Methods of promoting thymic epithelial cell and thymic epithelial cell progenitor differentiation of pluripotent stem cells

Abstract

To provide methods of promoting differentiation of pluripotent stem cells into thymic epithelial cells or thymic epithelial cell progenitors as well as the cells obtained from the methods, and solutions, compositions, and pharmaceutical compositions comprising such cells.SOLUTION: The present invention provides a method of inducing differentiation of human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEC progenitors) (TEPs), including the steps of: differentiating human pluripotent stem cells into endoderm cells; and culturing the resulting endoderm cells and differentiating the endoderm cells into anterior foregut cells by contacting the endoderm cells with an agent which inhibits BMP and an agent which inhibits TGFβ signaling, and further contacting the cells with an agent which stimulates the expression of HOXA3 and an agent which stimulates the expression of TBX1.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority to U.S. Patent Application No. 62 / 827,383, filed April 1, 2019, which is incorporated herein by reference in its entirety.

[0002] Explanation of government support This invention was made with Government support under Grant Nos. DK104207, DK103585 and AI045897 awarded by the National Institutes of Health. The Federal Government has certain rights in this invention.

[0003] The present disclosure provides methods for promoting differentiation of pluripotent stem cells into thymic epithelial cells or thymic epithelial progenitor cells, cells obtained by the methods, and solutions, compositions, and pharmaceutical compositions comprising such cells. The present disclosure also provides methods of utilizing thymic epithelial cells or thymic epithelial progenitor cells for disease treatment and prevention, and to generate organs, as well as other uses and kits. [Background technology]

[0004] The thymus is the primary lymphoid organ responsible for the development and education of T cells. Thymic epithelial cells (TECs) are the major components of the thymic stroma. Thymic cortical TECs (cTECs) are specialized for the positive selection of T cells, whereas medullary TECs (mTECs) are involved in the negative selection of T cells. TEC-mediated selection promotes a self-tolerant and diverse T cell repertoire capable of recognizing foreign antigens presented by self MHC molecules. Normal thymopoiesis contains a highly lineage network of stromal and hematopoietic cell types in addition to TECs.

[0005] In vitro generation of functional TECs or TEC precursors (TEPs) from human pluripotent stem cells (hPSCs) may create cells, tissues or organs that support T cell reconstitution in patients with thymic insufficiency due to congenital disorders (e.g., DiGeorge syndrome) and acquired insufficiency (due to HIV infection, high-dose chemotherapy and radiation therapy, which themselves cause thymocyte proliferative dysfunction, graft-versus-host disease combined with aging and long-term immunosuppressive therapy). Generating TECs from pluripotent stem cells (PSCs) is an important goal, since the number of TECs in the adult thymus is limited and reliable methods to grow TECs from the postnatal thymus are difficult to find. Creating in vitro protocols for tightly controlled differentiation of hPSCs into TECs requires precise knowledge and application of the temporal and cytokine instructions of development. Although the generation of functional TEPs from mouse or human PSCs that support mouse (Parent et al., 2013; Sun et al., 2013; Soh et al., 2014; Bredenkamp et al., 2014) or human (Su et al., 2015) T cell development has been reported, reconstitution of large amounts of human naive T cells has not been demonstrated. Thus, there is a need in the art for methods to generate human TEPs and TECs. Summary of the Invention

[0006] Herein, we present an efficient method for inducing differentiation of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into thymic epithelial progenitor cells (TEC progenitors) in vitro, where the thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) can generate thymic organs and T cells in vivo.

[0007] This protocol achieved the largest in vitro expression of FOXN1 reported to date without protein transduction or genetic modification. After culture, the cells expressed the epithelial markers EpCam, keratin 5, and keratin 8. When mixed with human thymic mesenchymal cells (ThyMES), the in vivo transplanted cells expressed IL2Rgamma, a marker of FOXN1 expression in thymic NOD-scid mice intravenously injected with human hematopoietic stem cells (HSCs). null supported the reconstitution of naive human T cells in NSG mice ( Khosravi-Mahrarlooei et al., 2020 ).

[0008] One embodiment of the present disclosure is a method for inducing differentiation of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEPs), comprising: (1) differentiating human pluripotent stem cells into endoderm cells; (2) culturing the resulting endoderm cells and differentiating the endoderm cells into anterior foregut cells by contacting or incubating the endoderm cells with a factor that inhibits BMP and a factor that inhibits TGFβ signaling, and further contacting or incubating the cells with a factor that stimulates HOXA3 expression and a factor that stimulates TBX1 expression; (3) further culturing the obtained anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by contacting or incubating the anterior foregut cells with a factor that stimulates the expression of TBX1 and a factor that stimulates the expression of PAX9 and PAX1; (4) further culturing the obtained pharyngeal endoderm cells and differentiating the pharyngeal endoderm cells into cells specialized for the distal pharyngeal pouch (PP), thymic epithelial cells or thymic epithelial progenitor cells by contacting or incubating the pharyngeal endoderm cells with a factor that inhibits BMP, and then contacting or incubating the pharyngeal endoderm cells with BMP; and (5) contacting or incubating the TECs or TEPs at the end of the method with a survivin inhibitor; The method includes:

[0009] A further embodiment is a method of obtaining thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEPs) from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), comprising: (1) differentiating human pluripotent stem cells into endoderm cells; (2) culturing the obtained endoderm cells and differentiating the endoderm cells into anterior foregut cells by contacting or incubating the endoderm cells with a factor that inhibits BMP and a factor that inhibits TGFβ signaling, and by contacting or incubating the cells with a factor that stimulates HOXA3 expression and a factor that stimulates TBX1 expression; (3) further culturing the obtained anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by contacting or incubating the anterior foregut cells with a factor that stimulates the expression of TBX1 and a factor that stimulates the expression of PAX9 and PAX1; (4) further culturing the obtained pharyngeal endoderm cells, and differentiating the pharyngeal endoderm cells into cells specialized for the distal pharyngeal pouch (PP), thymic epithelial cells, or thymic epithelial progenitor cells, by contacting or incubating the pharyngeal endoderm cells with a factor that inhibits BMP, and then contacting or incubating the pharyngeal endoderm cells with BMP; and (5) contacting or incubating the TECs or TEPs at the end of the method with a survivin inhibitor; The method includes:

[0010] A further embodiment of the present disclosure is a method of inducing differentiation of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEPs), comprising: (1) differentiating the pluripotent stem cells into endoderm cells by culturing the pluripotent stem cells in a serum-free differentiation medium and contacting or incubating the cells with human bone morphogenetic protein (BMP), human basic fibroblast growth factor (bFGF) and human activin A; (2) differentiating the endoderm cells of step (1) into anterior foregut cells by culturing the endoderm cells in a differentiation medium and contacting or incubating the cells with Noggin, SB431542, retinoic acid and FGF8b; (3) differentiating the anterior foregut cells from step (2) into pharyngeal endoderm cells by culturing the anterior foregut cells from step (2) in a differentiation medium and contacting or incubating the cells with FGF8b and retinoic acid followed by FGF8b and sonic hedgehog (Shh); (4) differentiating the pharyngeal endoderm cells from step (3) into those specialized for the third pharyngeal pouch by culturing the pharyngeal endoderm cells in a differentiation medium and contacting or incubating the cells with Noggin; (5) further differentiating the cells into third pharyngeal pouch specialized cells, TEPs or TECs, by culturing the pharyngeal endoderm cells from step (3) or step (4) in a differentiation medium and contacting or incubating the cells with BMP; and (6) exposing the cells to a survivin inhibitor; The method includes:

[0011] A further embodiment is a method of obtaining thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEPs) from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), comprising: (1) differentiating the pluripotent stem cells into endoderm cells by culturing the pluripotent stem cells in a serum-free differentiation medium and contacting or incubating the cells with human bone morphogenetic protein (BMP), human basic fibroblast growth factor (bFGF) and human activin A; (2) differentiating the endoderm cells from step (1) into anterior foregut cells by culturing the endoderm cells in a differentiation medium and contacting or incubating the cells with Noggin, SB431542, retinoic acid and FGF8b; (3) differentiating the anterior foregut cells from step (2) into pharyngeal endoderm cells by culturing the anterior foregut cells from step (2) in a differentiation medium and contacting or incubating the cells with FGF8b and retinoic acid followed by FGF8b and sonic hedgehog (Shh); (4) differentiating the pharyngeal endoderm cells from step (3) into those specialized for the third pharyngeal pouch by culturing the pharyngeal endoderm cells in a differentiation medium and contacting or incubating the cells with Noggin; (5) further differentiating the pharyngeal endoderm cells from step (3) or step (4) into third pharyngeal pouch specialized cells, TEPs, or TECs, by culturing the pharyngeal endoderm cells from step (3) or step (4) in a differentiation medium and contacting or incubating the cells with BMP; and (6) exposing the cells to a survivin inhibitor; The method includes:

[0012] In some embodiments, contacting or incubating the cells with various factors is accomplished by culturing the cells in medium containing the factors.

[0013] The present disclosure also provides cells obtained using the methods described herein, as well as solutions, compositions, and pharmaceutical compositions comprising cells obtained using the methods described herein.

[0014] In some embodiments, the cells express FOXN1, EpCAM, keratin 5, and keratin 8.

[0015] In some embodiments, these cells are thymic epithelial cells (TECs). In some embodiments, these cells are thymic epithelial progenitor cells (TEC precursors) (TEPs).

[0016] Any of the above described embodiments of cells, cell-containing solutions, compositions, and pharmaceutical compositions may be used to treat and / or prevent disease.

[0017] In some embodiments, the disease is a thymus disease.

[0018] In further embodiments, the disease is an autoimmune disease, including, but not limited to, type 1 diabetes, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's disease, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease, vitiligo, and alopecia areata.

[0019] Any of the above embodiments of cells, cell-containing solutions, compositions, and pharmaceutical compositions can be used to restore or restore decreased thymus function, where the loss of functionality is due to aging or injury or infectious disease such as HIV.

[0020] Any of the above-described embodiments of cells, cell-containing solutions, compositions, and pharmaceutical compositions can be used for T cell reconstitution following bone marrow transplantation.

[0021] Any of the above embodiments of cells, solutions comprising cells, compositions, and pharmaceutical compositions can be utilized to generate a hybrid thymus comprising cells and a thymus or other cells or tissues comprising a thymus. In some embodiments, the thymus is from a different individual. In some embodiments, the thymus is from a different species. In some embodiments, the thymus is from a pig. In some embodiments, the pig is a fetal pig. In some embodiments, the pig is a young pig.

[0022] Any of the above embodiments of cells, cell-containing solutions, compositions, and pharmaceutical compositions can be used to develop mouse models and conduct drug testing.

[0023] Any of the above embodiments of cells, cell-containing solutions, compositions, and pharmaceutical compositions can be used to therapeutically develop the thymus in individuals with congenital abnormalities resulting in partial or complete deficiency of thymic function, such as DiGeorge syndrome, 22q11.2 deletion syndrome, or NUDE syndrome.

[0024] In yet another embodiment, the present disclosure relates to kits for carrying out the methods of the present disclosure to obtain the cells, solutions, compositions, and pharmaceutical compositions disclosed herein. The present disclosure also includes kits that include the cells, solutions, compositions, and pharmaceutical compositions.

[0025] As described herein, the methods, systems, and kits are suitable for the reproducible generation of large amounts of thymic epithelial cells or thymic epithelial progenitor cells (TEPs).

