Method for promoting differentiation of thymic epithelial cells and thymic epithelial cell progenitor cells of pluripotent stem cells

Abstract

The present disclosure provides methods of promoting differentiation of pluripotent stem cells into thymic epithelial cells or progenitor cells of thymic epithelial cells, as well as cells obtained from the methods, as well as solutions, compositions and pharmaceutical compositions comprising such cells. The present disclosure also provides methods of using thymic epithelial cells or thymic epithelial cell progenitor cells for the treatment and prevention of disease, organ generation, and for other uses, as well as kits.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080021893.1, application date March 31, 2020, and invention name “Method for Promoting Differentiation of Thymic Epithelial Cells and Thymic Epithelial Cell Progenitors of Pluripotent Stem Cells”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. patent application serial number 62 / 827,383 filed on April 1, 2019, the entire contents of which are incorporated herein by reference.

[0004] Government funding statement

[0005] This invention was made with government support under Grant Nos. DK104207, DK103585, and AI045897 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The present disclosure provides a method for promoting pluripotent stem cells to differentiate into thymic epithelial cells or thymic epithelial cell progenitor cells, as well as cells obtained from the method, and solutions, compositions and pharmaceutical compositions comprising such cells. The present disclosure also provides methods and kits for using thymic epithelial cells or thymic epithelial cell progenitor cells for treating and preventing diseases, producing organs and for other purposes. Background Art

[0007] The thymus is the primary lymphoid organ responsible for the development and culture of T cells. Thymic epithelial cells (TECs) are key components of the thymic stroma. TECs in the thymic cortex (cTECs) are specialized for T cell positive selection, whereas myeloid TECs (mTECs) participate in T cell negative selection. TEC-mediated selection promotes a self-tolerant and highly diverse pool of T cells that recognize foreign antigens presented by self-MHC molecules. In addition to TECs, normal thymopoiesis involves a highly organized network of stromal and hematopoietic cell types.

[0008] The in vitro generation of functional TECs or TEC progenitors (TEPs) from human pluripotent stem cells (hPSCs) can generate cells, tissues or organs that contribute to T cell reconstitution in patients with thymic dysfunction due to congenital disorders such as DiGeorge syndrome and acquired dysfunction due to HIV infection, high-dose chemotherapy and radiotherapy treatment, implant-versus-host disease and long-term immunosuppressive therapy combined with advanced age, which in itself leads to poor thymic production function. Since the number of TECs in the adult thymus is limited and reliable methods for expanding them from the postnatal thymus have been difficult to achieve, the generation of TECs from pluripotent stem cells (PSCs) is an important goal. Creating an in vitro protocol for the tightly controlled differentiation of hPSCs to TECs requires the knowledge and application of precise developmental timing and cytokine cues. Although the generation of functional TEPs from murine or human PSCs that support murine (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 described, high levels of reconstitution of naive human T cells have not been demonstrated. Therefore, there is a need in the art for a method to generate human TEPs and TECs. Summary of the invention

[0009] This article shows an effective method for inducing human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), to differentiate into thymic epithelial cell progenitors (TEC progenitors) in vitro, wherein thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) are capable of generating thymic organoids and T cells in vivo.

[0010] This protocol achieved the highest in vitro expression of FOXN1 described to date without protein transduction or genetic modification. After culture, the cells expressed epithelial markers EpCam, keratin 5, and keratin 8. When mixed with human thymic mesenchymal cells (ThyMES), the cells implanted in vivo in thymus-depleted NOD-scid IL2Rγ cells that received human hematopoietic stem cells (HSCs) intravenously null Supporting naive human T cell reconstitution in (NSG) mice (Khosravi-Mahrarlooei et al. 2020).

[0011] One embodiment of the present disclosure is a method for inducing human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to differentiate into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEC progenitors) (TEPs), the method comprising the following steps:

[0012] 1. Differentiate human pluripotent stem cells into endoderm cells;

[0013] 2. culturing the obtained endoderm cells, and differentiating the endoderm cells into anterior foregut cells by the following method: contacting or incubating the endoderm cells with an agent that inhibits BMP and an agent that inhibits TGFβ signaling, and further contacting or incubating the cells with an agent that stimulates HOXA3 expression and an agent that stimulates TBX1 expression;

[0014] 3. further culturing the obtained anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by the following method: contacting or incubating the anterior foregut cells with an agent that stimulates TBX1 expression and an agent that stimulates PAX9 and PAX1 expression;

[0015] 4. further culturing the obtained pharyngeal endoderm cells, and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP) specialized cells, thymic epithelial cells, or thymic epithelial cell progenitor cells by the following method: contacting or incubating the pharyngeal endoderm cells with an agent that inhibits BMP and then contacting or incubating the pharyngeal endoderm cells with BMP; and

[0016] 5. Contacting or incubating the TEC or TEP at the end of the method with a survivin inhibitor.

[0017] A further embodiment is a method of obtaining thymic epithelial cells (TEC) or thymic epithelial cell progenitors (TEP) from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the method comprising the steps of:

[0018] 1. Differentiate human pluripotent stem cells into endoderm cells;

[0019] 2. culturing the obtained endoderm cells, and differentiating the endoderm cells into anterior foregut cells by the following method: contacting or incubating the endoderm cells with an agent that inhibits BMP and an agent that inhibits TGFβ signaling, and further contacting or incubating the cells with an agent that stimulates HOXA3 expression and an agent that stimulates TBX1 expression;

[0020] 3. further culturing the obtained anterior foregut cells and differentiating the anterior foregut cells into pharyngeal endoderm cells by the following method: contacting or incubating the anterior foregut cells with an agent that stimulates TBX1 expression and an agent that stimulates PAX9 and PAX1 expression;

[0021] 4. further culturing the obtained pharyngeal endoderm cells, and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP) specialized cells, thymic epithelial cells by the following method: contacting or incubating the pharyngeal endoderm cells with an agent that inhibits BMP and then contacting or incubating the pharyngeal endoderm cells with BMP; and

[0022] 5. Contacting or incubating the TEC or TEP at the end of the method with a survivin inhibitor.

[0023] A further embodiment of the present disclosure is a method of inducing human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to differentiate into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEC progenitors) (TEPs), the method comprising the following steps:

[0024] 1. Differentiating pluripotent stem cells into endoderm cells by the following method: culturing 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;

[0025] 2. Differentiating the endoderm cells from the first step 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;

[0026] 3. differentiating the anterior foregut cells from the second step into pharyngeal endoderm cells by culturing the cells in a differentiation medium and contacting or incubating the cells with FGF8b and retinoic acid and subsequently contacting or incubating the cells with FGF8b and Sonic Hedgehog (Shh);

[0027] 4. Differentiating the pharyngeal endoderm cells from step 3 into third pharyngeal pouch specialized cells by culturing the cells in a differentiation medium and contacting or incubating the cells with noggin;

[0028] 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 cells in a differentiation medium and contacting or incubating the cells with BMP; and

[0029] 6. Exposing the cells to a survivin inhibitor.

[0030] A further embodiment is a method of obtaining thymic epithelial cells (TEC) or thymic epithelial cell progenitors (TEP) from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the method comprising the steps of:

[0031] 1. Differentiating pluripotent stem cells into endoderm cells by the following method: culturing 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;

[0032] 2. differentiating the endoderm cells from the first step 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;

[0033] 3. differentiating the anterior foregut cells from the second step into pharyngeal endoderm cells by culturing the cells in a differentiation medium and contacting or incubating the cells with FGF8b and retinoic acid, followed by contacting or incubating with FGF8b and sonic hedgehog (Shh);

[0034] 4. Differentiating the pharyngeal endoderm cells from step 3 into third pharyngeal pouch specialized cells by culturing the cells in a differentiation medium and contacting or incubating the cells with noggin;

[0035] 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 cells in a differentiation medium and contacting or incubating the cells with BMP; and

[0036] 6. Exposing the cells to a survival inhibitor.

[0037] In some embodiments, contacting or incubating cells with various agents is accomplished by culturing the cells in a culture medium comprising the agents.

[0038] 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.

[0039] In some embodiments, these cells express FOXN1, EpCAM, keratin 5 and keratin 8. In some embodiments, these cells are thymic epithelial cells (TEC). In some embodiments, these cells are thymic epithelial cell progenitors (TEC progenitors) (TEPs).

[0040] All of the aforementioned embodiments including cells, solutions, compositions and pharmaceutical compositions comprising the cells can be used to treat and / or prevent diseases.

[0041] In some embodiments, the disease is a thymic disease.

[0042] 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 ( syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease, vitiligo, and alopecia areata.

[0043] All of the foregoing embodiments, including cells, solutions, compositions and pharmaceutical compositions comprising the cells, can be used to restore or rejuvenate impaired thymic function, where the impaired function is caused by aging or injury or an infectious disease such as HIV.

[0044] All of the foregoing embodiments, including cells, solutions, compositions and pharmaceutical compositions comprising the cells, can be used to reconstitute T cells after bone marrow transplantation.

[0045] All of the foregoing embodiments, including cells, solutions comprising the cells, compositions, and pharmaceutical compositions, can be used to produce hybrid thymuses comprising the cells and thymus or other cells or tissues comprising the thymus. In some embodiments, the thymuses are from different individuals. In some embodiments, the thymuses are from different species. In some embodiments, the thymuses are from pigs. In some embodiments, the pigs are fetal pigs. In some embodiments, the pigs are young pigs.

[0046] All of the foregoing embodiments, including cells, solutions, compositions, and pharmaceutical compositions comprising the cells, can be used to develop mouse models and perform drug testing.

[0047] All of the foregoing embodiments, including cells, solutions comprising the cells, compositions and pharmaceutical compositions, can be used to develop the thymus for use in treating individuals with congenital anomalies in which thymic function is partially or completely impaired, such as individuals with DiGeorge syndrome, 22q.11.2 deletion syndrome or nude syndrome.

[0048] In yet further embodiments, the present disclosure relates to kits for practicing the methods of the present disclosure to obtain the cells, solutions, compositions, and pharmaceutical compositions disclosed herein. The present disclosure also includes kits comprising the cells, solutions, compositions, and pharmaceutical compositions.

