Thymocytes and method for producing same

Abstract

The present disclosure provides methods for generating thymocytes by differentiation of pluripotent stem cells. Also provided herein are compositions and lineages of cell populations that include thymocytes. The methods of the present disclosure also include methods of maintaining thymocytes and therapeutic methods using the thymocytes of the present disclosure.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 256,443, filed October 15, 2021, U.S. Provisional Patent Application No. 63 / 296,251, filed January 4, 2022, U.S. Provisional Patent Application No. 63 / 321,136, filed March 18, 2022, and U.S. Provisional Patent Application No. 63 / 388,407, filed July 12, 2022, the entire contents of which are incorporated by reference into this application.

[0002] Government Funding Statement This invention was made with Government support under Grant No. 1R44AI170266-01 awarded by the National Institute of Allergy and Infectious Diseases (NIAID) Small Business Innovation Research (SBIR). The Government has certain rights in the invention. [Background technology]

[0003] background The thymus is a major lymphoid organ that plays a central role in the immune system. The thymic microenvironment provides a unique training ground for the development and maturation of effector cells such as lymphocytes (e.g., T cells). As is well known in the art, complex interactions between thymocytes and effector cells can determine the phenotype and functionality of effector cells. Some thymocyte-effector cell interactions are regulated such that recognition of factors expressed by thymocytes promotes survival of effector cells. In contrast, other thymocyte-effector cell interactions can cause the death of effector cells. By controlling such interactions, the thymus plays a pivotal role in establishing a repertoire of effector cells that can mount an activated immune response against foreign invaders while establishing tolerance to self.

[0004] There remains a need for improved methods for generating thymocytes and cell populations enriched for functional thymocytes that can differentiate into functional thymic epithelial cells. Summary of the Invention

[0005] overview The present disclosure provides thymocytes, methods of producing thymocytes, and / or methods of maintaining thymocytes in culture.

[0006] The present disclosure provides a method for inducing differentiation of pluripotent stem cells into thymocytes. Such a method may include multiple steps of differentiating the pluripotent stem cells into definitive endoderm cells. DE cells may be differentiated into anterior foregut endoderm (AFE) cells by culturing and contacting or incubating with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, and / or a combination thereof. AFE cells may be differentiated into ventral pharyngeal endoderm (VPE) cells by culturing and culturing in a first VPE medium and / or a second VPE medium. The first VPE medium may include ascorbic acid, retinoic acid, FGF, and / or a TGFβ inhibitor. In some embodiments, the first VPE medium may further include a WNT inhibitor. The second VPE medium may include noggin, a WNT activator, FGF, retinoic acid, and / or ascorbic acid. In some embodiments, the second VPE medium can further comprise a BMP inhibitor, an SHH inhibitor, or a combination thereof. The VPE cells can be further differentiated into thymocytes, such as thymic epithelial progenitor cells (TEPs), by contacting or incubating with ascorbic acid, FGF, BMP, and / or WNT activators.

[0007] In some embodiments, the thymocytes may be thymic epithelial progenitor cells (TEPs) and / or thymic epithelial cells (TECs). TEPs can be further differentiated into TECs by culturing with interleukins, WNT activators, RANKL, FGFs, BMPs, and / or ascorbic acid.

[0008] In some embodiments, pluripotent stem cells can be differentiated into DE cells by contacting or culturing the pluripotent stem cells in a first growth medium containing activin A, PI-103, and / or CHIR99021. Differentiation into DE cells can further include culturing the cells in a second growth medium containing activin A, a BMP inhibitor, PI-103, and / or CHIR99021.

[0009] In some embodiments, the BMP inhibitor can be LDN193189. In some embodiments, the TGFβ inhibitor can be SB431542. In some embodiments, the FGF can be FGF8b, FGF7, FGF10, FGF1, bFGF, or a combination thereof. In some embodiments, the WNT activator can be CHIR99021. In some embodiments, the BMP can be BMP2, BMP4, or a combination thereof. In some embodiments, the interleukin can be IL22. In some embodiments, the WNT inhibitor can be IWR-1. In some embodiments, the BMP inhibitor can be LDN193189. In some embodiments, the SHH inhibitor can be SANT-1.

[0010] The pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes of the present disclosure may be cultured in suspension. In some embodiments, the pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes may be cultured as aggregates in suspension.

[0011] In some embodiments, the pluripotent stem cells, the DE cells, the AFE cells, the VPE cells, or the thymocytes may be attached to a solid substrate. In some embodiments, the pluripotent stem cells, the DE cells, the AFE cells, the VPE cells, or the thymocytes may be attached to a solid substrate comprising an extracellular matrix-based medium.

[0012] The methods of the present disclosure may be performed for about 15 to 30 days. In some embodiments, the methods may be performed for about 18 to 25 days. In some embodiments, the pluripotent stem cells may be differentiated into definitive endoderm cells for about 5 days. In some embodiments, the DE cells may be differentiated into AFE cells for about 2 to 3 days. In some embodiments, the AFE cells may be differentiated into VPE cells in the first VPE medium for about 2 to 4 days and into VPE cells in the second VPE medium for about 2 to 3 days. In some embodiments, the VPE cells may be differentiated into thymocytes for about 3 to 12 days.

[0013] The disclosure also provides thymocytes, eg, TEPs and TECs, produced by the methods described herein.

[0014] Also provided herein is a method of culturing thymocytes in vitro. Such a method may include culturing or incubating the thymocytes in a thymocyte medium comprising FGF10, BMP4, FGF8b, CHIR99021, and / or ascorbic acid. In some embodiments, the thymocyte medium may further comprise FGF7 and RANKL. The method may also include culturing the thymocytes in suspension. As a non-limiting example, the thymocytes may be cultured as aggregates in suspension.

[0015] In some embodiments, the present disclosure provides a method of increasing FOXN1 expression in a population of thymocytes. Such a method may include freezing the population of thymocytes, thawing the population of thymocytes, and measuring and comparing FOXN1 expression in the population of thymocytes before freezing and comparing FOXN1 expression after thawing the population of thymocytes. In some embodiments, FOXN1 expression may be increased by about 10- to 100-fold. The population of thymocytes may be cultured as aggregates in suspension.

[0016] Also provided herein is a pharmaceutical composition comprising a population of thymocytes prepared by the methods described herein. The present disclosure also provides a method of treating or preventing a condition in a subject by administering a pharmaceutical composition of the present disclosure. In some embodiments, the condition is associated with a lack, reduction, or abnormality in the function of the thymus in the subject. The condition may be an immune deficiency, cancer, an autoimmune disease, an infectious disease, or graft-versus-host disease (GvHD). In some embodiments, the pharmaceutical composition is administered parenterally. For example, the composition may be implanted or injected into one or more lymph nodes of the subject. [Brief description of the drawings]

[0017] The above and other objects, features and advantages will become apparent from the following description of specific embodiments of the present disclosure, as illustrated in the accompanying drawings, which are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure. [Figure 1] Culture groups 1 to 4 (G1 to G4) showing increasing frequencies of expression levels of key biomarkers (SOX17 and FOXA2) of definitive endoderm (DE). [Figure 2-1] 1 shows the effect of the growth factor BMP4 on the expression of developmental genes. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3-1] Analysis of TEC biomarkers after plating on Matrigel is shown. [Figure 3-2] See description of Figure 3-1. [Figure 4-1] 1 shows TEC biomarker analysis after freeze-thaw cycles. [Figure 4-2] See description of Figure 4-1. [Figure 5A] 5A-5D: FIG. 5A shows HOXA3 expression under different culture conditions. FIG. 5B shows PAX1 expression under different culture conditions. FIG. 5C shows PSMB11 expression under different culture conditions. FIG. 5D shows FOXN1 expression under different culture conditions. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figure 6A-6B: Figure 6A shows expression of FOXN1 using primers targeting the cocoding region (left panel), the noncoding region (middle panel), and the HA tag of exogenously expressed FOXN1 (right panel). Figure 6B shows expression of the FOXN1 target gene in mock-transfected or FOXN1-targeted cells 1 day (D1) or 2 days (D2) after transfection. [Figure 6B] See legend to Figure 6A. [Figure 7] 1 shows the expression of FOXN1 in derivatives of iPSC-derived thymocytes. [Figure 8] 1 shows the expression of FOXN1 in derivatives of Experiment 29 and Experiment 30. [Figure 9] 1 shows the expression of FOXN1 in thymocytes after the cells were frozen and thawed. [Figure 10A] Figures 10A-10E: Figures 10A and 10B show the percentage of CD8 positive cells among CD45 positive cells in mice that received thymocyte transplants. Figures 10C, 10D, and 10E show the percentages of different hematopoietic cells in mice that received thymocyte transplants at 3 weeks, 11-13 weeks, and 14-16 weeks, respectively. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 11-1] Sorted fractions of thymocytes analyzed by qPCR for various markers relative to GAPDH are shown. [Figure 11-2] See description of Figure 11-1. [Figure 11-3]See description of Figure 11-1. [Figure 12A] Figures 12A-12B: Figure 12A shows gene expression distribution of iPSC-derived thymocytes. Figure 12B shows quantification of the percentage of FOXN1+ cells, KRT8+ cells, and EPCAM+ cells. [Figure 12B] See legend to Figure 12A. [Figure 13] 1 is a histogram showing the frequency of CD8+ or CD4+ cells in peripheral blood. [Figure 14] 1 shows various levels of FOXN1 expression in different thymocyte populations. [Figure 15] 1 shows HOXA3 and Pax9 expression in cells at the VPE stage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description I. Introduction Thymic epithelial cells are important in T cell differentiation. Thymocytes prepared as described herein can enable the properties of thymic tissue and thymocytes, such as thymus-associated immune function, to be utilized for therapeutic applications. For example, it is known that the decline in immune function associated with aging is caused by changes in the constituents and functional capacity of thymocytes. In addition, the thymus itself atrophies and ages due to changes in sex hormones, including androgens and estrogens. The onset of thymic atrophy may begin with the onset of puberty. Thus, regeneration of thymic epithelial cells can provide compositions and methods for mitigating the decline in immune function associated with aging.

[0019] II. Composition cell Cells of the present disclosure may include, without limitation, thymocytes, effector cells, pluripotent stem cells, populations thereof and cells derived therefrom.

[0020] In some embodiments, the cells of the disclosure may be autologous, allogeneic, syngeneic, or xenogeneic with respect to a particular individual or subject. In some embodiments, the thymocytes may be autologous, allogeneic, syngeneic, or xenogeneic with respect to the subject that will ultimately benefit from their clinical application. In some embodiments, the cells of the disclosure may be mammalian cells, particularly human cells. The cells may be primary cells or immortalized cell lines. In some aspects, the cells of the disclosure may be prepared or derived from a syngeneic cell source. Any of the cells described herein may be characterized by markers known in the art for the relevant cell type.

[0021] thymocyte A thymocyte may be a cell that has one or more phenotypic or genotypic markers associated with a cell derived from the thymus or a cell that is destined to become a thymic cell. The thymus may be an embryonic, fetal / fetal, or adult thymus.

[0022] Thymocytes may be TECs or may be derived from TECs. During embryonic development, TECs may be derived from non-hematopoietic cells that are negative for CD45 expression and positive for the epithelial marker EpCAM. TECs may be cortical thymic epithelial cells (cTECs) and / or medullary thymic epithelial cells (mTECs). mTECs are characterized by cytokeratin 5 (K5) and cytokeratin 14 (K14) expression, but have low levels of cytokeratin 8 (K8) expression, whereas cTECs express K8 and K18. In some embodiments, thymocytes may be derived from TECs that express both K5 and K8 (K5+K8+). In some aspects, K5+K8+ cells may be precursors of mTECs and / or cTECs. mTECs may also be positive for cell surface expression of UEA-1 (UEA-1) rather than Ly51 (e.g., UEA-1+Ly51-), whereas cTECs may be UEA-1-Ly51+. In some embodiments, thymocytes may be or may be derived from mTECs. In some embodiments, mTECs may have high expression of markers, such as, but not limited to, cytokeratin 5, cytokeratin 14, UEA-1, CD80, cathepsin L, and / or cathepsin S. In some embodiments, thymocytes may be or may be derived from cTECs that have high expression of markers, such as, but not limited to, cytokeratin 8, cytokeratin 18, Ly51, CD205, cathepsin L, and / or thymus-specific serine protease. As a non-limiting example, thymocytes may be or may be derived from cTECs that express markers such as, for example, CCL25, and / or KRT5. mTECs can express markers such as CCL19, KRT8, and / or AIRE.

[0023] In some embodiments, the thymocytes may be or may be derived from TECs expressing one or more markers, such as, for example, FOXN1, PAX9, PAX1, DLIA, ISL1, EYA1, SIX1, IL7, K5, K8 and AIRE.

[0024] The thymocytes may be or may be derived from any of the cell types described in Park et al. 2020 Science Vol. 367, Issue 6480, the entire contents of which are incorporated herein by reference. For example, the thymocytes may be derived from myoid cells, such as MYOD1 and MYOG-expressing myoid cells (referred to herein as TEC(myo)), and / or NEUROD1, SYP, CHGA-expressing TECs (referred to herein as TEC(neuro)).

[0025] The thymocytes may be derived from the cell types described in Bautista et al. 2021 Nat Commun 12, 1096, the entire contents of which are incorporated herein by reference. The thymocytes may be derived from the cell types described in Bautista et al. 2021 "cTEC 低 " cells, with lower levels of functional genes (HLA class II) and more KI67 + - may be characterized by the inclusion of proliferating cells. Thymocytes may be characterized as "mTEC" cells, as described by Bautista et al. 2021. 低 The thymocytes may be derived from "mTEC" cells and may be characterized by expression of CLDN4, lower levels of HLA class II, PSMB11, PRSS16, CCL25, and high levels of the chemokine CCL21. The thymocytes may be derived from "mTEC" cells as described by Bautista et al. 2021. 高 " cells and may be characterized by expression of SPIB, AIRE, FEZF2, higher levels of HLA class II. Thymocytes may be or be derived from keratinocyte-like mTECs as described in Bautista et al. 2021 and may be characterized by expression of KRT1, and / or IVL. In some embodiments, thymocytes may be or be derived from immature TECs (iTECs) as described in Bautista et al. 2021 that express canonical TEC identity genes, e.g., FOXN1, PAX9, SIX1.

[0026] In some embodiments, the thymocytes may be or may be derived from TECs that express one or more markers, for example, but not limited to, KRT5, KRT8, AIRE, PSMB11, and / or PRSS16.

[0027] In some embodiments, the thymocytes may be or may be derived from TECs that express one or more markers, for example, but not limited to, AIRE, CK5, CK8, CXCL12, CCL25, DLL4, and / or HLA-DR.

[0028] In some embodiments, thymocytes may be prepared from cells destined to become thymocytes. During embryonic development, pluripotent stem cells may differentiate into thymocytes through a stepwise differentiation process. In vitro, thymocytes may be prepared by differentiation of pluripotent stem cells into thymic stem cells by one or more of the following steps: PSCs may differentiate and / or be induced to differentiate into cells resembling definitive endoderm (DE). Definitive endoderm cells may differentiate and / or be induced to differentiate into cells resembling third pharyngeal pouch endoderm (PPE). Definitive endoderm and / or PPE cells may differentiate and / or be induced to differentiate into cells resembling ventral pharyngeal endoderm (AFE). AFEs may differentiate and / or be induced to differentiate into cells resembling third pharyngeal pouch endoderm (PPE). Thymic epithelial progenitor cells (TEPCs) may be generated from PPE cells. TECs may be derived from TEPCS. Each of the cell types described herein may be characterized by one or more markers. In some embodiments, pluripotent stem cells may be associated with increased expression of markers, such as, but not limited to, OCT4, SOX2, and / or Nanog. In some embodiments, definitive endoderm may be associated with increased expression of markers, such as, but not limited to, SOX17, FOXA2, CXCR4, and / or CER1. In some embodiments, anterior foregut cells (AFE) may be associated with increased expression of markers, such as, but not limited to, FOXA2, SOX2, and / or PAX9. In some embodiments, third pharyngeal pouch endoderm cells may be associated with increased expression of markers, such as, but not limited to, HOXA3, TBX1, PAX9, EYA1, SIX1, PBX1, and / or PAX1. In some embodiments, thymic epithelial progenitor cells may be associated with increased expression of markers, such as, but not limited to, FOXN1, EPCAM, K5, K8, and / or HOXA3. In some embodiments, the thymocytes may be derived from DE cells, third PPE cells, AFE cells, TEPCs, and / or TEC cells.