[0026] For the purpose of illustrating the invention, there is shown in the drawings certain embodiments of the invention. However, the invention is not limited to the detailed arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0027] [Figure 1]Establishment of a protocol for direct differentiation of hESCs into pharyngeal endoderm biased to the third pharyngeal pouch. Figure 1A is a schematic representation of the envisioned hESC differentiation steps to the desired cell fates, reflecting the treatment aims shown in Figure 1B. Figure 1B is an overview of the tested protocol for differentiation of hESCs into pharyngeal endoderm biased to the third pharyngeal pouch by day 15. Protocol #1 (denoted as "1" in Figure 1B) (FGF8b+RA250) was considered as the control protocol, whereas protocols #2 (denoted as "2" in Figure 1B) (FGF8b) (#1 vs. #2) and #3 (denoted as "3" in Figure 1B) (FGF8b+RA250 to FGF8b+Shh) are compared in Figure 1D (#1 vs. #3). In Figure 1B, "NS" stands for Noggin and SB431542. Figure 1C shows a representative flow cytometry analysis of expression of EpCAM and CXCR4 (endodermal markers) in embryoid body dissociations at day 4.5. Figure 1D is a graph showing a comparative analysis of gene expression in differentiated hESCs at day 15 under the protocol conditions shown in Figure 1B. The graph represents the fold change in RNA expression as measured by qPCR. (n=3-11; values ​​represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; ratio paired two-tailed t-test). Figure 1E shows a comparison of pharyngeal pouch marker expression at day 15 in hESCs differentiated using protocol #1 ("liver conditions" (Gouon-Evans et al., 2006)) with hESCs differentiated into "liver". Bar graphs represent fold changes in RNA expression as measured by qPCR (n=6; values ​​represent mean + SEM; *p<0.05, **p<0.01, ***p<0.001; two-tailed t-test with paired ratios). Figure 1F is a graph showing comparative analysis of gene expression in differentiated hESCs at day 15 under the protocol conditions shown in Figure 1B. Graphs represent fold changes in RNA expression as measured by qPCR (n=9-11; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001; two-tailed t-test with paired ratios). [Diagram 2]Development of protocols for the distalization of the third pharyngeal pouch and / or TECs. Figure 2A is an overview of the protocols tested for the distalization of cells biased towards the third pharyngeal pouch by day 30. In Figure 2A, "3b" and "3c" show modifications based on protocol #3 in Figure 1B, and "4b" and "4c" show modifications based on protocol #4 in Figure 1B. Figure 2B is a schematic representation of multiple hESC differentiation protocols tested under various culture conditions from day 6.5 onwards. hESCs were differentiated into defined endoderm (DE) for 4.5 days and then anteriorized with Noggin+SB (NS) and retinoic acid (RA). Cells were then patterned with different combinations of RA and display factors for 8.5 days until day 15. Figure 2C is a graph of the analysis of FOXA2, HOXA3, SIX1, TBX1, EYA1, PAX9 and PAX1 expression in hESC-derived cells from cultures containing RA and FGF8b (protocol #1) versus cultures containing RA and another factor instead of FGF8b as shown in Figure 2B. Bar graphs represent fold changes in RNA expression as measured by qPCR. (n=3; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Dunnett's multiple comparison test). Figure 2D shows the effect of Noggin exposure on PAX9 expression at day 30. Bar graphs show fold changes in PAX9 expression between protocols #3b vs #3c, and #4b vs #4c (n=4; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, two-tailed t-test with paired ratios). Figure 2E shows the fold change in FOXN1 expression at day 30 (protocol #3c vs #4c) after starting FGF8b treatment at day 4.5 vs day 6.5 as measured by qPCR (n=4-8; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001; two-tailed t-test with paired ratios). Figure 2F shows the fold change in FOXN1 expression at day 21 vs day 30 (pre- and post-exposure to BMP4) for protocol #4c as measured by qPCR (n=4-8; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001; two-tailed t-test with paired ratios).Figure 2G shows the fold change in FOXN1 expression at day 15 vs. day 30 of protocol #4c as measured by qPCR (n=4-8; values ​​represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 paired two-tailed t-test). [Diagram 3] Characterization of in vitro differentiated TEC progenitors at day 30. Figure 3A shows the expression of TEC markers in cultured cells (d30; protocol #4c) compared to fetal thymus (FTHY). (Ct relative to β-actin; n=3-22; values ​​represent mean + SEM; *p<0.05, **p<0.01, ***p<0.001; unpaired two-tailed Welch t-test). Each point represents an independent experiment. Figure 3B shows the expression of third pharyngeal pouch markers in H9 cells cultured for 30 days under protocol #4c conditions compared to fetal thymus. Bar graphs show mean Ct values ​​+ SEM (n=3-6) relative to β-actin. Unpaired two-tailed Welch t-test. Each point represents an independent experiment. Figure 3C shows the Pearson correlation analysis graph of gene expression levels of FOXN1 and GCM2, FOXN1 and IL7, and FOXN1 and CD205. Both axes show Ct values ​​relative to β-actin. Each point represents an independent experiment. [Figure 4]Treatment of day 30 hES-TEP cultures with the survivin inhibitor YM155 depletes pluripotent cells. Figure 4A is a schematic representation of protocol #4c showing the duration of YM155 treatment. This schematic also shows the full differentiation protocol. Figure 4B is a Pearson correlation analysis graph of FOXN1 and OCT4 expression. Both axes show Ct values ​​relative to β-actin. Each point represents an independent experiment. Figure 4C is a graph of the fold change in OCT4 expression at day 30 after pluripotent cell depletion (protocol #4c vs. #4c+YM155; n=5; values ​​represent mean+SEM; *p<0.05; two-tailed t-test with paired proportions). Figure 4D is a graph showing the percentage of survival without overt teratoma formation weeks after hES-TEP implantation. Mice implanted with hES-TEP from day 15 of protocol #4c (n=8, grey line) compared to mice implanted with hES-TEP from 30 day cultures with YM155 treatment (n=15, dotted black line) or no treatment (n=12, solid black line). Log-rank-Mantel-Cox test showed that day 15 survival of hES-TEP was p<0.005 compared to either day 30 hES-TEP alone or day 30 hES-TEP + YM155 treatment. [Diagram 5]Reaggregation of hES-TEPs and thymic mesenchymal cells prepared using the protocol shown in Figure 4A forms thymic organoids that support thymus formation. Figure 5A shows the percentage of T cells present when native thymic primordia were surgically removed (ATX) or not from NSG mice injected with human HSCs. ACK (ammonium-chloride-potassium) hemolysis of peripheral blood generated leukocytes that were stained for HuCD45+CD3+ T cells at the indicated weeks after HSC injection. NSG, n=12; ATX NSG, n=4. Figure 5B shows representative FACS plots gated on HuCD45+CD19-CD14- cells. NSG, n=10; ATX, n=14. Figures 5C-5F show the frequency of various cells when cultured hES-TEP clusters mixed with thymic mesenchymal cells (TMC) or TMCs alone were transplanted under the kidney capsule of ATX NSG mice injected with human HSCs. Figure 5C shows the frequency of HuCD45+ cells among all mouse+human CD45+ cells in PBMCs for individual hES-TEC / TMC mice and the average (gray line) for TMC-transplanted mice (n=6). Figure 5D shows the frequency of CD3+ cells among all mouse+human CD45+ cells in PBMCs for individual hES-TEP / TMC mice and the average (gray line) for TMC-transplanted mice (n=6). Figure 5E shows the frequency of CD4+ cells among all mouse+human CD45+ cells in PBMCs for individual hES-TEP / TMC mice and the average (gray line) for TMC-transplanted mice (n=6). Figure 5F shows the frequency of CD4+ cells stained for CD45RA+CD45RO- naive cells. Time points with less than 100 CD4+ events were excluded. Figure 5G shows human T cells in PBMCs from healthy PBMCs (left), hES-TEC / TMC mice (middle) and TMC mice 30 weeks after humanization (right). The hES-TEC / TMC plot is representative of mice that developed CD4+ and CD8+ T cells (n=4), and the TMC plot is representative of n=6. Figure 5H shows the expression of CD4+ and CD8+ in cells from hES-TEC / TMC (n=3). Cell suspensions were gated on HuCD45+CD19-CD14- cells. [Figure 6]hES-TECs generated from TEPs prepared using the protocol shown in Figure 4A survive in the porcine thymus and promote thymocyte proliferation. Figure 6A outlines the protocol for testing hES-TECs in vivo. Pig thymi were either injected with or without hES-TEPs and transplanted under the renal capsule of ATX NSG mice that had been intravenously injected with human HSCs. Figure 6B shows the results of flow cytometric analysis of thymic grafts 18-22 weeks after transplantation. Single cell suspensions derived from the stromal fraction of half of the thymic grafts digested with Liberase were stained and analyzed by flow cytometry. A human pediatric thymus was prepared as a control. Non-hematopoietic cells were gated on huCD45-HLA-ABC+. Thymic fibroblast marker (CD105+) and epithelial cell marker EpCAM are shown. Figure 6C is a graph of the frequency of huCD45-HLA-ABC+CD105-EpCAM+ epithelial cells in SwTHY+hES-TECs grafts (left bar, squares) and SwTHY grafts (right bar, triangles). Figure 6D is a representative flow cytometry plot of thymocytes gated on huCD45+CD19-CD14- cells and CD4 / CD8 distribution for human pediatric thymuses and pig thymuses with and without hES-TEPs injection (left to right). Figure 6E is a graph of absolute numbers of thymocytes from half of the thymic graft among double positive CD4+CD8+ cells, single positive CD4+CD8- cells, and CD4-CD8+ cells, showing further division into immature CD45RO+ compared to more mature CD45RA+ thymocytes. Means + SEM are shown for SwTHY+hES-TEC (n=6, squares) and SwTHY (n=5, triangles) from two independent experiments. Thymic grafts that yielded less than 6x105 cells (n=1 each for SwTHY+hES-TEC and SwTHY) were excluded from the analysis. P values ​​were determined comparing SwTHY+hES-TEC groups to SwTHY groups using the Mann-Whitney test, with p<0.05 considered significant. +p=0.05, *p<0.05, **p<0.005. Figure 6F is a graph of human immune cells assayed for total human (huCD45+) cells in PBMCs at the indicated weeks after humanization.Means + SEM are shown for pig thymus only (n=9, black line with triangles) and pig thymus injected with hES-TEP (n=11, green line with squares) from two independent hES-TEC differentiations. Figure 6G is a graph of human immune cells assayed for total B cells (huCD19+) in PBMCs at the indicated weeks after humanization. Means + SEM are shown for pig thymus only (n=9, black line with triangles) and pig thymus injected with hES-TEP (n=11, green line with squares) from two independent hES-TEP differentiations. Figure 6H is a graph of total human CD45+ immune cells in spleens 18-22 weeks after humanization analyzed by flow cytometry. Means + SEM are shown for pig thymus injected with hES-TEP (n=7, squares) and pig thymus only (n=6, triangles) from two independent hES-TEC differentiations. Figure 6I is a graph of total human CD19+ B cells in spleens analyzed by flow cytometry 18-22 weeks after humanization. Mean + SEM is shown for hES-TEP-injected pig thymus (n=7, squares) and pig thymus only (n=6, triangles) from two independent hES-TEP differentiations. Figure 6J is a graph of total human CD14+ myeloid cells in spleens analyzed by flow cytometry 18-22 weeks after humanization. Mean + SEM is shown for hES-TEP-injected pig thymus (n=7, squares) and pig thymus only (n=6, triangles) from two independent hES-TEP differentiations. [Figure 7]hES-TEP prepared using the protocol in Figure 4A and injected into pig thymus increased the percentage of CD4+ T cells in the blood and increased the number of naive T cells and CD4+ recent thymic emigrants in the spleen compared to control mice transplanted with pig thymus. Figures 7A-7C show the results of human immune cells assayed in PBMCs at the indicated weeks after humanization. Means + SEM are shown for pig thymus only (n=9, black line with triangles) and pig thymus injected with hES-TEP (n=11, green line with squares) from two independent hES-TEP differentiations. Figure 7A shows CD3+ cells. Figure 7B shows CD8+ cells. Figure 7C shows CD4+ cells. A significant effect of TEP injection was revealed by two-way ANOVA with p<0.05 considered significant in CD3+ and CD4+ kinetics. Bonferroni post hoc multiple comparisons at each time point, p<0.05 is indicated with an asterisk (*). Figure 7D shows the absolute number of CD3+ T cells in the spleen 18-22 weeks after humanization. Figure 7E shows the absolute number of CD8+ T cells in the spleen 18-22 weeks after humanization. Figure 7F shows the absolute number of CD4+ T cells in the spleen 18-22 weeks after humanization. Figure 7G shows CD45RA versus CCR7 used to distinguish naive cells, effector memory (EM) cells, central memory (CM) cells and terminally differentiated effector memory cells (EMRA) that re-express CD45RA (left panel) in CD8+ T cells (middle panel) or CD4+ T cells (right panel). Figure 7H shows absolute numbers of recent thymic emigrant CD31+CD4+ naive cells, defined as CD45RA+CCR7+ cells among splenic mononuclear cells. Means + SEM are shown for hES-TEP-injected pig thymus (n=7, squares) and pig thymus only (n=6, triangles) from two independent hES-TEP differentiations. P values ​​were determined by comparing the SwTHY only group with the SwTHY hES-TEP-injected group using the Mann-Whitney test, with p<0.05 considered significant. *p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition The terms used herein generally have their ordinary meaning in the art within the context of the present invention and the specific context in which each term is used. Certain terms are described below or elsewhere in this specification to provide further guidance to the practitioner in describing the methods of the present invention and their uses. It will be further understood that there may be more than one way of expressing the same thing. Thus, alternative and synonymous terms may be used for one or more terms described herein, and it is not critical whether a term is detailed or explained in detail in this specification. Synonymous terms are provided for certain terms. The provision of one or more synonymous terms does not exclude the use of other synonymous terms. The use of examples provided anywhere in this specification, including examples of terms described herein, is for illustrative purposes only and does not limit the scope and meaning of the invention or the exemplified terms. Similarly, the invention is not limited to only its preferred embodiments.

[0029] As used herein, the term "induced pluripotent stem cells", commonly abbreviated as iPS cells or iPSCs, refers to a type of pluripotent stem cell that is artificially created from non-pluripotent cells, typically adult somatic cells, or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neural cells, epithelial cells, etc.).

[0030] As used herein, the terms "differentiation" and "cell differentiation" refer to the process by which less specialized cells (i.e., stem cells) develop or mature or differentiate into more specialized or differentiated cells (i.e., thymic epithelial cells) with a more distinct morphology and / or function.

