Construction method and application of thymus organoid

Abstract

The invention discloses a construction method and application of thymus organoid, and the method comprises the following steps: differentiating pluripotent stem cells into thymus organoid under a suspension culture condition through combined and matched use of small molecule compounds and small molecule proteins, according to the method disclosed by the invention, the thymus organoid of which the structure and the function are closer to those of natural thymus can be efficiently obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids, and specifically relates to a construction method and application of thymic organoids. Background Art

[0002] The thymus is an important immune organ in the human body and the site of T cell differentiation and maturation. Aging or damage can easily lead to decreased immunity or autoimmune diseases or even malignant tumors. Therefore, when the thymus is diseased, it will have a serious impact on the body's immune function, causing the body's immune homeostasis to be disordered and accompanied by autoimmune diseases. T cell defects caused by thymic dysfunction make patients extremely susceptible to infection, causing malignant tumors and / or autoimmune diseases. In addition to thymic degeneration with aging, damage to thymic epithelial cells (TECs) can be caused by many external factors, including infection, radiation, immunosuppressant therapy, and graft-versus-host disease after hematopoietic stem cell transplantation.

[0003] Organoids can differentiate into a variety of organ-specific cell types and reproduce some of the functions and spatial structures of the corresponding organs. Their birth has injected new impetus into life medicine research and clinical applications, and they have shown broad application prospects in the fields of basic and clinical research on cancer, regenerative medicine, etc. Previously, people have successfully cultured organoids such as brain, intestine, liver, lung, kidney, tongue, and retina. However, progress in the field of thymic organoids has been slow. For many years, researchers have mainly explored the development process of thymic epithelial cells (TEC) through in vitro two-dimensional (2D) culture systems to reveal the mechanisms of thymic damage and regeneration. However, the monolayer 2D culture system can neither reproduce the unique three-dimensional (3D) epithelial reticular structure of the thymus, nor can it fully provide other supporting cell-derived cytokines and growth factors required for the directed differentiation of hematopoietic stem cells into functional T cells; there has been no report on the technology of constructing 3D thymic organoids using human pluripotent stem cells in vitro.

[0004] At present, there are three major bottlenecks in academic research in this field: first, it is impossible to simulate the positive selection and negative selection process of the human thymus in vitro; second, the in vitro preparation of allogeneic T cells cannot achieve complete HLA matching between allogeneic cells, resulting in immune rejection; third, it is difficult to prepare functional CD4-positive T cells in vitro. However, there has been no report on the use of human pluripotent stem cells to differentiate into truly functional thymic organoids, especially in vivo functions.

[0005] Previously, only a few researchers isolated thymic epithelial cells (TEC) and thymic stromal cells (TIC) from natural human thymus tissue, and then constructed tissue-engineered thymus with various scaffold materials, such as collagen scaffolds [1, 2, 3], thymic decellularized scaffolds [4, 5], and tantalum metal scaffolds [6, 7] to obtain a reassembled thymus-like structure in vivo. However, due to the very limited source of natural human thymus samples, thymus assembled from scaffolds or natural thymic epithelial cells or thymic stromal cells cannot be mass-produced to meet scientific research and clinical needs.

[0006] Currently, some researchers have differentiated human embryonic stem cells (hESCs) into thymic epithelial progenitor cells (TEPs)[8]. The differentiation process is entirely two-dimensional (2D) cell culture. After differentiation into thymic epithelial progenitor cells in vitro, the cells are transplanted into nude mice and eventually mature into thymic epithelial cells.

[0007] However, this differentiation process does not take into account the natural thymic structure and cell composition. The thymus is composed of a variety of thymic stromal cells, not a single thymic epithelial cell. In addition to thymic epithelial cells, the thymic stroma also includes fibroblasts, endothelial cells, etc. They are components of the thymic substance and vascular structure, and play an indispensable role in the development and function of thymic epithelial cells [9], and are also necessary to ensure the function of the thymus. A portion of thymic endothelial cells are surrounded by pericytes, which are specialized contractile fibroblast-like cells that express smooth muscle actin (α-SMA)

[10] . They promote the maturation of thymic epithelial cells through cell-to-cell interactions.

[0008] The disadvantages of the above-mentioned existing technologies are: 1. Human pluripotent stem cells can only be differentiated into thymic epithelial progenitor cells in vitro, and then the differentiated cells need to be transplanted into the body of immunodeficient mice to rely on the in vivo environment to further mature into thymic epithelial cells; 2. The differentiation process uses a large number of expensive cell growth factors, so the reagent cost is high; 3. The use of 2D plate differentiation method does not take into account the three-dimensional developmental niche of the natural thymic organ; 4. This method differentiates into a single thymic epithelial progenitor cell, and does not take into account the support of other stromal cells of the thymus, such as mesenchymal cells, fibroblasts, endothelial cells, etc. for thymic epithelial cells and the necessity of persistent cell signal regulation for the function of the thymus.

[0009] References

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[0011] [2] H,Sousa AE.Repairing thymic function.Curr Opin OrganTransplant,2013,18(3):363-368.

[0012] [3]Gostynska N,Shankar Krishnakumar G,Campodoni E,et al.3D porouscollagen scaffolds reinforced by glycation with ribose for tissue engineeringapplication.Biomed Mater,2017,12(5):055002.

[0013] [4]Campinoti S,Gjinovci A,Ragazzini R,et al.Reconstitution of afunctional human thymus by postnatal stromal progenitor cells and naturalwhole-organ scaffolds.Nat Commun,2020,11(1):6372.

[0014] [5]Schmitt TM, JC.Induction of T cell development fromhematopoietic progenitor cells by delta-like-1in vitro.Immunity,2002,17(6):749-756.

[0015] [6]Bobyn JD,Stackpool GJ,Hacking SA,et al.Characteristics of boneingrowth and interface mechanics of a new porous tantalum biomaterial.J BoneJoint Surg Br,1999,81(5):907-914.

[0016] [7]Marshall D, Bagley J, Le P, et al. T cell generation including positive and negative selection ex vivo in a three-dimensional matrix. JHematother Stem Cell Res, 2003, 12(5):565-574.

[0017] [8] Parent AV, Russ HA, Khan IS, et al. Generation of functional thymicepithelium from human embryonic stem cells that supports host T cell development. Cell Stem Cell. 2013; 13(2): 219-229.

[0018] [9] Nitta T, Takayanagi H. Non-Epithelial Thymic Stromal Cells: UnsungHeroes in Thymus Organogenesis and T Cell Development. Front Immunol. 2021; 11: 620894.

[0019]

[10] Ushiki T, Takeda M. Three-dimensional ultrastructure of the perivascular space in the rat thymus. Arch Histol Cytol (1997) 60:89–99. Summary of the invention

[0020] The purpose of the present application is to provide a method for constructing thymic organoids, which fully simulates the development of the natural thymus, and the tissue structure, cell composition and function of the constructed thymic organoids are highly consistent with those of the natural thymus.

[0021] The first aspect of the present invention provides a culture medium combination, which includes a first culture medium and a second culture medium; the first culture medium includes a first basic culture medium supplemented with Activin A, a GSK-3 inhibitor and a ROCK inhibitor; the second culture medium includes a second basic culture medium supplemented with Activin A and a B-27 additive.

[0022] In some embodiments of the present invention, the GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0023] In some embodiments of the present invention, the GSK-3 inhibitor is CHIR99021.

[0024] In some embodiments of the present invention, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447 and Azaindole 1.

[0025] In some embodiments of the present invention, the ROCK inhibitor is Y-27632.

[0026] In some embodiments of the present invention, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL.

[0027] In some embodiments of the present invention, the concentration of CHIR99021 added to the first basal culture medium is 4-6 μM.

[0028] In some embodiments of the present invention, the concentration of Y-27632 added to the first basal culture medium is 8-12 μM.

[0029] In some embodiments of the present invention, the concentration of Activin A added to the second basal culture medium is 80-120 ng / mL.

[0030] In some embodiments of the present invention, the concentration of B-27 additive added to the second basal culture medium is 0.5-1.5×.

[0031] In some embodiments of the present invention, the culture medium combination further comprises a third culture medium; the third culture medium comprises a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist.

[0032] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0033] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0034] In some embodiments of the invention, the Notch receptor agonist is selected from DLL4.