[0029] Pluripotent stem cells (PSC) In some embodiments, the cells of the present disclosure may be derived from pluripotent stem cells.

[0030] Pluripotent stem cells have the ability to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent stem cells may include stem cells such as embryonic stem cells, nuclear transfer-derived embryonic stem cells, induced pluripotent stem cells (iPSCs), etc. Pluripotent stem cells may have a stem cell phenotype that includes (i) the ability of self-renewal and (ii) pluripotency. Pluripotency-associated genes may include, but are not limited to, Oct-3 / 4, Sox2, Nanog, GDF3, REXI, FGF4, ESGI, DPPA2, DPPA4, hTERT, and SSEAI.

[0031] The cells described herein may be derived from embryonic stem cells. ES cells may include cells that (a) self-renew, (b) differentiate to produce all cell types in an organism, and / or (c) are derived from a developing organism. ES cells may be derived from the inner cell mass of a developing organism's blastula. ES cells may also be derived from blastomeres generated by single blastomere biopsy (SBB), which involves the removal of a single blastomere from the 8-cell stage of a developing organism. ES cells may be characterized by expression of markers, such as, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and / or alkaline phosphatase. Methods for generating and characterizing ES cells are known in the art and can be found, for example, in U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, U.S. Pat. No. 6,200,806 (the entire contents of each of which are incorporated herein by reference).

[0032] Induced pluripotent stem cells (iPSCs) can also be used to generate the cells of the present disclosure. iPSCs can include cells that have one or more properties, for example, but not limited to (a) self-renewal, (b) the ability to differentiate to produce all types of cells in an organism, and / or (c) somatic origin. iPSCs can express markers, for example, but not limited to, SSEA3, SSEA4, SOX2, OCT3 / 4, Nanog, TRA160, TRA1818, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, Zpf42. Methods of generating and characterizing iPSC cells can be found, for example, in U.S. Patent Publication Nos. US20090047263, US20090068742, US2009191159, US20090227032, US20090246875, and US20090304646, the entire contents of each of which are incorporated herein by reference. In some embodiments, iPSCs can be derived from T cells or non-T cells, B cells, or any other cells from peripheral blood mononuclear cells, hematopoietic progenitor cells, or any other somatic cell type.

[0033] In some embodiments, the pluripotent stem cells may be derived from adult stem cells. The adult stem cells may be obtained from a subject, such as the subject's inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood. The PSCs may also include embryonic stem cells from the placenta or umbilical cord, and progenitor cells (e.g., progenitor cells derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood).

[0034] Effector cells "Effector cell" refers to any cell or cell type that acquires the ability to execute, initiate or propagate a signal or cell death trigger upon contact or proximity to a thymocyte. "Contact or proximity" can refer to sufficient spatiotemporal proximity to allow cell-intrinsic or cell-extrinsic (e.g., cell-cell) signaling or other communication or interaction.

[0035] The effector cells described herein may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, thymus, or the stem or progenitor cells may be differentiated in vitro from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The stem or progenitor cells from primary tissue or ESCs or iPSCs may be human or non-human animal (e.g., mouse) in origin.

[0036] In some embodiments, the effector cell may be a hematopoietic cell. In some embodiments, the effector cell may be a lymphocyte. In some embodiments, the lymphocyte may be a CD45 positive lymphocyte.

[0037] Effector cells are CD4+CD8-T cells, CD4-CD8+T cells, CD34+CD7+CDla+ cells, CD3+TCRab+ cells, CD3+TCRgd+ cells, CD3+TCRab+CD4+CD8- cells, CD3+TCRab+ CD8+CD4- cells, CD3+TCRab+CD4+CD8-CD45RO-CD45RA+ cells, CD3+TCRab+CD8+CD4-CD45RO-CD45RA+ cells, CD3+TCRab+CD4+CD8-CD45RO-30 CD45RA+CCR7+ cells, CD3+TCRab+CD8+CD4-CD45RO-CD45RA+CCR7+ cells, CD3+TCRab+CD4+CD8-CD45RO-CD45RA+CD27+ cells, CD3+TCRab+CD8+CD4-CD45 ROCD45RA+ cells, CD27+, CD34+CD7+CD1a+ cells, CD34+CD5+CD7+ cells, CD34+CD5+CD7- cells, natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphoid T cells, or CD45+, CDI lb+, CDI lb-, CD15+, CD15-, CD24+, CD24-, CDI The cells may be positive for CD14+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CDIIa+, CDIIa-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, Foxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD3 I+, CD3 I-, CD30+, CD30-, CD38+, and / or CD38- cells, and / or combinations thereof.

[0038] In some embodiments, the effector cells may be T cells. The T cells may be cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. If obtained from a mammal, the effector cells may be obtained from a number of sources, including but not limited to, blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or fluids. The effector cells may be enriched or purified. The T cells may be any type of T cell and may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells (TILs), memory T cells, naive T cells.

[0039] In some embodiments, effector cells can be CCRXA-, CD3+, CD69-, MHC-1+, CD62L+, and / or CCR7+.

[0040] Effector cells may have a naive T cell (TN) phenotype, a central memory T cell (TcM) phenotype, or an effector memory T cell (TEM) phenotype. The phenotypes of TN, TcM and TEM cells are known in the art. For example, CCR7 and CD62L are expressed by TN and TcM cells, but not by TEM cells. Transcription factors LEFl, FOXPl and KLF7 are expressed by TN and TcM cells, but not by TEM cells. CD45RO and KLRGl are not expressed by TN cells, but are expressed by TEM cells (Gattinoni et al., Nat. Rev. Cancer, 12: 671-84 (2012)). Alternatively or additionally, TN and TcM cells may be characterized by longer telomeres compared to TEM cells.

[0041] In some embodiments, the effector cell can be a TCRα+TCRβ+ cell. The TCRα+TCRβ+ effector cell can be a T cell expressing a receptor that expresses an alpha (α) chain and / or a beta (β) chain. TCR alpha and beta chains are known in the art.

[0042] The effector cells may be further modified. In a further embodiment, the stem cells or progenitor cells may be genetically modified. For example, the stem cells or progenitor cells may express an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR), or both. In a further embodiment, the stem cells or progenitor cells may express an exogenous invariant natural killer T cell (iNKT)-associated TCR. In yet a further embodiment, the stem cells or progenitor cells express an exogenous antigen-specific TCR or have an exogenous genetic modification of a gene that regulates the differentiation, proliferation or function of T cells.

[0043] In some embodiments, the effector cells can be FOXP3+ Tregs. Tregs can be generated by clonal conversion of mTECs, whereby expression of Aire in mTECs causes expression of tissue-specific antigens that are presented on the surface (i.e., on antigen-presenting cells (APCs)). Autoreactive T cells that recognize tissue-specific antigens generate FOXP3+ Tregs that can mediate peripheral tolerance (see Husebye, Eystein S., Mark S. Anderson, and Olle Kampe. "Autoimmune polyendocrine syndromes." New England Journal of Medicine 378.12 (2018): 1132-1141, which is incorporated herein by reference in its entirety).

[0044] supporting cells In some embodiments, the cells of the present disclosure may include or be cultured with supporting cells that aid in the generation of thymocytes and / or the maintenance of thymocytes in culture. Non-limiting examples of supporting cells include hematopoietic non-T cell precursors such as macrophages and dendritic cells (DCs), non-hematopoietic cells such as epithelial cells and fibroblasts, stromal cells such as precursors of skeletal tissues, bone, cartilage, hematopoietic support stroma, and components such as adipocytes. In some embodiments, supporting cells promote the proliferation, survival, maturation, or function of thymocytes. In some embodiments, supporting cells may be mesenchymal in origin.

[0045] Supportive cells can be non-immune cells that can be present in the thymic microenvironment. For example, supportive cells can be fibroblasts, vascular smooth muscle cells (VSMCs), endothelial cells, and / or lymphatic endothelial cells.

[0046] In some embodiments, the supporting cells may be neuroendocrine cells (expressing BEX1, NEUROD1), myosinoid cells (expressing MYOD1, DES), and myelin-positive epithelial cells (expressing SOX10, MPZ) as described in Bautista et al. 2021 Nat Commun 12, 1096 (2021), the entire contents of which are incorporated herein by reference. In some embodiments, the mesenchymal cells may be associated with markers such as, but not limited to, LAMA2, LAMA4, PDGFRA, PDGFRB, LUM, CSPG4, COL1A2, COL3A1, IGF1, FGF7, FGF10, FST, BMP4, SFRP2, WNT5A. Mesenchymal cells may be positive or negative for one or more of these markers. In some embodiments, mesenchymal cells may be positive for some markers described herein, but negative for others.

[0047] In some embodiments, endothelial cells may be associated with one or more markers, such as, but not limited to, VEGFC, PECAM1, APLNR, PROX1, LYVE1, ACKR1, SELE, SELP, FN1, and / or TGFB1. Endothelial cells may be positive or negative for one or more of these markers. In some embodiments, endothelial cells may be positive for some markers described herein, but negative for others.

[0048] 3D culture In some embodiments, the cells of the present disclosure can be cultured in a three-dimensional culture (3D) system. In 3D culture, cells are cultured three-dimensionally with a surrounding extracellular framework. Pluripotent stem cells, DE cells, AFE cells, VPE cells, TEPs, and / or TECs can be cultured in 3D. The cells of the present disclosure can be cultured with or without a supporting scaffold. In some embodiments, the cells of the present disclosure can be cultured in a scaffold-free 3D cell culture. The cells can be cultured as organoids or spheroids. The cells can be cultured as spheroids. In spheroid culture, cells grow as an aggregation of cells and become round cell clusters in a three-dimensional structure. The cells can be round and uniform in shape. In some embodiments, the cells of the present disclosure can be cultured as organoids. As used herein, the term organoid refers to an artificial model of living cells in a three-dimensional or multi-layered structure, and can include cells other than thymocytes, such as effector cells and supporting cells. In some embodiments, the organoids can form an ordered structure.

[0049] In some embodiments, the composition of the present disclosure can comprise thymus organoid.Organoid is a miniature in vitro three-dimensional reproduction of an organ.Thymus organoid can be an in vitro three-dimensional miniature version of the thymus organ, which can mimic the physiology and function of human thymus.The method for preparing thymus organoid is described in International Patent Publication WO2019060336, the entire contents of which are incorporated herein by reference.

[0050] In some embodiments, the effector cells can be prepared by differentiating pluripotent stem cells or progenitor cells into lymphocytes by culturing PSC or progenitor cells with thymocytes. In some embodiments, the thymocytes can express a Notch ligand. In some embodiments, the Notch ligand can be Delta-like 1 (DLL1). In some embodiments, the Notch ligand is Delta-like 4 (DLL4). In some embodiments, the Notch ligand is described herein or in the art, for example, in U.S. Patent No. 7,795,404, the entire contents of which are incorporated herein by reference. The effector cells of the present disclosure can be prepared using a thymic organoid cell culture system. In some embodiments, the method further comprises contacting the co-cultured stem or progenitor cells and stromal cells with Flt-3 ligand and / or IL-7 and / or stem cell factor / Kit ligand and / or thrombopoietin. In some embodiments, differentiating stem or progenitor cells into T cells comprises culturing three-dimensional (3D) cell aggregates comprising a) a selected population of feeder cells that endogenously or exogenously express a Notch ligand, and b) a selected population of stem or progenitor cells with serum-free medium comprising B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem or progenitor cells described in International Patent Publication WO2017075389, the entire contents of which are incorporated herein by reference, may be useful in the present disclosure.

[0051] In some embodiments, the thymic organoids may be based on the artificial thymic organoids described in Seet CS, et al. Nat Methods. 2017;14(5):521-530, the entire contents of which are incorporated herein by reference. To prepare thymic organoids, thymocytes may be harvested by trypsinization and resuspended in serum-free culture medium ("RB27") that may contain RPMI1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate reconstituted in PBS (Sigma-Aldrich, St. Louis, MO), 1% penicillin / streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng / ml rhFLT3L, and 5 ng / ml rhIL-7 (Peprotech, Rocky Hill, NJ). Different ratios of thymocytes and effector cells may be prepared in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 minutes at 4° C. in a swinging bucket centrifuge. The supernatant was carefully removed and the cell pellet resuspended by vortexing briefly. For each organoid, a 0.4 μm Millicell transwell insert (EMD Millipore, Billerica, MA. Cat. No. PICM0RG50) may be placed into a 6-well plate containing 1 ml of RB27 per well. To plate the organoids, the insert was removed and placed on the edge of the plate to drain off excess medium. The cell slurry may be adjusted to 5 μl per organoid and plated by drawing up with a 20 μl pipette tip and forming a droplet on the edge of the pipette tip that is gently attached onto the cell insert. The cell insert may be placed back into the well containing 1 mL of RB27. The medium may be changed completely every 3-4 days by aspirating from around the cell insert and then replacing with 1 ml containing fresh RB27 / cytokines.In some embodiments, the organoids may be cultured in this manner for up to 10 weeks, 15 weeks, 20 weeks, 25 weeks, or 30 weeks.

[0052] FACS buffer (PBS / 0.5% bovine serum albumin / 2mM EDTA) was added to each well and the organoids were briefly dissociated by pipetting with a 1 ml "P1000" pipette, followed by passage through a 50 μm nylon strainer to harvest the organoid cells at the indicated times. In some experiments, single cell suspensions of MS5-hDLL1 cells were γ-irradiated at the indicated doses prior to use in organoids.

[0053] Thymic organoid effector cell co-cultures can be prepared as described in Seet CS, et al. Nat Methods. 2017;14(5):521-530, the entire contents of which are incorporated herein by reference. Thymocytes can be seeded onto 12-well plates coated with 0.1% gelatin 1-2 days prior to use to achieve 70-80% confluence. Media can be aspirated from the monolayer and 1.5 x 10 4 FACS purified effector cells (CD34+CD3- hematopoietic cells) can be plated with thymic organoids in 2 ml of medium composed of MEMα, 20% FBS, 30 μM L-ascorbic acid, 5 ng / ml rhFLT3L and 5 ng / ml rhIL-7. In some embodiments, effector cells can be transferred to thymic organoids every 4-5 days by harvesting the cells, filtering through a 50 μm nylon strainer, and replating in fresh medium. Once confluent, the cells were dispensed into multiple wells containing fresh stromal layers.

[0054] In some embodiments, the thymocytes of the present disclosure can be combined with double negative day 14 T cells to form cell clusters, which can then be attached to transwells as "organoids" and maintained in air-liquid interface culture conditions. In some embodiments, cells can be harvested from culture medium every few days to assess T cell maturation.

[0055] III. Method The present disclosure provides methods for differentiating pluripotent stem cells into thymocytes. In some embodiments, the present disclosure provides methods for differentiating induced pluripotent stem cells into thymocytes.