[0049] As described herein, the methods, systems and kits are suitable for large-scale, reproducible production of thymic epithelial cells or thymic epithelial progenitors (TEPs). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] For the purpose of illustrating the invention, there are shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0051] Figure 1 - Establishment of a protocol for direct differentiation of hESCs into 3PP-biased pharyngeal endoderm. Figure 1A is a schematic representation of the putative steps of hESC differentiation toward a desired cell fate, reflecting Figure 1B The purposes of the processing are as indicated in . Figure 1B Schematic diagram of the protocols tested to differentiate hESCs into 3PP-biased pharyngeal endoderm until day 15. Protocol #1 (in Figure 1B indicated as "1" in (FGF8b+RA 250 ) is considered as a reference scheme. Figure 1D In the solution #2 (in Figure 1B indicated as "2" in (FGF8b) (#1 vs. #2) and #3 (in Figure 1B In the comparison, FGF8b+RA250 and FGF8b+Shh (indicated as "3") (#1 vs. #3) were compared. Figure 1B In the figure, “NS” indicates Noggin and SB431542. Figure 1C Shown are representative flow cytometric analyses of EpCAM and CXCR4 (endodermal marker) expression on embryoid bodies isolated at day 4.5. Figure 1D It is shown on the 15th day Figure 1B Graph of comparative analysis of gene expression in hESCs differentiated under indicated protocol conditions. The graph represents fold changes in RNA expression as measured by qPCR. (n=3-11, values ​​represent mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test). Figure 1EComparison of PP marker expression in hESCs differentiated using protocol #1 versus hESCs differentiated to 'liver' at day 15 ('liver pathology' (Gouon-Evans et al. 2006)). Bar graph represents fold change of RNA expression as measured by qPCR (n=6, values ​​represent mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test). Figure 1F It is shown that on the 15th day Figure 1B Graph of comparative analysis of gene expression in hESCs differentiated under indicated protocol conditions. The graph represents 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 ratio paired t-test).

[0052] Figure 2 - Development of a protocol for distalization of the 3PP and / or TEC. Figure 2A Schematic diagram of the tested protocols for distalization of 3PP-biased cells before day 30. Figure 2A In the “3b” and “3c” instructions, the Figure 1B Modification of Scheme #3 in ; "4b" and "4c" indicate based on Figure 1B A modification of Scheme #4 in . Figure 2B Schematic diagrams of various hESC differentiation protocols tested under different culture conditions starting from day 6.5 are shown. hESCs were differentiated into definitive endoderm (DE) for 4.5 days and then pre-cultured with noggin + SB (NS) and retinoic acid (RA). Cells were then patterned for 8.5 days with different combinations of RA and indicated factors until day 15. Figure 2C FOXA2, HOXA3, SIX1, TBX1, EYA1, PAX9, and PAX1 in hESC-derived cells from cultures containing RA and FGF8b (protocol #1) compared to Figure 2B Figure 2. Expression analysis of RA+ factors replacing FGF8b as shown in Figure 2. Bar graph represents fold change of 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 comparisons test). Figure 2DShown is the effect of Noggin exposure on PAX9 expression at day 30. Bar graphs represent the fold change in PAX9 expression between regimens #3b compared to #3c and #4b compared to #4c. (n=4, values ​​represent mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test). Figure 2E Shown are the fold changes in FOXN1 expression at day 30 after initiation of FGF8b treatment at day 4.5 versus day 6.5 (regimen #3c versus #4c) as measured by qPCR (n=4-8, values ​​represent mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test). Figure 2F Shown are the fold changes in FOXN1 expression at day 21 compared to day 30 of regimen #4c (before and after BMP4 exposure) as measured by qPCR (n=4-8, values ​​represent mean±SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test). Figure 2G Shown are the fold changes in FOXN1 expression on day 15 compared to day 30 of regimen #4c as measured by qPCR (n=4-8, values ​​represent mean±SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed ratio paired t-test).

[0053] FIG3 - Characterization of in vitro differentiated TEC progenitors at day 30. Figure 3A Shown is 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, two-tailed unpaired Welch's t-test). Each point represents an independent experiment. Figure 3B Shown is the expression of the 3PP marker in H9 cells cultured for 30 days under protocol #4c conditions compared to fetal thymus. Bar graphs represent mean Ct values ​​+ SEM relative to β-actin (n=3-6). Two-tailed unpaired Welch's t-test. Each point represents an independent experiment. Figure 3C Graph of Pearson correlation analysis of gene expression levels of FOXN1 and GCM2, FOXN1 and IL7, and FOXN1 and CD205. Both axes depict Ct values ​​relative to β-actin. Each point represents an independent experiment.

[0054] FIG. 4 - Treatment of day 30 hES-TEP cultures with the survivin inhibitor YM155 depletes pluripotent cells. Figure 4A is a schematic diagram of protocol #4c showing the time period of YM155 treatment. The schematic diagram also shows the complete differentiation protocol. Figure 4B is a graph of Pearson correlation analysis of FOXN1 and OCT4 expression. The two axes depict 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 depletion of pluripotent cells (regimen #4c compared to #4c+YM155; n=5, values ​​represent mean±SEM, *p<0.05, two-tailed ratio paired t-test). Figure 4D Graph showing percentage of survival without overt teratoma formation within weeks after hES-TEP transplantation. Mice implanted with hES-TEP from day 15 of protocol #4c (n=8, grey line) were compared with mice implanted with hES-TEP from day 30 cultures treated with (n=15, black dashed line) or without (n=12, black solid line) YM155. Log-rank Mantel Cox test showed p<0.005 for hES-TEP day 15 survival compared to hES-TEP day 30 alone or hES-TEP day 30 + YM155 treatment.

[0055] Figure 5 - Re-aggregation use Figure 4A hES-TEPs prepared according to the protocol shown in and thymic mesenchymal cells formed thymic organoids that supported thymopoiesis. Figure 5A Shown are the percentages of T cells when native thymic rudiments were surgically removed (ATX) or not from NSG mice injected with human HSCs. ACK lysis of peripheral blood yielded white blood cells (WBCs) that were stained for HuCD45+CD3+ T cells at the indicated weeks after HSC injection. NSG n=12, ATX NSG n=4. Figure 5B Representative FACS plots gated on HuCD45+CD19-CD14- cells. NSG n=10, ATX n=14. Figures 5C-5F Shown are the frequencies of various cells when cultured hES-TEP clusters mixed with one or more thymic mesenchymal cells (TMCs) were implanted individually under the kidney capsule of ATXNSG mice injected with human HSCs. Figure 5C The frequency of HuCD45+ cells among total mouse+human CD45+ cells in PBMCs of individual hES-TEC / TMC mice and the mean value of mice implanted with TMCs (n=6) are shown (grey line). Figure 5DThe frequency of CD3+ cells among total mouse+human CD45+ cells in PBMCs of individual hES-TEP / TMC mice and the mean value of mice implanted with TMCs (n=6) are shown (grey line). Figure 5E The frequency of CD4+ cells among total mouse+human CD45+ cells in PBMCs of individual hES-TEP / TMC mice and the mean value of mice implanted with TMCs (n=6) are shown (grey line). Fig. 5F The frequency of CD4+ cells stained for CD45RA+CD45RO- naive cells is shown. Time points with less than 100 CD4+ events were excluded. Figure 5G Human T cells in PBMCs from healthy humans (left), hES-TEC / TMC (middle), and TMC mice (right) 30 weeks after humanization are shown. The hES-TEC / TMC graph represents n=4 mice that developed CD4+ and CD8+ T cells, and the TMC graph represents n=6. Figure 5H CD4+ and CD8+ expression on cells from hES-TEC / TMC (n=3) is shown. Cell suspensions were gated on HuCD45+CD19-CD14- cells.

[0056] Figure 6 - Use Figure 4A hES-TECs produced by TEPs prepared according to the protocol shown in persist in porcine thymus and promote thymopoiesis. Fig. 6A is a schematic diagram of the protocol for testing hES-TECs in vivo. Porcine thymus were injected with or without hES-TEPs and implanted under the kidney capsule of ATX NSG mice injected intravenously with human HSCs. Figure 6B The results of flow cytometric analysis of thymic implants 18-22 weeks after transplantation are shown. Single cell suspensions of the matrix portion of the liberase digestion from half of the thymic implants were stained and analyzed by flow cytometry. Human children's thymus was prepared as a control. Non-hematopoietic cells were gated as huCD45-HLA-ABC+. Markers for thymic fibroblasts (CD105+) and the 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-TEC (left bar, squares) and SwTHY (right bar, triangles) implants. Fig.6D Representative flow cytometry plots of thymocytes gated on huCD45+CD19-CD14- cells for CD4 / CD8 distribution of human child thymus and porcine thymus injected or not with hES-TEP (left to right).

[0057] Fig. 6EFigure 2 is a graph showing the absolute counts of thymocytes from half of the thymic implants in double positive CD4+CD8+, single positive CD4+CD8-, and CD4-CD8+, which further differentiate into immature CD45RO+ compared to more mature CD45RA+ thymocytes. Mean ± SEM of SwTHY+ hES-TECs (n=6, squares) and SwTHY (n=5, triangles) from two independent experiments are shown. Cells that produced less than 6x10 5 (n=1, thymic implants from SwTHY+hES-TEC and SwTHY) cells, respectively. The p value was determined using the Mann-Whitney test, comparing the SwTHY+hES-TEC to the SwTHY group, with p<0.05 considered significant. +p=0.05, *p<0.05, **p<0.005. Fig. 6F Figure 2 is a graph of human immune cells measured against total human (huCD45+) cells in PBMC at the indicated weeks after humanization. Mean ± SEM of porcine thymus alone (n=9, black line with triangles) and porcine thymus injected with hES-TEP (n=11, green line with squares) from two independent hES-TEC differentiations are shown. Figure 6G Figure 2 is a graph of human immune cells measured for total B cells (huCD19+) cells in PBMC at the indicated weeks after humanization. Mean ± SEM of individual porcine thymus differentiated from two independent hES-TEPs (n=9, black line with black triangles) and porcine thymus injected with hES-TEPs (n=11, green line with squares) are shown. Figure 6H is a graph of total human CD45+ immune cells in spleen 18-22 weeks post-humanization analyzed by flow cytometry. Mean ± SEM of porcine thymus injected with hES-TEP (n=7, squares) and porcine thymus alone (n=6, triangles) from two independent hES-TEC differentiations are shown. Fig.6I is a graph of total human CD19+ B cells in spleen 18-22 weeks post-humanization analyzed by flow cytometry. Mean ± SEM of hES-TEP-injected porcine thymus (n=7, squares) and porcine thymus alone (n=6, triangles) from two independent hES-TEP-differentiated porcine thymus (n=7, squares) are shown. Figure 6J is a graph of total human CD14+ bone marrow cells in spleen 18-22 weeks post-humanization analyzed by flow cytometry. Mean ± SEM of hES-TEP-injected porcine thymus (n=7, squares) and porcine thymus alone (n=6, triangles) from two independent hES-TEP differentiations are shown.