[0035] In some embodiments of the present invention, the concentration of SB431542 added to the third basal culture medium is 8-12 μM.

[0036] In some embodiments of the present invention, the concentration of DLL4 added to the third basal culture medium is 8-12 ng / mL.

[0037] In some embodiments of the present invention, the culture medium combination further includes a fourth culture medium, wherein the fourth culture medium includes a fourth basal culture medium supplemented with a TGF-β signaling pathway inhibitor, a Notch receptor agonist, and a cytokine.

[0038] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0039] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0040] In some embodiments of the invention, the Notch receptor agonist comprises DLL4.

[0041] In some embodiments of the invention, the cytokines include FLT3L, TPO, and SCF.

[0042] In some embodiments of the present invention, the concentration of SB431542 added to the fourth basal culture medium is 8-12 μM.

[0043] In some embodiments of the present invention, the concentration of DLL4 added to the fourth basal culture medium is 8-12 ng / mL.

[0044] In some embodiments of the present invention, the added concentration of TPO in the fourth basal culture medium is 4-6 ng / mL.

[0045] In some embodiments of the present invention, the concentration of FLT3L added to the fourth basal culture medium is 4-6 ng / mL.

[0046] In some embodiments of the present invention, the concentration of SCF added to the fourth basal culture medium is 40-60 ng / mL.

[0047] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are stem cell culture media;

[0048] The third basal culture medium and the fourth basal culture medium are endothelial cell culture medium, and the endothelial cell culture medium is selected from any one of EGM2 culture medium, ECM culture medium, and VascuLife VEGF Medium Complete Kit culture medium.

[0049] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium.

[0050] In some embodiments of the present invention, the third basal culture medium and the fourth basal culture medium are EGM2 culture medium.

[0051] The second aspect of the present invention provides a kit, which comprises the culture medium combination provided by the first aspect of the present invention.

[0052] In some embodiments of the present invention, trophoblast cells are further included.

[0053] The third aspect of the present invention provides the use of the culture medium combination provided in the first aspect of the present invention or the kit provided in the second aspect of the present invention: (1) preparing mesendoderm cells; (2) preparing third pharyngeal pouch endoderm cells; (3) preparing thymic organoids; 4) preparing products of inducing pluripotent stem cells to differentiate into mesendoderm cells; (5) preparing products of inducing pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; (6) preparing products of inducing pluripotent stem cells to differentiate into thymic organoids.

[0054] The fourth aspect of the present invention provides a method for preparing mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids, wherein pluripotent stem cells are cultured using the kit provided by the second aspect of the present invention to obtain mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids; the culture is a suspension culture.

[0055] In some embodiments of the present invention, the method specifically comprises:

[0056] (1) culturing pluripotent stem cells in the first culture medium and the second culture medium in the kit provided in the second aspect of the present invention successively to obtain mesendoderm cells; and / or

[0057] (2) co-culturing the mesendoderm cells and the trophoblast cells in the third culture medium in the kit provided by the second aspect of the present invention to obtain third pharyngeal pouch endoderm cells; and / or

[0058] (3) Cultivating the third pharyngeal pouch endoderm cells in the fourth culture medium in the kit provided in the second aspect of the present invention to obtain thymic organoids.

[0059] The fifth aspect of the present invention provides the use of thymic organoids prepared by the method provided in the fourth aspect of the present invention in any of the following items: 1) for constructing humanized blood and / or immune system animals; 2) for preparing products for constructing humanized blood and / or immune system animals; 3) for simulating human diseases; 4) for preparing products for simulating human diseases; 5) for preparing human T cells; 6) for preparing products for preparing human T cells; 7) for preparing products for inducing transplant tolerance; 8) for preparing products for restoring thymic function; 9) for producing humanized antibodies; 10) for producing humanized antibodies; 11) thymus-related drug screening, development and / or toxicity analysis; 12) for studying the pathogenic mechanism of thymic diseases; 13) for preparing products for treating thymic diseases; 14) for constructing thymic disease models; 15) thymus toxicology testing.

[0060] The beneficial effects of the present invention are:

[0061] The present invention provides a culture medium combination for inducing directional differentiation of pluripotent stem cells into thymic organoids. By using a combination of small molecule compounds and small molecule proteins, pluripotent stem cells are differentiated into thymic organoids, and thymic organoids with structures and functions closer to natural thymus can be efficiently obtained. During the entire differentiation process, pluripotent stem cells are first differentiated into mesendoderm, and then the formation of the third pharyngeal pouch endoderm is induced, and then the third pharyngeal pouch endoderm is differentiated into thymic organoids.

[0062] The present invention also provides a method for inducing directional differentiation of pluripotent stem cells into thymic organoids. The present invention establishes an efficient 3D suspension culture method for inducing pluripotent stem cells to differentiate into thymic organoids by combining chemical small molecules and small molecule proteins at different concentrations. Compared with the 2D adherent culture induced differentiation system, the cell 3D suspension culture induced differentiation system has the advantage of better simulating the three-dimensional microenvironment of the human body. Therefore, the cell 3D suspension culture induced differentiation system can efficiently obtain thymic organoids whose structure and function are closer to the natural thymus. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Diagram of the method for directing the differentiation of human pluripotent stem cells into thymic organoids.

[0064] Figure 2 The relevant detection results of the mesendoderm cell differentiation stage in Example 2, wherein: Figure 2 Middle A is the immunofluorescence staining of human pluripotent stem cell stemness genes SOX2 and OCT4; Figure 2 Middle B is the flow cytometry detection of human pluripotent stem cell stemness gene SSEA4; Figure 2 Middle C is the RT-qPCR detection of stemness (Pluripotency) genes, ectoderm (Ectoderm), mesoderm (Mesoderm) and endoderm (Endoderm) marker genes during mesendoderm differentiation; Figure 2 Middle D is an image of cell morphological changes during directional differentiation of mesendoderm cell spheres, scale bar = 50 μm; Figure 2 Middle E is the immunofluorescence image of FoxA2 and BRA in mesendodermal cell spheres; Figure 2 Middle F is the flow cytometry detection of CXCR4 and SOX17 in mesendoderm cell spheres; Figure 2 Middle G is a flow cytometry analysis of CXCR4 and SOX17 co-staining in mesendoderm cell spheres.

[0065] Figure 3 The relevant test results of the third pharyngeal pouch endoderm differentiation stage in Example 2, wherein: Figure 3 Middle A is the RT-qPCR detection of key genes of the third pharyngeal pouch endoderm; Figure 3 Middle B is the third pharyngeal pouch endoderm key genes SIX1 and PAX9 cell flow cytometry detection.

[0066] Figure 4 The relevant test results of the thymic organoid differentiation stage in Example 2, wherein: Figure 4 Middle A is the RT-qPCR detection of the key genes K5, K8, and EPCAM of thymic epithelial cells in thymic organoids; Figure 4 Middle B is the flow cytometry detection of thymic epithelial cells expressing stemness CD90 and EPCAM in thymic organoids; Figure 4Middle C is the RT-qPCR detection and cell flow cytometry analysis of the thymic epithelial cell transcription factor FOXN1 in thymic organoids; Figure 4 Middle D is the RT-qPCR detection and cell flow cytometry analysis of the autoimmune regulatory factor AIRE in thymic organoids; Figure 4 Middle E is the RT-qPCR detection and cell flow cytometry detection of MHC-II in thymic organoids; Figure 4 Middle F is the RT-qPCR detection of representative genes of thymic organoid stromal cells; Figure 4 Middle G is the representative functional gene RT-qPCR detection of thymic organoids; Figure 4 Middle H is the immunofluorescence image of the key proteins K5 and K8 of thymic epithelial cells in thymic organoids, scale bar = 50 μm; Figure 4 Middle I is the immunofluorescence staining of FOXN1, a key transcription factor of thymic epithelial cells, and PDGFRa, a mesenchymal cell marker, in thymic organoids. Scale bar = 50 μm.

[0067] Figure 5 The test results of thymic organoids in Example 3, wherein: Figure 5 A in the middle is the UAMP diagram of natural thymus samples and thymic organoids integrated at different time points; Figure 5 Middle B is the correlation analysis between natural thymus samples and thymic organs at different time points; Figure 5 Middle C is a UAMP image of the 19-week fetal thymus sample and thymic organoid integration; Figure 5 Middle D is a bar graph showing the proportions of various cell populations in the 19-week fetal thymus and thymic organoids.

