thymic epithelial cells) or a cell population that contains the target cells in a high ratio (high purity), it is characterized by the target cells.
  • the cultured cell population may be selected and sorted using a typical cell surface marker as an index.
  • the cells may be seeded at a cell density of about 1 ⁇ 10 4 cells / cm 2 to 1 ⁇ 10 6 cells / cm 2 .
  • cells are cultured two-dimensionally using a culture dish or the like, but three-dimensional culture using a gel-like culture substrate or the like may be performed.
  • BSA bovine serum albumin
  • KSR serum substitutes
  • reducing agents 2-mercaptoethanol, etc.
  • SF-03 Sudo Junyaku Co., Ltd.
  • culture conditions such as the culture temperature may be those generally employed in animal cell culture. That is, for example, it may be cultured in an environment of 37 ° C. and 5% CO 2 .
  • TRIzol Reagent invitrogen
  • Glycogen Roche
  • cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) according to the attached protocol.
  • PCR was performed using Takara Ex Taq (TaKaRa). The used primers are as follows.
  • ⁇ -actin Fw forward
  • ⁇ -actin Rv reverse
  • FoxN1 Fw actcttcccaaagcccatct: SEQ ID NO: 3
  • FoxN1 Rv agcaatggggtctttcctct: SEQ ID NO: 4
  • Pax1 Fw gcagccggctacctatctc: SEQ ID NO: 5
  • Pax1 Rv ggcagtccgtgtaagctacc: SEQ ID NO: 6
  • Pax3 Fw ctgcactcaagggactcctc: SEQ ID NO: 7
  • Patxc Pax9 Fw
  • the ECCD2 monoclonal antibody was labeled by standard methods using allophycocyanin (APC).
  • APC allophycocyanin
  • Biotinylated anti-mouse EpCAM antibody was purchased from BioLegend (California, USA).
  • MTS24 was provided by Boyd, RL (Monash Immunology and Stem Cell Laboratories, Monash University, Australia).
  • ES cells were provided by Dr. Sakurai (Kyoto University).
  • IPS (APS0001) cells were purchased from Riken Cell Bank. These cells were maintained using 2 mM L-glutamine (GIBCO) and 0.1 mM Non-Essential MEM (GIBCO), 50 units / ml-50 mg / ml penicillin-streptomycin (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma Aldrich ), 10 3 units / ml LIF (15% FBS / KNOCKOUT DMEM (invitrogen) medium containing leukemia inhibitory factor) .Cells were passaged once every two days.
  • a culture dish coated with 0.1% Gelatin from porcine skin (Type A) (SIGMA) / DW was used.
  • SIGMA porcine skin
  • a culture dish coated with 0.1% Gelatin from porcine skin (Type A) (SIGMA) / DW was used.
  • cells were stored in liquid nitrogen and used as early as possible after thawing culture for the experiments, and were collected by dissociating the cells with 0.25% Trypsin-EDTA (GIBCO) at the time of passage. After the treatment, the supernatant was removed and 4 ⁇ 10 5 cells were seeded on a 10 cm culture dish.
  • B Reagent used for differentiation induction
  • serum-free SF-03 (Sanko Junyaku) containing 0.2% BSA was used.
  • Factors used for differentiation induction were activin A (R & D systems), FGF8b (R & D systems), FGF7 (R & D systems), FGF10 (R & D systems), BMP4 (R & D systems), LiCl (Sigma Aldrich) and 2-mercaptoethanol.
  • a culture dish coated with collagen type IV (Nitta gelatin) was used. Cell number upon induction of differentiation was 2 ⁇ 10 5 / 10cm dish.
  • Tissue analysis Preparation of frozen section
  • the transplanted collagen sponge was recovered after 3 weeks, embedded in OCT compound (Sakura), and solidified with liquid nitrogen.
  • a frozen microtome for light microscopy (CW3050-4, common equipment) was used to prepare a frozen section having a thickness of 5 ⁇ m and attached to a slide glass (MATSUNAMI) with an APS coat.
  • Another method is a monolayer culture method, which is simply a method of culturing cells on a culture dish. Since this method has a two-dimensional (planar) structure, unlike EB, it is considered that a factor acts uniformly on all cells, and it is possible to increase the efficiency of inducing differentiation into specific cells. In addition, the single layer culture method has little interaction with surrounding cells, and by changing the factors added to the medium, the factors necessary for the developmental stage can be determined, which is also useful as a developmental research tool. it is conceivable that. Therefore, the monolayer culture method was adopted in this experiment.
  • inducers include Nordal, activin, and BMP, which are known from early blastocyst to late gastrulation embryo.