[0058] Figure 7 - Compared with control mice implanted with porcine thymus, Figure 4A Injection of hES-TEP prepared according to the protocol shown in into porcine thymus promoted an increase in the proportion of CD4+ T cells in the blood and an increase in the number of naive T cells and CD4+ recent thymic migrants in the spleen. Figures 7A-7C Results for human immune cells assayed in PBMCs at indicated weeks after humanization are shown. Mean ± SEM of individual porcine thymus differentiated from two independent hES-TEPs (n=9, black line with triangles) and porcine thymus injected with hES-TEPs (n=11, green line with squares) are shown. Fig. 7A CD3+ cells are shown. Figure 7B CD8+ cells are shown. Figure 7C CD4+ cells are shown. Significant effects of TEP injection were revealed by two-way ANOVA, where p<0.05 was considered significant on CD3+ and CD4+ kinetics. * Post-hoc Bonferroni multiple comparisons indicating p<0.05 for each time point. Fig.7D Shown are the absolute numbers of CD3+ T cells in the spleen 18-22 weeks after humanization. Fig. 7E Shown are the absolute numbers of CD8+ T cells in the spleen 18-22 weeks after humanization. Figure 7F Shown are the absolute numbers of CD4+ T cells in the spleen 18-22 weeks after humanization. Figure 7G Shown is a comparison of CD45RA and CCR7 for distinguishing naive, effector memory (EM), central memory (CM) and terminally differentiated effector memory cells (EMRA) re-expressing CD45RA (left panel) in CD8+ (middle panel) or CD4+ T cells (right panel). Figure 7H The absolute number of recent thymic migrants CD31+CD4+ naive cells, defined as CD45RA+CCR7+ cells in spleen mononuclear cells, is shown. Mean ± SEM of porcine thymus injected with hES-TEP (n=7, squares) and porcine thymus alone (n=6, triangles) from two independent hES-TEP differentiations are shown. The p value was determined using the Mann-Whitney test, comparing SwTHY alone to the SwTHY hES-TEP injected group, where p<0.05 was considered significant. *p<0.05. DETAILED DESCRIPTION

[0059] definition

[0060] The terms used in this specification generally have their ordinary meaning in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners in describing the inventive method and how to use it. In addition, it should be understood that the same thing can be described in more than one way. Therefore, alternative wording and synonyms can be used for any one or more terms in the terms discussed herein, regardless of whether the terms are elaborated or discussed in detail herein without any special meaning. Synonyms for certain terms are provided. The description of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in the specification (including examples of any terms discussed herein) is only illustrative, and in no way limits the scope and meaning of the present invention or any exemplary term. Similarly, the present invention is not limited to its preferred embodiments.

[0061] As used herein, the term "induced pluripotent stem cell", often abbreviated as iPS cell or iPSC, refers to a type of pluripotent stem cell artificially generated from non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.).

[0062] As used herein, the terms "differentiation" and "cell differentiation" refer to the process by which a less specialized cell (ie, a stem cell) develops or matures or differentiates into a more specialized or differentiated cell (ie, a thymic epithelial cell) having a more distinct form and / or function.

[0063] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard to the number of transfers. It will also be understood that not all progeny will have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. When different names are intended, this will be clear from the context.

[0064] With respect to cells, the term "isolated" refers to cells that have been separated from their natural environment (e.g., from a tissue or subject). The term "cell line" refers to a cell population capable of continuous or prolonged growth and division in vitro. Often, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes in karyotype may occur during storage or transfer of such clonal populations. Thus, cells derived from a referenced cell line may not be exactly identical to the progenitor cell or culture, and referenced cell lines include such variants. As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid (such as RNA).

[0065] abbreviation

[0066] hPSC - human pluripotent stem cells

[0067] ES or ESC - Embryonic Stem Cell

[0068] iPSC - Induced Pluripotent Stem Cell

[0069] TEC - thymic epithelial cells

[0070] TEP - thymic epithelial progenitor cells

[0071] PE - pharyngeal endoderm

[0072] DE - Definitive endoderm

[0073] AFE - anterior foregut or anterior foregut endoderm

[0074] PA - pharyngeal arch

[0075] 3PP - Third pharyngeal pouch

[0076] Shh- Sound Hedgehog Factor

[0077] RA - Retinoic acid

[0078] SP - Single Positive

[0079] DP - Double Positive

[0080] To differentiate DE into the third PP, a combination of FGF8 and retinoic acid (RA) was used to induce co-expression of TBX1 and HOXA3. RA treatment has previously been shown to enhance HOXA3 activity (Parent et al. 2013; Diman et al. 2011), but the potential of TBX1 to upregulate FGF8 is a novel finding disclosed herein. FGF8 is believed to play a dual role in the disclosed differentiation protocol: i) FGF8 signaling immediately after activin exposure drives Tbx1, thereby anteriorizing DE into the pharyngeal-biased AFE (Green et al. 2011). Early exposure to FGF8 (day 4.5 vs. day 6.5; protocol #3c vs. #4c) strongly pushes the culture toward the pharyngeal AFE, significantly increasing the number of FOXN1+ cells at day 30; ii) After anteriorization, FGF8b promotes the development of PE, now acting downstream of and binding to TBX1 (Vitelli et al. 2002; Vitelli et al. 2010).

[0081] Another cytokine that plays a key role in PE development is sonic hedgehog (Shh) (Moore-Scott and Manley 2005). Reducing RA exposure and replacing it with Shh (protocol #1 compared to #3) served as another innovation. This upregulated PAX9, PAX1, and TBX1, but downregulated HOXA, which is consistent with previous reports showing that Shh signaling induces Tbx1 in PE (Garg et al. 2001). High levels of HOXA3 are essential for early pharyngeal patterning, but its expression is attenuated at later stages. Indeed, Pax1 expression is reduced in Hoxa3 null mutants, while Hoxa3 expression is normal in Pax1;Pax9 double mutant embryos (Moore-Scott and Manley 2005). Hoxa3 expression is upregulated in Shh - / - Thus, in the disclosed scheme, the contribution of the temporally opposite gradients of HOXA3 and Pax1-Pax9 to the development of the third PP further justifies the initial use of RA followed by Shh treatment.

[0082] In the final part of the protocol, cells were exposed to noggin and then to BMP4. Although BMP signaling has been shown to be required for FOXN1 expression (Patel et al. 2006; Swann et al. 2017), this is the first report of in vitro thymic differentiation using noggin (BMP4 antagonist and / or inhibitor). The presence of noggin in the 3rd PP endoderm is associated with the parathyroid domain expressing BMP4 rather than the thymus (Patel et al. 2006). In the published protocol, the addition of ectopic noggin to the culture further enhanced the expression of PAX9 at day 30. Since BMP4 expression begins at E10.5 in cells of the 3rd PP endoderm just after noggin is expressed at E9.5 (Patel et al. 2006), cells were exposed to BMP4 (immediately after noggin) from day 21 to day 30. This caused an increase in FOXN1 at day 30 compared to day 21 and day 15. Interestingly, BMP4 treatment without prior exposure to Noggin did not induce any increase in FOXN1, confirming that Noggin exposure is required to generate sensitivity to BMP4.

[0083] Several groups have reported the ability to generate murine 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, implants composed of these cells, often together with supporting mesenchymal or EPCAM-cells from TEP cultures, reconstituted murine T cells in nude mice, but robust, continuous thymopoiesis in normally appearing thymic architecture was not demonstrated. Indeed, the possibility that a wave of thymopoiesis is followed by peripheral lymphopenia-driven expansion of mature T cells cannot be excluded. In one report, recovery of human T cell populations in peripheral tissues and human thymopoiesis in implanted tissues were demonstrated, but thymic architecture of the implanted cells was not demonstrated. In addition, peripheral markers of recent thymic migration were not included in the study, so it is unclear how robust or durable thymopoiesis was.

[0084] Here, we clearly demonstrate the hPSC-TEC-dependent emergence of naive human T cells in the periphery of mice implanted with hPSC-TEPs plus thymic mesenchymal cells and receiving human HSCs. Because the NSG mouse thymus is also capable of supporting human thymopoiesis, all NSG mice were thymectomized prior to implantation of hPSC-TEPs (Khosravi et al. 2020), thus ensuring that all peripheral T cells were derived from the implanted tissue. The phenotype of peripheral human T cells in these mice was ultimately converted to a memory phenotype.

[0085] The inability to generate a durable, structured thymus from a "stand-alone" cell implant has led to the development of a new approach for assessing the thymic production function of hPSC-TEPs in vivo. It has been previously demonstrated that fetal porcine thymic tissue supports normal human thymic production (Nikolic and Sykes 1999) with different TCR libraries (Shimizu et al. 20008) and robust peripheral naive T cell populations in NSG mice, but with some subtle differences observed for T cells developed in human thymic implants (Kalscheuer et al. 2014). These fetal porcine thymic fragments grow significantly and contain up to hundreds of millions of human thymocytes in a normally occurring thymic structure (Nikolic and Sykes 1999; Kalscheuer et al. 2014). Disclosed herein is a method of injecting hPSC-TEPs into a fragment of fetal porcine thymic tissue, which maintains human cells near porcine thymic tissue and ultimately causes human cells to be incorporated into the porcine thymus as it grows. Human TEPs incorporated into porcine thymus clearly expressed human cTEC and mTEC-associated cytokeratin and appeared to be integrated into the highly organized thymic structure of the implant. Most importantly, they had a significant functional effect, significantly increasing the total number of human thymocytes and the number of peripheral naive human T cells, including CD4+CD34RA+T cells with a CD31+RTE phenotype.

[0086] Methods and systems for obtaining thymic epithelial cells and / or thymic epithelial cell progenitor cells

[0087] The methods and systems described herein not only provide a reproducible method for obtaining thymic epithelial cells (TEC) or TEC progenitors (TEPs) by inducing human pluripotent stem cells to differentiate into thymic epithelial cells (TEC) or TEC progenitors (TEPs), but also provide an increase in the purity and homogeneity of thymic epithelial cells (TEC) or TEC progenitors (TEPs), thereby increasing functionality.

[0088] The methods and systems described herein produce defined and reproducible populations of cells that are fully functional following transplantation.In addition, the methods and systems described herein provide a substantially homogenous population of thymic epithelial cells (TECs) or TEC progenitors.

[0089] Human pluripotent stem cells are the starting material for the method of the present invention. Human pluripotent stem cells (hPSC) can be embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

[0090] The steps and timing of the method are described in Table 1 and Figure 4A middle.

[0091] Table 1 - Timeline of differentiation methods

[0092]

[0093]

[0094] The first step of the method is to differentiate hPSC into definitive endoderm (DE) cells using any method known in the art. Exemplified here is a previously published protocol using a serum-free differentiation medium containing BMP4, bFGF, and activin A. However, other protocols known in the art may be used.

[0095] The next step of the method is to cultivate the definitive endoderm cells obtained from the first step so that they are further differentiated into anterior foregut endoderm (AFE). Any culture medium used for differentiation scheme can be used to culture cells in this step. Serum-free differentiation medium is preferred. In addition, growth factors (such as EGF and FGF) can be added to the culture medium to promote cell growth.