[0068] Figure 6 Example 4: Schematic diagram and test results of the co-culture system, wherein: Figure 6 Middle A is a schematic diagram of the co-culture system of thymic organoids and hematopoietic stem / progenitor cells; Figure 6 Figure B shows the CD45 expression in the co-culture system at the indicated time points. + Representative kinetics of cell differentiation, DAPI - Total viable cells are shown in the gate; Figure 6 C shows the T cell progenitors (CD5 + CD7 + ) Representative kinetics of differentiation, DAPI - CD45 + Total viable cells are shown in the gate; Figure 6 D is the mature T in the co-culture system (CD3 + TCRαβ + ) Representative graphs of cell differentiation ratios; Figure 6 Middle E is the mature T (CD3 + TCRαβ+ ) Statistical graph of cell differentiation ratio; Figure 6 F is the CD3 in the co-culture system + Microscope images and immunofluorescence staining of T cells, representative immunofluorescence images at the 6th week of co-culture, green is CD3 staining; blue is DAPI (nucleus) staining, scale bar = 20 μm; Figure 6 G is a representative kinetic of the differentiation of mature CD4SP and CD8SP T cells in the co-culture system at the indicated time points, DAPI - CD45 + Total viable cells are shown in the gate; Figure 6 H is the time point in the co-culture system CD4SP(CD3 + TCRαβ + CD45RA + CD45RO - CCR7 + CD4 + CD8 - ) and CD8SP (CD3 + TCRαβ + CD45RA + CD45RO - CCR7 + CD4 - CD8 + ) cell ratio statistics (for DAPI - CD45 + Cells were gated); Figure 6 Figure 1 shows the CD3 in the co-culture system at week 6. + TCRγδ + Representative images of T cell differentiation, total live cells are shown in DAPI - CD45 + Gated; Figure 6 The middle J is CD3 in the co-culture system at week 6 + TCRγδ + T cell ratio statistics (gated on DAPI - CD45 + on cells); Figure 6 K is a CD4 + CD25 + The proportion of T cells in the co-culture system at week 6 was CD4 + CD25 + Representative images of T cell differentiation, total live cells are shown in DAPI - CD45 + CD3 + TCRαβ +Gated.

[0069] Figure 7 The relevant test results in Example 5, wherein, Figure 7 Figure A is the flow cytometric analysis of human CD45 in the peripheral blood of hETO mice 8 weeks after HSPCs transplantation. + CD3 + The proportion of T cells; Figure 7 Figure B is the flow cytometric analysis of human CD45 in the peripheral blood of hETO mice 12 weeks after HSPCs transplantation. + CD3 + The proportion of T cells; Figure 7 Figure C shows the expression of human CD45 in the peripheral blood of hETO mice 16 weeks after HSPCs transplantation by flow cytometry. + CD3 + The proportion of T cells; Figure 7 Middle D is the flow cytometry analysis of the expression of CD45, CD8, CD4, CD3, TCRαβ, CD25, FOXP3 and TCRγδ in the bone marrow of hETO mice 11 months after HSPCs transplantation from severely immunodeficient mice; Figure 7 Middle E is the cell flow cytometry analysis of the expression of CD45, CD8, CD4, CD3, TCRαβ, CD25, FOXP3 and TCRγδ in the spleen of hETO mice 11 months after HSPCs transplantation from severely immunodeficient mice.

[0070] Figure 8 is the relevant test result in Example 6, wherein, Figure 8 A in the middle is ELISA analysis showing the levels of human ovalbumin-specific IgM (left) and IgG (right) after one week; Figure 8 Middle (B) is an ELISA analysis showing the levels of human ovalbumin-specific IgM (left) and IgG (right) after two weeks.

[0071] Fig. 9 The relevant test results in Example 7, wherein, Fig. 9 A in the middle is the ratio of human T cells after allogeneic skin transplantation as detected by flow cytometry; Fig. 9 Middle B is a HE-stained image of the transplanted skin piece collected on day 6, scale bar = 300 μm; Fig. 9 Middle C is immunofluorescence staining showing the expression of human CD3 (green) and IL-10 (red) in the transplanted skin, and the cell nucleus was stained with DAPI, scale bar = 20 μm; Fig. 9 D is the flow cytometry analysis showing the CD3 + T cells express the human cytokines IFN-γ, TNFα, and IL-2.

[0072] Fig.10 qRT-PCR was used to detect the relative expression levels of endoderm and mesoderm marker gene mRNA from day 0 to day 3 of differentiation, where A: Activin A; A+B+W: Activin A, B-27 supplement, Wnt3A; A+B+C: Activin A, B-27 supplement and CHIR99021.

[0073] Fig.11 The relative expression levels of thymic marker gene mRNA at day 17 of differentiation were detected by qRT-PCR, where A: ActivinA; A+B+W: Activin A, B-27 supplement, Wnt3A; A+B+C: ActivinA, B-27 supplement and CHIR99021. DETAILED DESCRIPTION

[0074] In a first aspect of the present invention, a culture medium combination is provided, which comprises a first culture medium and a second culture medium; the first culture medium comprises a first basic culture medium supplemented with Activin A, a GSK-3 inhibitor and a ROCK inhibitor; the second culture medium comprises a second basic culture medium supplemented with Activin A and a B-27 additive.

[0075] The first basal medium and the second basal medium in the present invention refer to commercial culture media that can be used for stem cell culture, wherein the "first basal medium supplemented with Activin A, GSK-3 inhibitor and ROCK inhibitor" is obtained by additionally adding Activin A, GSK-3 inhibitor and ROCK inhibitor to a commercial culture medium that can be used for stem cell culture, and the "second basal medium supplemented with Activin A and B-27 additive" is obtained by additionally adding Activin A and B-27 additives to a commercial culture medium that can be used for stem cell culture.

[0076] In some embodiments of the present invention, the GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0077] In some embodiments of the present invention, the GSK-3 inhibitor is CHIR99021. In the present invention, CHIR99021 includes CHIR99021 and salts thereof, especially pharmaceutically acceptable salts.

[0078] In some embodiments of the present invention, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, FasudilHCl, GSK429286A, RKI-1447 and Azaindole 1.

[0079] In some embodiments of the present invention, the ROCK inhibitor is Y-27632. Y-27632 in the present invention includes Y-27632 and salts thereof, especially pharmaceutically acceptable salts.

[0080] In some embodiments of the present invention, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL. Here, the added concentration = the mass of Activin A added / the volume of the first basal culture medium.

[0081] In some embodiments of the present invention, the concentration of CHIR99021 added to the first basal medium is 4-6 μM. Here, the added concentration = the amount of CHIR99021 added / the volume of the first basal medium.

[0082] In some embodiments of the present invention, the concentration of Y-27632 added to the first basal medium is 8-12 μM. Here, the added concentration = the amount of Y-27632 added / the volume of the first basal medium.

[0083] In some embodiments of the present invention, the concentration of Activin A added to the second basal medium is 80-120 ng / mL. Here, the added concentration = the mass of Activin A added / the volume of the second basal medium.

[0084] In some embodiments of the present invention, the concentration of the B-27 additive in the second basal medium is 0.5-1.5×. Here, the added concentration = B-27 additive stock solution multiple * (volume of added B-27 additive / volume of the second basal medium), for example, if the B-27 additive is provided in a 50× liquid form, the original multiple is 50×.

[0085] In some embodiments of the present invention, the culture medium combination further comprises a third culture medium; the third culture medium comprises a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist.

[0086] The third basal culture medium in the present invention refers to a commercial culture medium that can be used for endothelial cell culture, wherein the "third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist" is obtained by additionally adding a TGF-β signaling pathway inhibitor and a Notch receptor agonist to a commercial culture medium that can be used for endothelial cell culture.

[0087] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0088] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0089] In some embodiments of the invention, the Notch receptor agonist is selected from DLL4.

[0090] In some embodiments of the present invention, the concentration of SB431542 added to the third basal medium is 8-12 μM. Here, the added concentration = the amount of SB431542 added / the volume of the third basal medium.

[0091] In some embodiments of the present invention, the concentration of DLL4 added to the third basal medium is 8-12 ng / mL. Here, the added concentration = the mass of DLL4 added / the volume of the third basal medium.