  • activin a method for inducing mesendoderm by activin A is actually known (references 2 and 8).
  • BMP an induction system to mesoderm using BMP4 is known.
  • lithium suppresses the activity of GSK-3b and stimulates the canonical Wnt pathway (Wnt- ⁇ -catenin pathway) via ⁇ -catenin, which is also known as an early development pathway (Reference 28). ).
  • activin A (10 ng / ml) alone differentiated into endoderm and definitive endoderm (FIG. 1).
  • FIG. 1 it was examined whether the difference in differentiation efficiency was caused by the difference in cell concentration during differentiation induction.
  • activin A concentration was 10 ng / ml
  • the cell concentration per area was The lower the cell, the more efficiently differentiated, and as the cell concentration increased, the proportion of cells in the endoderm and definitive endoderm decreased (results not shown).
  • LiCl alone did not differentiate into endoderm and definitive endoderm (results not shown), but when activin A (10 ng / ml) and LiCl were used together, differentiation into endomesoderm was promoted, The optimal LiCl concentration was 5 mM ( Figure 2). Under this condition, the cell group of the inner mesoderm increased about 1.6 times compared with the case of activin A alone (13.2%) (FIGS. 1 and 2). Also when iPS cells were used, endoderm cell groups were efficiently induced by the combined use of activin A (10 ng / ml) and LiCl (5 mM) (FIG. 3). When serum was added to a serum-free medium containing activin A, differentiation into endomesoderm and definitive endoderm did not occur (results not shown). From this result, it was suggested that the component contained in the serum works to suppress activin A.
  • Wnt stimulation involved in early development is a factor that determines the position of the dorsal side.
  • activin and Nordal are induced to differentiate the inner mesoderm.
  • the failure to induce in the cells is considered to be correct embryologically.
  • the differentiation into endomesoderm was promoted by induction with both activin A and LiCl when the endomesoderm was induced by activin. It suggests that there is some relationship.
  • NCCs neural crest cells
  • Reciprocal signals between endoderm and mesenchymal cells control primordial positioning and outgrowth, and cascades including patterning are expected.
  • signal pathways including FGFs, Wnt, BMPs, and SHH, which are major developmental signal pathways, are involved in thymic development, the details have not been elucidated.
  • FGF8 is expressed in pharyngeal sac endoderm at E10.5, and this expression is presumably a signal to surrounding NCC mesenchymal cells.
  • FGFR2-IIIb is expressed in TEC from about E13.5, and is thought to receive FGF7 and FGF10 signals released by surrounding cells (reference documents 24, 27, 29).
  • the expression of FGFR2-IIIb was also detected in the mesenchymal cells in E14 as a result of RT-PCR.
  • BMP4 also currently supports models that promote both FoxN1 and FGFR2-IIIb expression and promote TEC reactivity to FGF7 and FGF10 signals from mesenchymal cells.
  • the Wnt signal has also been suggested to have a function in the differentiation of TEC itself and the differentiation of thymic T cells (Reference Documents 11 and 16). Wnt induces FoxN1 expression, and is thought to promote TEC differentiation through regulation and regulation of FoxN1 expression through both autocrine and paracrine mechanisms. It is known that the interaction with the mesenchyme-derived mesenchymal tissue is important for thymic differentiation, so both ES cells and iPS cells are induced into the inner mesoderm and only the endoderm is not removed. We tried to regenerate thymus by inducing differentiation while mixing them.
  • FGF8 expression was confirmed in the second, third, and fourth pharyngeal sac in E9.5, and expression was also confirmed in several other sites (such as the head).
  • the function of FGF8 is thought to induce mesenchymal cells around the third pharyngeal sac, but differentiation was induced by FGF8 in consideration of the possibility of acting on self by the autocrine mechanism.
  • Activin A (10 ng / ml) and LiCl (5 mM) were added, and after induction of differentiation into endoderm and definitive endoderm, FGF8 (4 ng / ml) and LiCl (5 mM) was added and cultured for 3 days.
  • BMP4 is not expressed in the E9.5 third pharyngeal sac endoderm, but expression has been confirmed from E10.5 to the anterior part of the third pharyngeal sac (Reference 4).
  • Noggin a BMP4 inhibitor
  • Noggin a BMP4 inhibitor
  • FGF7 and FGF10 are known to be involved in thymic primordium development, and expression of FGF7 and FGF10 was confirmed by RT-PCR results in mesenchymal cells around the thymic primordium in E13.5 Has been.
  • FGFR2-IIIb which is a receptor for FGF7 and FGF10, has been confirmed to be expressed at the thymic primordia site at E13.5, which is the same period, and FGFR2-IIIb is activated by stimulation with FGF7 and FGF10.