[0096] Then the endoderm cells are contacted or incubated with an agent that inhibits BMP and an agent that inhibits TGFβ signaling to promote the differentiation of definitive endoderm cells into anterior foregut progenitor cells. The most effective method to achieve this is to add the agent to the culture medium of the cultured cells. However, any other method known in the art for contacting or incubating the cells with the agent can be used. Cells can be contacted or incubated with the agent simultaneously or in parallel.

[0097] Agents that inhibit BMP include, but are not limited to, Noggin and Dorsomorphin. Agents that inhibit TGFβ signaling include, but are not limited to, SB431542.

[0098] Doxoromorphine may be used in an amount ranging from about 0.5 μM to about 2 μM.

[0099] Noggin may 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 about 200 ng / ml being a preferred amount.

[0100] The agent for inhibiting TGFβ signaling is SB431542 in an amount ranging from about 1 μM to about 50 μM, or from about 2 μM to about 30 μM, or 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.

[0101] However, other agents that inhibit TGF[beta] signaling may be used in the methods.

[0102] In addition, it was found that the combined stimulation of TBX1 and HOXA3 expression at the AFE stage is essential for physiological 3PP endoderm development. Therefore, the cells are further contacted or incubated with agents that stimulate the expression of these genes. The agent used to stimulate TBX1 is FGF8b, which can be used 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. In some embodiments, FGF8b can be used at about 50 ng / ml.

[0103] From about day 4.5 to about day 15, the cells are contacted or incubated with the agent.

[0104] The agent used to stimulate HOXA3 is retinoic acid (RA) 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 can be used in an amount of about 0.6 μM. From about day 4.5 to about day 7.5, the cells can be contacted or incubated with such an agent. In addition to day 4.5 to day 7.5, stimulation of HOXA3 can be performed during any other 3-day period during the first 15 days.

[0105] like Figures 1D-1F As shown in , this scheme produces an AFE with high efficiency.

[0106] In any serum-free medium (referred to herein as " differentiation medium " or " serum-free differentiation medium ") for cell differentiation, continue to cultivate cells. In addition, growth factors (such as EGF and FGF) can be added in differentiation medium to promote cell growth. When this step starts about one to two days, the cells are contacted with RA from about 0.1 μM to about 0.6 μM, or about 0.2 μM to about 0.5 μM in the amount of or hatched together. In some embodiments, cells are contacted with about 0.25 μM RA or hatched together. In this whole step, the cells are also continued to contact or hatch together with the FGF8b 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 in the amount of or hatched together. As a non-limiting example, cells can be contacted with about 50 ng / ml FGF8b.

[0107] The next step is to promote the differentiation of the anterior foregut cells into pharyngeal endoderm (PE) cells.

[0108] In this step, the cells are contacted or incubated with an agent that induces the expression of PAX9 and PAX1. The most effective way to achieve this is to add the agent to the culture medium of the cultured cells. However, any other method known in the art for contacting or incubating the cells with the agent can be used. The cells can be contacted or incubated with the agent simultaneously or in parallel. The agent used to stimulate both PAX9 and PAX1 is sonic hedgehog (Shh) in an amount ranging from about 10 ng / ml to about 400 ng / ml, or from about 25 ng / ml to about 300 ng / ml, or from about 50 ng / ml to about 200 ng / ml. In some embodiments, Shh can be used at about 100 ng / ml.

[0109] Throughout this step, the cells are also contacted or incubated with an amount of FGF8b 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. In some embodiments, the cells may be contacted or incubated with about 50 ng / ml FGF8b.

[0110] Noggin can also be used to induce the expression of PAX9 and PAX1. Noggin can be used in an amount ranging from about 50ng / ml to about 400ng / ml, or from about 60ng / ml to about 300ng / ml, or from about 75ng / ml to about 200ng / ml. In some embodiments, Noggin can be used in an amount of about 100ng / ml.

[0111] This step is performed for about 4 to about 10 days.

[0112] The next step is to differentiate the PE cells into distal third PP / TEC. This step is divided into two steps: First, the cells are contacted or incubated with an agent that inhibits BMP. Agents that inhibit BMP include but are not limited to noggin and dorsomorphin.

[0113] Doxoromorphine may be used in an amount ranging from about 0.5 μM to about 2 μM.

[0114] Noggin can be used in an amount ranging from about 50ng / ml to about 400ng / ml, or from about 60ng / ml to about 300ng / ml, or from about 75ng / ml to about 200ng / ml. As a non-limiting example, noggin can be used in an amount of about 100ng / ml.

[0115] This portion of the step is performed for about 5 days to about 7 days.

[0116] The second part of the step contacts or incubates the cells with BMP4 in an amount ranging from about 5 ng / ml to about 300 ng / ml, or ranging from about 15 ng / ml to about 200 ng / ml, or ranging from about 25 ng / ml to about 100 ng / ml, or about 50 ng / ml. This part of the step is performed for about 5 days to about 10 days.

[0117] The final cells obtained according to the method can show gene expression of TEC markers including FOXN1, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL7, K5, K8 and AIRE. Figure 3A and 3B .

[0118] Although the method described above is a new, reproducible and robust method for inducing hPSC to differentiate into TEC or TEP, the method of the present invention also provides a further step of reducing and eliminating pluripotent cells that can cause teratomas in cells that are ultimately implanted. In this step, the cells are contacted or incubated together with a survivin inhibitor (such as YM155) of an amount ranging from about 5nM to about 50nM in the approximately last 24 hours of the method. As a non-limiting example, the cells can be contacted or incubated together with 20nM YM155. The cells can also be contacted or incubated together with a survivin inhibitor in parallel with BMP4 treatment. In some embodiments, the cells can be contacted or incubated together with a survivin inhibitor during the first 24 to 48 hours of the parallel BMP4 incubation.

[0119] The present invention also includes systems for practicing the disclosed methods of obtaining TEC or TEP from hPSC. These systems may include subsystems, wherein the subsystems include differentiation medium and agents that inhibit BMP and TGFβ signaling, agents that stimulate HOXA3, TBX1, PAX1 and PAX9 expression, agents that inhibit survival, and BMP4. These systems may include subsystems, wherein the subsystems include differentiation medium, and noggin, retinoic acid, FGF8b, sonic hedgehog, BMP and YM155.

[0120] cell

[0121] A further embodiment of the present disclosure is a thymic epithelial cell (TEC) or TEC progenitor (TEP) produced by the differentiation protocol described herein.

[0122] In some embodiments, these cells express FOXN1, EpCAM, keratin 5 and keratin 8. In some embodiments, these cells are thymic epithelial cells (TEC). In some embodiments, these cells are thymic epithelial cell progenitors (TEC progenitors) (TEPs).

[0123] Thus, one aspect of the present disclosure is a thymic epithelial cell (TEC) or TEC progenitor (TEP) produced by the methods described herein that is suitable for administration, transplantation, and implantation into a subject.

[0124] On the other hand, there is provided herein a composition comprising thymic epithelial cells or TEC progenitors (TEPs) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject. In some embodiments, the composition is a pharmaceutical composition further comprising any pharmaceutically acceptable carrier or excipient.

[0125] 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 1 x 106, at least 5 x 106, at least 1 x 107, at least 5 x 107, at least 1 x 108, at least 5 x 108, at least 1 x 109, at least 5 x 109, or at least 1 x 1010 thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods as described herein. In some embodiments, these cells are suitable for administration, transplantation, and implantation into a subject.

[0126] In certain embodiments, the present disclosure provides cryopreserved compositions or solutions of thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods as described herein. In some embodiments, these cells are suitable for administration, transplantation, and implantation into a subject.

[0127] In certain embodiments, the refrigerated composition or solution comprises at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1 x 106, at least 5 x 106, at least 1 x 107, at least 5 x 107, at least 1 x 108, at least 5 x 108, at least 1 x 109, at least 5 x 109, or at least 1 x 1010 thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation, and implantation into a subject.

[0128] In certain embodiments, the disclosure provides a cell culture comprising thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by the methods described herein. In certain embodiments, the cell culture comprises at least 1x 107, at least 5x 107, at least 1x 108, at least 5x 108, at least 1x 109, at least 5x 109 or at least 1x 1010 thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject.

[0129] In certain embodiments, the present disclosure provides therapeutic uses of thymic epithelial cells (TEC) or TEC progenitors (TEPs) produced by the methods as described herein, and compositions, solutions, and cell cultures comprising such cells, suitable for administration, transplantation, and implantation into a subject.

[0130] In other embodiments, the disclosure provides a population of substantially homogeneous thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject. In some embodiments, the cell colony comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thymic epithelial cells (TEC) or TEC progenitors (TEP).

[0131] On the other hand, a composition is provided herein, comprising a population of substantially homogeneous thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject. In some embodiments, the composition is a pharmaceutical composition further comprising any pharmaceutically acceptable carrier or excipient.

[0132] 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 1 x 106, at least 5 x 106, at least 1 x 107, at least 5 x 107, at least 1 x 108, at least 5 x 108, at least 1 x 109, at least 5 x 109, or at least 1 x 1010 thymic epithelial cells (TEC) or TEC progenitors (TEP) produced by the methods described herein. In some embodiments, these cells are suitable for administration, transplantation, and implantation into a subject.

[0133] In certain embodiments, the disclosure provides a refrigerated composition or solution of a colony of substantially homogeneous thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by methods as described herein. In certain embodiments, the refrigerated composition or solution comprises at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1x 106, at least 5x 106, at least 1x 107, at least 5x107, at least 1x 108, at least 5x 108, at least 1x 109, at least 5x 109 or at least 1x 1010 thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by methods as described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject.

[0134] In certain embodiments, the disclosure provides a cell culture comprising a population of substantially homogeneous thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by the invention as described herein. In certain embodiments, the cell culture comprises at least 1x 107, at least 5x 107, at least 1x108, at least 5x 108, at least 1x 109, at least 5x 109 or at least 1x 1010 thymic epithelial cells (TEC) or TEC progenitor cells (TEP) produced by the methods as described herein. In some embodiments, these cells are suitable for administration, transplantation and implantation into a subject.

[0135] In certain embodiments, the present disclosure provides therapeutic uses of substantially homogeneous populations of thymic epithelial cells (TEC) or TEC progenitors (TEPs) suitable for transplantation and implantation into a subject produced by the methods as described herein, and compositions, solutions, and cell cultures comprising such cells.

[0136] A further embodiment is a thymic organoid comprising a TEC or TEP disclosed herein in combination with other cells that make up the thymus.

[0137] Therapeutic Uses

[0138] The novel methods described herein for generating TECs or TEC progenitors (TEPs) from stem cells, and the cells and substantially homogeneous cell populations generated by this method, provide new treatments for diseases.