[0092] In some embodiments of the present invention, the fourth culture medium includes a fourth basal culture medium supplemented with a TGF-β signaling pathway inhibitor, a Notch receptor agonist, and a cytokine.

[0093] The fourth basal culture medium in the present invention refers to a commercial culture medium that can be used for endothelial cell culture, wherein the "fourth basal culture medium supplemented with TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines" is obtained by additionally adding TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines to a commercial culture medium that can be used for endothelial cell culture.

[0094] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0095] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0096] In some embodiments of the invention, the Notch receptor agonist comprises DLL4.

[0097] In some embodiments of the invention, the cytokines include FLT3L, TPO, SCF.

[0098] In some embodiments of the present invention, the concentration of SB431542 added to the fourth basal medium is 8-12 μM. Here, the added concentration = the amount of SB431542 added / the volume of the fourth basal medium.

[0099] In some embodiments of the present invention, the concentration of DLL4 added to the fourth basal medium is 8-12 ng / mL. Here, the added concentration = the mass of DLL4 added / the volume of the fourth basal medium.

[0100] In some embodiments of the present invention, the concentration of TPO added to the fourth basal medium is 4-6 ng / mL. Here, the added concentration = the mass of TPO added / the volume of the fourth basal medium.

[0101] In some embodiments of the present invention, the concentration of FLT3L added to the fourth basal medium is 4-6 ng / mL. Here, the added concentration = the mass of FLT3L added / the volume of the fourth basal medium.

[0102] In some embodiments of the present invention, the concentration of SCF added to the fourth basal medium is 40-60 ng / mL. Here, the added concentration = the mass of SCF added / the volume of the fourth basal medium.

[0103] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium.

[0104] In some embodiments of the present invention, the third basal culture medium and the fourth basal culture medium are EGM2 medium.

[0105] The second aspect of the present invention provides a kit, which comprises the culture medium combination provided by the first aspect of the present invention.

[0106] In some embodiments of the present invention, the kit comprises the first culture medium and the second culture medium of the first aspect of the present invention, which are used to induce pluripotent stem cells to differentiate into mesendoderm cells.

[0107] "Pluripotent stem cell" refers to a stem cell having the pluripotency of differentiating into all cells present in a living body, i.e., the three germ layers of endoderm, mesoderm and ectoderm, and also having the ability to proliferate. As the above-mentioned pluripotent stem cell, there is no particular limitation, and examples thereof include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells, etc. Pluripotent stem cells are preferably at least one selected from the group consisting of ES cells, ntES cells and iPS cells. In one aspect of an embodiment of the present invention, pluripotent stem cells are preferably iPS cells or ES cells. The organism that becomes the source of pluripotent stem cells is not particularly limited, preferably derived from mammalian pluripotent stem cells, more preferably derived from primate pluripotent stem cells. Specifically, pluripotent stem cells derived from people can be enumerated.

[0108] The human embryonic stem cells in the present invention are not human embryonic stem cells separated or obtained from human embryos that have undergone in vivo development, and are not stem cells separated or obtained from human embryos that have not undergone in vivo development and are fertilized for more than 14 days.

[0109] In some embodiments of the present invention, the kit further comprises a third culture medium for inducing the mesendoderm cells to differentiate into third pharyngeal pouch endoderm cells.

[0110] In some embodiments of the present invention, the kit further comprises a fourth culture medium and feeder cells for inducing the third pharyngeal pouch endoderm cells to differentiate into thymic organoids.

[0111] In some embodiments of the present invention, the trophoblast cells include at least one of OP9 cells, MS5 cells, HS-5 or other fibroblasts.

[0112] The third aspect of the present invention provides the use of the culture medium combination provided in the first aspect of the present invention or the kit provided in the second aspect of the present invention: (1) preparing mesendoderm cells; (2) preparing third pharyngeal pouch endoderm cells; (3) preparing thymic organoids; (4) preparing products of inducing pluripotent stem cells to differentiate into mesendoderm cells; (5) preparing products of inducing pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; (6) preparing products of inducing pluripotent stem cells to differentiate into thymic organoids.

[0113] The fourth aspect of the present invention provides a method for preparing mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids, by culturing the mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids using the kit provided in the second aspect of the present invention.

[0114] In some embodiments of the invention, the culture is a suspension culture.

[0115] In some embodiments of the present invention, the method specifically comprises:

[0116] (1) culturing pluripotent stem cells in the first culture medium and the second culture medium in the kit provided in the second aspect successively to obtain mesendoderm cells; and / or

[0117] (2) co-culturing the mesendoderm cells and the trophoblast cells in the third culture medium of the kit provided in the second aspect to obtain third pharyngeal pouch endoderm cells; and / or

[0118] (3) Cultivating the third pharyngeal pouch endoderm cells in the fourth culture medium in the kit provided in the second aspect to obtain thymic organoids.

[0119] In some embodiments of the present invention, the culture time for inducing pluripotent stem cells to differentiate into mesendoderm cells is 2-4 days.

[0120] In some embodiments of the present invention, the culture time using the first culture medium is 0.5-1.5 days.

[0121] In some embodiments of the present invention, the culture time using the second culture medium is 1.5-2.5 days.

[0122] In some embodiments of the present invention, the culture time for inducing mesendoderm cells to differentiate into third pharyngeal pouch endoderm cells is 6-8 days.

[0123] In some embodiments of the present invention, the culture time for inducing the third pharyngeal pouch endoderm cells to differentiate into thymic organoids is 6-10 days.

[0124] The fifth aspect of the present invention provides the use of thymic organoids prepared by the preparation method provided by the fourth aspect of the present invention in any of the following items: 1) for constructing humanized blood and / or immune system animals; 2) for preparing products for constructing humanized blood and / or immune system animals; 3) for simulating human diseases; 4) for preparing products for simulating human diseases; 5) for preparing human T cells; 6) for preparing products for preparing human T cells; 7) for preparing products for inducing transplant tolerance; 8) for preparing products for restoring thymic function; 9) for producing humanized antibodies; 10) for producing humanized antibodies; 11) thymus-related drug screening, development and / or toxicity analysis; 12) for studying the pathogenic mechanism of thymic diseases; 13) for preparing products for treating thymic diseases; 14) for constructing thymic disease models; 15) thymus toxicology testing.

[0125] The thymic organoids prepared by the present invention can have multiple uses, for example:

[0126] 1) Establishment of humanized blood and immune system animals

[0127] At present, the humanized mouse model of blood and immune system is a mouse model that reconstructs the human blood and immune system by implanting human hematopoietic stem cells and natural thymus tissue into immunodeficient mice. They can effectively reconstruct the human immune system and better simulate the human immune characteristics. However, the natural thymus resources of humans are scarce and cannot meet the needs of humanized animals. The present invention can directly induce human pluripotent stem cells to produce unlimited yields of thymic organoids, which can meet the needs of preparing humanized immune system model animals.

[0128] 2) Research on immunity and tumor immunity based on humanized immune system animal models

[0129] The immune system of wild-type mice has a strong rejection effect on xenogeneic cells and tissues from humans. Although human tumor transplantation models that rely on immunodeficient mice have been widely used in tumor immunity and new therapy research and development, the lack of human immune system and tumor immune microenvironment has greatly limited the translational research of immune mechanism and immunotherapy. Therefore, the humanized immune system animal model of the present invention can truly simulate the interaction between human tumor cells and the immune system, which is of great significance for tumor immunity research, human diseases and the development of new therapies.

[0130] 3) Virology research based on animal disease models with humanized immune systems

[0131] In the field of pathogenic microorganisms, especially in the study of viruses, humanized mice are widely used, such as HIV, EBV, dengue virus, HCV, etc. Humanized mice can be used for drug testing of human inflammatory responses and test the efficacy or toxicity of drugs in mouse models. The immune system of ordinary mice is still of mouse origin. This genus difference makes it impossible to replicate human viral infectious diseases in ordinary mice, and it is impossible to truly simulate the actual situation of the human body. Although primates can accurately simulate most human viral infectious diseases, their high price and complex operation limit their widespread application. Therefore, the immune system humanized mouse model constructed by the present invention can be widely used in human virology research.