[0056] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymocytes may include activation of WNT signaling. As a non-limiting example, an activator of WNT signaling may be CHIR99021.

[0057] In some embodiments, one or more of the steps involved in differentiation of iPSCs into thymocytes may include inhibition of WNT signaling. As a non-limiting example, an inhibitor of WNT signaling may be IWR1 (or IWR-1).

[0058] In some embodiments, one or more of the steps involved in differentiating iPSCs into thymocytes may include inhibition of BMP signaling. In some embodiments, inhibition of BMP signaling may be achieved using the BMP pathway inhibitor LDN193189.

[0059] In some embodiments, one or more of the steps involved in differentiating iPSCs into thymocytes may include inhibition of SHH signaling. In some embodiments, inhibition of SHH is achieved using the SHH antagonist SANT-1.

[0060] In some embodiments, one or more of the steps involved in differentiating iPSCs into thymocytes may include inhibition of TGFβ signaling. In some embodiments, inhibition of TGFβ signaling is achieved using the TGFβ inhibitor SB431542.

[0061] In some embodiments, one or more of the steps involved in differentiating iPSCs into thymocytes may include cell culture medium containing insulin-transferrin-selenium (ITS), knockout serum replacement (KSR), penicillin-streptomycin (also referred to herein as "Pen Strep") and non-essential amino acids (NEAA).

[0062] Preparation and maintenance of thymocytes Provided herein are methods of differentiating pluripotent stem cells into thymocytes. Such methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-target P13K inhibitor). In some embodiments, the first growth medium includes DMEM-F12, activin A, CHIR99021, insulin-transferrin-selenium (ITS), and knockout serum replacement (KSR). In some aspects, the second growth medium includes DMEM-F12, bFGF, activin A, LDN193189, ITS, and KSR. In some embodiments, the cells are cultured in the presence of PI-103. The concentration of PI-103 may be about 1 nM to 1000 nM. In one embodiment, the concentration of PI-103 may be 50 nM.

[0063] The definitive endoderm cells can be further differentiated into anterior foregut cells by culturing and contacting or incubating with at least one of SB431542, LDN-193189, and KSR. In some embodiments, the anterior foregut cells can be differentiated into pharyngeal endoderm cells by culturing and contacting or incubating with at least one of EGF, retinoic acid, FGF8B, and SHH. In some embodiments, the pharyngeal endoderm cells can be differentiated into thymic epithelial cells by culturing and contacting or incubating with at least one of BMP4, FGF8b, EGF, SANT, CHIR99021, ascorbic acid, or combinations thereof. In some embodiments, the differentiation is performed for about 14 to 25 days. For example, the differentiation is performed for about 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days.

[0064] In some embodiments, the disclosure provides methods for preparing one or more cells or cell types described herein. In some embodiments, the cells may be thymocytes.

[0065] A series of data accumulated in public databases provides single cell transcriptomes of primary human and mouse thymuses (see Bautista et al. 2021 Nat Commun 12, 1096; Kernfeld, et al. Immunity. 2018 Jun 19;48(6):1258-1270.e6; Zeng et al. Immunity. 2019 Nov 19;51(5):930-948.e6, the entire contents of each of which are incorporated herein by reference). This provides a rich source of material for identifying factors that promote differentiation and / or maturation of the cells of the present disclosure into thymocytes. Through analysis of scRNA sequencing data, the present disclosure identifies potential factors and / or supporting cells that may promote and / or maintain the thymocyte phenotype.

[0066] In some embodiments, the cells of the present disclosure may be isolated from an organism. In some embodiments, the organism may be a mammal. The mammalian cells may be isolated from human, rodent, porcine, and / or bovine sources. The human source of the cells of the present disclosure may be autologous or allogeneic. In some embodiments, tissues containing the cells of the present disclosure may be harvested and used intact for the applications described herein. The cells of the present disclosure may be obtained from embryonic, fetal / fetal, or adult organisms. In some aspects, the organism may be a living or cadaveric organism.

[0067] The cells described herein may be derived from other cell types. As a non-limiting example, the cells of the present disclosure may be derived from pluripotent stem cells (PSCs). In some embodiments, the cells of the present disclosure may be derived from progenitor cells. In some embodiments, the cells of the present disclosure may be derived from the differentiation of PSCs and / or progenitor cells.

[0068] In some embodiments, thymocytes may be prepared from PSCs. In this regard, the method may include culturing pluripotent stem cells for a period of time under conditions sufficient to differentiate the pluripotent stem cells into thymocytes. For example, the method may include culturing the pluripotent stem cells in the presence of factors and / or inhibitors that drive the differentiation of PSCs into thymocytes. Methods for differentiating PSCs into thymocytes are known in the art. Methods for differentiating PSCs into thymocytes may include the use of one or more parameters or combinations thereof for differentiation known in the art. The parameters include, but are not limited to, (i) differentiation factors, (ii) differentiation inhibitors, (iii) duration of differentiation, (iv) temperature, (v) substrate, and / or (vi) support cells that promote differentiation. Any method or parameters for differentiating PSCs into thymocytes described in the following references may be used herein: Parent et al. Cell Stem Cell. 2013 Aug 1;13(2):219-29; Soh et al. Stem Cell Rep. 2014 Vol. 2 j 925-937; Sun et al. Cell Stem Cell. 2013 Aug 1;13(2):230-6; Okabe et al. Cell. Reprog. 2015 Vol 17, No. 5; Su et al. Sci.Rep. 2015 5, 9882; Otsuka et al. Sci Rep 2020 10:224, International Patent Publications WO2019060336, WO2020205859, WO2020220040, WO2014134213, WO2010143529, WO2011139628, and Chinese Patent Publication CN201110121243, the entire contents of each of which are incorporated herein by reference.

[0069] Preparation and maintenance of definitive endoderm cells The present disclosure provides methods for preparing definitive endoderm cells that can subsequently differentiate into thymocytes. In some embodiments, the embryonic somatic cells may be prepared by culturing the cells in two-dimensional or three-dimensional culture. Such methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-targeted P13K inhibitor). In some embodiments, the first growth medium includes activin A, CHIR99021, insulin-transferrin-selenium (ITS), and / or knockout serum replacement (KSR).

[0070] In some aspects, the second growth medium comprises basic fibroblast growth factor (bFGF), activin A, LDN193189, ITS and KSR. In some embodiments, the second growth medium comprises CHIR99021.

[0071] In some embodiments, the concentration of CHIR99021 is about 0.1 μM to 100 μM. In some embodiments, the concentration of CHIR99021 is about 2 μM to 3 μM.

[0072] In some embodiments, the cells are cultured in the presence of PI-103. The concentration of PI-103 can be about 1 nM to 1000 nM. In one embodiment, the concentration of PI-103 can be 50 nM. In some aspects, the concentration of PI-103 can be 25 nM. In some embodiments, the pluripotent stem cells can be cultured for about 3 to 5 days. PI-103 can be added for 1 to 2 days. The pluripotent stem cells can be embryonic stem cells or induced pluripotent stem cells.

[0073] In some embodiments, the pluripotent stem cells may be cultured for about 3-5 days. The stem cells may be cultured for about 1-2 days in the first growth medium and for about 2-3 days in the second growth medium. The pluripotent stem cells may be cultured for 2 days in the first growth medium and for 3 days in the second growth medium. In some embodiments, the concentration of Activin A may be about 100 ng / ml. In some embodiments, the concentration of CHIR99021 may be 2 μM. In some embodiments, the concentration of bFGF may be 10 ng / ml. In some embodiments, the concentration of LDN193189 may be 200 nM. In some embodiments, CHIR99021 may be added to the second growth medium for about 1 day.

[0074] Provided herein are methods of differentiating pluripotent stem cells into thymocytes. Such methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-target P13K inhibitor). In some embodiments, the first growth medium includes DMEM-F12, activin A, CHIR99021, insulin-transferrin-selenium (ITS), and knockout serum replacement (KSR). In some aspects, the second growth medium includes DMEM-F12, bFGF, activin A, LDN193189, ITS, and KSR. In some embodiments, the second growth medium includes CHIR99021. In some embodiments, the concentration of CHIR99021 is about 0.1 μM to 100 μM. In some embodiments, the concentration of CHIR99021 is 2 μM. In some embodiments, the cells are cultured in the presence of PI-103. The concentration of PI-103 can be about 1 nM to 1000 nM. In one embodiment, the concentration of PI-103 can be 50 nM.

[0075] Preparation of anterior foregut endoderm (AFE) cells The definitive endoderm cells can be further cultured and differentiated into anterior foregut cells. In some embodiments, AFE cells can be prepared by culturing the cells in two-dimensional or three-dimensional culture. The definitive endoderm cells can be differentiated into AFE cells by contacting them with a BMP inhibitor, a TGFβ inhibitor, at least one FGF, and / or ascorbic acid. In some embodiments, the cell culture medium utilized for differentiation of DE cells into AFE cells can include N2-supplement (GIBCO, Waltham, Mass.), Eagle's Basal Medium (BME), GLUTAMAX (GIBCO, Waltham, Mass.), B27™ serum-free supplement, non-essential amino acids, KSR, and / or ITS.

[0076] In some embodiments, the BMP inhibitor may be LDN193189. In some embodiments, the concentration of LDN193189 is about 0.1 nM to about 1000 nM. In some embodiments, the concentration of LDN193189 is about 100 to 200 nM.

[0077] In some embodiments, the TGFβ inhibitor may be SB431542. In some embodiments, the concentration of SB431542 is about 1 μM to about 100 μM. As a non-limiting example, the concentration of SB431542 is 10 μM.

[0078] In some embodiments, the FGF may be FGF8. In some embodiments, the concentration of FGF8 is about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF8b is about 25 to 50 ng / ml.

[0079] DE cells can be differentiated into AFE cells for about 1 day, 2 days, 3 days, 4 days, or 5 days.

[0080] Preparation of ventral pharyngeal endoderm (VPE) cells The differentiation of AFEs into VPE cells is performed as a single-step process or a multi-step process. The multi-step process can be a two-step process. In a first step, the AFEs are cultured in VPE1 medium, and in a second step, the cells are cultured in VPE2 medium. In some embodiments, the VPE cells can be prepared by culturing the cells in a two-dimensional or three-dimensional culture. The VPE1 step can include culturing the cells for about 1 day, 2 days, 3 days, 4 days, or 5 days. The VPE2 step can include culturing the cells for about 2 days, 3 days, 4 days, 5 days, or 6 days.

[0081] In some embodiments, VPE1 medium can include retinoic acid, at least one FGF, a WNT inhibitor, a TGFβ inhibitor, and / or ascorbic acid.

[0082] In some embodiments, VPE2 medium may include noggin, a BMP inhibitor, a WNT activator (eg, CHIR99021), at least one FGF, retinoic acid, an SHH antagonist, and / or ascorbic acid.

[0083] In some embodiments, the FGF may be FGF8, FGF7, and / or FGF10. In some embodiments, the concentration of FGF8 is about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF8b is about 25 to 50 ng / ml.

[0084] In some embodiments, the WNT inhibitor is IWR1. The concentration of IWR1 may be about 0.01 to 10 μM. As a non-limiting example, the concentration of IWR1 is 2.5 μM.

[0085] In some embodiments, the TGFβ inhibitor may be SB431542. In some embodiments, the concentration of SB431542 is about 1 μM to about 100 μM. As a non-limiting example, the concentration of SB431542 is 10 μM.

[0086] In some embodiments, the concentration of ascorbic acid is about 0.1 to 30 μM. As a non-limiting example, the concentration of ascorbic acid can be 10 μM.

[0087] In some embodiments, the BMP inhibitor can be LDN193189. In some embodiments, the concentration of LDN193189 is about 0.1 nM to about 1000 nM. In some embodiments, the concentration of LDN193189 is about 100 to 200 nM.

[0088] In some embodiments, the SHH inhibitor can be SANT-1. In some embodiments, the concentration of SANT-1 is about 0.01 μM to about 10 μM. As a non-limiting example, the concentration of SANT-1 is 0.25 μM.

[0089] In some embodiments, anterior foregut cells can be cultured and differentiated into pharyngeal endoderm cells by contacting or incubating with at least one of EGF, retinoic acid, FGF8B, and / or SHH.

[0090] In some embodiments, VPE1 and / or VPE2 medium may contain N2-supplement (GIBCO, Waltham, Massachusetts), Basal Eagle's Medium (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement (with or without vitamin A), non-essential amino acids, KSR and / or ITS.

[0091] Preparation of thymic epithelial progenitor (TEP) cells The differentiation of VPE cells into TEP cells can be performed by culturing the cells in TEP medium. In some embodiments, TEP cells can be prepared by culturing cells in two-dimensional culture or three-dimensional culture. The TEP step can include culturing the cells for about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0092] In some embodiments, VPE cells can be differentiated into TEP cells using BMPs (eg, BMP4, BMP2), WNT activators such as CHIR99021, at least one FGF, and / or ascorbic acid.

[0093] In some embodiments, TEP medium may contain N2-supplement (GIBCO, Waltham, Mass.), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Mass.), B27™ serum-free supplement (with or without vitamin A), non-essential amino acids, KSR, and / or ITS.

[0094] In some embodiments, the BMP can be BMP2 or BMP4. The concentration of the BMP can be from 1 ng / ml to about 100 ng / ml. In some aspects, the concentration of the BMP can be 50 ng / ml.

[0095] In some embodiments, the FGF may be FGF8, FGF7, FGF1, and / or FGF10. In some embodiments, the concentration of FGF is about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF is about 25 to 50 ng / ml.

[0096] In some embodiments, pharyngeal endoderm cells can be cultured and differentiated into thymic epithelial cells by contacting or incubating with at least one of BMP4, FGF8b, EGF, SANT-1 (a SHH antagonist), CHIR99021, ascorbic acid, or a combination thereof.

[0097] Preparation of thymic epithelial cells (TECs) TEP cells can be further differentiated in vitro into TECs. Differentiation into TECs can be performed in 2D or 3D cultures. In some embodiments, differentiation of TEPs can be performed for about 2 days, 3 days, 4 days, 5 days, or 6 days.

[0098] In some embodiments, differentiation of TEPs into TECs is performed in TEC medium.

[0099] The TEC medium may contain RANKL, an interleukin, such as (IL22), at least one FGF, at least one BMP (eg, BMP4), a WNT activator, and / or ascorbic acid.

[0100] In some embodiments, the concentration of RANKL may be from about 1 ng / ml to about 100 ng / ml, hi some embodiments, the concentration of RANKL may be from about 20 ng / ml to about 50 ng / ml.

[0101] In some embodiments, the FGF may be FGF8, FGF7, FGF1, and / or FGF10. In some embodiments, the concentration of FGF is about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF is about 25 to 50 ng / ml.

[0102] In some embodiments, the concentration of the interleukin is about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of IL22 is about 20 ng / ml.

[0103] TEC medium may contain N2-supplement (GIBCO, Waltham, Mass.), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Mass.), B27™ serum-free supplement (with or without vitamin A), non-essential amino acids, KSR and / or ITS.

[0104] In some embodiments, differentiation is performed for about 14 to 17 days. In some embodiments, the cells of the present disclosure can be cultured as aggregates. In some embodiments, the cells of the present disclosure can be cultured in an extracellular matrix-based medium, such as Geltrex.