[0031] The expressions "cell", "cell line" and "cell culture" used herein are used interchangeably, and all such names include progeny cells. Thus, the words "transformant" and "transformed cell" include the subject primary cell and the culture derived therefrom, regardless of the number of passages. It is also understood that not all progeny cells have exactly the same DNA content due to intentional or unintentional mutation. Mutant progeny cells that have the same function or biological activity as screened for in the originally transformed cell are included. If a different name is intended, it will be clear from the context.

[0032] With respect to cells, the term "isolated" refers to cells that are separated from their natural environment (e.g., derived from a tissue or subject). The term "cell line" refers to a population of cells capable of continuous or sustained growth and division in vitro. In many cases, a cell line is a cloned population derived from a single progenitor cell. It is further known in the art that spontaneous or induced karyotypic changes may occur during storage or passage of such cloned populations. Thus, cells derived from a cell line may not be exactly identical to their ancestor cells or cultures, and cell lines include such variants. As used herein, the term "recombinant cell" refers to a cell into which exogenous DNA fragments (such as DNA fragments that result in the transcription of a biologically active polypeptide or the production of a nucleic acid such as a biologically active RNA) have been introduced.

[0033] Abbreviation hPSC ~ human pluripotent stem cells ES or ESC ~ Embryonic Stem Cells iPSC ~ induced pluripotent stem cells TEC ~ Thymic epithelial cells TEP ~ Thymic epithelial progenitor cells PE ~ pharyngeal endoderm DE ~ definitive endoderm AFE ~ anterior foregut or anterior foregut endoderm PA ~ Pharyngeal arch 3rd PP ~ 3rd pharyngeal pouch Shh ~ Sonic Hedgehog RA ~ Retinoic Acid SP ~ Single positive DP ~ double positive

[0034] To differentiate the definite endoderm (DE) into the third pharyngeal pouch, a combination of FGF8 and retinoic acid (RA) was used to induce co-expression of TBX1 and HOXA3. Although RA treatment has previously been shown to increase HOXA3 activity (Parent et al., 2013; Diman et al., 2011), the potential ability of FGF8 to upregulate TBX1 was a novel finding disclosed herein. FGF8 appears to play two roles in the disclosed differentiation protocol: (i) FGF8 signaling immediately following activin exposure activates Tbx1 to anteriorize the definite endoderm (DE) into the pharynx-biased AFE (Green et al., 2011). Early exposure to FGF8 (day 4.5 vs. day 6.5; protocol #3c vs. #4c) strongly directs cultures towards the pharyngeal AFE and significantly increases the number of FOXN1+ cells at day 30; (ii) after anteriorization, FGF8b contributes to the development of the pharyngeal endoderm (PE), where it acts downstream of and together with TBX1 (Vitelli et al., 2002; Vitelli et al., 2010).

[0035] Another cytokine that plays an important role in pharyngeal endoderm (PE) development is sonic hedgehog (Shh) (Moore-Scott and Manley, 2005). RA exposure was reduced and replaced with Shh as an innovation (Protocol #1 vs. #3). This upregulated PAX9, PAX1, and TBX1, but downregulated HOXA, which is consistent with previous reports showing that Shh signaling induces Tbx1 in pharyngeal endoderm (PE) (Garg et al., 2001). High levels of HOXA3 are important for early pharyngeal field patterning, but its expression is reduced at later stages. Indeed, Pax1 expression is reduced in Hoxa3 null mutants, whereas Hoxa3 expression is normal in Pax1 and Pax9 double mutant embryos (Moore-Scott and Manley, 2005). Hoxa3 expression is downregulated by Shh - / - Thus, the contribution of HOXA3 and the Pax1-Pax9 temporal inverse gradient to the third pharyngeal pouch development further justifies the early use of RA followed by Shh treatment in the disclosed protocol.

[0036] In the final part of the protocol, cells are exposed to Noggin and then to BMP4. Although it has been shown that BMP signaling is required for FOXN1 expression (Patel et al., 2006; Swann et al., 2017), this disclosure is the first report to utilize Noggin, a BMP4 antagonist and / or inhibitor, for in vitro differentiation of the thymus. The presence of Noggin in the third pharyngeal pouch endoderm has been associated with the thyroid region rather than the thymus where BMP4 is expressed (Patel et al., 2006). In the protocol disclosed herein, adding ectopic Noggin to the culture further enhanced PAX9 expression at day 30. Since BMP4 expression begins at E10.5, shortly after Noggin expression at E9.5 in cells of the third pharyngeal pouch endoderm (Patel et al., 2006), cells were exposed to BMP4 from day 21 to day 30 (shortly after Noggin). This resulted in an increase in FOXN1 at day 30 compared to days 21 and 15. Interestingly, BMP4 treatment without prior exposure to Noggin did not result in an increase in FOXN1, confirming that Noggin is required for the emergence of sensitivity to BMP4.

[0037] Several groups have reported on the ability to generate mouse and human TEPs from PSCs (Parent et al., 2013; Sun et al., 2013; Soh et al., 2014; Su et al., 2015; Lai and Jim, 2009). In three reports, grafts consisting of these cells, often together with supporting mesenchymal or EPCAM-cells derived from TEP cultures, reconstituted mouse T cells in nude mice, but did not show stable and sustained thymocyte proliferation in normal-appearing thymic structures. Indeed, the possibility that thymocyte proliferation could be followed by amplification of mature T cells by peripheral lymphopenia was not excluded. One report demonstrated human T cell repopulation in peripheral tissues and human thymocyte proliferation in transplanted tissues, but no thymic structures were shown for the transplanted cells. Also, peripheral markers of recent thymic emigration were not included in the study, so it is unclear how robust and sustained the thymocyte proliferation is.

[0038] Described herein, we clearly demonstrated hPSC-TEC-dependent emergence of human naive T cells in the periphery of mice transplanted with hPSC-TEPs and thymic mesenchymal cells and receiving human HSCs. Because the thymus of NSG mice can also support human thymocyte proliferation, all NSG mice were thymectomized prior to transplantation of hPSC-TEPs (Khosravi et al., 2020), ensuring that all peripheral T cells were generated from the transplanted tissue. The phenotype of peripheral human T cells in these mice eventually converted to a memory type.

[0039] The inability to generate a sustained structured thymus from a "stand-alone" cell graft led to the development of a novel approach to evaluate the thymocyte proliferation function of hPSC-TEPs in vivo. Fetal pig thymus tissue has previously been demonstrated to support the proliferation of phenotypically normal human thymocytes with diverse TCR repertoires (Shimizu et al., 2008) and stable peripheral naive T cell populations in NSG mice (Nikolic and Sykes, 1999), with some subtle differences from what is observed in T cells developing in human thymic grafts (Kalscheuer et al., 2014). These fetal pig thymus fragments proliferate remarkably and contain up to hundreds of millions of human thymocytes in a normal-appearing thymic structure (Nikolic and Sykes, 1999; Kalscheuer et al., 2014). Disclosed herein is a methodology for injecting hPSC-TEPs into fragments of fetal porcine thymic tissue, which maintained human cells in close proximity to the porcine thymic tissue and led to their integration into the porcine thymus as it developed. Human TEPs integrated into the porcine thymus clearly expressed human cTEC- and mTEC-associated cytokeratins and appeared to integrate into the highly organized thymic structures of the grafts. Most importantly, they had a striking functional effect, significantly increasing the total number of human thymocytes and the number of peripheral human naive T cells, including CD4+CD34RA+ T cells with a CD31+RTE phenotype.

[0040] Method and system for obtaining thymic epithelial cells and / or thymic epithelial progenitor cells

[0041] The methods and systems described herein not only provide a reproducible method for obtaining thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEPs) by inducing differentiation of human pluripotent stem cells into thymic epithelial cells (TECs) or thymic epithelial progenitor cells (TEC precursor cells) (TEPs), but also provide improved purity and homogeneity, and thus enhanced ability, of thymic epithelial cells (TECs) or TEC precursor cells (TEPs).

[0042] The methods and systems described herein generate fully functional and reproducible specific cell populations upon transplantation. Moreover, the methods and systems described herein provide substantially homogenous populations of thymic epithelial cells (TECs) or TEC progenitor cells.

[0043] Human pluripotent stem cells are the starting material for the method of the invention. Human pluripotent stem cells (hPSCs) may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0044] The steps and timing of the method are shown in Table 1 and Figure 4A.

[0045] [Table 1]

[0046] The first step of the method is to differentiate the hPSCs into defined endoderm (DE) cells using any method known in the art. Exemplified herein is the use of a previously reported protocol that uses serum-free differentiation medium containing BMP4, bFGF and activin A. However, other protocols known in the art can also be used.

[0047] The next step of the method is to culture the definitive endoderm cells obtained in the first step to further differentiate into anterior foregut endoderm (AFE). Any medium from the differentiation protocol can be used for cell culture in this step. A serum-free differentiation medium is preferred. In addition, growth factors such as EGF and FGF can be added to the medium to promote cell proliferation.

[0048] To promote the differentiation of the definite endoderm cells into anterior foregut cell precursor cells, the endoderm cells are then contacted or incubated with a factor that inhibits BMP and a factor that inhibits TGFβ signaling. The most efficient way to achieve this is to add the factor to the medium in which the cells are cultured. However, any other method known in the art for contacting or incubating cells with a factor can be used. The cells can be contacted or incubated with multiple factors simultaneously or in parallel.

[0049] Factors that inhibit BMP include, but are not limited to, noggin and dorsomorphin.Factors that inhibit TGFβ signaling include, but are not limited to, SB431542.

[0050] Dorsomorphin can be used in amounts ranging from about 0.5 μM to about 2 μM.

[0051] Noggin can be used in an amount ranging from about 25 ng / ml to about 500 ng / ml, or from about 50 ng / ml to about 400 ng / ml, or from about 100 ng / ml to about 300 ng / ml, with a preferred amount being about 200 ng / ml.

[0052] The agent for inhibiting TGFβ signaling is SB431542 in an amount ranging from about 1 μM to about 50 μM, or in an amount ranging from about 2 μM to about 30 μM, or in an amount ranging from about 5 μM to about 20 μM. In some embodiments, the agent for inhibiting TGFβ signaling is SB431542 in an amount of about 10 μM.

[0053] However, other factors that inhibit TGFβ signaling can also be used in this method.

[0054] Furthermore, it has been shown that the combined stimulation of TBX1 and HOXA3 expression at the AFE stage is essential for physiological third pharyngeal pouch endoderm development. Therefore, the cells are further contacted or incubated with factors that stimulate the expression of these genes. The factor for TBX1 stimulation is FGF8b, which may be used in an amount ranging from about 10 ng / ml to about 200 ng / ml, or in an amount ranging from about 20 ng / ml to about 150 ng / ml, or in an amount ranging from about 30 ng / ml to about 100 ng / ml. In some embodiments, FGF8b may be used at about 50 ng / ml.

[0055] The cells are contacted or incubated with the agent from about day 4.5 to about day 15.

[0056] The agent for HOXA3 stimulation is retinoic acid (RA) and is used in an amount ranging from about 0.1 μM to about 0.6 μM, or from about 0.2 μM to about 0.5 μM. In some embodiments, retinoic acid may be used in an amount of about 0.6 μM. The cells can be contacted or incubated with the agent from about day 4.5 to about day 7.5. Stimulation of HOXA3 can be performed at other times, other than days 4.5 to 7.5, consisting of 3 days during the first 15 day period.

[0057] As shown in Figures 1D-1F, this protocol produces AFEs with high efficiency.

[0058] The cells are subsequently cultured in any serum-free medium (herein referred to as "differentiation medium" or "serum-free differentiation medium") used for differentiation of cells. In addition, growth factors such as EGF and FGF can be added to the differentiation medium to promote cell proliferation. Early in the process, the cells are contacted or incubated with retinoic acid (RA) in an amount ranging from about 0.1 μM to about 0.6 μM, or in an amount ranging from about 0.2 μM to about 0.5 μM, for about 1 to 2 days. In some embodiments, the cells are contacted or incubated with about 0.25 μM RA. The cells are subsequently contacted or incubated with FGF8b in an amount ranging from about 10 ng / ml to about 200 ng / ml, or in an amount ranging from about 20 ng / ml to about 150 ng / ml, or in an amount ranging from about 30 ng / ml to about 100 ng / ml throughout the process. As a non-limiting example, the cells may be contacted with about 50 ng / ml of FGF8b.

[0059] The next step promotes the differentiation of anterior foregut cells into pharyngeal endoderm (PE) cells.

[0060] In this step, the cells are contacted or incubated with a factor that induces the expression of PAX9 and PAX1. The most efficient way to accomplish this is to add the factor to the medium in which the cells are cultured. However, any other method known in the art of contacting or incubating the cells with the factor can be utilized. The cells may be contacted or incubated with multiple factors simultaneously or in parallel. One factor for stimulation of both PAX9 and PAX1 is Sonic Hedgehog (Shh) in an amount ranging from about 10 ng / ml to about 400 ng / ml, or in an amount ranging from about 25 ng / ml to about 300 ng / ml, or in an amount ranging from about 50 ng / ml to about 200 ng / ml. In some embodiments, Shh may be used at about 100 ng / ml.