[0139] The ability to generate functional TECs from human pluripotent stem cells will have important applications in modeling human immune responses in mice and in modeling and treating thymus deficiency syndromes (such as DiGeorge syndrome, Noord syndrome, and immunodeficiency complicated by bone marrow transplantation for leukemia). The cells can also be used clinically for cell therapy and transplanted into patients to achieve T cell reconstitution, or to generate immune tolerance to prevent implant rejection after organ transplantation, or to restore impaired thymus function due to injury or aging.

[0140] Thus, one embodiment is a method of treating or preventing a thymic disease in a subject in need thereof, comprising the step of administering, transplanting or implanting a therapeutically effective amount of a cell of the present disclosure, a solution comprising a cell of the present disclosure, a composition comprising a cell of the present disclosure, or a pharmaceutical composition comprising a cell of the present disclosure to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0141] A further embodiment is a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising the step of administering, transplanting or implanting a therapeutically effective amount of a cell of the present disclosure, a solution comprising a cell of the present disclosure, a composition comprising a cell of the present disclosure, or a pharmaceutical composition comprising a cell of the present disclosure to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0142] Another embodiment is a method of restoring or rejuvenating impaired thymic function in a subject in need thereof, comprising the step of administering, transplanting or implanting a therapeutically effective amount of a cell of the present disclosure, a solution comprising a cell of the present disclosure, a composition comprising a cell of the present disclosure, or a pharmaceutical composition comprising a cell of the present disclosure to a subject in need thereof. The subject is preferably a mammal, and most preferably a human. In some embodiments, the impairment is due to injury. In some embodiments, the impairment is due to aging. In some embodiments, the impairment is due to congenital abnormalities.

[0143] Yet further embodiments are methods of reconstituting T cells after bone marrow transplantation in a subject in need thereof, comprising the step of administering, transplanting or implanting a therapeutically effective amount of a cell of the disclosure, a solution comprising a cell of the disclosure, a composition comprising a cell of the disclosure, or a pharmaceutical composition comprising a cell of the disclosure to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0144] Cells obtained using the methods disclosed herein can be used to produce hybrid thymus. In some embodiments, the hybrid thymus comprises thymic epithelial cells obtained using the methods disclosed 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 disclosed 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 young pig. In some embodiments, the pig is a fetus. A method for obtaining such a hybrid pig is disclosed in co-owned patent application number PCT / US2019 / 051865.

[0145] A further embodiment is to use the cells to develop a mouse model. Since the discovery of cell reprogramming (iPSC), pluripotent stem cells representing countless genetic diseases can now be produced from patient tissues, creating a new era of disease modeling. IPSCs from patients with different autoimmune diseases involving central tolerance can be differentiated into TEC (or TEP), which are then injected or implanted into mice, where the cells can regenerate and develop into various morbidities or illnesses. Humanized mouse models can be produced by TECs of patients with autoimmune diseases (such as multiple sclerosis), or type I diabetes, or congenital abnormalities (congenic abnormality) (such as DiGeorge syndrome). The in vivo environment of the mouse can then be used to study the progress of illnesses that could not originally develop in vitro.

[0146] In addition, personalized humanized mouse models can be produced using the cells described herein. So far, the most developed humanized mouse models contain samples of human hematopoietic stem cells (HSC) and children or human fetal thymus implanted under the kidney capsule. The limitation of these mouse models is that the HLA from these two types of cell populations (HSC and TEC) do not match because they come from two different individuals. Using the differentiation scheme disclosed herein, TEC (or TEP) can be differentiated from iPSC identical to HSC, so immune system cell HLA will match the HLA on the human TEC transplanted on mice. This technology can be used for individual patients, thereby producing personalized immune (PI) mice.

[0147] A further embodiment is the use of cells for in vivo (using the mouse models described previously, including but not limited to the personalized immunity (PI) mouse model) or in vitro drug testing. In vitro differentiated TEC cultures can be used to test the efficacy of drugs against different pathologies affecting TEC, such as cancer (thymoma) or infectious or autoimmune diseases.

[0148] Reagent test kit

[0149] The present disclosure also provides kits.

[0150] In one embodiment, the kit includes one or more components, including human pluripotent stem cells, a culture medium for culturing and differentiating hPSCs, such culture medium comprising growth factors and an agent that inhibits BMP and TGFβ signaling, an agent that stimulates the expression of HOXA3, TBX1, PAX1 and PAX9, an agent that inhibits survival, and BMP4.

[0151] In another embodiment, a kit includes one or more components, including human pluripotent stem cells, a medium for culturing and differentiating hPSCs, the medium comprising growth factors and noggin, retinoic acid, FGF8b, sonic hedgehog, BMP, and YM155.

[0152] In further embodiments, the kit may include TEC or TEC progenitor cells (TEP) obtained by the current methods and systems of the present disclosure. The kit may also include reagents for culturing the cells.

[0153] In further embodiments, a kit may include a pharmaceutical composition comprising TEC or TEC progenitor cells (TEPs) obtained by the current methods and systems of the present disclosure.

[0154] In further embodiments, a kit may include a cryopreserved composition comprising TEC or TEC progenitor cells (TEPs) obtained by the present methods and systems of the present disclosure.

[0155] The kit may further include a package insert containing information about the pharmaceutical composition and dosage form in the kit. For example, the following information about the combination of the invention may be provided in the insert: how supplied, appropriate storage conditions, reference materials, manufacturer / distributor information, and patent information.

[0156] Example

[0157] The present invention may be better understood by reference to the following non-limiting examples which are given to more fully illustrate preferred embodiments of the invention. They should in no way be construed as limiting the broad scope of the invention.

[0158] Example 1 - Materials and Methods

[0159] Maintenance of hPSCs

[0160] RUES2 were cultured on mouse embryonic fibroblasts (Rockefeller University Embryonic Stem Cell Line 2, NIH approval number NIHhESC-09-0013, accession number 0013; pp. 13-24) as previously described (Green et al. 2011). Approximately 25,000 cells / cm 2 Mouse embryonic fibroblasts (GlobalStem, Rockville, MD) were plated at a density of 1:2.5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5 .5

[0161] Induction of endoderm

[0162] Differentiation was performed as described by Huang et al., 2014 in serum-free differentiation (SFD) medium 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). The cells were then briefly trypsinized (0.05%, 1 min at 37°C) into a single cell suspension and plated onto a low-adhesion 6-well plate [Costar 2 (Corning Incorporated, Tewksbury MA)] to form embryoid bodies in a serum-free differentiation medium containing human BMP4 0.5ng / ml, human bFGF 2.5ng / ml (R&D Systems) and human activin A 100ng / ml (R&D Systems) on a low-adhesion plate within 84 hours (about 3.5 days). The embryoid bodies were then collected, briefly trypsinized (0.05%, 1 min at 37°C) into 3-10 small cell masses, and resuspended again in endoderm induction medium for another 24 hours. The cells were fed (depending on density) every 24-48 hours and maintained in a 5% CO2 / 5% O2 / 90% N2 environment.

[0163] Induction of the anterior foregut endoderm, pharyngeal endoderm, and the distal third pharyngeal pouch

[0164] After a total of 108 hours on low-adhesion plates containing endoderm induction medium (as described above), embryoid bodies were collected and plated without trypsinization on Matrigel-coated 24-well tissue culture plates (approximately 50,000-70,000 cells / well) in SFD medium supplemented with 200 ng / mL recombinant human (rh) Noggin and 10 μM SB431542 (NS) (as described in the established protocol Green et al. 2011) with retinoic acid (0.25 μM) and FGF8b 50 ng / mL (as a new modification of this protocol) for 48 hours. For pharyngeal endoderm, the resulting cells were then treated with FGF8b (50 ng / mL) and retinoic acid (0.25 μM) for 24 hours, followed by FGF8b (50 ng / mL) and Sonic Hedgehog (Shh) (100 ng / mL) for 8 days ( Figure 1BFor the third pharyngeal pouch specification, the cells were then exposed to recombinant human Noggin (200 ng / mL) for 6 days and then to BMP4 (10 ng / mL) until day 30 of differentiation ( Figure 2A To avoid teratoma formation after implantation in mice, cells were also exposed to the survivin inhibitor YM155 (20 nM) (Lee et al. 2013) for 24 h during the last step of subsequent experiments ( Figure 4A ). Throughout the process, the cell cultures were maintained at 37°C in a 5% CO2 / air environment. The cells were fed every 24 hours.

[0165] Quantitative real-time PCR

[0166] Using Trizol (Invitrogen) and Direct-zol RNA Miniprep kit (Zymo Research), extract the total RNA from the ES cell cluster of indicated time differentiation with indicated culture method according to the manufacturer's instructions. Use NanoDrop 2000 spectrophotometer (ThermoFisher Scientific) to measure RNA concentration. Using Superscript III kit (Invitrogen), according to the manufacturer's instructions, 500ng RNA is amplified by random hexamer by reverse transcription. Use ABI Power SYBR Green PCR master mix to perform real-time quantitative PCR on ABI ViiA7 thermal cycler (Applied Biosystems Life Technologies) in 20ul volume. PCR cycle conditions are set to 50°C for 2 minutes, 95°C for 10 minutes, followed by 95°C for 15 seconds and 60°C for 1 minute, for 40 cycles. For all reactions and primer pairs, verify the single peak dissociation / melting curve. Quantification of each gene transcript was obtained by comparing the mean of the experimental CT values ​​of triplicates to a standard curve of serially diluted genomic DNA of each primer target and then normalized by dividing it by the CT housekeeping gene b-actin. Primer sequences are listed in Table 2.

[0167] Table 2 - Quantitative PCR Primers

[0168]

[0169]

[0170]

[0171] Immunohistochemistry and immunofluorescence

[0172] hES cultures in 24-well tissue culture plates were fixed with paraformaldehyde in PBS (4%) for 10 minutes at room temperature. Cells were washed twice in PBS, permeabilized in PBS containing 0.1% triton for 20 min, and blocked in 5% fetal donkey serum for 1 hour at room temperature.

[0173] Thymic implants were extracted, embedded in OCT (Tissue-Tec, Torrance CA) medium, frozen and 5-7 μm thick sections were cut for immunostaining. The sections were stained with H&E to observe the gross histology and interface of the thymic implants with the mouse kidney tissue. For immunofluorescence staining, tissue sections were fixed and permeabilized in 100% ice-cold acetone and allowed to dry completely. Tissue sections were blocked in PBS supplemented with 0.1% Tween and 0.1% bovine serum albumin. Slides were washed in PBS 0.1% Tween and stained with primary antibodies for 2 hours at room temperature, then washed and incubated in secondary antibodies for 2 hours at room temperature.