[0105] Preparation of effector cells Also provided herein are methods for preparing effector cells. In some embodiments, the effector cells can be lymphocytes. The effector cells can be obtained from primary cells from a mammal or established cell lines. If obtained from a mammal, the effector cells can be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or fluids. The effector cells can be enriched or purified. In some embodiments, the effector cells can be T cells. The T cells can be any type of T cell and at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells (TILs), memory T cells, and naive T cells. Methods for isolating and / or enriching lymphocytes are known in the art. Methods for enriching a population of lymphocytes obtained from a mammal or donor can be completed by any suitable separation method, including but not limited to the use of separation media (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte Enrichment Cocktail, or Lymphocyte Separation Medium (LSA) (MP Biomedical Catalog No. 0850494X), etc.), cell size, shape or density separation by filtration or elutriation, immunomagnetic separation (e.g., Magnetic Activated Cell Sorting System, MACS), fluorescent separation (e.g., Fluorescence Activated Cell Sorting System, FACS), and / or bead-based column separation.

[0106] In some embodiments, the effector cells described herein may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, thymus, or the stem or progenitor cells may be differentiated in vitro from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The stem or progenitor cells from primary tissue or ESCs or iPSCs may be derived from human or non-human animals (e.g., mice) in origin.

[0107] In some embodiments, the effector cells can be prepared by differentiating pluripotent stem cells or progenitor cells into lymphocytes by culturing the PSC or progenitor cells with supporting cells that ectopically express a Notch ligand. In some embodiments, the supporting cells can be OP9 cells. In some embodiments, the Notch ligand is Delta-like 1 (DLL1). In some embodiments, the Notch ligand is Delta-like 4 (DLL4). In some embodiments, the Notch ligand is described herein or in the art, for example, in U.S. Patent No. 7,795,404, the entire contents of which are incorporated herein by reference. The effector cells of the present disclosure can be prepared using an artificial thymic organoid (ATO) cell culture system that utilizes supporting cells that ectopically express OP9-DLL1. In some embodiments, the method further comprises contacting the co-cultured stem or progenitor cells and stromal cells with Flt-3 ligand and / or IL-7 and / or stem cell factor / Kit ligand and / or thrombopoietin. In some embodiments, differentiating stem or progenitor cells into T cells comprises culturing three-dimensional (3D) cell aggregates comprising a) a selected population of feeder cells expressing an exogenous Notch ligand, and b) a selected population of stem or progenitor cells with serum-free medium comprising B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem or progenitor cells described in International Patent Publication WO2017075389, the entire contents of which are incorporated herein by reference, may be useful in the present disclosure.

[0108] In some embodiments, the effector cells can be or can be derived from hematopoietic cells. Methods for preparing hematopoietic cells from pluripotent stem cells are known in the art, for example, as described in U.S. Pat. No. 9,834,754, and can include one or more of the following steps: (i) inducing hematopoietic differentiation in a population of human pluripotent stem cells, where activin / nodal signaling is inhibited between days 1 and 4 of differentiation, (ii) sorting the induced population based on expression of CD34 and CD43, and / or (iii) selecting a fraction of the CD34 positive and / or CD43 negative cell population, where sorting and cell fraction selection are performed on a day selected from about days 6-11 of differentiation (the entire contents of U.S. Pat. No. 9,834,754 are incorporated herein by reference).

[0109] In some embodiments, thymocytes and / or effector cells can be cultured in the presence of extracellular vesicles (e.g., exosomes) derived from thymocytes and / or effector cells. Methods for preparing thymocyte exosomes are described in U.S. Patent Publication US2020299641, the entire contents of which are incorporated herein by reference. In some embodiments, exosomes can be derived from thymocytes engineered to ectopically express DLL1.

[0110] In some embodiments, effector cells such as T cells may be derived from the differentiation of other cell types. T cell differentiation may include four stages: 1) mesoderm induction (about days 1-4), 2) hematopoietic specification (about days 4-8), 3) hematopoietic commitment and expansion (about days 8-10), and / or 4) T lymphocyte differentiation. PSCs (iPSCs or ESCs) can be used as the starting cell population for mesoderm differentiation. These cells can be differentiated into mesoderm cells. Mesoderm cells can be further differentiated into hematopoietic cells, which may expand in number. Cell culture systems for use in the present disclosure include, but are not limited to, a first cell culture medium for mesoderm induction, a second cell culture medium for hematopoietic specification and expansion, and a third cell culture medium for T lymphocyte differentiation. The first cell culture medium may include BMP4 (e.g., human BMP4) and bFGF (e.g., human bFGF). PSCs or ESCs can be used as the starting cell population. Undifferentiated PSCs or ESCs can be transferred to low attachment plates to allow for the formation of embryoid bodies (EBs). The formation of EBs during the first stage can be promoted by overnight incubation in the presence of hBMP4. The EBs can then be cultured with BMP4 and bFGF up to day 4 to allow for mesoderm induction. The success of mesoderm induction can be tested, for example, by measuring the percentage of KDR+PDGFR- cells. The second cell culture medium can include VEGF (e.g., hVEGF), and a cocktail of hematopoietic cytokines. The cocktail of hematopoietic cytokines can include SCF (e.g., hSCF), Flt3L (e.g., hFlt3L), at least one cytokine, and bFGF for hematopoietic specification. The cytokines can be Th1 cytokines, including but not limited to IL3, IL15, IL7, IL12, and IL21. The EBs can be cultured in the second cell culture medium for hematopoietic specification up to about day 10. EBs can be immunophenotyped by FACS for expression of CD34, CD31, CD43, CD45, CD41a, c-kit, Notch1, IL7Ra. In some embodiments, CD34+ cells from EBs at about day 1 express the highest levels of key transcription factors for lymphoid differentiation, such as CD127 (IL7Ra) and Notch1.The third cell culture medium can include feeder cells and SCF, Flt3L, and at least one cytokine. The cytokine can be a Th1 cytokine, including but not limited to IL3, IL15, IL7, IL12, and IL21. In some embodiments, at about day 10, the EBs can be dissociated and the hematopoietic progenitor cells can be transferred onto feeder cells to induce T lymphocyte differentiation in the presence of SCF, Flt3L, and Th1 cytokine(s) (e.g., IL-7) in the established co-culture system. In some embodiments, the co-culture system can include thymocytes and / or feeder cultures, such as OP9-DL11 feeder cells.

[0111] In some embodiments, the co-culture may be performed using a co-culture medium. In some embodiments, the co-culture medium may include StemSpan SFEM II and StemSpan™ T Cell Progenitor Maturation Supplement. In some embodiments, the co-culture medium may include αMEM, 4% B27 supplement, 30 uM ascorbic acid, 50 ng / ml IL7, 50 ng / ml FLT3L, 50 ng / ml TPO, 50 ng / ml SCF, and / or 1X Pen Strep. In some embodiments, the co-culture medium may include DMEM / F12, 1% B27 supplement without vitamin A, 50 μM ascorbic acid, 50 ng / ml FGF8b, 50 ng / ml BMP, 50 ng / ml FGF10, 2 uM CHIR99021, 0.1% ITS, 0.0025% KSR, 0.5X Pen Strep, 1x NEAA, 1% N2, 1% Glutamax, 1% β-ME, 50 ng / ml IL7, 50 ng / ml FLT3L, 50 ng / ml TPO, and / or 50 ng / ml SCF.

[0112] Aggregate Size In some embodiments, the cells of the present disclosure may be cultured in a three-dimensional culture. In some embodiments, the cells of the present disclosure may be in the form of aggregates or spheroids. The term "spheroid" refers to a cluster of cells and / or a cell colony. Spheroids can be formed from various cell types, such as thymocytes, pluripotent cells, effector cells, stem cells, and / or support cells. Spheroids may be spherical or irregular in shape. Spheroids may contain heterogeneous cell populations, cell types, cells of different states, such as proliferating cells, quiescent cells, and necrotic cells.

[0113] In some embodiments, the size of the spheroids / aggregates can be adjusted. For example, the size of aggregates in pluripotent stem cells can be important during expansion, since the size of the aggregates can determine the oxygen distribution within the cell spheroid, resulting in the formation of discrete zones consisting of outer, middle and inner spheroid regions in descending order of oxygen supply, which exhibit the core characteristics of proliferation, quiescent survival and apoptosis, respectively (Langan et al. Plos One. 2016;11(2), the entire contents of which are incorporated herein by reference). In some embodiments, the aggregates can be about 50 μm to 500 μm, about 100 μm to 1000 μm, about 200 μm to 2000 μm, about 250 μm to 2500 μm, about 300 μm to 3000 μm, about 400 μm to 4000 μm. In embodiments, the size of the spheroids / aggregates can be 250 μm.

[0114] How to use The present disclosure provides a method of treating or preventing a condition in a subject. The method includes administering to a subject any of the cell populations described herein or a pharmaceutical composition comprising any of the cell populations described herein in an amount effective to treat or prevent the condition in the subject. The condition can be cancer, an immune deficiency, an autoimmune condition, an infection, or a hematological condition. The condition can be associated with a lack, reduced or abnormal function of the thymus in the subject. For example, the condition can be DiGeorge syndrome, thymoma (such as thymoma type A or thymoma type B), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymic tumor, thymic atrophy (such as age-related thymic atrophy), thymic cyst, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, thymic radiation, myasthenia gravis, thymic carcinoma, or thymic hyperplasia, thymic radiation, age- or infection-related thymic hypofunction.

[0115] In certain embodiments, the cells described herein can be used to treat subjects who can undergo thymectomy. In certain embodiments, the subject can have congenital heart disease, can undergo open-chest surgery, or has undergone open-chest surgery. The subject can undergo thymectomy to treat one or more indications related to the thymus, such as myasthenia gravis or thymoma.

[0116] Various cancers can be treated with the pharmaceutical compositions of the present disclosure. The cancer can be a tumor or hematological malignancy, including, but not limited to, all types of lymphoma / leukemia, carcinoma and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid, uterus.

[0117] The cells, compositions and pharmaceutical compositions of the present disclosure can be used to treat infectious diseases, which may be caused by organisms such as, but not limited to, bacteria, viruses, protozoa, and / or fungi.

[0118] IV. Pharmaceutical Compositions A pharmaceutical composition of the present disclosure may include a composition having one or more cells described herein and one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, and the like; proteins, amino acids such as polypeptides or glycine, and the like; antioxidants; chelating agents such as EDTA or glutathione, and the like; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure are prepared in one embodiment for intravenous administration.

[0119] In some embodiments, the pharmaceutical formulation may include any isotonic carrier, such as, for example, saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In one embodiment, the pharma- ceutically acceptable carrier may be supplemented with human serum albumin.

[0120] In certain embodiments, the pharmaceutical composition may be substantially free, e.g., free of detectable levels of contaminants selected from, e.g., endotoxins, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria, and fungi.

[0121] buffer solution In some embodiments, a pharmaceutical composition of the present disclosure is prepared with one or more buffering agents.

[0122] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, hydroxide calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, dihydrogen potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, dihydrogen sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and / or combinations thereof.

[0123] Non-limiting examples include aqueous formulations such as a phosphate buffered formulation at pH 7.4, or a citrate buffered formulation at pH 6.2, formulations for lyophilization such as a citrate buffered formulation at pH 6.2 containing 3% mannitol, a citrate buffered formulation at pH 6.2 containing 4% mannitol / 1% sucrose, or the like, or formulations prepared by the process disclosed in U.S. Pat. No. 8,883,737 to Reddy et al., the entire contents of which are incorporated herein by reference.

[0124] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated in a parenteral dosage form. The parenteral formulation may be an aqueous solution containing carriers or excipients such as salts, carbohydrates, and buffers (e.g., pH 3-9), or a sterile non-aqueous solution, or a dry form that can be used in combination with a suitable vehicle such as sterile, pyrogen-free water. For example, an aqueous solution of a therapeutic agent of the present disclosure may include isotonic saline, 5% glucose, or other pharma- ceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters, and amides, as disclosed, for example, in U.S. Pat. No. 7,910,594 by Vlahov et al. (Endocyte), the entire contents of which are incorporated herein by reference. In another example, an aqueous solution of a therapeutic agent of the present disclosure may include a phosphate buffered formulation (pH 7.4) for intravenous administration, as disclosed, for example, in Example 23 of WO2011014821 by Leamon et al., the entire contents of which are incorporated herein by reference. The parenteral dosage form may be in the form of a reconstitutable lyophilizate containing a dose of a therapeutic agent of the present disclosure. Any extended release dosage form known in the art can be utilized, such as the biodegradable carbohydrate matrices described in U.S. Pat. Nos. 4,713,249, 5,266,333, and 5,417,982, the disclosures of which are incorporated herein by reference, or, alternatively, a slow speed pump (e.g., an osmotic pump) can be used.

[0125] Nutrients In some embodiments, a pharmaceutical composition of the present disclosure comprises one or more nutrients that improve the health, survival, and / or proliferation of a cell as described herein.

[0126] In some embodiments, the pharmaceutical formulation comprises a vitamin. In some embodiments, the pharmaceutical composition comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range derivable therein): biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the pharmaceutical composition comprises a combination thereof or a salt thereof. In some embodiments, the pharmaceutical composition comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof.

[0127] In some embodiments, the pharmaceutical composition further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the pharmaceutical composition comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof.

[0128] In some embodiments, the pharmaceutical composition comprises an amino acid, an inorganic ion, and / or a monosaccharide. In some embodiments, the amino acid comprises arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some embodiments, the inorganic ion comprises sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some embodiments, the pharmaceutical composition further comprises one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In some embodiments, the pharmaceutical composition further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0129] Preservatives Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®.

[0130] V, Usage and Administration The cells and pharmaceutical compositions of the present disclosure described above can be administered by any delivery route, systemic or local, that results in a therapeutically effective result.

[0131] In some embodiments, thymocytes and effector cells may be co-delivered to the same anatomical location of a subject. In some embodiments, thymocytes and effector cells may be delivered to different anatomical locations of a subject.

[0132] In some embodiments, the thymocytes and effector cells can be delivered to a subject simultaneously, via the same delivery route or different delivery routes.

[0133] In some embodiments, thymocytes may be administered to a subject prior to administration of the effector cells.

[0134] In some embodiments, thymocytes may be administered to the subject following administration of the effector cells.

[0135] Non-limiting examples of routes of administration include enteral (into the gut), gastrointestinal, epidural (into the dura), oral (through the mouth), transdermal, intracerebral (into the brain), intraventricular (into the ventricles of the brain), epidermal (application to the skin), endodermal (into the skin itself), subcutaneous (deep under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous continuous infusion, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous injection (into the bone marrow), intrathecal (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the abdominal cavity), intravesical infusion, intravitreal (through the eye), Intracavernous injection (into the affected cavity), intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), vaginal, insufflation (snorted), sublingual, sublabial, enema, eye drops (onto the conjunctiva) or ear drops, auricular (in or through the ear), buccal (towards the cheek), conjunctival, dermal, dental (to one or more teeth), electroosmotic, intracervical, endosinusial, intratracheal, extracorporeal, hemodialysis, infiltrating, interstitial, intraabdominal, intra-amniotic, intra-articular, intra-biliary, intra-bronchial, intra-synovial, intrachondral (intrachondral) ), intracaudal (in the cauda equina), intracisternal (in the posterior cerebellum-medullary cistern), intracorneal (in the cornea), dental, intracoronary (in the coronary arteries), intracavernosal (in the distensible space of the corpus cavernosum), intradiscal (in the intervertebral disc), intraductal (in a glandular duct), intraduodenal (in the duodenum), intradural (in the dura or subdural), intraepidermal (towards the epidermis), intraesophageal (towards the esophagus), intragastric (in the stomach), intragingival (in the gums), intraileal (in the distal part of the small intestine), intralesional (in a localized lesion or directly introduced into a lesion), intraluminal (in the lumen), intralymphatic (in the lymphatic vessels), intramedullary (in the medullary cavity of a bone), intrameningeal (in the meninges), cardiac intramuscular (inside the heart muscle), intraocular (inside the eye), intraovarian (inside the ovary), intrapericardial (inside the pericardium), intrapleural (inside the pleura), intraprostatic (inside the prostate), intrapulmonary (inside the lungs or their bronchi), intrasinus (in the nose or orbital sinuses), intraspinal (inside the spine), intracerebrospinal fluid (inside the cerebrospinal fluid cavities of a joint), intratendon (inside a tendon), intratesticular (inside the testicles), intrathecal (inside the cerebrospinal fluid at any level of the neuraxis), intrathoracic (inside the thorax), intratubular (inside the tubules of an organ), intratumoral (inside a tumor), intratympanic (inside the middle ear), intravascular (in one or more blood vessels), intraventricular (inside the ventricles of the heart), iontophoresis (using an electric current(to transfer soluble salt ions into body tissues), lavage (to wash or flush an open wound or body cavity), laryngeal (just above the larynx), nasogastric (through the nose and into the stomach), occlusive dressing (local administration using a bandage to occlude the affected area), ophthalmic (to the outside of the eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (through the mouth or nose for a local or systemic effect). including intratracheal, by inhalation), retrobulbar (behind the pons or behind the eye), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (through or across the tympanic cavity), ureteral (towards the ureter), urethral (towards the urethra), vaginal, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, cyclophototherapy, or spinal.