[0061] The cells are subsequently contacted or incubated with FGF8b in an amount ranging from about 10 ng / ml to about 200 ng / ml, or from about 20 ng / ml to about 150 ng / ml, or from about 30 ng / ml to about 100 ng / ml throughout the entire incubation period. In some embodiments, the cells may be contacted or incubated with about 50 ng / ml of FGF8b.

[0062] Noggin may also be used to induce expression of PAX9 and PAX1. Noggin may be used in an amount ranging from about 50 ng / ml to about 400 ng / ml, or from about 60 ng / ml to about 300 ng / ml, or from about 75 ng / ml to about 200 ng / ml. In some embodiments, Noggin may be used in an amount of about 100 ng / ml.

[0063] This process is carried out for about 4 to about 10 days.

[0064] The next step is the differentiation of PE cells into distal third pharyngeal pouch / TECs. This step is divided into two parts: in the first part, the cells are contacted or incubated with factors that inhibit BMPs, including but not limited to noggin and dorsomorphin.

[0065] Dorsomorphin may be used in an amount ranging from about 0.5 μM to about 2 μM. Noggin may be used in an amount ranging from about 50 ng / ml to about 400 ng / ml, or from about 60 ng / ml to about 300 ng / ml, or from about 75 ng / ml to about 200 ng / ml. As a non-limiting example, Noggin may be used in an amount of about 100 ng / ml.

[0066] This portion of the process is carried out for about 5 days to about 7 days.

[0067] In a second part of the process, the cells are contacted or incubated with BMP4 in an amount ranging from about 5 ng / ml to about 300 ng / ml, or in an amount ranging from about 15 ng / ml to about 200 ng / ml, or in an amount ranging from about 25 ng / ml to about 100 ng / ml, or about 50 ng / ml. This part of the process is carried out for about 5 days to about 10 days.

[0068] The final cells obtained according to the method will show gene expression of TEC markers including FOXN1, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL7, K5, K8 and AIRE, see Figures 3A and 3B.

[0069] Although the above method is a novel, reproducible and reliable method for inducing differentiation of hPSCs into TECs or TEPs, the method also provides an additional step to reduce or eliminate pluripotent cells that may give rise to teratomas in the final transplanted cells. In this step, the cells are contacted or incubated with a survivin inhibitor (such as YM155) in an amount ranging from about 5 nM to about 50 nM for about the last 24 hours of the method. As a non-limiting example, the cells may be contacted or incubated with 20 nM YM155. The cells may also be contacted or incubated with a survivin inhibitor simultaneously with the BMP4 treatment. In some embodiments, the cells may be contacted or incubated with a survivin inhibitor during the first 24 to 48 hours of the simultaneous incubation with BMP4.

[0070] The invention also includes systems for carrying out the disclosed methods for obtaining TECs or TEPs from hPSCs. These systems may include subsystems, the subsystems including differentiation medium, and a factor that inhibits BMP and TGFβ signaling, a factor that stimulates expression of HOXA3, TBX1, PAX1 and PAX9, a factor that inhibits survivin, and BMP4. These systems may include subsystems, the subsystems including differentiation medium, and noggin, retinoic acid, FGF8b, sonic hedgehog, BMP, and YM155.

[0071] cell A further embodiment of the present disclosure are thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the differentiation protocols described herein.

[0072] In some embodiments, these cells express FOXN1, EpCAM, keratin 5, and keratin 8. In some embodiments, these cells are thymic epithelial cells (TECs). In some embodiments, these cells are thymic epithelial progenitor cells (TEC precursors) (TEPs).

[0073] Thus, one aspect of the disclosure is thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) suitable for administration, transplantation and grafting into a subject, produced by the methods described herein.

[0074] In another aspect, provided herein is a composition comprising thymic epithelial cells or TEC progenitor cells (TEPs) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and grafting into a subject. In some embodiments, the composition is a pharmaceutical composition further comprising any pharma- ceutically acceptable carrier or excipient.

[0075] In certain embodiments, the composition or pharmaceutical composition comprises at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods described herein. 6 Pieces, at least 5x10 6 Pieces, at least 1x10 7 Pieces, at least 5x10 7 Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0076] In certain embodiments, the present disclosure provides cryopreserved compositions or solutions of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and grafting into a subject.

[0077] In certain embodiments, the cryopreservation composition or solution contains at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. 6 Pieces, at least 5x10 6 Pieces, at least 1x10 7 Pieces, at least 5x10 7 Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0078] In certain embodiments, the disclosure provides a cell culture comprising thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. In certain embodiments, the cell culture comprises at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. 7 Pieces, at least 5x10 7 Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0079] In certain embodiments, the present disclosure provides therapeutic uses of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) produced by the methods described herein, suitable for administration, transplantation and grafting into a subject, and compositions, solutions and cell cultures comprising such cells.

[0080] In other embodiments, the present disclosure provides a substantially homogeneous population of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject. In some embodiments, the cell population comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thymic epithelial cells (TECs) or TEC progenitor cells (TEPs).

[0081] In another aspect, provided herein is a composition comprising a substantially homogeneous population of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) produced by the method as described herein.In some embodiments, these cells are suitable for administration, transplantation and grafting into a subject.In some embodiments, the composition is a pharmaceutical composition further comprising any pharma- ceutically acceptable carrier or excipient.

[0082] In certain embodiments, the population or composition or pharmaceutical composition comprises at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by a method as described herein. 6 Pieces, at least 5x10 6 Pieces, at least 1x10 7 Pieces, at least 5x10 7 Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0083] In certain embodiments, the present disclosure provides a cryopreservation composition or solution of a substantially homogenous population of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. In certain embodiments, the cryopreservation composition or solution contains at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. 6 Pieces, at least 5x10 6 Pieces, at least 1x10 7 Pieces, at least 5x10 7Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0084] In certain embodiments, the present disclosure provides a cell culture comprising a substantially homogenous population of thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods described herein. In certain embodiments, the cell culture comprises at least 1x10 thymic epithelial cells (TECs) or TEC progenitor cells (TEPs) generated by the methods as described herein. 7 Pieces, at least 5x10 7 Pieces, at least 1x10 8 Pieces, at least 5x10 8 Pieces, at least 1x10 9 Pieces, at least 5x10 9 pcs, or at least 1x10 10 In some embodiments, these cells are suitable for administration, transplantation, and grafting into a subject.

[0085] In certain embodiments, the present disclosure provides therapeutic uses of substantially homogenous populations of thymic epithelial cells (TECs) or thymic epithelial progenitor cells suitable for transplantation or grafting into a subject, as well as compositions, solutions, and cell cultures comprising such cells, produced by the methods as described herein.

[0086] A further embodiment is a thymic organ comprising the TECs or TEPs disclosed herein in combination with other cells that comprise the thymus.

[0087] therapeutic use The novel methods described herein for generating TECs or TEC precursors (TEPs) from stem cells, and the cells and substantially homogenous cell populations generated thereby, provide novel methods for treating disease.

[0088] The ability to generate functional TECs from human pluripotent stem cells may have important applications in modeling human immune responses in mice, and in modeling and treating thymic insufficiency syndromes (such as DiGeorge syndrome, NUDE syndrome, and the immunodeficiencies that complicate bone marrow transplantation in leukemia). The cells may also be utilized clinically for cell therapy purposes, transplanted into patients to achieve T cell reconstitution, or to generate immune tolerance to prevent graft rejection after organ transplantation, or to restore thymic function that has been reduced by injury or aging.

[0089] Thus, one embodiment is a method of treating or preventing a thymic disorder in a subject in need thereof, comprising administering, transplanting, or grafting to a subject in need thereof a therapeutically effective amount of cells of the present disclosure, a solution comprising cells of the present disclosure, a composition comprising cells of the present disclosure, or a pharmaceutical composition comprising cells of the present disclosure. The subject is preferably a mammal, and most preferably a human.

[0090] A further embodiment is a method of treating or preventing an autoimmune disease in a subject in need thereof comprising administering, transplanting or grafting to a subject in need thereof a therapeutically effective amount of cells of the present disclosure, a solution comprising cells of the present disclosure, a composition comprising cells of the present disclosure, or a pharmaceutical composition comprising cells of the present disclosure. The subject is preferably a mammal, and most preferably a human.

[0091] Another embodiment is a method of restoring or restoring decreased thymic function in a subject in need thereof, comprising administering, transplanting, or grafting to a subject in need thereof a therapeutically effective amount of cells of the present disclosure, a solution comprising cells of the present disclosure, a composition comprising cells of the present disclosure, or a pharmaceutical composition comprising cells of the present disclosure. The subject is preferably a mammal, and most preferably a human. In some embodiments, the decline is due to injury. In some embodiments, the decline is due to aging. In some embodiments, the decline is due to a congenital abnormality.

[0092] A further embodiment is a method of reconstituting T cells in a subject in need thereof comprising administering, transplanting or grafting to a subject in need thereof a therapeutically effective amount of cells of the present disclosure, a solution comprising cells of the present disclosure, a composition comprising cells of the present disclosure, or a pharmaceutical composition comprising cells of the present disclosure. The subject is preferably a mammal, and most preferably a human.

[0093] The cells obtained using the methods disclosed herein can be used to create a hybrid thymus. In some embodiments, the hybrid thymus comprises thymic epithelial cells obtained using the methods described herein and thymic tissue from a second individual of the same species. In some embodiments, the hybrid thymus comprises thymic epithelial cells obtained using the methods described herein and thymic tissue from a second species. In some embodiments, the second species is a pig. In some embodiments, the second species is a miniature pig. In some embodiments, the pig is a juvenile pig. In some embodiments, the pig is a fetus. Methods for obtaining such hybrid pigs are disclosed in commonly owned patent application PCT / US2019 / 051865.

[0094] A further embodiment is the use of the cells to develop mouse models. With the discovery of cellular reprogramming (iPSCs), a new era of disease modeling utilizing pluripotent stem cells indicates that a myriad of genetic diseases can now be created from patient tissue. iPSCs derived from patients with several different autoimmune diseases involving central tolerance can be differentiated into TECs (or TEPs) and then injected or transplanted into mice, where the cells can recapitulate or develop various pathologies or disorders. Humanized mouse models can be created from TECs derived from patients with autoimmune diseases such as multiple sclerosis, or type 1 diabetes, or congenital abnormalities such as DiGeorge syndrome. Mice can then be used in an in vivo environment to examine the progression of disorders that would not occur in vitro.

[0095] Furthermore, personalized humanized mouse models can be created using the cells described herein. The most developed humanized mouse models to date include human hematopoietic stem cells (HSCs) and pediatric or human fetal thymus samples transplanted under the kidney capsule. The limitation of these mouse models is that the HLA of the two types of cell populations (HSCs and TECs) is not compatible because they are derived from two different individuals. Using the differentiation protocol disclosed herein, it is possible to differentiate TECs (or TEPs) from the same iPSCs as HSCs, where the HLA of the immune system cells is compatible with the HLA of the human TECs transplanted into the mouse. This technology can be used for individual patients, resulting in personalized immune (PI) mice.

[0096] A further embodiment is the use of the cells for in vivo drug testing (using the mouse models described above, including but not limited to the personalized immunity (PI) mouse model) or in vitro drug testing. In vitro cultures of differentiated TECs can be used to test drugs against different pathologies that affect TECs, such as cancer (thymoma), or infectious diseases, or autoimmune diseases.

[0097] kit The present disclosure also provides kits.

[0098] In one embodiment, the kit comprises one or more components, such as human pluripotent stem cells; media for culturing and differentiating hPSCs (such as media containing growth factors; and factors that inhibit BMP and TGFβ signaling; factors that stimulate expression of HOXA3, TBX1, PAX1 and PAX9; factors that inhibit survivin; and BMP4).

[0099] In another embodiment, the kit comprises one or more components, including media for the culture and differentiation of human pluripotent stem cells, hPSCs, such as media containing growth factors and noggin, retinoic acid, FGF8b, sonic hedgehog, BMP, and YM155.

[0100] In further embodiments, the kit may include TECs or TEC progenitor cells (TEPs) obtained by the methods and systems of the present disclosure. The kit may also include reagents for culturing the cells.

[0101] In further embodiments, the kit may include a pharmaceutical composition comprising TECs or TEC progenitor cells (TEPs) obtained by the methods or systems of the present disclosure.

[0102] In further embodiments, the kit may include a cryopreservation composition comprising TECs or TEC progenitor cells (TEPs) obtained by the methods or systems of the present disclosure.

[0103] The kit may further include a package insert containing information regarding the pharmaceutical compositions and dosage forms in the kit. For example, the following information regarding the combination of the present invention may be provided in the package insert: how it is supplied, suitable storage conditions, references, manufacturer / distributor information, and patent information.