  • the expression of FGF7 and FGF10 occurs after the formation of thymic primordia from the pharyngeal sac where FGF8 is expressed. After culturing in a medium supplemented with FGF8 and LiCl (Day 4 (D4) -7) On the first day (D7)), BMP4, FGF7, FGF10 and LiCl were subsequently added and cultured.
  • IPS cells were used to examine the relationship between the concentration of each factor added and differentiation induction.
  • culture was carried out on the 7th day (D7) to the 12th day (D12) while changing only the concentration of FGF10 (2 to 20 ng / ml) and keeping other factors constant.
  • FGF10 2 to 20 ng / ml
  • the optimum concentration of FGF10 was 5 ng / ml.
  • the same experiment was carried out by changing only the concentration of BMP4 (1 to 10 ng / ml) and keeping the other factors constant.
  • Dll1 and Dll4 (reference document 33), which are required for thymocyte differentiation, was weakly expressed even when undifferentiated, but the expression of Dll4 slightly increased from day 7 of culture (D7) ( Results not shown).
  • Other factors known for thymic development include Tbx1 involved in developmental stage, p63 involved in stem cell maintenance, FGFR2 as a receptor for FGFs, and K5 as a marker for thymic medulla (Refs. 9, 10, 16) Expression also increased according to differentiation induction (FIG. 5).
  • the expression of Nanog decreased with time, suggesting that differentiation is progressing (FIG. 5), and the expression of Nanog and Oct3 / 4 did not disappear completely, and the expression of p63 increased.
  • stem cells other than ES cells When the differentiation-induced cells were subcultured in serum-containing medium, colonies appeared and were considered to be holoclones because they grew, and this cell also expressed genes related to the thymus. Not shown). From these results, it was suggested that epithelial stem cells can be recovered from cells containing mature cells with low proliferation ability by passage of differentiated cells. In addition, observation of the subcultured cells and the results of FCM showed that not only completely uniform cells but also some cell groups were mixed.
  • GSC the marker gene of endomesoderm
  • EpCAM1 which is a surface marker of thymic epithelial cells
  • MTS24 expression which is known to regulate the subpopulation of thymic epithelial progenitor cells in the early developmental stage
  • Thymic epithelial progenitor cells are precursor cells of cortical thymic epithelial cells (cTEC) and medullary thymic epithelial cells (mTEC). Therefore, an attempt was made to differentiate thymic epithelial progenitor cells generated by the above differentiation induction method into medullary thymic epithelial cells.
  • RANK ligand and CD40 ligand were reported as important factors in the development of medullary region and self-tolerance in the thymus, and it was suggested that RANK ligand is more important in the construction of early thymic medulla region (Reference 18). Therefore, in order to induce differentiation into medullary thymic epithelial cells, the cells were cultured in a medium supplemented with RANK ligand (10 ng / ml) and LiCl (5 mM) from day 12 of culture (FIG. 7).
  • E-cad positive cells and PDGFR- ⁇ positive cells were densely present at different sites.
  • the E-cad positive cell group is a cell derived from the endoderm system, and the PDGFR- ⁇ positive cell group can be considered to be a cell derived from the mesoderm (reference documents 33 and 34). It was suggested that at least endoderm cells and mesodermal cells differentiated by the method, and each formed a population.
  • the cell group obtained by adding activin A from the fourth day of culture (D4) to the seventh day of culture (D7) will be referred to as A + cell group, and the cell group without activin A added is the A ⁇ cell group. I decided to call it.
  • this A + cell group strongly expressed the target gene (FIG. 12).
  • FGF7, FGF10, BMP4 and LiCL from the seventh day of culture (D7) and continuing the culture for seven days, the expression of many genes related to thymic development increases, and FoxN1 is a marker for thymic epithelial cells. Also significantly increased (FIG. 12).
  • the addition of activin A induces most cell groups to endoderm cell groups, with the result that no factor from other cells is present in the medium (ie, added FGF7, FGF10, It is thought that the differentiation progressed efficiently in the direction of thymic epithelial cells.
  • thymic epithelial progenitor cells or medullary thymic epithelial cells can be prepared in vitro. These cells can be used as a therapeutic material for thymic disorders. It is also expected that a thymus-like tissue / organ is constructed using cells obtained by the method of the present invention and used as an educational organ for T cells.