[0136] In some embodiments, pharmaceutical compositions containing the cells of the present disclosure may be delivered intrathymic (into the thymus).

[0137] In some embodiments, pharmaceutical compositions containing the cells of the present disclosure may be surgically placed into a subject. As non-limiting examples, the cells may be surgically placed into the kidney capsule or quadriceps muscle.

[0138] In some embodiments, the cells and pharmaceutical compositions may be administered via intrahepatic administration, intrasplenic injection, or intraportal injection.

[0139] The cells and pharmaceutical compositions described herein can be provided to a subject by direct injection into bone marrow (referred to herein as intraosseous injection). The bone can be a long bone, such as the tibia, fibula, femur, metatarsal, phalanges of the lower leg, humerus, radius, ulna, metacarpals, and / or phalanges of the upper leg.

[0140] Parenteral and parenteral administration In some embodiments, the cells and pharmaceutical compositions described herein can be administered parenterally.

[0141] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations can be, for example, sterile injectable solutions, suspensions, and / or emulsions in parenterally acceptable non-toxic diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0142] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0143] In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material that is poorly water soluble. The rate of absorption of the active ingredient depends on its rate of dissolution, which in turn may depend on the size of the crystals and the crystalline system. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0144] Lymph node injection In certain embodiments, the thymocytes and / or pharmaceutical compositions of the present disclosure may be delivered into a subject by engraftment into a lymph node. In some embodiments, the thymocytes and / or pharmaceutical compositions of the present disclosure may be delivered to a subject in an amount effective to form ectopic thymic tissue in a lymph node. In certain embodiments, the methods and compositions described herein are used to deliver the cells and / or pharmaceutical compositions of the present disclosure into a lymph node of a subject to allow the thymocytes to engraft in the lymph node and produce an ectopic thymus. In certain embodiments, the ectopic thymus can restore thymic function in a subject, for example, to supplement or enhance one or more functions that a normal healthy thymic organ can perform. For example, the ectopic thymus can participate in immune regulation of the body by promoting T cell growth, development, maturation, selection and / or function, but is not limited thereto.

[0145] Non-limiting examples of lymph nodes to which the cells and pharmaceutical compositions can be delivered include jejunal, popliteal, axillary, periportal, abdominal, celiac, para-aortic, splenic hilar, hepatic hilar, left gastric, right gastric, left gastroepiploic, right gastroepiploic, retroperitoneal, pyloric, pancreatic, and / or pyloric lymph nodes. lineal) lymph nodes), splenic lymph nodes, hepatic lymph nodes (e.g., cystic lymph nodes, foramen lymph nodes, Winslow's foramen), pancreaticoduodenal lymph nodes (e.g., superior pancreaticoduodenal lymph nodes, inferior pancreaticoduodenal lymph nodes), superior mesenteric lymph nodes, ileocolic lymph nodes, prececal lymph nodes, retrocecal lymph nodes, appendix lymph nodes, mesenteric lymph nodes (e.g., paracolic lymph nodes, left colonic lymph nodes, middle colonic lymph nodes, right colonic lymph nodes, inferior mesenteric lymph nodes, S colonic lymph nodes, superior rectal lymph nodes), common iliac lymph nodes (e.g., medial common iliac nodes, middle common iliac nodes, lateral common iliac nodes, subaortic lymph nodes, promontory common iliac nodes), and external iliac lymph nodes (e.g., medial external iliac nodes, middle external iliac nodes, lateral external iliac nodes, medial hiatal femoral nodes, middle hiatal femoral nodes, lateral hiatal femoral nodes, interiliac nodes, obturator-external iliac obturator lymph nodes).

[0146] As a non-limiting example, any of the methods for transplanting thymic tissue into lymph nodes described in International Patent Publication WO2021026195 (the entire contents of which are incorporated herein by reference) may be useful in the present disclosure.

[0147] Depot Administration As described herein, in some embodiments, cells and compositions, including pharmaceutical compositions of the present disclosure, are formulated as depots for sustained release. Generally, administration is targeted to a specific organ or tissue ("target tissue"). In some embodiments, localized release is affected by the use of a biocompatible device. For example, the biocompatible device can limit the diffusion of cells in the subject.

[0148] In some embodiments of the present disclosure, the cells, compositions, and pharmaceutical compositions are spatially retained in or proximal to a target tissue. A method is provided for providing a pharmaceutical composition to a target tissue of a mammalian subject by contacting the target tissue (including one or more target cells) with the pharmaceutical composition under conditions in which the target tissue is substantially retained within the target tissue, i.e., at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99.99% of the composition is retained within the target tissue. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or greater than 99.99% of the pharmaceutical composition administered to a subject is present for a period of time following administration.

[0149] Dosage and Regimen The present disclosure provides methods of administering the cells, compositions and pharmaceutical compositions of the present disclosure to a subject in need thereof. The pharmaceutical compositions comprising the described cells may be administered to a subject using any dosage and any route of administration effective for the prevention, treatment, management, or diagnosis of a disease, disorder, and / or condition. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, mammal, or animal. The specific therapeutically effective, prophylactically effective, or appropriate diagnostic dose level for any particular individual will depend on a variety of factors, including the disorder and severity of the disorder being treated, the activity of the specific payload used, the specific composition used, the age, weight, general health, sex, and diet of the patient, and the time and route of administration.

[0150] In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein is about 1x10 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , or 5x10 8 In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein is at least about 1x10 cells / kg. 6 , 2x10 6 , 3x10 6 , 5x10 6 , 1x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , or 5x10 8In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein can be up to about 1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , or 5x10 8 In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein can be about 1x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein can be about 1x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein can be about 1x10 7、 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein can be about 1x10 7 , 1.5x10 7 , 2x10 7 , 2.5x10 7 , 3x10 7 , 3.5x10 7 , 4x10 7 , 5x10 7 , 1x10 8 , 1.5x10 8 , 2x10 8 , 2.5x10 8 , 3x10 8 , 3.5x10 8 , 4x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 In some embodiments, the dose of the cells, compositions and / or pharmaceutical compositions described herein may be about 1-3x10 cells / kg. 7 From 1 to 3 x 10 8 It may be cells / kg.

[0151] In certain embodiments, the cells described herein or the pharmaceutical compositions disclosed herein may be administered at about 10 to 600 μl / site, 50 to about 500 μl / site, 100 to about 400 μl / site, 120 to about 300 μl / site, 140 to about 200 μl / site, or about 160 μl / site.

[0152] The desired dose may be delivered at least once, three times a day, twice a day, once a day, every other day, every third day, every week, every other week, every three weeks, or every four weeks. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

[0153] The desired dose of cells of the present disclosure can be administered once or multiple times. The cells, compositions and pharmaceutical formulations can be administered periodically at a set frequency over a period of time, or can be administered continuously as a "continuous flow". The total daily dose, i.e., the amount administered or prescribed within a 24-hour period, can be administered by any of these methods, or a combination of these methods.

[0154] In some embodiments, delivery of cells to a subject results in a therapeutic effect of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years.

[0155] The cells of the present disclosure can be used in combination with one or more other therapeutic, preventive, research or diagnostic agents or medical procedures, either sequentially or simultaneously. Generally, each agent is administered at a dose and / or time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, preventive, research or diagnostic compositions in combination with agents that can improve bioavailability, reduce and / or modify metabolism, inhibit excretion and / or modify distribution in the body.

[0156] For example, the cells of the present disclosure are administered as a biocompatible device that limits diffusion within the subject's body to improve bioavailability in the therapeutic target area. The cells of the present disclosure may be administered by localized delivery.

[0157] The term "conditioning regimen" refers to the course of therapy that a patient undergoes prior to stem cell transplantation. For example, prior to hematopoietic stem cell transplantation, a patient may undergo myeloablative therapy, non-myeloablative therapy, or reduced intensity conditioning to prevent rejection of the stem cell transplant, even if the stem cells originate from the patient. The conditioning regimen may involve the administration of a cytotoxic agent. The conditioning regimen may also include immunosuppression, antibodies, and irradiation. Other possible conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318 (5854) Science 1296-9 (2007); Palchaudari et al., 34(7) Nature Biotechnology 738-745 (2016); Chhabra et al., 10:8(351) Science Translational Medicine 351ra105 (2016)) and CAR-T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2018), each of which is incorporated herein by reference in its entirety). The conditioning regimen is also designed to create a niche "space" that allows the transplanted cells to have a place in the body to engraft and grow. In hematopoietic stem cell transplantation, for example, the conditioning regimen creates a niche space in the bone marrow for the transplanted hematopoietic stem cells to engraft. Without a conditioning regimen, the transplanted hematopoietic stem cells are unable to engraft. In some embodiments, a subject may be administered the cells, compositions and / or pharmaceutical preparations of the present disclosure following a conditioning regimen.

[0158] VI, definition Expression: As used herein, "expression" and its grammatical equivalents, in the context of a marker, refers to the production of a marker, and the level or amount of the marker. For example, the expression of a marker in a cell or the presence of a marker or a cell being positive for a marker refers to the expression of the marker at a level similar to a positive control level. The positive control level may be determined by the level of the marker expressed by a cell known to have a cell fate associated with the marker. Similarly, the absence of expression of a marker or a cell being negative for a marker refers to the expression of the marker at a level similar to a negative control level. The negative control level may be determined by the level of the marker expressed by a cell known not to have a cell fate associated with the marker. Thus, the absence of a marker does not simply mean that the expression of the marker is at an undetectable level, but in certain cases, a cell may express the marker, but the expression may be low compared to the positive control or at a level similar to that of the negative control.

[0159] Effector cell: As used herein, "effector cell" refers to any cell or cell type that acquires the ability to execute, initiate or propagate a signal or cell death trigger upon contact or proximity to a thymocyte. "Contact or proximity" refers to sufficient spatiotemporal proximity to allow cell-intrinsic or cell-extrinsic (e.g., cell-cell) signaling or other communication or interaction.

[0160] Lymphocyte: As used herein, "lymphocyte" encompasses the meaning and use as one of ordinary skill in the art would understand the term, and further refers to a type of immune cell derived from bone marrow that is found in lymphatic tissue or blood. In some embodiments, lymphocytes mature in the thymus gland.

[0161] Negative: As used herein, the term "negative" (sometimes abbreviated as "-"), when used herein in reference to expression of an indicated cell marker, means that the cells do not express the indicated cell marker at detectable levels.

[0162] Positive: As used herein, the term "positive" (sometimes abbreviated as "+"), when used herein in reference to expression of an indicated cell marker, means that a cell expresses the indicated cell marker at a detectable level, and can include, for example, expression at a low (but detectable) level as well as expression at a high (high) level.

[0163] Pre-T cell: As used herein, "pre-T cell" refers to a lymphocyte that can mature or differentiate into a T cell.

[0164] Soluble Factor: As used herein, "soluble factor" refers to any protein or peptide that can bind to a cell surface molecule or be taken up by a cell. Uptake by a cell can be by passive diffusion, transporters, and / or endocytosis.

[0165] Thymocyte or origin or lineage: As used herein, "thymocyte or thymic origin or thymic lineage" refers to a cell that has one or more phenotypic or genotypic markers associated with a cell derived from the thymus or a cell that is destined to become a cell of the thymus. As used herein, the thymus can be an embryonic, fetal / fetal, or adult thymus.

[0166] Variant: The term "variant" as used in reference to a biomolecule (e.g., a training factor or an end factor) refers to a biomolecule that is related to or derived from a parent molecule. A variant may be, for example, a modified, truncated, mutated, homologous, or otherwise altered version of the parent molecule. The term variant can be used to describe either a polynucleotide or a polypeptide.

[0167] The details of one or more embodiments of the present disclosure are described in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the present description. In this description, the singular form includes the plural form unless the context indicates otherwise. Unless otherwise defined, all technical and scientific terms used in this description have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present description controls.

[0168] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0169] Example 1 Preparation of pluripotent stem cells On day 1, 2.5 million single iPSCs were placed in suspension at day 1 and the ROCK inhibitor Y27632 (10 μM) was added. The cells were placed on a shaker at 70 RPM. Differentiation continued on days 2-3, with half of the medium replaced with fresh medium every day on days 2 and 3. At this point, the spheroids were approximately 250 μm in diameter. The cells were placed on a shaker at 70 RPM. On day 4, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 g for 5 min at room temperature and the supernatant was aspirated.

[0170] The spheroids were washed with PBS, centrifuged at 250g for 5 min, and the PBS was aspirated (into the hood). 3-4 ml of pre-warmed Accutase was added to the 15 ml conical tube. The tube was tapped to agitate the aggregates and the tube was placed in the incubator for 5-7 min. The spheroids were agitated every 2-3 min in the 15 ml tube. After 5 min, the aggregates were gently agitated using a 1000 μl pipette to convert the spheroids to single cells. 7-8 ml of DMEM-F12 medium was added to each 15 ml tube, the spheroids were centrifuged at 250g for 5 min at room temperature, and the supernatant was aspirated.

[0171] Example 2 Generation of Definitive Endoderm from Pluripotent Stem Cells On day 2, the iPSCs are split into four different groups to test different conditions. Tables 1 and 2 show the culture media utilized and Table 3 shows the various culture conditions tested.

[0172] (Table 1) Medium A TIFF2024536496000002.tif31128

[0173] (Table 2) Medium B TIFF2024536496000003.tif37128

[0174] (Table 3) Protocol optimization TIFF2024536496000004.tif45142

[0175] Example 3 Generation of Thymocytes from Pluripotent Stem Cells Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the right time. Differentiation was performed as described in Example 2 or as follows. On day 1, PSCs in spheroids were cultured in medium A / A 50% / 50% (Stem Scale and DMEM-F12) containing Activin A (100ng / mL), 2μM CHIR99021, KSR (0.05%), Pen strep (100X), PI-103 (25nM). On days 2 and 3, cells were cultured in DMEM-F12 containing Activin A (100ng / mL), 2μM CHIR99021, ITS (1:1000), KSR (0.05%), Pen strep (100X), PI-103 (25nM), non-essential amino acids (NEAA) (100X). On days 4–5, cells were cultured in DMEM-F12 containing activin A (100 ng / mL), LDN193189 (100 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100x).

[0176] Differentiation of definitive endoderm (DE) to anterior foregut endoderm (AFE) was performed as follows: On days 6–7, cells were cultured in AFE medium prepared by adding SB431542 (10 μM), LDN193189 (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), penicillin-streptomycin (100X), and NEAA (100X) to DMEM-F12.