[0104] Working Example The present invention will be better understood by reference to the following non-limiting examples, which are set forth to more fully illustrate preferred embodiments of the invention, but are not intended to limit the broad scope of the invention in any way.

[0105] Example 1 - Methods and Materials Maintenance of hPSCs RUES2 (Rockefeller University Stem Cell Line 2; National Institutes of Health (NIH) approval number: NIHhESC-09-0013, registration number: 0013; passage number: 13–24) were cultured on mouse embryonic fibroblasts as previously described (Green et al., 2011). Mouse embryonic fibroblasts (GlobalStem, Rockville, MD) were cultured at approximately 25,000 cells / cm. 2 hPSCs were plated at a density of 1000 μg / ml. hPSCs were cultured in DMEM / F12 containing 20% ​​KnockOut Serum Replacement [Gibco (Life Technologies, Grand Island, NY)], 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 20 ng / ml FGF-2 (R&D Systems, Minneapolis, MN). Medium was changed daily and cells were passaged at a 1:24 dilution every 4 days using Accutase / EDTA (Innovative Cell Technologies, San Diego, CA). Undifferentiated hPSCs were maintained in a 5% CO2 atmosphere. Human H9 embryonic stem cell lines were also processed by protocol #4c. Cell lines were karyotyped every 6 months and checked for the absence of mycoplasma contamination using PCR.

[0106] Endoderm induction Differentiation was performed using serum-free differentiation medium (SFD) consisting of DMEM / F12 (3:1) (Life Technologies) supplemented with N2 [Gibco (Life Technologies)], B27 (Gibco), ascorbic acid (50 μg / ml, Sigma), Glutamax (2 mM, Life Technologies), monothioglycerol (0.4 μM, Sigma), 0.05% bovine serum albumin (BSA) (Life Technologies) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA) as described by Huang et al. (2014). Cells were then briefly trypsinized (0.05%, 37°C, 1 min) into a single cell suspension and seeded into low-attachment 6-well plates [Costar 2 (Corning Incorporated, Tewksbury MA)] and allowed to form embryoid bodies for 84 h (approximately 3.5 days) in low-attachment plates containing serum-free differentiation medium containing 0.5 ng / ml human BMP4, 2.5 ng / ml human bFGF, (R&D Systems) and 100 ng / ml human activin A (R&D Systems). Embryoid bodies were then collected, briefly trypsinized (0.05%, 37°C, 1 min) into 3-10 small cell clumps and resuspended in endoderm induction medium for an additional 24 h. Cells were maintained in a 5% CO2 / 5% O2 / 90% N2 atmosphere with medium changes every 24-48 h (depending on cell density).

[0107] Induction of anterior foregut endoderm, pharyngeal endoderm, and distal third pharyngeal pouch After a total of 108 hours on low-adherence plates containing endoderm induction medium (above), embryoid bodies were collected and, without trypsinization, plated (approximately 50,000-70,000 cells / well) into Matrigel-coated 24-well tissue culture plates for 48 hours in SFD medium supplemented with 200 ng / mL recombinant human (rh) Noggin and 10 μM SB431542 (NS) (as described in the protocol established by Green et al. (2011)), retinoic acid (0.25 μM) and 50 ng / mL FGF8b (as a novel modification of this protocol). To obtain pharyngeal endoderm, the prepared cells were then treated with FGF8b (50 ng / mL) and retinoic acid (0.25 μM) for 24 h, followed by FGF8b (50 ng / mL) and sonic hedgehog (Shh) (100 ng / mL) for 8 days (Figure 1B). To specialize into the third pharyngeal pouch, the cells were then exposed to rhnoggin (200 ng / mL) for 6 days, followed by BMP4 (10 ng / mL) until differentiation day 30 (Figure 2A). To prevent teratoma formation after transplantation into mice, the cells were also exposed to the survivin inhibitor YM155 (20 nM) (Lee et al., 2013)) for 24 h at the final stage of the subsequent experiment (Figure 4A). Throughout the entire process, the cell cultures were maintained at 37 °C in a 5% CO2 atmosphere. The cells were given a medium change every 24 h.

[0108] Real-time quantitative PCR Total RNA from clusters of ES cells differentiated in the indicated culture methods for the indicated times was extracted using Trizol (Invitrogen) and Direct-zol RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. A spectrophotometer NanoDrop 2000 (ThermoFisher Scientific) was used to determine RNA concentration. 500 ng of RNA was reverse transcribed and amplified with random hexamers using the Superscript III kit (Invitrogen) according to the manufacturer's instructions. Real-time quantitative PCR was performed in a volume of 20 μl using ABI Power SYBR Green PCR Master Mix in a thermal cycler ABI ViiA7 (Applied Biosystems Life Technologies). PCR cycle conditions were set at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Single-peak dissociation / melting curves were determined for all reactions and all primer pairs. Quantitative values ​​for each gene transcript were obtained by comparing the average C values ​​of triplicate experiments for each primer target against a standard curve of serially diluted genomic DNA, and then normalizing by dividing by the C values ​​of the housekeeping gene β-actin. A list of primer sequences is shown in Table 2.

[0109] [Table 2] TIFF2024153768000004.tif54162

[0110] Immunohistochemistry and immunofluorescence hES cultures in 24-well tissue culture plates were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were washed twice with PBS, permeabilized with 0.1% triton in PBS for 20 min, and blocked in 5% fetal donkey serum for 1 h at room temperature.

[0111] Thymus grafts were removed, embedded and frozen in OCT compound (Tissue-Tec, Torrance CA), and 5-7 μm thick sections were cut for immunostaining. Sections were stained with hematoxylin-eosin to visualize gross histology and the interface between the thymus graft and mouse kidney tissue. For immunofluorescence staining, tissue sections were fixed, permeabilized with ice-cold 100% acetone, and dried completely. Tissue sections were blocked in PBS supplemented with 0.1% Tween and 0.1% bovine serum albumin. Slides were washed with PBS containing 0.1% Tween, stained with primary antibodies for 2 h at room temperature, then washed and incubated with secondary antibodies for 2 h at room temperature.

[0112] Cultures or tissue sections were incubated with one or a combination of two or three of the primary antibodies shown in Table 3 below, followed by incubation with the appropriate secondary antibodies. Images were collected on a Leica slide scanner (SCN 400 whole slide scanning platform) for hematoxylin-eosin stained sections, and immunofluorescence images were collected on a Leica two-photon excitation laser scanning microscope TCS SP8.

[0113] [Table 3] TIFF2024153768000006.tif141162

[0114] Animal and human tissues NOD-scidIL2Rgamma nu11(NSG, stock 005557) mice were obtained from Jackson Laboratory and housed and bred in microisolator cages with Helicobacter-free and Pasteurella-free SPF barriers. Human fetal thymus and liver tissues (gestational age 17-20 weeks) were obtained from Advanced Biosciences Resource. Fetal liver tissue was minced into small pieces and incubated at 37°C in 199 medium (Corning) supplemented with 0.01 mg / ml bovine pancreatic DNase I (Sigma), 2.5 mM HEPES, 4 μg / ml gentamicin (Gibco), and 1 WU / ml Liberase® (Roche) to generate a single cell suspension. Cells were filtered through a 70 μm mesh cell strainer and brought to 100 ml with 199 medium supplemented as listed above without Liberase. Human mononuclear cells were enriched by density gradient centrifugation over 15 ml of Ficoll (Histopaque-1077, Sigma) with the hepatocyte suspension. Mononuclear cells were collected, washed, resuspended in MACS buffer, and CD34+ cells were enriched to approximately 80% pure CD34+ using magnetic activated cell sorting (MACS) according to the manufacturer's protocol (Miltenyi). CD34+ cells were aliquoted and frozen in human AB serum (GEMCell) containing 10% DMSO (Sigma).

[0115] 3-5mm human pediatric thymus from patients undergoing cardiac surgery 3 Fragments were cryopreserved in human AB serum containing 10% DMSO. To generate primary thymic mesenchyme, thymic fragments were thawed, disaggregated by Liberase® digestion as described above, and cultured at approximately 2×10 4 cells / cm 2Cells were seeded at 100°C for 30 min at 37°C. Medium was replaced after 48 h to remove unattached cells and was replaced every 3–4 days for up to 3 weeks until cells reached maximum surface confluence. Cells at passages 7–10 were used for experiments, and cell type was confirmed by flow cytometry (CD45-CD105+CD90+EpCAM-) (Siepe et al., 2009). The use of human tissues / cells was approved by the Columbia University Irving Medical Center (CUIMC) Institutional Review Board, and all experiments were performed in accordance with the approved protocols.

[0116] Humanized mice NSG mice aged 6–10 weeks were thymectomized as described by Khosravi-Maharlooei et al. (2020) and allowed to recover for at least 3 weeks. After recovery, animals were conditioned by X-ray total body irradiation (TBI) at 1.8 Gy. Cryopreserved fetal porcine thymus (gestational age 60–90 days) was thawed in medium 199 supplemented with DNAse, gentamicin and HEPES as described above. Fetal porcine fragments (l–2 mm) were then cut into 100 pieces. 3 ) with 2xL0 in a 28 gauge syringe 5 hES-derived TEPs were either injected or not with cells and coated with 50% Matrigel (Corning) in medium 199. At 4–24 h after total body irradiation (TBI), 1×10 6 ~2xl0 6 1x10 thymic mesenchymal cells mixed 6 ~2xl0 6 hES-derived TEPs, 1x10 6 ~2xl0 6 Thymic mesenchymal cells alone, fetal porcine thymus injected with hES-TEPs, or fetal porcine thymus alone were transplanted under the kidney capsule. 5Human fetal CD34+ cells were injected intravenously. Peripheral human immune reconstitution was assayed every 2-3 weeks after transplantation after full recovery as indicated. Blood samples were taken from the tail vein and immune cell populations were enriched by density gradient centrifugation over Ficoll as described above. At the time of euthanasia, thymus, spleen and peripheral blood were collected for analysis. Thymus grafts were excised from the kidneys of mice and divided into two. One thymus fragment was disrupted to develop thymocytes and the remaining stromal elements were digested with Liberase® as described above to prepare single cell suspensions for flow cytometry analysis. The second thymus fragment was embedded in OCT. Spleens were disrupted and filtered through a 70 μm nylon filter and red blood cells were lysed with hypotonic lysis buffer (ACK Gibco). Peripheral blood from cardiac puncture was enriched for white blood cells by density gradient centrifugation over Ficoll. All animal experiments were performed under protocols approved by the Columbia University Institutional Animal Care and Use Committee.

[0117] Flow cytometry Human immune reconstitution and differentiation efficiency of hES-TEP cultures were determined by multiparametric flow cytometry. To assay human immune reconstitution, single cell suspensions prepared from thymus grafts, tissue from the anterior mediastinum, spleen and peripheral blood were prepared as described above. Day 4.5 embryoid bodies from hES-TEP cultures were dissociated into single cells with 0.05% trypsin / EDTA. Cells were stained with fluorochrome-conjugated monoclonal antibodies against mouse and human cell surface antigens (Table 4). Cells were captured with an LSRII or Fortessa (BD Biosciences) and data analysis was performed using the software FlowJo (TreeStar, Ashland OR).

[0118] [Table 4]

[0119] statistics Statistical analysis and comparisons were performed using Graph-Pad Prism 7.0 (GraphPad Software). Values ​​for individual mice are shown as bar graphs, with bar heights representing the mean + standard error of the mean. For qPCR data, Ct values ​​normalized to the internal control β-actin were graphed, and relative gene expression was compared using a two-tailed Student's t test with paired ratios. For multiple comparisons (triplicates or more) of multiple experimental groups against a single control group, one-way ANOVA with Dunnett's multiple comparison test was used. Gene correlations were assessed by Pearson correlation coefficients, with p<0.05 considered significant, and linear regression was also performed to determine the coefficient of determination. Euthanasia due to teratoma growth was plotted on Kaplan-Meier plots and analyzed by the Mantel-Cox log-rank test to determine p values. Comparisons between mouse groups were performed by the nonparametric Mann-Whitney U test. Effects between transplantation groups were determined by calculation of two-way ANOVA. If the two-way ANOVA was significant (p<0.05), Bonferroni's multiple comparison test was performed for each individual time point. P<0.05 was considered significant.

[0120] Example 2 - Direct differentiation of hESCs into pharyngeal endoderm cells that are polarized to the third pharyngeal pouch The thymus is derived from the pharyngeal endoderm (PE), the anterior-most part of the endoderm. Direct differentiation of ESCs into TECs involves the sequential induction of definitive endoderm (DE), anterior foregut (AFE) and PE, followed by the formation of the third pharyngeal pouch (3 rdPP) requires the specification of the thymic region (Gordon and Manley, 2011) (Figures 1A and 2A). ESCs were differentiated into the DE and then into the AFE as described above with Activin A, followed by Noggin and SB431542 (NS) (Kubo et al., 2004; D'Amour et al., 2005; Green et al., 2011) (Figure 1B). Flow cytometry analysis showed co-expression of the endoderm markers EpCAM and CXCR4 in 98.3% of day 4.5 cells dissociated from embryoid bodies (Figure 1C). Inhibition of both BMP / TGFβ after DE induction resulted in AFE with high efficiency (>90%) (Soh et al., 2014). Concordantly, immunofluorescence staining at day 9 showed that the majority of cells expressed FOXA2 (endoderm) and SOX2 (foregut), confirming efficient specification into AFE (FOXA2+SOX2+) (results not shown).