[0174] Cultures or tissue sections were incubated with one, or a combination of two or three of the following primary antibodies and appropriate secondary antibodies listed in Table 3. Images of H&E stained sections were collected on a Leica SCN 400 whole slide scanning platform, and immunofluorescence images were collected on a Leica TCS SP82 Photon laser scanning microscope.

[0175]

[0176]

[0177] Animal and human tissue

[0178] NOD-scid IL2Rγ null (NSG, stock 005557) mice were obtained from Jackson Laboratory and were bred and housed in microisolator cages in SPF barriers free of Helicobacter and Pasteurella pneumotropica. Human fetal thymus and liver tissue (gestational age 17-20 weeks) were obtained from Advanced Biosciences Resource. Fetal liver tissue was cut into small pieces and incubated at 37°C in an RT-PCR reaction mixture supplemented with 0.01 mg / ml DNase I from bovine pancreas (Sigma), 2.5 mM HEPES, 4 ug / ml Gentamicin (Gibco) and 1 WU / ml Liberase TM(Roche) culture medium 199 (Corning) incubate to produce single cell suspension.Cells are filtered through a 70um mesh cell strainer into up to 100ml culture medium 199, which is supplemented as listed above without releasing enzymes.Human monocytes are enriched by density gradient centrifugation by layering the hepatocyte suspension on 15ml Ficoll (Histopaque-1077Sigma).Mononuclear cells are collected, washed, resuspended in MACS buffer, and enriched for CD34+ cells by magnetic activated cell sorting (MACS) according to the manufacturer's protocol (Miltenyi), to a purity of about 80% CD34+.CD34+ cells are frozen in human serum AB (GEMCell) containing 10% DMSO (Sigma) with aliquots.

[0179] Three to five mm of human thymus from patients undergoing cardiac surgery 3 The fragments were cryopreserved in human AB serum containing 10% DMSO. To generate primary thymic mesenchyme, thymic pieces were thawed and treated with Liberase as described above. TM Digestion and dissociation, and at about 2x10 4 cells / cm 2 Plates were seeded in DMEM medium (Gemini Bio-Products) supplemented with 10% fetal bovine serum. The medium was changed after 48 hours to remove non-adherent cells and every 3-4 days until 3 weeks, until the cells were confluent. Cells at passages 7-10 were used for experiments, and the identity of the cells was verified by flow cytometry (CD45-CD105+CD90+EpCAM-) (Siepe et al. 2009). The use of human tissues / cells was approved by the Institutional Review Board of Columbia University Irving Medical Center (CUIMC), and all experiments were performed according to the approved protocol.

[0180] Humanized mice

[0181] Six to ten week old NSG mice were thymectomized as described (Khosravi-Maharlooei et al. 2020) and allowed to recover for at least 3 weeks. After recovery, animals were conditioned with 1.8 Gy total body irradiation (TBI) by X-ray. Cryopreserved fetal porcine thymus (60-90 days gestation) was thawed in Medium 199 supplemented with DNAse, gentamicin, and HEPES as described above. Fetal porcine fragments (1-2 mm 3 ) injection or no injection 2x10 5hES-derived TEPs were plated with 50% Matrigel (Corning) in Medium 199. 4-24 hours after TBI, 1-2 × 10 6 1-2x10 thymic mesenchymal cells mixed 6 hES-derived TEP, single 1-2x10 6 Thymic mesenchymal cells, fetal porcine thymus injected with hES-TEP, or fetal porcine thymus alone were implanted under the renal capsule and injected intravenously with 2x10 5 Fetal human CD34+ cells were added. As indicated, after full recovery, peripheral human immune reconstitution was determined every 2-3 weeks after implantation. Blood was collected from the tail vein and the immune population was enriched by density gradient centrifugation with Ficoll as described above. At euthanasia, thymus, spleen and peripheral blood were collected for analysis. Thymic implants were excised from mouse kidneys and divided into two pieces. One thymic fragment was crushed to separate thymocytes, and the remaining matrix components were purified by Liberase as described above. TM Digestion to produce a single cell suspension for flow cytometry analysis. The second thymus fragment is embedded in OCT. The spleen is crushed, filtered through a 70um nylon filter and lysed with a hypotonic lysis buffer (ACK Gibco) for erythrocyte lysis. By density gradient centrifugation on Ficoll, peripheral blood from cardiac puncture is enriched with leukocytes. All animal experiments were performed according to the protocol approved by the Columbia University Institutional Animal Care and Use Committee.

[0182] Flow cytometry

[0183] Human immune reconstitution and differentiation efficiency of hES-TEP cultures were determined by multi-parameter flow cytometry. To determine human immune reconstitution, single cell suspensions prepared from thymic implants, tissue from the anterior mediastinum, spleen, and peripheral blood were prepared as described above. 0.05% trypsin / EDTA was used to dissociate embryoid bodies from hES-TEP cultures on day 4.5 into single cells. Cells were stained with fluorescent dye-labeled monoclonal antibodies against mouse and human cell surface antigens (Table 4). Cells were obtained on LSRII or Fortessa (BD Biosciences), and data analysis was completed using FlowJo software (TreeStar, Ashland OR).

[0184] Table 4 - Monoclonal antibodies against mouse and human cell surface antigens

[0185]

[0186] statistics

[0187] Statistical analysis and comparison were performed using Graph-Pad Prism 7.0 (GraphPad Software). The values ​​for individual mice are shown in bar graphs, where the height of the bar depicts the mean + standard error of the mean. For qPCR data, Ct values ​​normalized to the internal control β-actin were plotted, and relative gene expression was compared using a two-tailed ratio paired student t-test. For multiple comparisons (more than two) from several experimental groups to a single control group, a one-way ANOVA with Dunnett's multiple comparison test was used. Gene correlations were assessed using the Pearson correlation coefficient, where p<0.05 was considered significant; linear regression was also performed and r-squared was determined. Euthanasia due to teratoma growth was plotted on a Kaplan-Meyer plot and analyzed by the Mantel-Cox log-rank test to determine the p-value. Comparisons between mouse groups were performed using the nonparametric Mann-Whitney U test. Effects between transplant groups were resolved by calculating a two-way analysis of variance (ANOVA). When two-way ANOVA was significant (p<0.05), Bonferonni's multiple comparison test was performed at each time point. P<0.05 was considered significant.

[0188] Example 2 - Direct differentiation of hESC into 3PP-biased pharyngeal endoderm

[0189] The thymus is derived from the pharyngeal endoderm (PE), the most anterior part of the endoderm. Directed differentiation of TEC from ESCs requires the sequential induction of the definitive endoderm (DE), anterior foregut (AFE), and PE, followed by the specialization of the thymic domain of the third pharyngeal pouch (3PP) (Gordon and Manley 2011)( Figure 1A and 2A ESCs were differentiated from DE to AFE using activin A followed by noggin plus SB431542 (NS) as previously described (Kubo et al. 2004; D'Amour et al. 2005; Green et al. 2011) ( Figure 1B Flow cytometric analysis showed co-expression of endoderm markers EpCAM and CXCR4 in 98.3% of cells from dissociated embryoid bodies at day 4.5 ( Figure 1C). Dual BMP / TGF-β inhibition after DE induction produced AFE with high efficiency (>90%) (Soh et al. 2014). Consistently, immunofluorescence staining at day 9 showed that most cells expressed FOXA2 (endoderm) and SOX2 (foregut), confirming efficient specification into AFE (FOXA2+SOX2+) (results not shown).

[0190] Secondly, the differentiation of AFE towards a thymic fate is concentrated on HOXA3\TBX1\PAX9\PAX1\SIX1 and EYA1, which are genes involved in PE development and 3PP formation (Manley and Condie 2010). Therefore, their expression was used as a readout at day 15 of culture.

[0191] In humans, HOXA3 is observed throughout the 3rd PP endoderm and surrounding mesenchyme, whereas TBX1 is expressed in the core mesenchyme of the 1st, 2nd, and 3rd pharyngeal arches (PA) and in the 3rd PP endoderm (Farley et al. 2013). In the PE, expression of these two genes overlaps only in the 3rd PP (Farley et al. 2013). Retinoic acid (RA), a factor essential for PA (Kopinke et al. 2006) and PP (Wendling et al. 2000) morphogenesis, has been associated with Hoxa3 expression (Diman et al. 2011), whereas the prevalence of Fgf8 in the PP overlaps with Tbx1 at E10.5 in mice (Vitelli et al. 2002). To mimic physiological 3rd PP endoderm development, simultaneous expression of TBX1 and HOXA3 was induced by combined RA and FGF8b stimulation of AFE cells in protocol #1 ( Figure 1B To confirm the effect of RA, the absence of RA was tested in protocol #2. The addition of RA was necessary for HOXA3 expression ( Figure 1D , scheme #1 compared to #2), which is consistent with the results shown by Parent et al. 2013.

[0192] FGF10, FGF7, CHIR (activator of Wnt signaling), 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 effects of replacing FGF8 alone with these cytokines were investigated in protocol #1. Not only did FGF8b+RA induce the highest expression of most readout genes, it was also able to drive TBX1 expression ( Figure 2B and 2C ) is the only combination of ( Figure 2B Addition of BMP4, CHIR, FGF7, and FGF10 to the regimen with FGF8b+RA did not improve the expression of any 3PP markers (not shown).

[0193] Despite expression of the majority readout gene 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 PPs and are restricted to a subset of TECs after birth (Wallin et al. 1996; Hetzer-Egger et al. 2002). Therefore, in addition to being AFE markers, Pax1 and Pax9 are also TEC markers. Although the expression of PAX9 and PAX1 was statistically higher in protocols #1 and #2 than in the negative control (liver, 'liver pathology' (Gouon-Evans et al. 2006)) Figure 1E ), but Shh was introduced at culture day 7.5 as a strategy to further upregulate PAX9 and PAX1, because Shh induces the expression of Pax1 and Pax9 in the ventral somites (Furumoto et al. 1999). Both Shh and its receptor PTC1 are expressed in human TECs and have been reported to contribute to TEC differentiation (Saldana et al. 2016; Sacedon et al. 2003).

[0194] Since Shh enhances 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 approached significance in PAX1 (5-fold; p=0.053) ( Figure 1D , regimen #1 compared to #3). TBX1 expression was also significantly increased, consistent with reports showing that Shh induces Tbx1 expression in PE ( Figure 1D )(Garg et al. 2001).

[0195] We next tested whether increased exposure to FGF8b, beginning at day 4.5 of culture, could play a role in shifting AFE development toward PE and enhancing expression of 3PP genes. Equivalent expression of 3PP markers was observed in both protocols #3 and #4, leading us to pursue efforts to optimize these two markers in parallel to explore their potential beyond day 15 of differentiation ( Figure 1F ).