[0177] Differentiation of anterior foregut endoderm (AFE) to ventral pharyngeal endoderm (VPE) was performed as follows: From days 8 to 13, cells were cultured in VPE medium prepared by adding retinoic acid (0.1 μM), FGF8b (50 ng / ml), SHH(SAG) (100 ng / ml), EGF (50 ng / ml), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), penicillin-streptomycin (100X), and NEAA (100X) to DMEM-F12.

[0178] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells was performed using one of two protocols. In protocol 1, from days 14-16, cells were cultured in DMEM-F12 containing BMP4 (50 ng / ml), FGF8b (50 ng / ml), EGF (50 ng / ml), SANT-1 (0.25 μM), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100X). In the second protocol, on days 14–16, cells were cultured in DMEM F12 containing Noggin (50 ng / ml), FGF8b (50 ng / ml), EGF (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100X). On days 17–21, cells were cultured in DMEM-F12 containing BMP4 (50 ng / ml), FGF8b (50 ng / ml), EGF (50 ng / ml), SANT-1 (0.25 μM), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100X).

[0179] The medium was changed completely every day. The cells were collected and placed in a laminar flow hood. The supernatant was aspirated and freshly prepared medium was added. In addition, between the differentiation steps, the cells were washed with DMEM-F12 and placed for 5 minutes.

[0180] Example 4 3D Suspension Culture of Cell Lines Three pluripotent cell lines were established to be used to optimize the differentiation protocol: a human ES FOXN1-GFP reporter line, a human iPSC line (A15849 GIBCO), and a research-grade iPSC line from a GMP-grade therapeutic iPSC cell line. All three cell lines were adapted to in vitro 3D culture using Matrigel. The pluripotency of these cell lines was analyzed by flow cytometry analysis for the expression of stem cell markers TRA-1-60 and SSEA-4. More than 95% of pluripotent stem cells were found to express TRA-1 and SSEA-4.

[0181] Example 5. Characterization of DE cells Definitive endoderm (DE) cells obtained as G1-G4 from the four different culture conditions described in Example 2 were analyzed for markers of DE cells, namely SOX17 and FOXA2. Culture groups 1 (G1)-4 (G4) showed a relative increase in the expression levels of key biomarkers of DE, SOX17 and FoxA2 (see Figure 1).

[0182] The cells were also positive for the expression of markers associated with DE cells, namely EPCAM and SOX17. The numbers of double positive cells in G1, G2, G3 and G4 were 18.2%, 16.2%, 54.3% and 69.6%, respectively. In this study, G4 produced the highest number of positive cells.

[0183] Manipulation of the starting size of spheroids increased the frequency of DE cells to >95%.

[0184] Example 6 Effects of various growth factors Differentiation of DE cells into thymocytes can be promoted by adding factors known to be important for thymic differentiation from DE. One example is BMP, which is known to be important for the development and maturation of thymic progenitors from the third pharyngeal pouch. The effect of the timing of BMP addition was investigated (Figure 2). Spheroids were induced to differentiate from an iPSC state to TEP and the timing of BMP addition was varied. Spheroids were collected at different stages (DE, AFE, VPE, early and late TEP) and analyzed for biomarkers associated with the different stages. Conditions with and without BMP were compared at early and late TEP stages. BMP suppressed PAX1 and PAX9, but upregulated TBX1 and HOXA3.

[0185] Example 7 Equivalence of in vitro to in vivo transfer We expect that thymocytes generated by the methods described herein will differentiate into mTECs and cTECs upon in vivo transplantation. To test whether 3D spheroids spread into epithelial sheets in vivo, spheroids were attached on Matrigel and further differentiated in 2-D. After 4-5 days of culture on Matrigel, the expression of genes associated with TECs was investigated. As shown in Figure 3, DLL4 and CK8 expression was downregulated, whereas FOXN1 expression was upregulated. FOXN1 is a key regulator of thymic differentiation.

[0186] Example 8 Effect of Freezing and Thawing on TEP Phenotype TEPs may need to be frozen, thawed, and recultured for various clinical applications. The expression of thymocyte-associated markers was examined after freezing and thawing.

[0187] TEPs frozen, thawed, and resuspended in 3D culture were able to re-form spheroids in approximately 3-4 days. Expression of genes associated with the late TEP stage was compared before freezing and after thawed spheroid formation. Expression levels of genes, namely HOXA3, PAX9, EPCAM, and CXCL12, were similar before freezing and after thawing, indicating that TEPs can be frozen, thawed, and recultured for transplantation (see Figure 4).

[0188] Example 9 Thymocyte Biomarkers Several putative markers have been associated with TEP cells, including CD205 (Mohtashami M et al 2013 Int Immunol 25:601 and Campinoti S et al 2020 Nat Comm 11:1), EPCAM (Parent et al 2013 Cell Stem Cell 13:219), and claudins 3 and 4 (Hamazaki Y et al 2016 Immunol Reviews 271:38).

[0189] Double positive CD205+ / EPCAM+ cells have been used to quantify progenitor cells in the thymus (Campinoti S et al 2020 Nat Comm 11:1). This combination of markers was used to characterize phenotypic TEPs.

[0190] When we analyzed TEPs at early and late stages of differentiation using CD205 / EPCAM double staining, we found that the frequency of CD205+ / EPCAM+ cells increased from <1% to >25%. These data indicate that CD205+ / EPCAM+ can be used as a marker for TEP cells.

[0191] Example 10 In vitro assay of T cell differentiation To compare and correlate the in vitro function of iPSC-TEPs with the functional output observed after in vivo transplantation, an in vitro assay was developed to differentiate T cells from CD34+ HSCs. As a positive control, double positive (DN) pro-T cells were generated from CD34+ HSCs using the StemSpan T-cell kit (StemCell Technology), which were then matured into double positive CD4 and CD8 T cells. On day 21, the percentage of CD4+ / CD8+ cells was 5.3%, increasing to 49.4% of cells on day 28, 53.2% on day 35, and 86.8% on day 42.

[0192] Example 11 Preparation of thymocytes from pluripotent stem cells A protocol for preparing thymocytes from pluripotent stem cells was optimized.

[0193] Experiments 16A and 16B In this experiment, Experiment 16 Medium A / A was added 2 days prior to the addition of Experiment 16 Medium A to demonstrate the utility of adding Experiment 16 Medium A. In Experiment 16A, spheroids were seeded on Geltrex in 2D culture medium at the DE induction stage, whereas in Experiment 16B, a single cell suspension was prepared prior to seeding on Geltrex.

[0194] On day 1, 2.5 million single iPSCs were placed in day 1 suspension and ROCK inhibitor Y27632 (10 μM) was added. The shaker was set at 70 RPM. On day 2, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 g for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS and centrifuged again. The supernatant was removed and 1 ml of medium was added to each well of a 6-well low attachment plate. 6 ml of Experiment 16 Medium A was added to the 1-day old iPSC aggregates and mixed gently before adding 1 ml to each well of the 6-well plate.

[0195] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the right time. On day 1, cells were treated with Experiment 16 Medium A / A containing 50% / 50% (Stem Scale and DMEM-F12) and the factors / small molecules Activin A (100ng / mL), CHIR99021 (2μM), KSR (0.05%), Penicillin-Streptomycin (100X), PI-103 (25nM). On days 2-3, Experiment 16 Medium A was added containing DMEM-F12, Activin A (100ng / mL), 2μM CHIR99021, ITS (1:1000), KSR (0.05%), Penicillin-Streptomycin (100X), PI-103 (25nM), and NEAA (100X). On days 4–5, add Experiment 16 Medium B containing DMEM-F12, activin A (100 ng / mL), LDN193189 (100 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), penicillin-streptomycin (100X) and NEAA (100x).

[0196] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On days 6-7, Experiment 16 AFE medium was added containing DMEM-F12 and the factors / small molecules SB431542 (10 μM), LDN193189 (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (100X), vitamin A free B27 (0.5X), and NEAA (100X). On day 7, only half of the medium was changed.

[0197] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On days 8-9, Experiment 16 VPE1 medium was added containing DMEM-F12 and the factors / small molecules SB431542 (10 uM / ml), retinoic acid (0.1 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (100X), NEAA (100X) and vit A-free B27 (0.5X). On day 9, only half of the medium was changed. On days 10-11, VPE2 medium containing DMEM-F12, retinoic acid (0.1 μM), FGF8b (50 ng / ml), Noggin (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (100X), NEAA (100X), and VIT A-free B27 (0.5X) was added. On day 11, only half of the medium was replaced.

[0198] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On days 12-14, Experiment 16 TEP medium was added containing DMEM-F12 and the factors / small molecules FGF8b (50ng / ml), BMP4 (50ng / ml), CHIRR99 (2μM), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (100X), NEAA (100X). On days 13 and 14, only half of the medium was changed. On days 14-15, TEC1 medium containing DMEM-F12, BMP4 (50 ng / ml), FGF8b (50 ng / ml), ascorbic acid (30 μM), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), and NEAA (100X) was added. On day 16, cells were seeded onto Geltrex-coated 24-well plates as aggregates (experiment 16A) or single cells (experiment 16B). From days 16 to 27, TEC2 medium containing DMEM-F12, BMP4 (50 ng / ml), FGF8b (50 ng / ml), ascorbic acid (30 μM), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), vitamin A-free B27 (0.5X), N2 (100X), Glutamax (100X), and BME (100X) was added. The medium was changed daily.

[0199] No improvement in DE differentiation was observed. Seeding the aggregates in 2D allowed the cells to grow better in 2D and induced differentiation more strongly.

[0200] Test 18 In this experiment, we tested the transfer of cells into 2D culture at the VPE stage.

[0201] Expansion of iPSC lines in suspension culture On day 1, 2.5 million single iPSCs were placed in day 1 suspension and ROCK inhibitor Y27632 (10 μM) was added. The shaker was set at 70 RPM. On day 2, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 g for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS and centrifuged again. The supernatant was removed and 1 ml of medium was added to each well of a 6-well low attachment plate. 6 ml of Experiment 18 Medium A was added to the 1-day old iPSC aggregates and mixed gently before adding 1 ml to each well of the 6-well plate.

[0202] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the right time. On day 1, Experiment 18 Medium A / A containing 50% / 50% (Stem Scale and DMEM-F12) was added. On days 2-3, Experiment 18 Medium A containing DMEM-F12 and with factors / small molecules Activin A (100ng / mL), 2μM CHIR99021, ITS (1:1000), KSR (0.05%), Pen strep (100X), PI-103 (25nM), NEAA (100X) was added. On days 4-5, Experiment 18 Medium B was added containing DMEM-F12 and the factors / small molecules Activin A (100 ng / mL), LDN193189 (100 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100x).

[0203] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On days 6-7, Experiment 18 AFE medium was added containing DMEM-F12 and the factors / small molecules SB431542 (10 μM), LDN193189 (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen Strep (100X), Vitamin A-free B27 (0.5X) and NEAA (100X). On day 7, only half of the medium was changed.

[0204] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 8, aggregates were seeded on Geltrex-coated 24-well plates. On days 8-9, cells were transferred to Experiment 18 VPE1 medium containing DMEM-F12 and the factors / small molecules SB431542 (10 uM / ml), retinoic acid (0.1 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), vit A-free B27 (0.5X). On day 9, only half of the medium was changed.

[0205] On days 10-11, Experiment 18 VPE2 medium was added containing DMEM-F12 and the factors / small molecules retinoic acid (0.1 μM), FGF8b (50 ng / ml), Noggin (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), and Vitamin A-free B27 (0.5X). On day 11, only half of the medium was replaced.

[0206] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On days 12-14, Experiment 18 TEP medium was added containing DMEM-F12 and the factors / small molecules FGF8b (50ng / ml), BMP4 (50ng / ml), CHIRR99 (2μM), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), and NEAA (100X). On days 13 and 14, only half of the medium was changed. On days 15-20, Experiment 18 TEC1 medium was added containing DMEM-F12 and the factors / small molecules BMP4 (50 ng / ml), FGF8b (50 ng / ml), ascorbic acid (30 μM), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), and NEAA (100X).

[0207] On days 21-22, Experiment 18 TEC2 medium was added containing DMEM-F12 and BMP4 (50ng / ml), CHIRR9901 (2μM), FGF8b (50ng / ml), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A-free B27 (0.5X), N2 (100X), Glutamax (100X) and BME (100X). The medium was changed completely every day.

[0208] The TEPs generated in this experiment showed good FOXN1 expression.

[0209] Experiments 21 and 22 In this experiment, the effect of adding PI-103 to experiment 21 / 22 medium B was examined. The effect of transitioning to 2D culture at the AFE stage was also examined.

[0210] Expansion of iPSC lines in suspension culture On day 1, 2.5 million single iPSCs were placed in day 1 suspension and ROCK inhibitor Y27632 (10 μM) was added. The shaker was set at 70 RPM. On day 2, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 g for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS and centrifuged again. The supernatant was removed and 1 ml of medium was added to each well of a 6-well low attachment plate. 6 ml of Experimental 21 / 22 Medium A was added to the 1-day old iPSC aggregates and mixed gently before adding 1 ml to each well of the 6-well plate.

[0211] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the appropriate time. On days 1-2, experimental 21 / 22 medium A was added, containing DMEM-F12 and the factors / small molecules Activin A (100ng / mL), 2μM CHIR99021, ITS (1:1000), KSR (0.05%), Pen strep (100X), PI-103 (25nM) and NEAA (100X). On days 3-5, experiment 21 / 22 medium B was added containing DMEM-F12 and the factors / small molecules Activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X) and NEAA (100x). In experiment 22, PI-103 (25 nM) was omitted.

[0212] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, aggregates were seeded onto Geltrex-coated 24-well plates. On day 6, cells were cultured in Experiment 21 / 22 AFE medium* containing DMEM-F12 and supplemented with factors / small molecules SB431542 (10 μM), LDN193189 (200 nM), ascorbic acid (30 μM), Pen Strep (100X), N2 (100X), Glutamax (100X), BME (100X), and BSA (0.05%).

[0213] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On days 9-10, DMEM-F12 and Experiment 21 / 22 VPE1 medium containing the factors / small molecules SB431542 (10uM / ml), retinoic acid (0.1μM), FGF8b (50ng / ml), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A free B27 (0.5X), Glutamax (100X), BME (100X) and N2 (100X) were added. On day 10, only half of the medium was changed. On days 11-12, DMEM-F12 and VPE2 medium containing the factors / small molecules retinoic acid (0.1 μM), FGF8b (50 ng / ml), Noggin (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A-free B27 (0.5X), Glutamax (100X), BME (100X) and N2 (100X) were added. On day 12, only half of the medium was changed.

[0214] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On days 12-14, DMEM-F12 and Experiment 21 / 22 TEP medium containing the factors / small molecules FGF8b (50ng / ml), FGF10 (50ng / ml), BMP4 (50ng / ml), CHIRR99 (2μM), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A free B27 (0.5X), Glutamax (100X), BME (100X) and N2 (100X) were added. On days 14 and 15, only half of the medium was changed.

[0215] On days 16-18, Experiment 21 / 22 TEC medium was added containing DMEM-F12 and the factors / small molecules BMP4 (50ng / ml), FGF8b (50ng / ml), ascorbic acid (30μM), retinoic acid (0.1μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A free B27 (0.5X), N2 (100X), Glutamax (100X) and BME (100X). The medium was changed in its entirety every day.

[0216] Addition of PI-103 to experiment 21 / 22 medium B enhanced DE differentiation. Shift to 2D culture at the AFE stage resulted in less FOXN1 expression compared to experiment 18.

[0217] Test 23 The effect of transitioning from 3D to 2D culture at the VPE stage was examined. In experiment 23, the effect of adding CHIR99021 to medium B was examined.