[0132] 4) In vitro preparation of CD8 positive T immune cells suitable for clinical treatment

[0133] During the development or differentiation process, human T lymphocytes must be tamed by the thymus for negative selection to screen out T lymphocytes that have immune responses to the human body. The human thymus organoids constructed by the present invention are co-cultured with human hematopoietic stem cells in vitro to tame human T lymphocytes differentiated from human hematopoietic stem cells, and produce CD8-positive T immune cells that can be used clinically in vitro, avoiding the risk of transplanted T cells causing autoimmune diseases in patients.

[0134] 5) In vitro preparation of CD4-positive T immune cells and immune regulatory cells suitable for clinical treatment

[0135] At present, there are technical difficulties in preparing CD4-positive T immune cells in vitro. After the human thymus organoids constructed by the present invention are co-cultured with human hematopoietic stem cells, the proportion of CD4-positive T immune cells differentiated from human hematopoietic stem cells is significantly higher than that of CD8-positive T immune cells. In addition, CD4-positive T cells prepared by co-culturing human hematopoietic stem cells with human thymus organoids can be induced into immunoregulatory cells, and human hematopoietic stem cells can also be co-cultured with human thymus organoids to directly differentiate into immunoregulatory cells. Therefore, CD4-positive T immune cells and immunoregulatory cells that can be used in clinic can be produced in vitro.

[0136] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0137] Example 1 A kit for directed induction of differentiation of thymic organoids

[0138] A kit comprises a first culture medium, a second culture medium, a third culture medium and a fourth culture medium.

[0139] The first culture medium is: X-VIVO 20 culture medium (purchased from Lonza) with 100 ng / mL Activin A (purchased from peprotech), 5 μM CHIR99021 (purchased from Selleckchem), and 10 μM Y-27632 (purchased from Selleckchem);

[0140] The second culture medium was: X-VIVO 20 medium with 1× B-27 supplement (purchased from Gibco) and 100 ng / mL Activin A;

[0141] The third culture medium is: EGM2 medium (purchased from Lonza) containing 10uM SB431542 (purchased from MCE), 10ng / mL DLL4 (purchased from Peprotech);

[0142] The fourth culture medium is: EGM2 medium containing 10 uM SB431542, 10 ng / mL DLL4, 5 ng / mL TPO (purchased from Peprotech), 5 ng / mL FLT3L (purchased from Peprotech), and 50 ng / mL SCF (purchased from Peprotech).

[0143] The kit further comprises OP9 bone marrow stromal cells (purchased from the Cell Bank of the Chinese Academy of Sciences).

[0144] Example 2: A method for directional induction of differentiation of thymic organoids from 3D suspension culture of human pluripotent stem cells (hPSCs)

[0145] This embodiment provides a method for differentiating pluripotent stem cells into thymic organoids: wherein the differentiation and formation of thymic organoids requires the induction of mesendoderm and third pharyngeal pouch endoderm in sequence, and finally the development of thymic organoids ( Figure 1 ).

[0146] In this differentiation process, 3D suspension culture was used to culture pluripotent stem cells for 3 days using Activin A, CHIR99021, Y-27632, and 1×B-27 supplement. Figure 1 The mesendoderm differentiation stage was completed on days 0-3 in the experiment. Then, the third pharyngeal pouch endoderm differentiation stage ( Figure 1 3-10 days in the culture medium), then supplement SCF, TPO, and FLT3L cytokines to the culture medium, and harvest after 7 days ( Figure 1 Days 10-17 in the study).

[0147] Compared with the existing methods, the differences are: first, the CHIR99021 small molecule is used to replace the expensive growth factor Wnt3a for mesendoderm cell differentiation, and a smaller combination of cytokines is used to differentiate thymic organoids; second, the use of 3D suspension culture can better simulate the developmental niche of natural thymic organs; third, no auxiliary materials such as scaffolds are required, and thymic organoids can produce their own extracellular matrix to provide support for thymic organoids; fourth, thymic organoids can be differentiated from a single human pluripotent stem cell to simultaneously produce thymic epithelial cells that exert thymic functions, as well as all supporting cells such as mesenchymal cells, fibroblasts, endothelial cells, and pericytes, perfectly simulating the process, components and structure of natural thymic cell development, forming thymic organoids with natural thymic functions in the true sense; whether in vitro or in vivo, they can promote the development of human hematopoietic stem and progenitor cells into a variety of functional T cells.

[0148] The specific steps are as follows:

[0149] 1. Mesendoderm cell differentiation stage:

[0150] S1. Mesendoderm cell differentiation stage

[0151] Digest hESCs (obtained from WiCell Research Institute) in the form of cell pellets. After washing the cells with PBS, add 1 mL of digestion solution Gentle Cell Dissociation Reagent (GCDR, STEM CELL TECHNOLOGIES), return to the 37°C carbon dioxide incubator and incubate for 3 minutes, then discard the GCDR in the well, add 1 mL of mesendoderm differentiation medium-1 (first medium), gently blow the cells, and collect them in a centrifuge tube. After supplementing a certain volume of mesendoderm differentiation medium-1, use a pipette to evenly inoculate the cell suspension in a low-adhesion six-well plate and culture overnight. On the second day, wash the cell spheres three times with PBS, and add fresh mesendoderm differentiation medium-2 (second medium). Change the same medium once a day. That is, after a total of 3 days of treatment, human pluripotent stem cells are induced to differentiate into mesendoderm cell spheres;

[0152] The specific operation of cell medium replacement is as follows: collect the cell spheres into a centrifuge tube, use natural sedimentation or low-speed centrifugation to sink the cell spheres to the bottom of the tube, discard the supernatant, add 1 mL of PBS to wash the organoids, discard it, and add the differentiation medium at the corresponding time point.

[0153] S2. Cell Harvesting

[0154] On the third day of culture, the mesendoderm cell spheres were subjected to flow cytometry, immunofluorescence and RT-qPCR. The mesendoderm cell spheres were collected, and after natural sedimentation, the supernatant was discarded and 1 mL of PBS was added to wash the mesendoderm cell spheres. Then 1 mL of TrypLE TM Express enzyme digestion at room temperature or 4% paraformaldehyde fixation.

[0155] For the first stage (days 0-3), the differentiation system can promote the formation of endoderm containing a small part of mesoderm, then transition to the third pharyngeal pouch endoderm, and finally directed differentiation into thymic organoids.

[0156] Before differentiation, the pluripotency of hPSCs at day 0 was tested by immunofluorescence and flow cytometry. The results showed that most cells co-expressed SOX2 and OCT4 ( Figure 2 Middle A), SSEA4 + More than 96% of cells ( Figure 2B); cells that have undergone mesendoderm differentiation stage show that pluripotency markers (OCT4 and NANOG) genes and neuroectoderm marker (SOX1) genes are gradually downregulated, and endoderm-related genes (FOXA2, SOX17, CXCR4 and GATA6) are rapidly upregulated; mesoderm (BRA, Brachyury) genes are first significantly upregulated, and then rapidly downregulated ( Figure 2 Middle C).

[0157] HE staining of mesendoderm cell spheres ( Figure 2 Middle D). Immunofluorescence on day 3 showed that most cells expressed the endoderm marker FOXA2, and a small number of cells expressed the mesoderm marker Brachyury (BRA) ( Figure 2 Middle E); Flow cytometry analysis further confirmed the expression of CXCR4, a key gene in endoderm + The cell ratio is 93.8%, SOX17 + The cell ratio was 51.3%, CXCR4 + SOX17 + The cell ratio is 50.2% ( Figure 2 Medium F, Figure 2 Middle G).

[0158] These results suggest that this 3D differentiation can successfully differentiate hPSCs into endoderm, accompanied by the formation of a small amount of mesoderm.

[0159] 2. The third pharyngeal pouch endoderm differentiation stage:

[0160] S1. The third pharyngeal pouch endoderm differentiation stage

[0161] Collect the mesendoderm cell spheres prepared in step 1 into a centrifuge tube, let the cell spheres sink to the bottom of the tube by natural sedimentation, discard the supernatant, and add 1 mL of PBS to wash the cells;

[0162] Before the experiment, OP9 cells were revived, treated with mitomycin, discarded and washed 5 times with PBS. TM Express Enzyme to digest OP9 cells. 4 OP9 cells were added to the third pharyngeal pouch endoderm differentiation medium (third medium), mixed with the washed mesendoderm cell spheres, and added to a low-adhesion well plate for culture;

[0163] S2. Cell Harvesting

[0164] On the 10th day of culture, the third pharyngeal pouch endoderm cell spheres were subjected to flow cytometry, immunofluorescence and RT-qPCR. The cell spheres were collected and naturally settled. The supernatant was discarded and 1 mL PBS was added to wash the cell spheres. Then 1 mL of TripLE TM Express enzyme digestion at room temperature or 4% paraformaldehyde fixation.