[0121] We next focused on the differentiation of thymus-bound AFEs, focusing on genes involved in PE development and third pharyngeal pouch formation, HOXA3, TBX1, PAX9, PAX1, SIX1 and EYA1 ( Manley and Condie, 2010 ), whose expression was used as readout genes at day 15 in culture.

[0122] In humans, HOXA3 is found throughout the third pharyngeal pouch endoderm and in the surrounding mesenchyme, whereas TBX1 is expressed in the central core mesenchyme of the first, second, and third pharyngeal arches (PAs) and in the third pharyngeal pouch endoderm (Farley et al., 2013). In PE, the expression of these two genes overlaps only in the third pharyngeal pouch (Farley et al., 2013). Retinoic acid (RA), an essential factor for morphogenesis of the PA (Kopinke et al., 2006) and PP (Wendling et al., 2000), correlates with Hoxa3 expression (Diman et al., 2011), while the extent of Fgf8 localization in the PP overlaps with Tbx1 at E10.5 in mice (Vitelli et al., 2002). To mimic physiological third pharyngeal pouch endoderm development, co-expression of TBX1 and HOXA3 was induced by stimulating AFE cells with a combination of RA and FGF8b in protocol #1 (Figure 1B). To confirm the role of RA, this protocol was tested with protocol #2, in which RA was omitted. Addition of RA was essential for HOXA3 expression (Figure 1D, protocol #1 vs. #2), consistent with the results shown by Parent et al. (2013).

[0123] FGF10, FGF7, CHIR (an activator of the Wnt signaling pathway), and BMP4 are also factors known to regulate readout genes (Parent et al., 2013; Sun et al., 2013; Soh et al., 2014; Su et al., 2015). The effect of substituting FGF8 with each of these cytokines individually was examined in protocol #1. FGF8b+RA was the only combination (Figure 2B) that not only maximized the expression levels of most readout genes, but also drove TBX1 expression (Figure 2B and Figure 2C). The addition of BMP4, CHIR, FGF7, and FGF10 to the protocol using FGF8b+RA did not improve the expression of any third pharyngeal pouch markers (not shown).

[0124] Despite the expression of most readout genes, FOXN1, a master regulator of TEC differentiation (Romano et al., 2013), was barely detectable at day 15 of culture (not shown), leaving room for improvement. In mice, Pax9 and Pax1 are expressed in the four pharyngeal pouches and become restricted to a subpopulation of TECs after birth (Wallin et al., 1996; Hetzer-Egger et al., 2002). Thus, in addition to being markers of AFE, Pax1 and Pax9 are also markers of TECs. Although the expression of PAX9 and PAX1 is statistically higher in protocols #1 and #2 than in the negative control (liver, "liver condition" (Gouon-Evans et al., 2006)) (Figure 1E), Shh was added at day 7.5 of culture as a strategy to further upregulate PAX9 and PAX1, since Shh induces the expression of Pax1 and Pax9 in the ventral somites (Furumoto et al., 1999). Both Shh and its receptor, PTC1, have been reported to be expressed in human TECs and contribute to TEC differentiation ( Saldana et al., 2016 ; Sacedon et al., 2003 ).

[0125] Because Shh promotes RA clearance (Probst et al., 2002), RA exposure was reduced and replaced with Shh on day 6.5 (Figure 1B). This resulted in a significant increase in PAX9 (2.5-fold; p<0.0001) and also an increase in PAX1 that approached significance (5-fold; p=0.053) (Figure 1D, protocol #1 vs. #3). TBX1 expression was also significantly increased, consistent with reports showing that Shh increases Tbx1 expression in PE (Figure 1) (Gar et al., 2001).

[0126] We next investigated whether increasing exposure to FGF8b starting on culture day 4.5 would bias AFE development toward the PE and promote expression of third pharyngeal pouch genes. Because comparable expression of third pharyngeal pouch markers was observed in both protocols #3 and #4, we continued our efforts to optimize both simultaneously to pursue this possibility beyond differentiation day 15 (Figure 1F).

[0127] Example 3 - Distalization of the Third Pharyngeal Pouch Although the addition of FGF8b during anteriorization and / or culture with Shh improved the expression of third pharyngeal pouch markers, cultures at day 15 in culture showed low FOXN1 expression (results not shown). In mice, Bmp4 is coexpressed with FoxN1 in the ventral / posterior presumptive thymic region of the third pharyngeal pouch endoderm at E11.5 (Moore-Scott and Manley, 2005; Bleul and Boehm, 2005). Therefore, it was thought that the addition of BMP4 would result in better expression of FOXN1. Therefore, cultures at day 15 were exposed to BMP4 (protocols #3b and #4b in Figure 2A). However, the addition of BMP4 failed to induce the expression of FoxN1 assayed at days 22 and 30 of culture in protocols #3b and #4b (results not shown). It was hypothesized that insufficient expression of PAX9, which is also expressed in TECs after thymic organogenesis (Manley and Condie 2010; Hetzer-Egger et al. 2002), might be responsible for the insufficient FOXN1 expression.

[0128] We next examined whether the addition of Noggin would enhance PAX9 expression. Noggin is a BMP4 antagonist and / or inhibitor expressed throughout the mesenchyme of the third pharyngeal arch at E9.5 in mice, immediately adjacent to the early third pharyngeal pouch endoderm (Patel et al., 2006). BMP4 expression begins at E10.5 in cells of the third pharyngeal pouch endoderm (Patel et al., 2006). It was expected that Noggin would diffuse from the mesenchyme to the third pharyngeal pouch endoderm cells just before BMP4 signaling occurs in this region. To mimic this event, BMP4 was replaced with Noggin from days 16 to 22 in protocols #3c and #4c (Figure 2A). PAX9 expression was significantly increased in both protocols by the addition of Noggin (Figure 2D).

[0129] A five-fold higher level of FOXN1 expression was observed in protocol #4c (FGF8b in the middle of anteriorization) compared to protocol #3c (Figure 2E). Therefore, protocol #4c was further optimized. To confirm that cells continue to produce FOXN1 after the addition of BMP4, FOXN1 expression was compared at days 21 and 30 using protocol #4c. Figure 2F shows that FOXN1 expression was significantly higher at day 30 than at day 21, confirming that BMP4 exposure can promote FOXN1 expression even after day 21. In protocol #4c, FOXN1 levels at day 30 were eight-fold higher than at day 15 (Figure 2G).

[0130] Gene expression of TEC markers at day 30 of culture compared to lysates of human fetal whole thymus is shown in Figure 3A. Although thymic stromal samples were diluted by the presence of thymocytes, protocol #4c achieved 76% of the FOXN1 expression observed in thymic lysates, which was significantly higher than values ​​reported by other research groups performing the same comparison (Parent et al., 2013; Sun et al., 2013; Su et al., 2015). Furthermore, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL7, K5, K8 and AIRE mRNAs were detectable at levels comparable or higher than in fetal thymus. To establish the reproducibility of this protocol in other hESC lines, the human H9 embryonic stem cell line was treated with protocol #4c. Expression of TEC markers ISL1, FOXN1, K5, K8, DLL4, AIRE and IL7 (Figure 3B) was demonstrable in H9 cells differentiated by this protocol.

[0131] Immunostaining of day 15 protocol #4c cultures revealed colonies positive for the PE markers TBX1, EYA, ISL1, and SIX, which also co-stained with the third pharyngeal pouch marker EpCAM (results not shown). At day 30 of culture, these colonies were still positive for the general epithelial marker EpCAM, and for K5 and UEA-1, which are associated with mTECs, and K8, which is associated with cTECs (results not shown). A strong correlation was also found between the expression levels of FOXN1 and GCM2, a thyroid marker also found in the third pharyngeal pouch (Figure 3C). This suggests the presence of cells destined to mature into thyroid precursors despite BMP4 exposure, indicating incomplete distalization of the third pharyngeal pouch (Gordon et al., 2001). IL7 is the most important cytokine produced by TECs and promotes thymocyte survival, differentiation, and proliferation (Zamisch et al., 2005), as well as CD205, which functions as an endocytic receptor in cTECs (Shakib et al., 2009). We found that expression of IL7 and CD205 correlated with FOXN1 expression (Figure 3C).

[0132] Example 4 - Functional capacity assessment of hES-TEPs hES-TEPs differentiated with protocol #4c were examined for their ability to support thymocyte proliferation from human hematopoietic stem cells transplanted into humanized mice. The persistent presence of undifferentiated pluripotent cells in culture is a major barrier to clinical translation when utilizing ES and iPSC derived cells. Transplantation experiments revealed the presence of pluripotent cells upon transplantation, which leads to rapid uncontrolled proliferation of graft-derived cells and formation of teratomas (results not shown). Consistent with these results, OCT4, a marker of pluripotent cells, was detected in hES-TEP cultures at day 30 (Figure 4C) (Pan et al., 2002). However, TEPs at day 30 in culture showed co-expression of OCT4 in EpCAM+ cells (results not shown), and qPCR analysis showed a correlation between FOXN1 and OCT4 expression levels (Figure 4B), suggesting that OCT4 expression may be part of the TEC differentiation program.

[0133] The survivin inhibitor YM155 has been reported to selectively eliminate pluripotent cells (Lee et al., 2013). YM155 treatment at the final 24-h culture was tested to determine whether it was sufficient to eliminate pluripotent cells (Figure 4A). OCT4 expression was significantly reduced by YM155 treatment (Figure 4C). Engraftment of untreated day 15 hES-TEPs led to teratomas in all animals by 11 weeks post-implantation (Figure 4D). hES-TEPs cultured for 30 days in the presence and absence of YM155 showed reduced teratoma formation compared to untreated controls transplanted with day 15 TEPs, with only 3 of 15 animals in the group receiving YM155-treated cells forming teratomas (results not shown).

[0134] The native thymic anlagen of NSG hosts was able to support low-level thymocyte proliferation from human fetal liver-derived HSCs. We developed a method to surgically remove both lobes of the native thymic anlagen from NSG mice and blocked T cell development in thymectomized (ATX) NSG animals transplanted with human HSCs (Khosravi-Maharlooei et al., 2020). Complete removal of the native thymic anlagen in ATX mice was confirmed by harvesting connective tissue from the anterior mediastinum and assaying for the absence of thymocytes exhibiting CD4+CD8+ (Figure 5A and Figure 5B). Therefore, all subsequent recipients were thymectomized to assess transplanted hES-derived TEPs functionality.

[0135] Example 5 - Formation of functional thymus organs using hES-TEP / TMCs To test the functionality of cultured hES-TEPs in supporting thymocyte proliferation, clusters of hES-TEPs mixed with human thymic mesenchymal cells (TMCs) (generated using protocol #4c) or TMCs alone were cultured at 2 x 10 5Human HSCs were transplanted under the kidney capsule of ATX NSG mice intravenously injected with hES-TEP / TMC. Total human CD45+ cells in peripheral blood were shown for all mice, with an average human chimerism of 61% + 21% 11-31 weeks after humanization in mice transplanted with hES-TEP / TMC and 81% + 13% in mice transplanted with TMCs (Figure 5C). Engraftment of human HSCs resulted in predominant B cell production (data not shown). As early as 9 weeks after transplantation of TEPs under the kidney capsule, human CD3+ T cells were detected in more than 1% of total human blood cells in two mice transplanted with hES-TEP / TMCs and eventually in six of seven hES-TEP-transplanted mice, whereas TMC-transplanted controls showed no peripheral T cell reconstitution (Figure 5D). Although T cells were biased toward the CD4+ lineage rather than the CD8+ lineage, four of the seven hES-TEP / TMC-transplanted mice developed both CD4+ and CD8+ cells (Figure 5E and Figure 5G). CD4+ cells were further assayed for expression of the naive T cell marker CD45RA and the effector / memory T cell marker CD45RO. In the four mice that developed CD4+ and CD8+ T cells, CD4+ T cells had a predominantly naive phenotype (CD45RA+CD45RO-), consistent with de novo thymocyte proliferation (Figure 5F). Over time, CD4+ T cells converted to an effector / memory phenotype (CD45RA-CD45RO+), consistent with thymocyte proliferation arrest and lymphopenia.

[0136] CD4+CD8+ double positive cells were present at low frequency in hES-TEP / TMC (Figure 5H). hES-TEP / TMC grafts were slightly expanded in volume and displayed a disorganized structure with no discernible cortical or medullary regions on hematoxylin and eosin staining (results not shown). Furthermore, cells derived from hES-TEC / TMC grafts appeared to invade into the renal parenchyma, suggesting the presence of multiple cell types differentiating from TEP-cultured cells in vivo. Despite the disorganized structure, some cells in hES-TEC / TMC grafts co-stained with TEC markers EpCAM, pan-cytokeratin, and human MHC II (HLA-DR), suggesting terminal differentiation and long-term survival of hES-TECs (results not shown).