[0218] Expansion of iPSC lines in suspension culture On day 1, 2.5 million single iPSCs were placed in day 1 suspension and ROCK inhibitor Y27632 (10 μM) was added. The shaker was set at 70 RPM. On day 2, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 g for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS and centrifuged again. The supernatant was removed and 1 ml of medium was added to each well of a 6-well low attachment plate. 6 ml of Experiment 23 Medium A was added to the 1-day old iPSC aggregates and mixed gently before adding 1 ml to each well of the 6-well plate.

[0219] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the appropriate time. On days 1-2, cells were cultured in Experimental 23 Medium A containing DMEM-F12 and the small molecules / factors Activin A (100ng / mL), 2μM CHIR99021, ITS (1:1000), KSR (0.05%), Pen strep (100X), PI-103 (25nM) and NEAA (100X). On days 3-4, add Experiment 23 Medium B containing DMEM-F12 and the small molecules / factors Activin A (100 ng / mL), CHIRR9901 (2 μM), LDN193189 (200 nM), PI-103 (25 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X) and NEAA (100x).

[0220] On day 5, Experiment 23 Medium B was added containing DMEM-F12 and the small molecules / factors Activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (100X), and NEAA (100x).

[0221] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) Aggregates were seeded onto Geltrex-coated 24-well plates on day 6. On days 6-8, experimental 23 AFE medium* containing DMEM-F12 and small molecules / factors SB431542 (10 μM), LDN193189 (200 nM), Pen Strep (100X), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), NEAA (100X), Vitamin A-free B27 (0.5X), and BME (100X) were added.

[0222] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 9, aggregates were seeded onto Geltrex-coated 24-well plates. On days 9-10, DMEM-F12 and Experiment 23 VPE1 medium containing the small molecules / factors SB431542 (10uM / ml), retinoic acid (0.1μM), FGF8b (50ng / ml), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A-free B27 (0.5X), and BME (100X) were added. On day 10, only half of the medium was added. On days 11-12, add experimental 23 VPE2 medium containing DMEM-F12 and the small molecules / factors retinoic acid (0.1 μM), FGF8b (50 ng / ml), Noggin (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (30 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A free B27 (0.5X), Glutamax (100X), BME (100X) and N2 (100X).

[0223] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On days 13-16, DMEM-F12 and Experiment 23 TEP medium containing the small molecules / factors FGF8b (50ng / ml), FGF10 (50ng / ml), BMP4 (50ng / ml), CHIRR99 (2μM), ascorbic acid (30μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), Vitamin A-free B27 (0.5X), Glutamax (100X), BME (100X) and N2 (100X) were added. On days 14, 15 and 16, only half of the medium was replaced. On days 17-20, TEC medium containing DMEM-F12 and the small molecules / factors BMP4 (50 ng / ml), FGF8b (50 ng / ml), ascorbic acid (30 μM), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), NEAA (100X), vitamin A-free B27 (0.5X), N2 (100X), Glutamax (100X), and BME (100X) was added. The medium was changed daily.

[0224] In experiment 23, the addition of CHIRR99 to medium B was found to enhance differentiation.

[0225] Example 12 Optimization of DE differentiation Experiments 21 and 23 described in Example 11 were designed to identify optimal conditions for 3D differentiation into DE. By including a Wnt activator in the differentiation medium in experiment 23, the differentiation efficiency into DE was increased compared to the absence of a Wnt activator in experiments 21 and 22. There were more Sox17 / FoxA2 double positive cells in experiment 23, while there were more Sox17 / FoxA2 negative cells in experiments 21 and 22.

[0226] The expression of higher EPCAM / FoxA2 double-positive cells was higher in experiment 23. Similarly, SOX2 / Tra1-60 expression was higher at the level of Tra1-60 / SOX2 double-negative cells, while a small population of Tra1-60 / Sox2 double-positive cells was persistently observed in experiments 21 / 22 (likely undifferentiated iPSC cells).

[0227] CXCR4 expression was lower in experiment 23 compared to experiments 21 and 22.

[0228] Taken together, differentiation of pluripotent state iPSCs proved to be more complete in experiment 23.

[0229] Example 13 2D culture of TEP Spheroids (also referred to herein as "3D cultures") were induced to differentiate iPSCs into DE and AFE in 3D conditions. A subsequent 2D culture step (i.e., culturing cells in Geltrex) was found to be important for the induction of final FoxN1-positive TEP cells. Expression of markers associated with iPSC-to-TEP differentiation was measured at advanced stages of differentiation for the various differentiation protocols described in Example 11. Using key developmental genes as markers, several conditions were found to be important. Comparison of the timing of the transition to 2D with AFE induction in 3D versus AFE induction in 2D showed that AFE induction in 3D outperformed AFE induction in 2D for further differentiation of iPSCs into TEPs, as marked by the upregulation of key VPE markers HOXA3 (Figure 5A), Pax1 (Figure 5B) and PSMB11 (Figure 5C) at the VPE1 stage. AFE in the 3D experiment 23 condition showed significantly higher expression of HOXA3, PAX1, and PSMB11 up to the VPE1 stage compared to AFE in the 2D culture in experiment 18.

[0230] Induction into FoxN1+ TEP was observed under 2D conditions, i.e., culture in Geltrex containing medium (Figure 5D). Transition of spheroids to 2D culture was performed by either adhering spheroids to Geltrex or dissociating spheroids into single cells and replating on Geltrex. This analysis showed that adhering spheroids to Geltrex resulted in stronger and more consistent differentiation into FOXN1+ TEP (Figure 5D). High expression of FOXN1+ TEP / TEC was observed only under 2D culture conditions. In experiment 16, 3D culture of TEP did not result in FOXN1 expression compared to 2D culture of TEP in experiment 18. Transition of TEP from 3D culture to 2D culture in experiment 16 showed induction of FOXN1 expression. In experiment 16, 3D spheroids were transferred to 2D culture conditions by either allowing the spheroids to adhere to Geltrex (experiment 16A) or by dissociating the spheroids into single cells and then replating them on Geltrex (experiment 16B). As shown in Figure 5D, the conditions in experiment 16A led to robust and consistent differentiation of FOXN1-positive TEPs.

[0231] Example 14 Ectopic Expression of FOXN1 The effect of ectopic expression of FOXN1 on TEP differentiation was tested. TEPs were transfected with FOXN1. Expression of FOXN1 was measured using primers targeting the coding region of FOXN1. As shown in Figure 6A, transfection of FOXN1 mRNA generated high levels of FOXN1 mRNA, including exogenous and endogenous mRNA molecules. When primers targeting only mRNA with the 3'UTR were used, strong expression of endogenous FOXN1 mRNA induced by exogenously expressed mRNA was observed. When primers targeting the HA tag added to the exogenous mRNA were used, expression of exogenous mRNA in TEPs was confirmed (Figure 6A).

[0232] High expression of exogenous and endogenous FOXN1 mRNA strongly induced direct targets of FoxN1, such as CCL25 and DLL4 ( Fig. 6B ).

[0233] Example 15 Differentiation of iPSCs into Thymocytes Test 27 Expansion of iPSC lines in suspension culture Before starting the differentiation protocol, iPSCs were cultured in suspension for 3-4 days until they reached a diameter of approximately 300-400 micrometers. The iPSCs were then passaged using Accutase to change the aggregates into single cells. 2.5 million single iPSC cells were placed in suspension in 6-well ultra-low attachment plates. The iPSCs were cultured at 37°C in stem scale medium containing 10 μM of the ROCK inhibitor Y27632.

[0234] 1 ml of the supernatant was removed from the plate and 1 ml of pre-warmed stem scale medium was added to the culture. On day 2, the supernatant was transferred together with the spheroids into a 15 ml conical tube and centrifuged at 250G for 5 minutes. The spheroids were washed with PBS and resuspended in 1 ml of medium A in each well of a 6-well low attachment plate.

[0235] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the right time. On days 1-2, iPSCs were cultured in medium A (basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), NEAA (1:400)) supplemented with PI-103 (25 nM). On day 2, 1 ml of medium was removed and 1 ml of freshly prepared medium A + PI-103 was added. On days 3–5, medium B (DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), NEAA (1:400)) and PI-103 (25 nM) were added. On day 3, CHIRR9901 (2 μM) was added to medium B. On days 3–5, 1 ml of medium was removed and 1 ml of freshly prepared medium was added.

[0236] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was removed. The spheroids were washed with PBS and plated into 6-well low attachment plates containing DMEM-F12 and AFE medium-2 containing FGF8b (50 ng / ml) plus factors / small molecules SB431542 (10 μM), LDN193189 (200 nM), ascorbic acid (10 μM), penicillin-streptomycin (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), NEAA (100X), B27 (no RA) (1:200).

[0237] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 8, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250 G for 5 minutes and the supernatant was aspirated. Spheroids were washed with PBS and resuspended in 50 μl of VPE1a medium containing DMEM-F12 and the factors / small molecules SB431542 (10 uM / ml), retinoic acid (0.1 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (1:200), NEAA (100X), and vitamin A-free B27 (0.5X), ascorbic acid (10 μM), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). Spheroids were then added to 24-well plates coated with Geltrex (1:100).

[0238] On day 9, only half of the medium was replaced. On days 10-11, VPE2b medium containing DMEM-F12, retinoic acid (0.1 μM), FGF8b (50 ng / ml), Noggin (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (100X), NEAA (100X), vit A-free B27 (0.5X), Glutamax (1:100), BME (1:100) and N2 (1:100) was added. On days 10 and 11, an equal volume of freshly made VPE2b medium (500 ul) was gently added without disturbing the aggregates.

[0239] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) and thymic epithelial cells (TEC) For this differentiation step, VPE cells were cultured in thymocyte medium, e.g., TEP medium or TEC medium. From day 12 to 19, cells were supplemented with Experiment 27 TEP medium containing DMEM-F12 and the factors / small molecules FGF10 (50 ng / ml), B27 (without VITA) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), plus FGF8b (50 ng / ml), BMP4 (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), penicillin-streptomycin (1:200), NEAA (100X).

[0240] On day 12, an equal volume of freshly made TEP medium was gently added without disturbing the aggregates. On days 13–19, 50% of the supernatant was removed and freshly prepared Experiment 27 TEP medium was added.

[0241] From days 20 to 23, cells were resuspended in DMEM-F12 and Experiment 27 TEC medium containing FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), CHIRR99 (2μM), plus BMP4 (50ng / ml), FGF8b (50ng / ml), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (100X), Vitamin A-free B27 (0.5X), N2 (100X), Glutamax (100X), BME (100X), and NEAA (100X).

[0242] Test 29 Expansion of iPSC lines in suspension culture Before starting the differentiation protocol, iPSCs were cultured in suspension for 3-4 days until they reached a diameter of approximately 300-400 micrometers. The iPSCs were then passaged using Accutase to change the aggregates into single cells. 2.5 million single iPSC cells were placed in suspension in 6-well ultra-low attachment plates. The iPSCs were cultured at 37°C in stem scale medium containing 10 μM of the ROCK inhibitor Y27632.

[0243] 1 ml of the supernatant was removed from the plate and 1 ml of pre-warmed stem scale medium was added to the culture. On day 2, the supernatant was transferred together with the spheroids into a 15 ml conical tube and centrifuged at 250G for 5 minutes. The spheroids were washed with PBS and resuspended in 1 ml of medium A in each well of a 6-well low attachment plate.

[0244] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at the right time. On days 1-2, iPSCs were cultured in medium A (basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), NEAA (1:400)). On day 2, 1 ml of medium was removed and 1 ml of freshly prepared medium was added. On days 3–5, cells were incubated with medium B (DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400). On days 4–5, 1 ml of medium B was removed and 1 ml of freshly prepared medium B was added.

[0245] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was removed. The spheroids were washed with PBS and plated into 6-well low attachment plates containing AFE medium-2 (Experiment 29A) or Geltrex-coated plates (Experiment 29B). The AFE medium used in the experiment contained the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100) and NEAA (1:400). On days 7 and 8, 1 ml of AFE medium was removed and 1 ml of freshly prepared medium was added.

[0246] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 9, for experiment 29A, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS, resuspended in VPE1 medium and plated onto 24-well plates coated with Geltrex (1:100). Cells in both experiments 29A and 29B were cultured in VPE1 medium containing the following basal medium: DMEM-F12, SB431542 (10 uM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400) from days 9-10. On day 9, an equal volume of freshly made VPE1 medium (500 ul) was gently added without disturbing the aggregates.

[0247] On day 10, 50% of the supernatant was removed and freshly prepared VPE1 medium (500ul) was added per well. On days 11-12, the medium was replaced with VPE2 medium containing the following components: basal medium: DMEM-F12, Noggin (50ng / ml), CHIR99021 (2μM), FGF8b (50ng / ml), retinoic acid (0.1μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100). On days 11 and 12, an equal volume of freshly made VPE2 medium (500 ul) was gently added without disturbing the aggregates.

[0248] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) For this differentiation step, VPE cells were cultured in thymocyte medium, e.g., TEP medium or TEC medium. On days 13-18, the medium was replaced with Experiment 29 TEP medium containing the following basal medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vit A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 13, an equal volume of freshly prepared TEP medium (500 ul) was gently added without disturbing the aggregates. On days 14–18, 50% of the supernatant was removed and freshly prepared TEP medium (500 ul) was added per well. On days 19-22, the medium was replaced with TEC medium containing basal medium: DMEM-F12, FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), and BME (100X). On days 20-22, 50% of the supernatant was removed and freshly prepared TEC medium (500ul) was added per well. On day 22, experimental 29B cells were re-seeded as single cells in suspension (3D) and on Geltrex-coated plates (2D). The remaining cells were frozen. Frozen experimental 29B cells were thawed in suspension and maintained in TEC medium.

[0249] Test 30 Expansion of iPSC lines in suspension culture Before starting the differentiation protocol, iPSCs were cultured in suspension for 3-4 days until they reached a diameter of approximately 300-400 micrometers. The iPSCs were then passaged using Accutase to change the aggregates into single cells. 2.5 million single iPSC cells were placed in suspension in 6-well ultra-low attachment plates. The iPSCs were cultured at 37°C in stem scale medium containing 10 μM of the ROCK inhibitor Y27632.

[0250] 1 ml of the supernatant was removed from the plate and 1 ml of pre-warmed stem scale medium was added to the culture. On day 2, the supernatant was transferred together with the spheroids into a 15 ml conical tube and centrifuged at 250G for 5 minutes. The spheroids were washed with PBS and resuspended in 1 ml of medium A in each well of a 6-well low attachment plate.

[0251] Differentiation of iPSCs into definitive endoderm (DE) Differentiation of iPSCs into DE requires the introduction of small molecules and growth factors at appropriate times. On days 1–2, iPSCs were cultured in medium A (basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400)). On days 3–5, cells were treated with medium B containing basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200) and NEAA (1:400).

[0252] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was removed. The spheroids were washed with PBS and plated into 6-well low attachment plates containing DMEM-F12 and AFE medium-2 containing FGF8b (50 ng / ml) plus factors / small molecules SB431542 (10 μM), LDN193189 (200 nM), ascorbic acid (10 μM), penicillin-streptomycin (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), NEAA (100X), B27 (no RA) (1:200).

[0253] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 9, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 min at room temperature and the supernatant was aspirated. The spheroids were washed with PBS, resuspended in VPE1 medium and plated onto 24-well plates coated with Geltrex (1:100). Cells were cultured in VPE1 medium containing the following components from days 9 to 11: basal medium: DMEM-F12, SB431542 (10 uM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 10, an equal amount of freshly made VPE1 medium (500 μl) was gently added without disturbing the aggregates. On day 11, 50% of the supernatant was removed and freshly prepared VPE1 medium (500 ul) was added per well. On days 12–13, the medium was removed and replaced with basal medium: DMEM-F12, Noggin (50 ng / ml), CHIR99021 (2 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), and VPE2 medium containing 2 (1:100).