[0165] RT-qPCR results showed that the key markers of the third pharyngeal pouch endoderm (PAX8, FGF10, SIX1, ETV5, FGF3, TBX1, PAX9, DUSP6, PDX1) were expressed at the highest level during the entire differentiation process ( Figure 3 Middle A). Similarly, flow cytometry was performed on day 10, and most cells expressed SIX1 and PAX9 ( Figure 3 (middle B).

[0166] 3. Thymic organoid differentiation stage:

[0167] For the third stage, based on in vivo developmental cues, SCF, TPO and FLT3L were added to the second stage culture medium to induce thymic organoids.

[0168] S1. Cell culture medium replacement

[0169] Collect the third pharyngeal pouch endoderm cell spheres cultured and differentiated in step 2 into a centrifuge tube, use natural sedimentation to sink the cell spheres to the bottom of the tube, discard the supernatant, add 5 mL PBS to wash the cells, and then add thymic organoid culture medium (fourth culture medium) on the 10th to 17th day for thymic organoid differentiation. The culture medium is changed once a day.

[0170] S2. Cell Harvesting

[0171] On the 17th day of culture, thymic organoids were analyzed by flow cytometry, immunofluorescence, and RT-qPCR. The cell spheres were collected and naturally settled. The supernatant was discarded and 1 mL of PBS was added to wash the cell spheres. Then 1 mL of TripLE TM Express enzyme digestion at room temperature or 4% paraformaldehyde fixation.

[0172] RT-qPCR was performed to detect the relative expression levels of marker genes for thymic epithelial cells (AIRE, Foxn1, K5, K8, EpCAM, MHC-II), mesenchymal cells (PDGFRA), endothelial cells (PECAM1), pericytes (PDGFRB), and vascular smooth muscle cells (ACTA2). The results showed that as the differentiation time went on, all of the above genes were significantly higher at day 17 of hETO development than those of undifferentiated cells (day 0) ( Figure 4 (A, CF) (p<0.05).

[0173] In addition, these cells have thymic features, FOXN1 + cell( Figure 4 Middle C), Aire + cell( Figure 4 Middle D), MHCII + cell( Figure 4 E) were 56.5%, 59.7%, and 58.8% respectively. Flow cytometry analysis confirmed that CD90 was expressed in EpCAM positive cells ( Figure 4 Middle B). Thymus function-related genes DLL4 and CCL25 are gradually upregulated during hETO development ( Figure 4 Middle G).

[0174] More importantly, immunofluorescence staining showed that hETO expressed markers of various thymic matrix cell types. On the one hand, hETO regionally expressed thymic cortical epithelial marker K8 and thymic medullary epithelial marker K5 ( Figure 4 On the other hand, hETO highly expressed the key transcription factor FOXN1 of thymic epithelial cells and the mesenchymal cell marker PDGFRA ( Figure 4 Therefore, differentiation day 17 was defined as the hETO developmental stage, when there was maturation and fusion of multiple cell types.

[0175] Example 3 Detection of thymic organoids

[0176] In this example, the single-cell transcriptome sequencing data obtained from the thymic organoids cultured in Example 2 were first subjected to multi-sample integrated analysis with the published large-scale transcriptomes of human thymus with developmental ages between 19 weeks of fetus and 6 years after birth.

[0177] The results showed that thymic organoids have cell clusters in each population of the natural thymus ( Figure 5 The Pearson parametric correlation test was used to analyze the single-cell sequencing data of these samples, and it was found that the strongest correlation was found between thymic organoids and natural thymus at 19 weeks of fetal development ( Figure 5 (middle B).

[0178] The anchors were identified using the Find Integration Anchors function, and these anchors were used by Integrate Data to integrate the thymic organoids and the natural thymus fetal period of 19 weeks. Visual analysis showed that the integrated cells could be divided into 11 cell groups, and each cluster was explored for its marker genes and annotated as a specific cell type. Based on published literature references, the various cell groups were defined as mesenchymal cells, hemogenic endothelial cells, immature thymic epithelial cells, pericytes, nerve-related cells, vascular smooth muscle cells, NKT cells, endothelial cells, fibroblasts, thymic epithelial cells-1, and thymic epithelial cells-2 ( Figure 5 Middle C).

[0179] The results showed that compared with the natural thymus, the thymic organoids were highly similar to the 19-week natural thymic fetal cell composition, with only a few cells having quantitative differences, such as fewer hematopoietic endothelial cells and a higher proportion of mesenchymal cells in the thymic organoids ( Figure 5 (middle D).

[0180] Example 4 hPSC-derived 3D thymic organoids co-cultured with human hematopoietic stem / progenitor cells (HSPCs) to generate T cells in vitro

[0181] S1.CD34 + Establishment of a co-culture system of human hematopoietic stem / progenitor cells (HSPC) and thymic organoids

[0182] OP9 cells were seeded into 0.1% gelatin-coated six-well plates one day before the experiment to reach a density of 70-80%. OP9 cells were treated with mitomycin before use; 3D thymic organoids hETO were washed 3 times with PBS and purified from human umbilical cord blood CD34 by magnetic bead sorting. + HSPCs were mixed, the supernatant was discarded after centrifugation, and the precipitate was resuspended with 20 μL culture medium; the cell mixture was sucked up with a 20 μL pipette tip and gently deposited onto a 0.4 μm Millicell transwell plug (EMD Millipore) by forming a droplet at the end of the pipette tip; the plug was placed on a six-well plate with OP9 to form an upper and lower chamber, and each well contained 1 mL of mRB27 culture medium in the lower chamber; the plug was transferred to a new monolayer of OP9 stromal cells every 4-5 days; single cells around thymic organoids in the upper chamber were collected every two weeks, hETO was filtered using a 40 μm cell strainer, and single cell suspensions were harvested for cell flow cytometry detection and cultured for at least 6 weeks, with the medium changed once a day.

[0183] The mRB27 culture medium consisted of RPMI 1640 (Thermo), 4% B-27 supplement (Gibco), 30 μM L-ascorbic acid-2-phosphate trisodium salt (Sigma), 1% penicillin-streptomycin-gentamicin mixed solution (Solarbio), 1% Glutamax (Thermo), 5 ng / mL FLT3L (Peprotech), 5 ng / mL IL-7 (Peprotech), and 10 ng / mL DLL4 (Peprotech). The culture medium needed to be freshly prepared every week.

[0184] Thymic organoids and HSPCs were placed in an organoid culture chamber for co-culture to establish an air-liquid culture interface to induce T cell lineage commitment ( Figure 6 Middle A). CD45 + At the second week, the proportions of HSPCs cultured alone and HSPCs co-cultured with hETO were 97.1% and 96.0%, respectively. + The proportion of cells reached more than 98% ( Figure 6 Middle B). CD5 + CD7 + Pro-T lineage cells appeared at the second week, and co-culture of HSPCs with hETO increased CD5 + CD7 + The proportion of T cells reached a maximum of 60.3% in the fourth week and decreased as T cells differentiated and matured. Figure 6 Middle C). More mature CD3 + TCRαβ + T cells appeared as early as week 2 and increased over time.

[0185] Compared with the HSPCs alone group, more mature CD3 + TCRaβ + The proportion of T cells was higher, reaching 11.5% at the sixth week. The HSPCs group produced CD3 + TCRaβ + The proportion of T cells is only 2.62% ( Figure 6 (middle D).

[0186] CD3 + TCRaβ + The proportion of T cells was statistically analyzed and the results showed that at each time point, the hETO group was significantly higher than the HSPCs group in terms of CD3 + TCRaβ + The proportion of T cells was at a high level ( Figure 6Middle E). Immunofluorescence showed that HSPCs differentiated into CD3 + T cells ( Figure 6 Middle F).

[0187] During the differentiation of HSPCs into T lineage, 16.1% CD3 + TCRaβ + CD4 single positive (SP) T and 9.52% CD3 + TCRaβ + CD8 SP T cells were preferentially induced to differentiate into mature CD3 + TCRαβ + CD4SP T cells accounted for 90.3%, while only HSPCs tended to be induced to differentiate into mature CD3 + TCRαβ + CD8 SP T cells accounted for 8.5%, and more than 90% of CD3 + TCRαβ + T cells were positively selected as CD4 SP or CD8 SP T cells ( Figure 6 Middle G).