[0196] Example 3 - Distalization of the 3rd PP

[0197] Although the addition of FGF8b during proximal and / or culturing with Shh increased the expression of 3PP markers, day 15 cultures showed low FOXN1 expression (results not shown). In mice, BMP4 is co-expressed with FoxN1 at E11.5 in the ventral / posterior prospective thymic domain of the 3PP endoderm (Moore-Scott and Manley 2005; Bleul and Boehm 2005). It was therefore hypothesized that the addition of BMP4 might result in better FOXN1 expression. Therefore, day 15 cultures were exposed to BMP4 ( Figure 2A , Protocols #3b and #4b). However, for Protocols #3b and #4b, addition of BMP4 failed to induce FoxN1 expression measured on days 22 and 30 of culture (results not shown). It is hypothesized that insufficient expression of PAX9, which is also expressed in TECs after thymic organogenesis (Manley and Condie 2010; Hetzer-Egger et al. 2002), may be responsible for the poor expression of FOXN1.

[0198] Next it was tested whether the addition of noggin would increase PAX9 expression. Noggin is a BMP4 antagonist and / or inhibitor that is expressed in the mesenchyme throughout the 3PA at E9.5 in mice, adjacent to the early 3PP endoderm (Patel et al. 2006). BMP4 expression begins at E10.5 in cells of the 3PP endoderm (Patel et al. 2006). It was hypothesized that noggin could diffuse from the mesenchyme to the 3PP endoderm cells just before BMP4 signaling occurs in this region. To mimic this event, noggin was substituted for BMP4 from day 16 to day 22 in protocols #3c and #4c ( Figure 2A ). In both regimens with the addition of Noggin, PAX9 expression increased significantly ( Figure 2D ).

[0199] Five-fold higher levels of FOXN1 expression were observed in regimen #4c (FGF8b during pro-induction) compared to regimen #3c ( Figure 2E ). Therefore, protocol #4c was further optimized. To confirm that the cells produced FOXN1 after BMP4 addition, FOXN1 expression was compared at day 21 and day 30 using protocol #4c. Figure 2F The results showed that FOXN1 expression was significantly higher on day 30 than on day 21, confirming that BMP4 exposure has the potential to enhance FOXN1 expression after day 21. In regimen #4c, FOXN1 levels on day 30 were 8-fold higher than those on day 15 ( Figure 2G ).

[0200] Gene expression of TEC markers at day 30 of culture compared to whole human fetal thymus lysate is shown in Figure 3AAlthough the thymic stromal samples were diluted due to the presence of thymocytes, protocol 4c achieved 76% of the FOXN1 expression seen in thymic lysates. This is significantly higher than levels reported by other groups making the same comparison (Parent et al. 2013; Sun et al. 2013; Su et al. 2015). In addition, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL7, K5, K8, and AIRE mRNAs were detectable at levels comparable to or higher than those in fetal thymus. To determine the reproducibility of this protocol in other hESC lines, the human H9 ES cell line was treated with protocol #4c. TEC markers ISL1, FOXN1, K5, K8, DLL4, AIRE, and IL7 ( Figure 3B ) expression could be demonstrated in H9 cells differentiated using this protocol.

[0201] Immunostaining of protocol #4c cultures at day 15 revealed colonies positive for the PE markers TBX1, EYA, ISL1, and SIX, which also co-stained with the 3PP marker EpCAM (results not shown). At day 30 of culture, these colonies were still positive for EpCAM (a universal epithelial marker), K5 and UEA-1 (which are associated with mTEC), and K8 (which is associated with cTEC) (results not shown). A strong correlation between the expression levels of FOXN1 and GCM2 was also found ( Figure 3C ), GCM2 is a parathyroid marker also found in the 3PP. This suggests that despite BMP4 exposure, there are cells destined to mature into parathyroid progenitors, demonstrating incomplete distalization of the 3PP (Gordon et al. 2001). IL7 is an essential cytokine produced by TECs that 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). IL7 and CD205 expression were found to correlate with the expression of FOXN1 ( Figure 3C ).

[0202] Example 4 - Determination of the functional capacity of hES-TEPs

[0203] hES-TEP differentiated with protocol #4c were tested for their ability to support thymic generation from human hematopoietic stem cells implanted in humanized mice. The retention of undifferentiated pluripotent cells in culture is a major clinical translation barrier using ES and iPSC derivatives. Implantation experiments revealed the presence of pluripotent cells at the time of transplantation, resulting in rapid uncontrolled growth and formation of teratomas from cells in the implants (results not shown). Consistent with these results, OCT4, a marker for pluripotent cells, was detected in hES-TEP cultures at day 30 ( Figure 4C)(Pan et al. 2002). However, TEPs at day 30 of culture showed co-expression of OCT4 in EpCAM+ cells (results not shown), and qPCR analysis showed a correlation between the expression levels of FOXN1 and OCT4 ( Figure 4B ), indicating that OCT4 expression may be part of the TEC differentiation program.

[0204] The survivin inhibitor YM155 has been reported to selectively eliminate pluripotent cells (Lee et al. 2013). Treatment with YM155 during the last 24 h of culture was tested to see if it was sufficient to eliminate pluripotent cells ( Figure 4A ). OCT4 expression was significantly reduced by YM155 treatment ( Figure 4C Implantation of untreated day 15 hES-TEP resulted in teratomas in all animals by 11 weeks post-implantation ( Figure 4D hES-TEPs cultured to day 30 in the presence and absence of YM155 showed reduced teratoma formation compared to untreated controls implanted with TEPs on day 15, with only 3 of 15 animals developing teratomas in the group receiving YM155-treated cells (results not shown).

[0205] The native thymic rudiments of NSG hosts are able to support low-level thymopoiesis from human fetal liver-derived HSCs. A method was developed to surgically remove both lobes of the native thymic rudiments from NSG mice to prevent T cell development in thymectomized (ATX) NSG animals engrafted with human HSCs (Khosravi-Maharlooei et al. 2020). Complete removal of the native thymic rudiments in ATX mice was confirmed by collecting connective tissue from the anterior mediastinum and determining the absence of CD4+CD8+ developing thymocytes ( Figure 5A and 5B ). Therefore, to assess the functional capacity of the implanted hES-derived TEPs, all subsequent recipients underwent thymectomy.

[0206] Example 5 - Functional thymic organogenesis using hES-TEP / TMC

[0207] To test the functional ability of cultured hES-TEPs to support thymopoiesis, hES-TEP clusters mixed with human thymic mesenchymal cells (TMCs) (generated using protocol #4c) or TMCs alone were implanted into 2×10 5 Figure 1 shows the total human CD45+ cells in the peripheral blood of all mice, with human chimeras averaging 61% + 21% in mice implanted with hES-TEP / TMC and 81% + 13% in mice implanted with TMC at 11-31 weeks post-humanization. Figure 5CHuman HSC implantation resulted in dominant B cell production (data not shown). As early as 9 weeks after subrenal TEP implantation, human CD3+ T cells accounting for more than 1% of total human blood cells were detected in two mice implanted with hES-TEP / TMC, and eventually in 6 of 7 mice implanted with hES-TEP, while controls implanted with TMC showed no peripheral T cell reconstitution ( Figure 5D ). T cells were biased towards the CD4+ rather than CD8+ lineage, however, 4 of 7 mice implanted with hES-TEP / TMC generated both CD4+ and CD8+ cells ( Figure 5E and 5G ). The expression of the naive T cell marker CD45RA and the effector / memory T cell marker CD45RO in CD4+ cells was further determined. In the four mice that generated CD4+ and CD8+ T cells, the CD4+ T cells had a predominantly naive phenotype (CD45RA+CD45RO-), consistent with de novo thymic generation ( Fig. 5F Over time, CD4+ T cells converted to an effector / memory phenotype (CD45RA-CD45RO+), consistent with thymopoietic arrest and lymphopenic expansion.

[0208] There are low frequencies of CD4+CD8+ double positive cells in hES-TEP / TMC ( Figure 5H hES-TEP / TMC implants were slightly expanded in volume and presented a disordered architecture, with no discernible cortical or medullary regions in hematoxylin and eosin staining (results not shown). In addition, cells from hES-TEC / TMC implants appeared to penetrate the renal parenchyma, indicating the presence of multiple cell types differentiated from TEP cultured cells in vivo. Despite the disordered architecture, some cells in hES-TEC / TMC implants were co-stained with TEC markers EpCAM, Pancytokeratin, and human MHC II (HLA-DR), indicating long-term terminal differentiation and survival of hES-TEC (results not shown).

[0209] Example 6 - Strategy for testing the effects of hES-TEC: evidence for integration into porcine thymic implants

[0210] It was hypothesized that the ability of hES-TEPs to generate true thymic tissue in vivo might be limited by the absence of the thymic structural scaffold or other cell types required to generate a functional thymus. To address this possibility, the survival and function of hES-TEPs (generated by protocol #4c) injected into porcine fetal thymus implants were studied in humanized mice. Fig. 6AIt has been previously demonstrated that fetal porcine thymus (SwTHY) supports robust thymopoiesis from human fetal liver-derived HSCs in NOD-scid or NSG mice (Kalcheuer et al. 2014; Nikolic and Sykes 1999; Nauman et al. 2019).

[0211] The presence of hES-TECs in SwTHY implants injected 18–22 weeks after transplantation was analyzed by flow cytometry and immunofluorescence. TM Stromal cells from half of the thymic implants were isolated 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 of SwTHY+hES-TEC and SwTHY+hES-TEC of huCD45-HLA-ABC+ cells is shown ( Figure 6B HuCD45-HLA-ABC+CD105-EpCAM+ was detected at a frequency of 1.6%+2.3% in the thymus injected with hES-TEC, whereas it was not detected in the uninjected SwTHY, as expected ( Figure 6B and 6C ). The whole thymus implant was stained with epithelial cell marker 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 and terminally differentiated human TEC (green) inoculated with thymus implants differentiated from human HSC in the bone marrow. Confocal microscopy showed co-localized HLA-DR and cytokeratin expressed by hES-TEC (yellow) in injected SwTHY, but not in uninjected SwTHY (results not shown). hES-TEC was detected in 6 of 7 SwTHY+hES-TEC thymus implants.

[0212] Example 7 - Injection of hES-TEP into porcine thymus improves human thymopoiesis

[0213] Thymocytes at the end of differentiation were measured by flow cytometry to determine whether hES-TECs support improved human thymopoiesis. The distribution of single positive (SP) CD4+, CD8+, and double positive (DP) CD4+CD8+ cells in SwTHY+hES-TEC and SwTHY implants was similar to that in human pediatric thymus ( Fig.6D hES-TECs in SwTHY resulted in a significant increase in the total number of thymocytes and CD4+CD8+ DP cells compared to SwTHY implants ( Fig. 6EThe frequencies and absolute numbers of CD4+SP, CD4+CD45RA+, and CD4+CD45RO+ developing T cells were significantly increased in SwTHY+hES-TECs compared with SwTHY implants ( Fig. 6E ). These data suggest that hES-TECs can promote human thymopoiesis by providing human MHC interactions required for survival of thymocytes from the double-positive stage to terminal differentiation.