[0137] Example 6 - Strategies to examine the impact of hES-TECs: Evidence of integration into porcine thymus grafts We hypothesized that the ability of hES-TEPs to generate true thymic tissue in vivo might be limited by the absence of thymic structural scaffolding or other cell types required to generate a functional thymus. To address this possibility, we examined the survival and function of hES-TEPs (generated by protocol #4c) injected into fetal pig thymuses transplanted into humanized mice. See Figure 6A. Previously, fetal pig thymus (SwTHY) has been shown to support robust thymocyte expansion from human fetal liver-derived HSCs in NOD-scid or NSG mice (Kalcheuer et al., 2014; Nikolic and Sykes, 1999; Nauman et al., 2019).

[0138] The presence of hES-TECs was analyzed by flow cytometry and immunofluorescence in injected SwTHY grafts 18-22 weeks after transplantation. Stromal cells from half of the thymic grafts were dissociated with Liberase® and stained for markers of human cells (huCD45 and HLA-ABC), thymic fibroblasts (CD105) and epithelial cells (EpCAM). The distribution of CD105 and EpCAM cells for SwTHY+hES-TEC and SwTHY is shown for huCD45-HLA-ABC+ cells (Figure 6B). HuCD45-HLA-ABC+CD105-EpCAM+ were detected at a frequency of 1.6%+2.3% in the hES-TEC-injected thymus, whereas they were not detectable in the non-injected SwTHY, as expected (Figures 6B and 6C). Intact thymic grafts were stained with the epithelial cell markers cytokeratin 14 and anti-human pan-MHCII (HLADR). Cytokeratin 14 is expressed on human and porcine epithelial cells (red). HLA-DR is expressed on human antigen-presenting cells differentiated from human HSCs in bone marrow that seed the thymic grafts, and on terminally differentiated human TECs (green). Confocal microscopy observed colocalization of HLA-DR and cytokeratin expressed by hES-TECs (yellow) in injected SwTHY but not in non-injected SwTHY (results not shown). hES-TECs were detected in six of seven SwTHY+hES-TEC thymic grafts.

[0139] Example 7 - hES-TEP injection into pig thymus improved human thymocyte proliferation End-stage differentiated thymocytes were assayed by flow cytometry to determine whether hES-TECs support improved human thymocyte proliferation. The distribution of single positive (SP) CD4+, CD8+, and double positive (DP) CD4+CD8+ cells in SwTHY+hES-TECs and SwTHY grafts was similar to that in human pediatric thymuses (Figure 6D). hES-TECs in SwTHY resulted in a significant increase in the total number of thymocytes and CD4+CD8+DP cells compared to SwTHY grafts (Figure 6E). In SwTHY+hES-TECs, the frequency and absolute number of CD4+ single positive CD4+CD45RA+ and CD4+CD45RO+ T cells were significantly increased in SwTHY+hES-TECs compared to SwTHY grafts (Figure 6E). These data suggested that hES-TECs promote human thymocyte proliferation by providing the human MHC interactions necessary for thymocyte survival from the double-positive stage to terminal differentiation.

[0140] We next investigated whether infusion of hES-TEPs into SwTHY altered the frequency and phenotype of T cells in the periphery of HSC-injected mice compared to non-injected SwTHY transplanted under the renal capsule (Figure 6A). Animals transplanted with SwTHY or SwTHY injected with hES-TEPs (SwTHY+hES-TEC) developed stable human chimerism with similar frequencies of B cells, which averaged approximately 30%+14% in peripheral blood 11–21 weeks after humanization (Figure 6F and Figure 6G). Comparative kinetics of T cell reconstitution demonstrated a significant increase in the CD3+ T cell population, with an increased CD4+ T cell frequency in the blood of the SwTHY+hES-TEC group compared to the SwTHY group (Figure 7A).

[0141] As the primary immune organ, immune cell populations in the spleen were assayed to determine whether hES-TEC infusion altered the frequency and absolute number of cells. The frequency and total number of human immune cells were similar between the SwTHY+hES-TEC and SwTHY groups (Figure 6H). Similarly, the number of CD19+ B cells and CD14+ monocytes did not differ between the groups (Figure 6I and Figure 6J). The frequency and total number of CD3+ T cells were increased in the SwTHY+hES-TEC group compared to SwTHY-transplanted animals (Figure 7E). Both CD8+ cytotoxic T cells and CD4+ helper T cells were increased in percentage (%) and absolute number in the SwTHY+hES-TEP-treated group compared to the SwTHY control (Figure 7F).

[0142] Phenotypic and functional subpopulations of CD4 and CD8 T cells were defined based on expression of the chemokine receptor CCR7 and expression of CD45RA to define naive (CD45RA+CCR7+), central memory (Tcm) (CD45RA-CCR7+), effector memory (Tern) (CD45RA-CCR7-), and terminally differentiated effector memory cells (TEMRA) (CD45RA+CCR7-) populations that re-express CD45RA (Figure 7G) (Thome et al., 2014). Consistent with the increased numbers of T cells in animals transplanted with SwTHY+hES-TEP, naive, Tcm, Tern, and TEMRA were significantly increased in both the CD4+ and CD8+ T cell compartments (Figure 7G). CD31 (platelet / endothelial cell adhesion molecule-1 or PECAM-1) is expressed by new naive CD4+ T cells that have recently migrated from the thymus. Animals injected with SwTHY+TEP showed a significant increase in the number of CD31+ cells in the naive CD4+ T cell population compared with SwTHY controls (Figure 7H), consistent with the interpretation that hES-TECs contribute to human T cell development. References Bleul and Boehm, BMP Signaling Is Required for Normal Thymus Development. The Journal of Immunology, 2005. 175(8): p. 5213. Bredenkamp et al., An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts. Nature cell biology, 2014. 16(9): p. 902-908. D‘Amour et al., Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature Biotechnology, 2005. 23: p. 1534. Diman et al., A Retinoic Acid Responsive Hoxa3 Transgene Expressed in Embryonic Pharyngeal Endoderm, Cardiac Neural Crest and a Subdomain of the Second Heart Field. PLoS ONE, 2011. 6(11): p. e27624. Farley et al., Dynamics of thymus organogenesis and colonization in early human development. Development (Cambridge, England), 2013. 140(9): p. 2015-2026. Furumoto et al., Notochord-Dependent Expression of MFH1 and PAX1 Cooperates to Maintain the Proliferation of Sclerotome Cells during the Vertebral Column Development. Developmental Biology, 1999. 210(1): p. 15-29. Garg et al., Tbx1, a DiGeorge Syndrome Candidate Gene, Is Regulated by Sonic Hedgehog during Pharyngeal Arch Development. Developmental Biology, 2001. 235(1): p. 62-73. Green et al., Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nature Biotechnology, 2011. 29: p. 267. Gordon et al., Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch. Mechanisms of Development, 2001. 103(1): p. 141-143. Gordon, Hox genes in the pharyngeal region: how Hoxa3 controls early embryonic development of the pharyngeal organs. Int J Dev Biol, 2018. 62(11-12): p. 775-783. Gordon and Manley, Mechanisms of thymus organogenesis and morphogenesis. Development (Cambridge, England), 2011. 138(18): p. 3865-3878. Gouon-Evans et al., BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nature Biotechnology, 2006. 24(11): p. 1402-1411. Hetzer-Egger et al., Thymopoiesis requires Pax9 function in thymic epithelial cells. European Journal of Immunology, 2002. 32(4): p. 1175-1181. Huang et al., Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol, 2014. 32(1): p. 84-91. Kischeuer et al., Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts. J Immunol, 2014. 192(7): p. 3442-50. Khosravi-Maharlooei et al., Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice. Eur J Immunol, 2020. 50(1): p. 138-141. Kopinke et al., Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification. Developmental Dynamics, 2006. 235(10): p. 2695-2709. Kubo et al., Development of definitive endoderm from embryonic stem cells in culture. Development, 2004. 131(7): p. 1651. Lai and Jin, Generation of thymic epithelial cell progenitors by mouse embryonic stem cells. Stem Cells, 2009. 27(12): p. 3012-20. Lee et al., Inhibition of pluripotent stem cell-derived teratoma formation by small molecules. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(35): p. E3281-E3290. Manley and Condie, Transcriptional Regulation of Thymus Organogenesis and Thymic Epithelial Cell Differentiation, in Progress in Molecular Biology and Translational Science, A. Liston, Editor. 2010, Academic Press. p. 103-120. Scott and Manley, Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs. Developmental Biology, 2005. 278(2): p. 323-335. Nauman et al., Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction. Xenotransplantation, 2019: p. e12558. Nikolic and Sykes, Porcine thymus supports development of human T cells that are tolerant to porcine xenoantigens. Transplant Proc, 1999. 31(1-2): p. 924. Pan et al., Stem cell pluripotency and transcription factor Oct4. Cell Research, 2002. 12(5): p. 321-329. Parent et al., Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development. Cell stem cell, 2013. 13(2): p. 10.1016 / j.stem.2013.04.004. Patel et al., Bmp4 and Noggin expression during early thymus and parathyroid organogenesis. Gene Expression Patterns, 2006. 6(8): p. 794-799. Probst et al., SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling. Development (Cambridge, England), 2011. 138(10): p. 1913-1923. Romano et al., FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program. Frontiers in immunology, 2013. 4: p. 187-187. Sacedon et al., Expression of Hedgehog Proteins in the Human Thymus. Journal of Histochemistry and Cytochemistry, 2003. 51(11): p. 1557-1566. Saldana et al., Sonic Hedgehog regulates thymic epithelial cell differentiation. Journal of Autoimmunity, 2016. 68: p. 86-97. Shakib et al., Checkpoints in the Development of Thymic Cortical Epithelial Cells. The Journal of Immunology, 2009. 182(1): p. 130. Shimizu et al., Comparison of human T cell repertoire generated in xenogeneic porcine and human thymus grafts. Transplantation, 2008. 86(4): p. 601-10. Siepe et al., Human neonatal thymus-derived mesenchymal stromal cells: characterization, differentiation, and immunomodulatory properties. Tissue Eng Part A, 2009. 15(7): p. 1787-96. Soh et al., FOXN1(GFP / w) Reporter hESCs Enable Identification of Integrin-β4, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1(+) Thymic Epithelial Progenitors. Stem Cell Reports, 2014. 2(6): p. 925-937. Su et al., Efficient in vitro generation of functional thymic epithelial progenitors from human embryonic stem cells. Sci Rep, 2015. 5: p. 9882. Sun et al., Directed Differentiation of Human Embryonic Stem Cells into Thymic Epithelial Progenitor-like Cells Reconstitutes the Thymic Microenvironment In Vivo. Cell Stem Cell, 2013. 13(2): p. 230-236. Swann et al., Cooperative interaction of BMP signalling and Foxn1 gene dosage determines the size of the functionally active thymic epithelial compartment. Scientific reports, 2017. 7(1): p. 8492-8492. Sykes and Sachs, Transplanting organs from pigs to humans. Sci Immunol, 2019. 4(41). Tanabe et al., Role of Intrinsic (Graft) Versus Extrinsic (Host) Factors in the Growth of Transplanted Organs Following Allogeneic and Xenogeneic Transplantation. Am J Transplant, 2017. 17(7): p. 1778-1790. Thome et al., Spatial map of human T cell compartmentalization and maintenance over decades of life. Cell, 2014. 159(4): p. 814-28. Vitelli et al., A genetic link between Tbx1 and fibroblast growth factor signaling. Development, 2002. 129(19): p. 4605. Vitelli et al., Partial Rescue of the Tbx1 mutant Heart Phenotype by Fgf8: genetic evidence of impaired tissue response to Fgf8. Journal of molecular and cellular cardiology, 2010. 49(5): p. 836-840. Wallin et al., Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation. Development, 1996. 122(1): p. 23. Wendling et al., Retinoid signaling is essential for patterning the endoderm of the third and fourth pharyngeal arches. Development, 2000. 127(8): p. 1553. Yamada et al., Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med, 2005. 11(1): p. 32-4. Zamisch et al., Ontogeny and Regulation of IL-7-Expressing Thymic Epithelial Cells. The Journal of Immunology, 2005. 174(1): p. 60.