[0188] In the thymus, CD3 + TCRαβ + CD8 SP and CD3 + TCRαβ + CD4 SP T cells from double positive (DP)-like "immature ” to “mature "Phenotype. CD3 was detected at week 6 + TCRαβ + CD45RA + CD45RO - CCR7 + Mature The ratio of T, hETO group CD4 SP and The proportion of CD8 SP was higher than that of HSPCs group (P<0.05) ( Figure 6 (H) CD3 in the hETO group + TCRγδ + The T cell ratio was highly consistent throughout the experiment, reaching 10.9% at 6 weeks, while the HSPCs group had only 1.76%, which further demonstrated that thymic organoids can promote the multidirectional differentiation potential of HSPCs into the T lineage ( Figure 6 Zhong I and Figure 6(J) At week 6 of differentiation, CD4 + CD25 + T in CD45 + CD3 + TCRαβ + The cell ratio was as high as 82.3%, while that of the HSPCs group was only 0.66%, which further proved that thymic organoids can promote the differentiation of HSPCs into Treg lineage and have immune tolerance function ( Figure 6 Middle K).

[0189] In summary, the in vitro T cell differentiation method of this embodiment provides a thymus-like developmental site for the formation of T lineages and can produce a very high proportion of mature CD3 + TCRαβ + CD4 SP T cells overcome the problem that they could only produce mature CD8 SP in vitro + This is also consistent with the mature CD4 T cells in the natural thymus and in vivo environment. + T is higher than CD8 + T ratio relationship.

[0190] Example 5: Establishment of a method for thymus reconstruction and immune system reconstruction in severely immunodeficient mice by transplantation of hPSC-derived 3D thymic organoids

[0191] Severe immunodeficient mice NSI (Shenzhen Invitro Biopharmaceutical Technology Co., Ltd.), NCG and NCG-X (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were isolated from umbilical cord blood CD34 + Sublethal irradiation (1.0-1.5Gry) was performed 6-8 hours before cell transplantation, and 300,000-500,000 CD34 cells were transplanted into each mouse. + Human umbilical cord blood hematopoietic stem / progenitor cells, and one month later, thymic organoid renal capsule transplantation surgery was performed on mice. The mice were anesthetized by breathing isoflurane through an anesthesia machine, the mouse hair was removed and the skin was prepared with iodine. The mouse skin and muscles were cut to expose the kidneys, and multiple thymic organoids were implanted under the renal capsule. The mouse kidneys were repositioned, the muscles and skin were sutured, and the wounds were disinfected. 0.02% neomycin was supplemented in the mouse drinking water for one week. The entire operation was performed aseptically and the tissues and organs were kept moist.

[0192] After blood was collected from the tail vein of the mice, they were euthanized and their spleen, kidney grafts and other tissues were removed. After washing with PBS, the cells were ground on a 200 μm mesh and the cell suspension was collected. Mouse CD45 was strictly gated to exclude negative controls. +Potential contamination of cells. After washing the cell suspension, use red blood cell lysis buffer to remove mouse red blood cells, then use flow cytometry antibody staining for 30 minutes, wash once with PBS and then test on the machine. Mice transplanted with human fetal thymus were used as positive controls. HSPCs transplanted mice 8 weeks ( Figure 7 Medium A), 12 weeks ( Figure 7 Medium B) and 16 weeks ( Figure 7 In C), human CD3 + The results showed that the proportion of human CD3 T cells in peripheral blood of mice transplanted with thymic organoids (hETO group) was + The T cell ratio was very close to that in mice transplanted with natural human fetal thymus (Thymus group).

[0193] Flow cytometry analysis was performed on severely immunodeficient mice in the hETO group 11 months after HSPCs transplantation, and it was found that in the hETO group, human CD45 + The cell ratio is 50.3%, CD3 + TCRαβ + The proportion of CD4 T cells was 21.0%, the proportion of CD4 SPT cells was 18.7%, and the proportion of CD8 SP T cells was 15.0%. + FOXP3 + Treg cells account for CD45 + CD3 + CD4 + 10.2% of T cells, CD3 + TCRγδ + The proportion of T cells was 3.86% ( Figure 7 In D), human CD45 + The cell ratio is 75.9%, CD3 + TCRαβ + The proportion of CD4 SP T cells was 6%, and the proportion of CD8 SP T cells was 58.2%. + FOXP3 + Treg cells account for CD45 + CD3 + CD4 + 7.28% of T cells, CD3 + TCRγδ + The proportion of T cells was 1.71% ( Figure 7 Middle E).

[0194] Example 6: Human immune system reconstituted by hPSC-derived 3D thymic organoids in severely immunodeficient mice has humoral immunity

[0195] To test whether the reconstituted T cells in mice were fully functional, human T lymphocytes in the transplanted mice were examined and their phenotype and functional properties were compared with those of T cells generated after transplantation of native human fetal thymus.

[0196] The mice were divided into four groups, namely, the blank control group (NC group), the group transplanted with human umbilical cord blood HSPCs only (HSPCs group), the group transplanted with human umbilical cord blood HSPCs and natural thymus (Thymus group), and the group transplanted with human umbilical cord blood HSPCs and thymus organoids (hETO group). Then, each group of mice was inoculated with chicken ovalbumin OVA antigen. After the first week after OVA re-immunization ( Figure 8 Middle A) and the second week ( Figure 8 (B) The levels of ovalbumin-specific human immunoglobulins M and G (IgM and IgG) in mouse peripheral blood serum were detected.

[0197] The results showed that compared with the Thymus group, higher IgG and IgM were also detected in the peripheral blood serum of mice in the hETO group, indicating that thymic organoids and natural thymus-trained T cells played a role in the synergistic process of B cell-mediated immunoglobulin class switching and antibody secretion, inducing functional humoral immune responses to protein antigens.

[0198] Example 7: Human immune system reconstituted by hPSC-derived 3D thymic organoids in severely immunodeficient mice has cellular immunity capability

[0199] To verify the ability of T cells to reject allogeneic skin and form a memory response in vivo, allogeneic skin from C57BL / 6 mice was transplanted into mice reconstituted with human T cells. Flow cytometry analysis showed that a large number of mature human CD8SP T cells and CD4SP T cells were generated in the peripheral blood of allogeneic skin transplant recipient mice ( Fig. 9 (A).

[0200] Six days after mouse skin transplantation, H&E and immunohistochemical staining were used to detect the infiltration of inflammatory cells in the transplanted skin. H&E staining showed that a large number of inflammatory cells infiltrated the dermis of the transplanted skin in the hETO co-transplantation group (hETO group) and the natural thymus co-transplantation group (Thymus group), and the necrotic area was large. However, the HSPCs transplantation group (HSPCs) had relatively few inflammatory cells infiltration ( Fig. 9 (middle B).

[0201] Similarly, immunofluorescence staining results also showed that CD3 + T lymphocyte infiltration was obvious, and most of them expressed IL-10 ( Fig. 9 Middle C).

[0202] The peripheral blood of mice was collected to detect the ratio of human IL-2, IFN-γ and TNF-α. The results showed that the mice in the hETO group could produce a large amount of cytokines, IFN-γ + TNF-α + The proportion is as high as 78.1%, IL2 + TNF-α + and IL-2 + IFN-γ + The proportions of the thymus group and the Thymus group were similar, 86.6% and 76.2% respectively. These results indicate that the T cells trained by thymus organoids can produce a large number of cytokines to reject allogeneic skin grafts ( Fig. 9 (middle D).

[0203] Taken together, these results indicate that mouse reconstitution of human T cells mediates rejection of allogeneic skin grafts and stimulates T cell proliferation and activation, which also proves that typical adaptive immune responses can occur in mice with a humanized immune system.

[0204] Comparative Example: A method for directional induction and differentiation of thymic organoids from 3D suspension culture of human pluripotent stem cells (hPSCs)

[0205] Specifically include the following steps (mesendoderm cell differentiation stage):

[0206] The formulation of the culture medium is as follows:

[0207] Method 1: Endoderm differentiation medium (X-VIVO 20 medium containing 100 ng / mL Activin A, 10 μM Y-27632).