[0214] We next tested whether injection of hES-TEPs into SwTHY altered T cell frequency and phenotype in the periphery of HSC-injected mice compared to uninjected SwTHY implanted under the kidney capsule ( Fig. 6A Robust human chimeras generated from animals implanted with SwTHY or with SwTHY injected with hES-TEP (SwTHY+hES-TEC) had similar B cell frequencies, which averaged approximately 30% ± 14% in peripheral blood from 11 to 21 weeks after humanization ( Fig. 6F and 6G Compared with the SwTHY group, the comparative kinetics of T cell reconstitution showed a significant increase in the proportion of CD3+ T cells due to the increased frequency of CD4+ T cells in the blood of the SwTHY+hES-TEC group ( Fig. 7A ).

[0215] The spleen immune population, as the primary immune organ, was determined to determine whether hES-TEC injection altered the frequency or absolute cell number. The frequency and total number of human immune cells were comparable between the SwTHY+hES-TEC group and the SwTHY group ( Figure 6H ). Similarly, there were no differences between the groups in the number of CD19+ B cells and CD14+ monocytes ( Fig.6I and 6J The frequency and total number of CD3+ T cells increased in the SwTHY+hES-TEC group compared to animals implanted with SwTHY ( Fig. 7E Compared with the SwTHY control, the percentage and absolute number of CD8+ cytotoxic and CD4+ helper T cells were increased in the SwTHY+hES-TEP injection group ( Figure 7F ).

[0216] Phenotypic and functional subsets of CD4 and CD8 T cells were defined based on the expression of the chemokine receptors CCR7 and CD45RA to delineate populations of naive (CD45RA+CCR7+), central memory (Tcm) (CD45RA-CCR7+), effector memory (Tem) (CD45RA-CCR7-), and terminally differentiated effector memory cells that re-express CD45RA (TEMRA) (CD45RA+CCR7-) ( Figure 7G)(Thome et al. 2014). Consistent with the increase in T cell numbers in animals implanted with SwTHY+hES-TEP, naive, Tcm, Tem, 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 naive CD4+ T cells compared to SwTHY controls ( Figure 7H ), which is consistent with the explanation that hES-TEC promotes human T cell development.

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Claims

1. A method for inducing pluripotent stem cells to differentiate into thymic epithelial cells or thymic epithelial cell progenitor cells, comprising the following steps: a. differentiating the pluripotent stem cells into definitive endoderm cells; b. culturing the definitive endoderm cells and differentiating the definitive endoderm cells into anterior foregut cells by contacting or incubating the definitive endoderm cells with an agent that inhibits BMP and an agent that inhibits TGFβ signaling, and further contacting or incubating the definitive endoderm cells with an agent that stimulates HOXA3 expression and an agent that stimulates TBX1 expression; 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 an agent that stimulates HOXA3 expression, an agent that stimulates TBX1 expression, and an agent that stimulates PAX9 and PAX1 expression; d. culturing the pharyngeal endoderm cells and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP) specialized cells, thymic epithelial cells or thymic epithelial progenitor cells by contacting or incubating the pharyngeal endoderm cells with an agent that inhibits BMP; and e. Cultivating the pharyngeal endoderm cells from step c or step d, and differentiating the pharyngeal endoderm cells into distal pharyngeal pouch (PP) specialized cells, thymic epithelial cells or thymic epithelial progenitor cells by contacting or incubating the pharyngeal endoderm cells with BMP.

2. The method of claim 1, wherein step a is performed for about one day to about six days.

3. The method of claim 1, wherein in step a., the pluripotent stem cells are cultured in a serum-free differentiation medium and contacted or incubated with human bone morphogenetic protein (BMP) in an amount of about 0.5 ng / ml, human basic fibroblast growth factor in an amount of about 2.5 ng / ml, and human activin A in an amount of about 100 ng / ml.

4. The method of claim 1, wherein step b. is performed starting from about day 3 to about day 5 and lasting for about 2 days to about 3 days.

5. The method of claim 1, wherein in step b., the agent that inhibits BMP is selected from the group consisting of noggin and doxorubicin, the agent that inhibits TGFβ signaling is SB431542, the agent that stimulates HOXA3 expression is retinoic acid, and the agent that stimulates TBX1 expression is FGF8b.

6. The method of claim 1, wherein step c. is performed starting from about day 5 to about day 8 and continuing for about 6 to about 10 days.

7. The method of claim 1, wherein in step c., the agent that stimulates HOXA3 expression is retinoic acid, the agent that stimulates TBX1 expression is FGF8b, and the agent that stimulates PAX9 and PAX1 expression is Sonic Hedgehog (Shh).

8. The method of claim 7, wherein in step c., at about 24 hours, 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, FGF8b is used in an amount of about 50 ng / mL and Shh is used in an amount of about 100 ng / ml.

9. The method of claim 1, wherein step d. is performed starting from about day 12 to about day 18 and continuing for about 4 days to about 7 days.

10. The method of claim 1, wherein in step d., the agent that inhibits BMP is selected from the group consisting of noggin and doxorubicin.

11. The method of claim 1, wherein step e. is performed starting from about day 19 to about day 25 and lasting for about 5 days to about 15 days.

12. The method of claim 1, wherein in step e., BMP is used in an amount of about 50 ng / ml.

13. The method of claim 1, further comprising the step of contacting or incubating the TEC or TEP at the end of the method with a survivin inhibitor.

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

15. A method for inducing pluripotent stem cells to differentiate into thymic epithelial cells or thymic epithelial cell progenitor cells, comprising the following steps: a. differentiating the pluripotent stem cells into definitive endoderm cells by the following method: 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 definitive endoderm cells from step a. into anterior foregut cells by: culturing the definitive endoderm cells in the serum-free differentiation medium and contacting or incubating the definitive 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 in the serum-free differentiation medium and contacting or incubating the anterior foregut cells with FGF8b and retinoic acid, followed by contacting or incubating with FGF8b and sonic hedgehog (Shh); d. differentiating the pharyngeal endoderm cells from step c. into third pharyngeal pouch specialized cells, thymic epithelial cells or thymic epithelial cell progenitor cells by culturing the pharyngeal endoderm cells in the serum-free differentiation medium and contacting or incubating the 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 progenitor cells by culturing the pharyngeal endoderm cells in the serum-free differentiation medium and contacting or incubating the pharyngeal endoderm cells with BMP.

16. The method of any one of claims 1 or 15, 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 step a. is performed for about one day to about six days.

18. The method of claim 15, wherein 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.

19. The method of claim 15, wherein step b. is performed starting from about day 3 to about day 5 and continuing for about 2 days to about 3 days.

20. The method of claim 15, wherein 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.

21. The method of claim 15, wherein step c. is performed starting from about day 5 to about day 8 and continuing for about 6 days to about 10 days.

22. The method of claim 15, wherein in step c., at about 24 hours from the start, FGF8b is used in an amount of about 50 ng / mL and retinoic acid is used in an amount of about 0.25 μM.

23. The method of claim 15, wherein in step c., at about 48 hours, FGF8b is used in an amount of about 50 ng / mL and Shh is used in an amount of about 100 ng / ml.

24. The method of claim 15, wherein step d. is performed beginning on about day 12 to about day 18 and continuing for about 4 days to about 7 days.

25. The method of claim 15, wherein in step d., noggin is used in an amount of about 100 ng / ml.

26. The method of claim 15, wherein step e. is performed beginning on about day 19 to about day 25 and continuing for about 5 days to about 15 days.

27. The method of claim 15, wherein in step e., BMP is used in an amount of about 50 ng / ml.

28. The method of claim 15, further comprising the step of contacting or incubating the TEC or TEP at the end of the method with a survival inhibitor.

29. The method of claim 28, wherein the survivin inhibitor is YM155.

30. Thymic epithelial cells or thymic epithelial cell progenitor cells obtained by the method of claim 1 or 15.

31. A method for preventing and / or treating a thymic disease, comprising administering a therapeutically effective amount of the cell of claim 30 to a subject in need thereof.

32. The method of claim 31 , wherein the disease is an autoimmune disease.

33. A method for restoring or rejuvenating impaired thymic function, comprising administering a therapeutically effective amount of the cells of claim 30 to a subject in need thereof.

34. The method of claim 33, wherein the impaired thymic function is due to injury, aging, or congenital abnormality.

35. A method for testing a drug on a subject using the cell of claim 30, wherein the TEC or TEP is derived from the subject.

36. A method for developing a mouse model using the cell of claim 30.

37. A method of reconstituting T cells after bone marrow transplantation, comprising administering a therapeutically effective amount of the cells of claim 30 to a subject in need thereof.

38. A method of producing a hybrid thymus comprising combining the thymic epithelial cell of claim 30 with another cell comprising the thymus.

39. A method for producing a hybrid thymus, comprising transplanting the thymic epithelial cells of claim 30 into a porcine thymus.

40. The method of claim 39, wherein the porcine thymus is from a pig selected from the group consisting of a piglet and a fetal pig.

41. A hybrid thymus produced by any one of the methods of claims 38 to 40.

42. A method for inducing pluripotent stem cells to differentiate into thymic epithelial cells or thymic epithelial cell progenitor cells, comprising the following steps: a. differentiating the pluripotent stem cells into definitive endoderm cells by the following method: 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 definitive endoderm cells from step a. into anterior foregut cells by: culturing the definitive endoderm cells in the serum-free differentiation medium and contacting or incubating the definitive endoderm cells with noggin and SB431542 (NS); c. differentiating the anterior foregut cells from step b. into pharyngeal endoderm cells by culturing the anterior foregut cells in the serum-free differentiation medium and contacting or incubating the anterior foregut cells with FGF8b and retinoic acid, followed by contacting or incubating with FGF8b and sonic hedgehog (Shh); and d. differentiating the pharyngeal endoderm cells from step c. into third pharyngeal pouch specialized cells or thymic epithelial cells by culturing the pharyngeal endoderm cells in the serum-free differentiation medium and contacting or incubating the pharyngeal endoderm cells with BMP4.

43. The method of claim 42, wherein step b. further comprises contacting or incubating the definitive endoderm cells with retinoic acid.

44. The method of claim 43, comprising contacting or incubating the definitive endoderm cells with FGF8b.

45. The method of claim 42, wherein step d further comprises contacting or incubating the pharyngeal endoderm cells with noggin.

46. ​​The method of claim 42, further comprising contacting or incubating the thymic epithelial cells with a survivin inhibitor.

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