Claims

1. An in vitro or ex vivo method for generating a hybrid thymus comprising combining thymic epithelial cells (TECs) with additional cells comprising the thymus, the thymic epithelial cells being a process for inducing differentiation of pluripotent stem cells into epithelial cells or thymic epithelial progenitor cells (TEPs); (a) differentiating pluripotent stem cells into committed endoderm cells; (b) culturing the committed endoderm cells and differentiating the committed endoderm cells into anterior foregut cells by contacting or incubating the committed endoderm cells with a factor that inhibits bone morphogenetic protein (BMP) and a factor that inhibits TGFβ signaling, and further contacting or incubating the committed endoderm cells with a factor that stimulates expression of HOXA3 and a factor that stimulates expression of TBX1; (c) culturing the anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by contacting or incubating the anterior foregut cells with a factor that stimulates expression of HOXA3, a factor that stimulates expression of TBX1, and a factor that stimulates expression of PAX9 and PAX1; (d) culturing pharyngeal endoderm cells and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP)-specialized cells, TECs or TEPs, by contacting or incubating the pharyngeal endoderm cells with a factor that inhibits BMP; and (e) culturing the pharyngeal endoderm cells from step (c) or step (d) and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP)-specialized cells, TECs or TEPs, by contacting or incubating the pharyngeal endoderm cells with BMP; The method is obtained by a process comprising:

2. A method according to claim 1, comprising: (A)(i) step (a) is carried out for about 1 day to about 6 days; and / or (ii) in step (a), the pluripotent stem cells are cultured in a serum-free differentiation medium and contacted or incubated with human BMP in an amount of about 0.5 ng / ml, human basic fibroblast growth factor (bFGF) in an amount of about 2.5 ng / ml, and human activin A in an amount of about 100 ng / ml; (B)(i) step (b) is performed for about 2 to about 3 days starting on about day 3 to about day 5; and / or (ii) in step (b) the factor that inhibits BMP is selected from the group consisting of Noggin and Dorsomorphin, the factor that inhibits TGFβ signaling is SB431542, the factor that stimulates expression of HOXA3 is retinoic acid, and the factor that stimulates expression of TBX1 is FGF8b; (C)(i) step (c) is carried out for about 6 to about 10 days starting from about day 5 to about day 8; and / or (ii) in step (c) the factor that stimulates the expression of HOXA3 is retinoic acid, the factor that stimulates the expression of TBX1 is FGF8b, and the factor that stimulates the expression of PAX9 and PAX1 is sonic hedgehog (Shh), wherein in step (c) at about 24 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / ml and retinoic acid is used in an amount of about 0.25 μM, and at 48 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / mL and Shh is used in an amount of about 100 ng / ml; (D)(i) step (d) is carried out for about 4 to about 7 days starting on about day 12 to about day 18; and / or (ii) the factor that inhibits BMP in step (d) is selected from the group consisting of noggin and dorsomorphin; and / or (E) The method, wherein: (i) step (e) is performed for about 5 to about 15 days beginning on about day 19 to about day 25; and / or (ii) in step (e) BMP is used in an amount of about 50 ng / ml.

3. The method according to claim 1, further comprising the step of contacting or incubating the TECs or TEPs with a survivin inhibitor at the end of the method.

4. The method according to claim 3, wherein the survivin inhibitor is YM155.

5. An in vitro or ex vivo method for generating a hybrid thymus comprising combining thymic epithelial cells (TECs) with additional cells comprising the thymus, the thymic epithelial cells being a process for inducing differentiation of pluripotent stem cells into thymic epithelial cells or thymic epithelial progenitor cells (TEPs); (a) differentiating the pluripotent stem cells into committed endoderm cells by culturing the pluripotent stem cells in a serum-free differentiation medium and contacting or incubating the pluripotent stem cells with human bone morphogenetic protein (BMP), human basic fibroblast growth factor (bFGF) and human activin A; (b) differentiating the committed endoderm cells from step (a) into anterior foregut cells by culturing the committed endoderm cells from step (a) in serum-free differentiation medium and contacting or incubating the committed endoderm cells with noggin, SB431542 (NS), retinoic acid and FGF8b; (c) differentiating the anterior foregut cells from step (b) into pharyngeal endoderm cells by culturing the anterior foregut cells from step (b) in a serum-free differentiation medium and contacting or incubating the anterior foregut cells with FGF8b and retinoic acid followed by FGF8b and sonic hedgehog (Shh); (d) differentiating the pharyngeal endoderm cells from step (c) into third pharyngeal pouch specialized cells, TECs, or TEPs by culturing the pharyngeal endoderm cells from step (c) in serum-free differentiation medium and contacting or incubating the cells with Noggin; and (e) further differentiating the pharyngeal endoderm cells into third pharyngeal pouch specialized cells, thymic epithelial cells, or thymic epithelial progenitor cells by culturing the pharyngeal endoderm cells from step (c) or step (d) in a serum-free differentiation medium and contacting or incubating the pharyngeal endoderm cells with BMP; The method is obtained by a process comprising:

6. The method according to claim 1, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

7. The method according to claim 5, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

8. The method of claim 5, comprising: (A)(i) step (a) is carried out for about 1 day to about 6 days; and / or (ii) in step (a) BMP is used in an amount of about 0.5 ng / ml, human basic fibroblast growth factor is used in an amount of about 2.5 ng / ml, and human activin A is used in an amount of about 100 ng / ml; (B)(i) step (b) is performed for about 2 to about 3 days beginning on about day 3 to about day 5; and / or (ii) in step (b) noggin is used in an amount of about 200 ng / ml, SB431542 is used in an amount of about 10 μM, retinoic acid is used in an amount of about 0.25 μM, and FGF8b is used in an amount of about 50 ng / mL; (C)(i) step (c) is carried out for about 6 to about 10 days beginning about day 5 to about day 8; and / or (ii) in step (c), at about 24 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / mL and retinoic acid is used in an amount of about 0.25 μM; and / or (iii) in step (c), at about 48 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / mL and Shh is used in an amount of about 100 ng / ml; (D)(i) step (d) is performed for about 4 to about 7 days beginning on about day 12 to about day 18; and / or (ii) in step (d) Noggin is used in an amount of about 100 ng / ml; and / or (E) The method, wherein: (i) step (e) is performed for about 5 to about 15 days beginning on about day 19 to about day 25; and / or (ii) in step (e) BMP is used in an amount of about 50 ng / ml.

9. The method of claim 5, further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor at the end of the method.

10. The method according to claim 9, wherein the survivin inhibitor is YM155.

11. A method for generating a hybrid thymus, comprising transplanting thymic epithelial cells into a porcine thymus, the thymic epithelial cells being derived from a process for inducing differentiation of pluripotent stem cells into epithelial cells or thymic epithelial progenitor cells (TEPs), comprising: (a) differentiating pluripotent stem cells into committed endoderm cells; (b) culturing the committed endoderm cells and differentiating the committed endoderm cells into anterior foregut cells by contacting or incubating the committed endoderm cells with a factor that inhibits bone morphogenetic protein (BMP) and a factor that inhibits TGFβ signaling, and further contacting or incubating the committed endoderm cells with a factor that stimulates expression of HOXA3 and a factor that stimulates expression of TBX1; (c) culturing the anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by contacting or incubating the anterior foregut cells with a factor that stimulates expression of HOXA3, a factor that stimulates expression of TBX1, and a factor that stimulates expression of PAX9 and PAX1; (d) culturing pharyngeal endoderm cells and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP)-specialized cells, TECs or TEPs, by contacting or incubating the pharyngeal endoderm cells with a factor that inhibits BMP; and (e) culturing the pharyngeal endoderm cells from step (c) or step (d) and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP)-specialized cells, TECs or TEPs, by contacting or incubating the pharyngeal endoderm cells with BMP; The method is obtained by a process comprising:

12. The method according to claim 11, comprising: (A)(i) step (a) is carried out for about 1 day to about 6 days; and / or (ii) in step (a), the pluripotent stem cells are cultured in a serum-free differentiation medium and contacted or incubated with human BMP in an amount of about 0.5 ng / ml, human basic fibroblast growth factor (bFGF) in an amount of about 2.5 ng / ml, and human activin A in an amount of about 100 ng / ml; (B)(i) step (b) is performed for about 2 to about 3 days starting on about day 3 to about day 5; and / or (ii) in step (b), the factor that inhibits BMP is selected from the group consisting of noggin and dorsomorphin, the factor that inhibits TGFβ signaling is SB431542, the factor that stimulates expression of HOXA3 is retinoic acid, and the factor that stimulates expression of TBX1 is FGF8b; (C)(i) step (c) is carried out for about 6 to about 10 days starting from about day 5 to about day 8; and / or (ii) in step (c) the factor that stimulates the expression of HOXA3 is retinoic acid, the factor that stimulates the expression of TBX1 is FGF8b, and the factor that stimulates the expression of PAX9 and PAX1 is sonic hedgehog (Shh), wherein in step (c), at about 24 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / ml and retinoic acid is used in an amount of about 0.25 μM, and at about 48 hours from the start of step (c), FGF8b is used in an amount of about 50 ng / ml and Shh is used in an amount of about 100 ng / ml; (D)(i) step (d) is carried out for about 4 to about 7 days starting on about day 12 to about day 18; and / or (ii) in step (d), the factor that inhibits BMP is selected from the group consisting of noggin and dorsomorphin; and / or (E) The method, wherein: (i) step (e) is performed for about 5 to about 15 days beginning on about day 19 to about day 25; and / or (ii) in step (e), BMP is used in an amount of about 50 ng / ml.

13. The method of claim 11, further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor at the end of the method.

14. The method according to claim 13, wherein the survivin inhibitor is YM155.

15. A method for generating a hybrid thymus, comprising transplanting thymic epithelial cells into a porcine thymus, the thymic epithelial cells being derived from a process for inducing differentiation of pluripotent stem cells into epithelial cells or thymic epithelial progenitor cells (TEPs), comprising: (a) differentiating the pluripotent stem cells into committed endoderm cells by culturing the pluripotent stem cells in a serum-free differentiation medium and contacting or incubating the pluripotent stem cells with human bone morphogenetic protein (BMP), human basic fibroblast growth factor (bFGF) and human activin A; (b) differentiating the committed endoderm cells from step (a) into anterior foregut cells by culturing the committed endoderm cells from step (a) in serum-free differentiation medium and contacting or incubating the committed endoderm cells with noggin, SB431542 (NS), retinoic acid and FGF8b; (c) differentiating the anterior foregut cells from step (b) into pharyngeal endoderm cells by culturing the anterior foregut cells from step (b) in a serum-free differentiation medium and contacting or incubating the anterior foregut cells with FGF8b and retinoic acid followed by FGF8b and sonic hedgehog (Shh); (d) differentiating the pharyngeal endoderm cells from step (c) into third pharyngeal pouch specialized cells, TECs or TEPs, by culturing the pharyngeal endoderm cells from step (c) in serum-free differentiation medium and contacting or incubating the pharyngeal endoderm cells with Noggin; and (e) further differentiating the pharyngeal endoderm cells from step (c) or step (d) into third pharyngeal pouch specialized cells, thymic epithelial cells, or thymic epithelial cell precursor cells by culturing the pharyngeal endoderm cells from step (c) or step (d) in a serum-free differentiation medium and contacting or incubating the pharyngeal endoderm cells with BMP; The method is obtained by a process comprising:

16. The method of claim 11, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

17. The method of claim 15, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

18. The method of claim 15, comprising: (A)(i) step (a) is carried out for about 1 day to about 6 days; and / or (ii) in step (a), BMP is used in an amount of about 0.5 ng / ml, human basic fibroblast growth factor (bFGF) is used in an amount of about 2.5 ng / ml, and human activin A is used in an amount of about 100 ng / ml; (B)(i) step (b) is performed for about 2 to about 3 days beginning on about day 3 to about day 5; and / or (ii) in step (b), noggin is used in an amount of about 200 ng / ml, SB431542 is used in an amount of about 10 μM, retinoic acid is used in an amount of about 0.25 μM, and FGF8b is used in an amount of about 50 ng / mL; (C)(i) step (c) is carried out for about 6 to about 10 days beginning about day 5 to about day 8; (ii) in step (c), at about 24 hours from the initiation of step (c), FGF8b is used in an amount of about 50 ng / mL and retinoic acid is used in an amount of about 0.25 mM; and / or (iii) in step (c), at about 48 hours from the initiation of step (c), FGF8b is used in an amount of about 50 ng / mL and Shh is used in an amount of about 100 ng / ml; (D)(i) step (d) is carried out for about 4 to about 7 days beginning on about day 12 to about day 18; and / or (ii) in step (d), Noggin is used in an amount of about 100 ng / ml; and / or (E) The method, wherein: (i) step (e) is performed for about 5 to about 15 days beginning on about day 19 to about day 25; and / or (ii) in step (e), BMP is used in an amount of about 50 ng / ml.

19. The method of claim 15, further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor at the end of the method.

20. The method according to claim 19, wherein the survivin inhibitor is YM155.

21. The method according to any one of claims 11 to 20, wherein the pig thymus is derived from a pig selected from a miniature pig, a young pig, and a fetal pig.

22. A hybrid thymus produced by the method according to any one of claims 1 to 20.

23. A hybrid thymus produced by the method of claim 21.

24. Use of the hybrid thymus described in claim 22 to increase human thymus formation.

25. Use of the hybrid thymus described in claim 23 to increase human thymus formation.

26. Use of the hybrid thymus described in claim 22 to improve human T cell development.

27. Use of the hybrid thymus described in claim 23 to improve human T cell development.

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