[0208] Method 2: Endoderm differentiation medium-1 (X-VIVO 20 medium containing 100 ng / mL Activin A, 50 ng / mL Wnt3A, 10 μM Y-27632) and endoderm differentiation medium-2 (X-VIVO 20 medium containing 1×B-27 supplement, 100 ng / mL Activin A).

[0209] Method 3, Endoderm differentiation medium-1 (X-VIVO 20 medium containing 100 ng / mL Activin A, 5 μM CHIR99021, 10 μM Y-27632) and endoderm differentiation medium Endoderm differentiation medium-2 (X-VIVO 20 medium containing 1×B-27 supplement, 100 ng / mL Activin A).

[0210] S1. Digestion of hPSCs with cell clusters: After aspirating the culture medium, add 1mL PBS to wash the cells, then add 1mL digestion solution GCDR, and return to the 37℃ CO2 incubator for incubation for 3 minutes. Then aspirate the GCDR in the well, add the 0th endoderm culture medium, and use a pipette to gently blow the cells until the small cell clusters fall off, and finally obtain a cell suspension. Inoculate the cell suspension in a low-adhesion six-well plate, add the culture medium, mix well, and place in the incubator.

[0211] S2. Cell medium replacement: Collect the cell spheres into a centrifuge tube, use natural sedimentation or low-speed centrifugation to sink the cell spheres to the bottom of the tube, discard the supernatant, add 1 mL of PBS to wash the human pluripotent stem cell spheres, discard the supernatant, and add the endoderm differentiation medium at the corresponding time point;

[0212] Method 1 uses 100 ng / mL Activin A and 10 μM Y-27632 from Day 0 to Day 3 (Day 0-Day 3) (marked as Method A); Method 2 uses 100 ng / mL Activin A, 50 ng / mL Wnt3A, and 10 μM Y-27632 from Day 0 to Day 1 (Day 0-Day 1), and culture medium 1×B-27 supplement and 100 ng / mL Activin A from Day 2 to Day 3 (Day 2-Day 3) (marked as Method A+B+W); Method 3 replaces Wnt3A in Method 2 with the chemical small molecule 5 μM CHIR99021 (marked as Method A+B+C).

[0213] S4. Cell harvesting: On the third day of culture, flow cytometry, immunofluorescence and RT-qPCR were performed on the endoderm cell spheres. The endoderm cell spheres were collected, and after natural sedimentation, the supernatant was discarded and 1 mL of PBS was added to wash the endoderm cell spheres. RNA extraction and RT-qPCR were then performed.

[0214] During this differentiation process, we explored three endoderm differentiation methods and strived to find an optimal endoderm-promoting thymic organoid differentiation pathway.

[0215] Cell spheres were collected from day 0 to day 3 (Day 0-Day 3), and qRT-PCR was used to detect the mesoderm marker gene Brachyury and the endoderm marker genes SOX17, FoxA2 and CXCR4. The results showed that there was no significant difference in the expression level of Brachyury mRNA on the third day of differentiation. The relative expression levels of SOX17, FoxA2, ​​and CXCR4 in the differentiation system with the addition of Wnt3A or the chemical small molecule CHIR99021 were slightly higher than those in the first combination (method A) ( Fig.10). As differentiation progressed, organoids were collected on the 17th day of differentiation to detect specific markers of epithelial cells (EpCAM, K8, K5, AIRE), mesenchymal cells (PDGFRA), endothelial cells (PECAM1) and smooth muscle cells (ACTA2) in human thymic organs. The results showed that the third endoderm differentiation method (A+B+C method) has more potential advantages for the development and maturation of thymic organoids ( Fig.11 ).

[0216] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A culture medium combination, comprising a first culture medium and a second culture medium; the first culture medium comprises a first basal culture medium supplemented with Activin A, a GSK-3 inhibitor and a ROCK inhibitor; the second culture medium comprises a second basal culture medium supplemented with Activin A and a B-27 additive.

2. The culture medium combination according to claim 1, characterized in that: The GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021; preferably, the GSK-3 inhibitor is CHIR99021; Preferably, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447 and Azaindole 1; preferably, the ROCK inhibitor is Y-27632; Preferably, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL; Preferably, the concentration of CHIR99021 added to the first basal medium is 4-6 μM; Preferably, the concentration of Y-27632 added to the first basal culture medium is 8-12 μM.

3. The culture medium combination according to claim 1, characterized in that: The concentration of Activin A added to the second basal medium is 80-120 ng / mL; Preferably, the concentration of B-27 additive added to the second basal culture medium is 0.5-1.5×.

4. The culture medium combination according to claim 1, characterized in that: The culture medium combination further includes a third culture medium; the third culture medium includes a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; preferably, the TGF-β signaling pathway inhibitor is SB431542; Preferably, the Notch receptor agonist is selected from DLL4; Preferably, the concentration of SB431542 added to the third basal medium is 8-12 μM; Preferably, the added concentration of DLL4 in the third basal culture medium is 8-12 ng / mL.

5. The culture medium combination according to claim 1, characterized in that: The culture medium combination further includes a fourth culture medium, wherein the fourth culture medium includes a fourth basal culture medium supplemented with a TGF-β signaling pathway inhibitor, a Notch receptor agonist, and a cytokine; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; preferably, the TGF-β signaling pathway inhibitor is SB431542; Preferably, the Notch receptor agonist comprises DLL4; Preferably, the cytokines include FLT3L, TPO, SCF; Preferably, the concentration of SB431542 added to the fourth basal medium is 8-12 μM; Preferably, the concentration of DLL4 added to the fourth basal medium is 8-12 ng / mL; Preferably, the added concentration of TPO in the fourth basal medium is 4-6 ng / mL; Preferably, the added concentration of FLT3L in the fourth basal medium is 4-6 ng / mL; Preferably, the added concentration of SCF in the fourth basal culture medium is 40-60 ng / mL.

6. The culture medium combination according to any one of claims 1 to 5, characterized in that: The first basal culture medium and the second basal culture medium are stem cell culture media; The third basal culture medium and the fourth basal culture medium are endothelial cell culture medium, and the endothelial cell culture medium is selected from any one of EGM2 culture medium, ECM culture medium, and VascuLife VEGF Medium Complete Kit culture medium; Preferably, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium; Preferably, the third basal culture medium and the fourth basal culture medium are EGM2 culture medium.

7. A kit, characterized in that: The kit comprises the culture medium combination according to any one of claims 1 to 6; Preferably, trophoblast cells are also included.

8. Use of the culture medium combination according to any one of claims 1 to 6 or the kit according to claim 7: (1) preparing mesendoderm cells; (2) preparing third pharyngeal pouch endoderm cells; (3) Preparation of thymic organoids; (4) Preparing products for inducing pluripotent stem cells to differentiate into mesendoderm cells; (5) preparing a product of inducing pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; (6) Prepare products for inducing pluripotent stem cells to differentiate into thymic organoids.

9. A method for preparing mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids, comprising culturing pluripotent stem cells using the kit according to claim 7 to obtain mesendoderm cells and / or third pharyngeal pouch endoderm cells and / or thymic organoids; the culturing is suspension culture; Preferably, the method specifically comprises: (1) culturing pluripotent stem cells in the first culture medium and the second culture medium in the kit of claim 7 to obtain mesendoderm cells; and / or (2) co-culturing the mesendoderm cells and the trophoblast cells in the third culture medium in the kit according to claim 7 to obtain third pharyngeal pouch endoderm cells; and / or (3) Cultivating the third pharyngeal pouch endoderm cells in the fourth culture medium in the kit of claim 7 to obtain thymic organoids.

10. Use of the thymic organoid prepared by the method according to claim 9 in any of the following: 1) Used to construct animals with humanized blood and / or immune system; 2) Products for preparing animals with humanized blood and / or immune system; 3) Simulate human diseases; 4) Used to prepare products that simulate human diseases; 5) Preparation of human T cells; 6) Products for preparing human T cells; 7) For the preparation of products for inducing transplantation tolerance; 8) Used for preparing products for restoring thymus function; 9) Producing humanized antibodies; 10) Used to produce humanized antibodies; 11) Thymus-related drug screening, development and / or toxicity analysis; 12) Study the pathogenic mechanism of thymic diseases; 13) Preparation of products for treating thymic diseases; 14) Constructing thymic disease models; 15) Thymus toxicology testing.

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