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Abstract

La présente invention concerne un moyen pour induire la différenciation d'une cellule-souche pluripotente en une cellule progénitrice épithéliale thymique ou une cellule épithéliale thymique médullaire. Le moyen comprend la mise en œuvre d'une première étape d'induction consistant à provoquer la différenciation d'une cellule-souche pluripotente en une cellule endodermique définitive et d'une seconde étape d'induction consistant à provoquer la différenciation de la cellule endodermique définitive en une cellule progénitrice épithéliale thymique ou une cellule épithéliale thymique médullaire utilisant une combinaison des FGF8, FGF7, FGF10, BMP4 et du chlorure de lithium, induisant de ce fait la différenciation de la cellule-souche pluripotente en la cellule progénitrice épithéliale thymique ou la cellule épithéliale thymique médullaire. Lors de la seconde étape d'induction, une première culture est mise en œuvre dans des conditions dans lesquelles le FGF8 est ajouté mais les FGF7 et FGF10 ne sont pas ajoutés à un milieu de culture et, à la suite de la première culture, une seconde culture est mise en œuvre dans des conditions dans lesquelles les FGF7 et FGF10 sont ajoutés à un milieu de culture.
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EP3853355A4 (fr) * 2018-09-20 2022-07-27 The Trustees of Columbia University in the City of New York Thymus hybride, procédés de fabrication, et méthodes d'utilisation pour induire une tolérance vis-à-vis d'une xénogreffe, et restaurer une immunocompétence et la fonction thymique
WO2023243627A1 (fr) * 2022-06-17 2023-12-21 国立大学法人京都大学 Procédé de production de cellules épithéliales thymiques

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US9850465B2 (en) 2013-02-27 2017-12-26 The Regents Of The University Of California Generation of thymic epithelial progenitor cells in vitro
WO2014134213A1 (fr) * 2013-02-27 2014-09-04 The Regents Of The University Of California Production in vitro de cellules progénitrices épithéliales thymiques
JP2016512031A (ja) * 2013-03-13 2016-04-25 ウイスコンシン アラムナイ リサーチ ファウンデーシヨンＷｉｓｃｏｎｓｉｎ Ａｌｕｍｎｉ Ｒｅｓｅａｒｃｈ Ｆｏｕｎｄａｔｉｏｎ 定義された条件下におけるヒト多能性幹細胞の造血内皮分化のための方法及び材料
WO2019060336A1 (fr) * 2017-09-20 2019-03-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Génération in vitro d'organoïde thymique à partir de cellules souches pluripotentes humaines
US11898166B2 (en) 2017-09-20 2024-02-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services In vitro generation of thymic organoid from human pluripotent stem cells
JP2020534004A (ja) * 2017-09-20 2020-11-26 アメリカ合衆国 ヒト多能性幹細胞由来の胸腺オルガノイドのインビトロ生成
JP7372910B2 (ja) 2017-09-20 2023-11-01 アメリカ合衆国 ヒト多能性幹細胞由来の胸腺オルガノイドのインビトロ生成
EP3853355A4 (fr) * 2018-09-20 2022-07-27 The Trustees of Columbia University in the City of New York Thymus hybride, procédés de fabrication, et méthodes d'utilisation pour induire une tolérance vis-à-vis d'une xénogreffe, et restaurer une immunocompétence et la fonction thymique
EP3947639A4 (fr) * 2019-04-01 2023-01-25 The Trustees of Columbia University in the City of New York Procédés pour favoriser la différenciation de cellules souches pluripotentes en cellules épithéliales thymiques et en progéniteurs de cellules épithéliales thymiques
CN113646423A (zh) * 2019-04-01 2021-11-12 纽约市哥伦比亚大学理事会 促进多能干细胞的胸腺上皮细胞和胸腺上皮细胞祖细胞的分化的方法
WO2020205859A1 (fr) 2019-04-01 2020-10-08 The Trustees Of Columbia University In The City Of New York Procédés pour favoriser la différenciation de cellules souches pluripotentes en cellules épithéliales thymiques et en progéniteurs de cellules épithéliales thymiques
CN113767167A (zh) * 2019-04-26 2021-12-07 科罗拉多大学董事会，法人 从诱导多能干细胞体内产生功能性和患者特异性胸腺组织
EP3959304A4 (fr) * 2019-04-26 2023-01-25 The Regents Of The University Of Colorado, A Body Corporate Génération de tissu thymique fonctionnel et spécifique au patient in vivo à partir de cellules souches pluripotentes induites
JP7176748B2 (ja) 2019-07-22 2022-11-22 学校法人関西医科大学 Ｔ細胞受容体γδ陽性細胞の誘導方法
JP2021016356A (ja) * 2019-07-22 2021-02-15 学校法人関西医科大学 Ｔ細胞受容体γδ陽性細胞の誘導方法
WO2023243627A1 (fr) * 2022-06-17 2023-12-21 国立大学法人京都大学 Procédé de production de cellules épithéliales thymiques

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