[0254] On day 13, an equal volume of freshly made VPE2 medium (500 μl) was gently added without disturbing the aggregates.

[0255] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) For this differentiation step, VPE cells were cultured in thymocyte medium, e.g., TEP medium or TEC medium. On days 14-16, cells were treated with Experiment 30 TEP medium containing DMEM-F12, FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vit A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On days 15 and 16, fresh TEP medium was added to the cells. From days 17 to 22, cells were treated with TEC medium containing DMEM-F12, FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR, (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), and BME (100X).

[0256] On days 17–22, 50% of the supernatant was removed and freshly prepared TEC medium (500 μl) was added per well. On day 22, the cells were treated with Accutase to prepare single cells, which were then transplanted into mice.

[0257] Example 16 FOXN1 expression in iPSC-derived thymocytes Cultivating cells in 2D versus 3D aggregates in suspension is yet another variable that needs to be adjusted to obtain optimal thymocyte populations from iPSCs. Other variables include the growth factors and small molecules utilized for culture at different stages. A transition from 3D aggregates to culture in 2D at the ventral pharyngeal endoderm (VPE) stage can promote the expression of markers such as HOXA3, PAX9, and PAX1. iPSC-derived thymocytes were consistently found to express the thymocyte marker FOXN1 in different derivatives (Figure 7). TEPs expressing FOXN1 at levels between 0.000025 and 0.0001 normalized to GAPDH were observed in all experiments tested. Thus, a 16-18 day protocol allows for the generation of FOXN1 positive cells compared to other protocols that can be as long as 30 days.

[0258] Example 17 Optimization of growth factors and culture conditions In experiments 16-23, expression of FOXN1 and other markers up to day 5 of TEP differentiation. In these experiments, culturing TEPs for an additional week results in downregulation of FOXN1. Growth factors such as FGF7 (KGF), FGF10 and RANKL have been described as important for late maturation of TEPs to TECs. Addition of FGF7, FGF10, KGF and RANKL to TEP medium was tested to support TEP culture beyond 5 days. In experiments 29 and 30, supplementing TEP medium with FGF7, FGF10 and RANKL extended the culture of TEPs beyond 5 days to 2 weeks while maintaining FOXN1 expression. To facilitate scale-up manufacturing of iPSC-derived thymocyte products, it is important to be able to establish differentiation protocols entirely in 3D suspension. The feasibility of maintaining TEPs in 3D suspension cultures was also investigated in these experiments. At the TEP stage, single cells were harvested from the culture plate and resuspended in TEP medium as 3D aggregates. FOXN1 expression levels in TEPs from different experiments are shown in Figure 8. In Exp29-TEP, FOXN1 expression was detected at week 1 of the TEP stage. When TEPs were reseeded in 2D cultures, no FOXN1 expression was detected after one week, but by week 2 FOXN1 was detected in the cultures. In Exp29B-TEP2-3D, where single-cell TEPs were resuspended in 3D-aggregate cultures, FOXN1 expression was already detectable by week 1 and was even higher by week 2. In experiment 30, comparable FOXN1 expression was detected even before extended culture in 2D or 3D.

[0259] Example 18 Effect of Freezing and Thawing TEP The ability to freeze, thaw, and recover iPSC-TEP provides significant logistical value in supporting the use and delivery of iPSC-TEP to clinical sites. Feasibility of freezing and thawing iPSC-TEP. Single cell suspensions of experimental 29B TEP1 cells were frozen and then thawed as 3D suspensions in TEP medium. Aggregates were observed on day 1 and increased in size and compactness. By day 8, the aggregates reached 200-300 μm in diameter. FOXN1 expression of Exp29B TEP under different culture conditions is shown in Figure 9. Frozen-thawed "Exp29B-FT-TEP" cells in 3D aggregates expressed FOXN1 at levels comparable to human thymus on day 8 after thawing. Exp29B-FT-TEP was compared to 29B-TEP in 3D for 3 weeks.

[0260] Example 19 In vivo transplantation of thymocyte populations 1~4x10 6 iPSC-derived TEPs were suspended in 25 μL of Geltrex and injected under the kidney capsule of 6-week-old nude mice. Three animals were transplanted with iPSC-derived thymocytes from experiment 18, and eight animals were transplanted with cells from experiment 21 / 22. Blood samples were taken from transplanted mice at weeks 3, 11, and 13, and the hematopoietic cell numbers of mice after iPSC-TEP transplantation were compared with fetal thymus transplanted mice and control non-transplanted animals. As shown in Figure 10A, the percentage of CD8+% of CD45 cells in mice transplanted with TEPs increased from week 3 to weeks 13 and 16. Overall, the percentage of CD8+ cells was higher than the control at week 16 in experiment 18 and at week 14 in experiment 21 / 22. It should be noted that the subset of mice shown in Figure 10B showing CD8+ cells in experiment 21 / 22 included two mice that did not incorporate transplanted cells due to technical reasons. In contrast to the CD8+% of CD45+ cells, other hematopoietic cell populations including CD4+ cells, B220+ and NK1.1+ cells did not increase in percentage values ​​from week 3 to weeks 11-13 and 14-16 in experiments 18 and 21 / 22 compared to controls (Figures 10C, 10D and 10E). Together, these data indicate that the iPSC-derived thymocytes described herein can promote lymphopoiesis.

[0261] Example 20. Characterization of iPSC-derived TEPs expressing FOXN TEPs generated by experiment 31 were classified into four different fractions: EPCAM high, medium, low, and negative using an anti-EPCAM antibody. The classified fractions were analyzed by qPCR for various markers as indicated (Figure 11). FOXN1 was expressed almost exclusively in the EPCAM high population. Genes involved in TEP development and maturation, including PAX9, SIX1, and claudin 4, were also expressed in the EPCAM high population. HOXA3, TBX1, and DLL4 were preferentially expressed in the EPCAM low to negative fraction.

[0262] Example 21. Frequency of markers in thymic epithelial cells Single-cell RNA-seq was performed on iPSC-derived TEPs prepared using the differentiation protocol described herein. Data from experiments 7 (A-C), 21, 22, 23, 27, 29, and 30 were analyzed. For experiments 21 and 23, sample replicates were also tested to assess technical reproducibility. For experiments 29 and 30, in addition to 3D to 2D protocol variations, the freeze-thaw (FT) effect of TEPs and the comparison of the effect of replating into 2D (Exp30FT-2D) versus replating into 3D (Exp30FT-3D) were tested. In experiment 29, a comparison of Exp29A vs 29B was tested in a range of conditions, including a comparison of seeding into 2D (Exp29B-2D) versus replating after freeze-thaw in 3D (29BFT-3D). Of these experiments, transplanted samples showed no significant differences in FOXN1 高 (Exp29BFT-3D), FOXN1 低 (Exp30, Exp30FT-3D), and FOXN1 - (Exp30FT-2D).

[0263] Single-cell transcriptomes of 122,436 cells from 16 iPSC-derived TEC samples (14 different experiments) were integrated by Harmony and UMAP analysis was performed to ensure removal of batch effects. Gene expression distributions of selected genes for TEC (EPCAM, KRT8, FOXN1, IVL, etc.) and pluripotency (POU5F1, NANOG) were then plotted. The results are shown in Figure 12A. EPCAM and Krt-8 were widely expressed with a high percentage in most experiments, while the others are expressed at different frequencies throughout the experiments. From the violin plot, it was observed that FOXN1 was more highly expressed in TECs of Exp29BFT-3D, which is consistent with the highest qPCR expression data. Exp29BFT-3D cells also showed the highest expression of markers KRT5 and involucrin (IVL), genes specific to mTEC cells and keratinoid mTEC cells. Exp30FT-2D and Exp30FT-3D were the only experiments in which cells expressing CCL21, a marker specific for mTEC cells, were detected. Finally, it should be noted that another mTEC marker, FEZF2, is also highly expressed in a subset of iPSC-TEPs, including those with thymopoietic activity.

[0264] Figure 12B shows quantification of the percentage of FOXN1+, KRT8+ and EPCAM+ cells in iPSC-differentiated thymocytes and thymus tissue samples. Consistent with the violin plot analysis, sample Exp29BFT-3D had the highest frequency of FOXN1+ cells. It should be noted that in the human thymus, the level of FOXN1+ cells is highest during prenatal and neonatal life (10-15%), and the frequency is significantly reduced to 2-3% by the age of 25 years.

[0265] Example 22 Detection limit of undifferentiated pluripotent stem cells To unbiasedly assess the state of pluripotency gene regulatory networks in the Exp29B3D sample, single-cell transcriptomes of 7166 cells analyzed from this sample were classified using the CellNet training dataset, which contains high-quality bulk RNA-seq-derived pluripotency gene regulatory networks (called embryonic stem cells, esc) and high-quality bulk RNA-seq data of 13 other cell types shown in the panel. According to the analysis, besides cTEC and mTEC cells, the other main cell types in iPSC-TEP were neuroendocrine cells. These data indicate a very high degree of sensitivity of this approach to detect cells with a pluripotent stem cell signature. In addition, the activation state of embryonic stem cell networks appears to be very low in iPSC cells, suggesting an overall downregulation of the pluripotency program.

[0266] The percentage of OCT4 (POU5F1) and Nanog positive cells was quantified based on all cells with log-normalized expression levels above zero. Overall, expression of POU5F1 and NANOG was less than 2% of cells from iPSC-TEP samples.

[0267] Example 23 Correlation between in vivo thymocyte regeneration activity and FOXN1 levels of transplanted iPSC-derived TEPs Three groups of iPSC-derived TEP cells were transplanted into the renal subcapsular space with Geltrex: high FOXN1 (18–20 times higher than GAPDH, x10 4 ), low FOXN1 (5-8x relative to GAPDH, x10 4 ) and cells negative for FOXN1. Control animals were sham-transplanted with Geltrex alone. Evidence of restored thymopoiesis was shown by the expression of single positive CD4 + and CD8 + Figure 13 shows the expression of CD8+ cells in the peripheral blood of animals at different time points after transplantation. + or CD4 + 1 is a histogram showing the frequency of cells expressing FOXN1. 高 iPSC-TEP and to a lesser extent FOXN1 低In animals transplanted with iPSC-TEPs, CD4 and CD8 cells were observed at levels significantly higher than in control animals. FOXN1-negative iPSC-derived TEPs showed no evidence of thymopoiesis. Thus, the data show a positive correlation between high levels of FOXN1 expression in iPSC-derived TEPs and their ability to restore thymopoiesis.

[0268] Example 24. Identity and frequency of subpopulations of iPSC-derived TECs compared to primary human thymus Single-cell transcriptomes of 122,436 cells from 16 iPSC-derived TEC samples (14 different experiments) were integrated by Harmony and subjected to UMAP analysis to ensure removal of batch effects. iPSC single-cell transcriptomics from all iPSC-derived TEC samples were then reference-mapped to the reference single-cell atlas of human thymocytes generated by Bautista et al. (Bautista, JL, et al. Nat Commun 12, 1096 (2021)). The average classification score calculated by Symphony was calculated for each cell type. An overall conservation of Symphony-predicted cell types across samples from different experiments was observed. For example, Exp29BFT-3D contains cells with high classification scores as keratinocyte-like mTECs, along with iPSC-derived thymocytes from samples Experiment 30 and Exp30FT-3D.

[0269] We then calculated the percentage of Symphony-predicted cell types across samples. In general, the most frequent cells found across all samples were cTEC high, cTEC low, and neuroendocrine cells. The most frequent cells in the human thymus are also cTEC high and cTEC low cells.

[0270] Example 25 Lymphocyte neogenesis in mice transplanted with iPSC-derived TEPs Fetal thymus fragments or iPSC-derived TEPs were transplanted into the subcapsular space of 6-week-old athymic nude mice. 6iPSC-derived TEPs were suspended in 15 μl of Geltrex and introduced under the kidney capsule of 6-week-old nude mice. For fetal thymus, E13.5 fetal lobes were cultured in 2DG for 5 days to deplete T progenitor cells. In one group of animals, three fetal lobes were transplanted as fragments into each kidney capsule. In another group, single cell suspensions were prepared from 2DG-treated fetal thymus and transplanted as cell suspensions. At different time points after transplantation, animals were bled for flow cytometric analysis of hematopoietic cells, including T, B, and NK cells. Here, we focus only on the data for CD4+ and CD8+ T cells. To establish a benchmark for iPSC-TEPs capable of reconstituting thymic lymphopoiesis, iPSC-TEPs expressing different levels of FOXN1 (Figure 14) were tested. Transplanted fetal mouse thymocyte suspensions restored detectable T lymphopoiesis at 6 weeks, but with slightly lower levels of CD8+ and CD4+ cells compared to fetal thymus fragments. FOXN1-high iPSC-TEPs showed CD8 and CD4 cells above 1% levels in control animals starting around week 6. CD8 levels ranged (2-4%) higher than CD4 (1-2%). FOXN1-low animals appeared to have consistent levels of CD4 and CD8 cells above background. De novo thymopoiesis by FOXN1-low iPSC-TEPs could not be determined due to outliers in Geltrex control animals. Frequencies of other hematopoietic cells, such as B cells (B220) and NK cells (NK1.1), were similar between nude controls and iPSC-TEP-transplanted animals.

[0271] To evaluate the functional response of T cells generated from thymopoiesis of transplanted iPSC-TEPs, splenocytes were harvested from sacrificed animals and prepared for cytokine release studies. Cells were stimulated with PMA / ionomycin, and cytokines produced in response to T cell activation signals were analyzed by flow staining. Under non-stimulated conditions, background staining levels were observed in splenocytes from transplanted mice. Activated splenocytes from Geltrex transplants showed low levels of IFNγ+TNFα+ producing cells at 3.63%. In contrast, CD4+ cells from iPSC-derived TECs and fetal thymus transplants showed 12.3% and 5.8% IFNγ+TNFα+ producing cells, respectively. IFNγ or TNF expressing cells in the CD8 population were also higher in both iPSC-TEC and fetal thymus transplants compared to Geltrex transplants. In conclusion, PMA / ionomycin stimulation resulted in a higher release of the cytokines IFNγ and TNFα compared to control splenocytes.

[0272] Example 26 Differentiation of iPSC cells into thymocytes (Experiment 31) Expansion of iPSC lines in suspension culture On day 0, cultured mature iPSCs (3-4 days in suspension (3D), 300-400 micrometers in diameter) were treated with pre-warmed Accutase to convert aggregates into single cells. Three million single iPSCs were plated in suspension in 6-well ultra-low attachment plates in Stem scale medium with the ROCK inhibitor Y27632 (10 μM). On day 1, pre-warmed stem scale medium was added to the cultures. On day 2, the supernatant and spheroids were transferred into a 15 ml conical tube. The spheroids were centrifuged at 250G for 5 min at room temperature, and the supernatant was aspirated. 1 ml of medium A was added to each well of the 6-well low attachment plate, and the plate was incubated in a 37 °C incubator. Medium A was prepared containing basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), penicillin-streptomycin (1:100), PI-103 (25 nM), and NEAA (1:400).

[0273] Differentiation of iPSCs into definitive endoderm (DE) Cells were cultured in medium A on days 1–2 and in medium B on days 3–5. Medium A was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), penicillin-streptomycin (1:100), PI-103 (25 nM), and NEAA (1:400). Medium B was prepared containing the following: basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400).

[0274] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, the supernatant and spheroids were transferred into a 15 ml conical tube. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS, resuspended in 1 ml of AFE medium and plated onto a 6-well low attachment plate. In this experiment, the cells were kept in suspension as aggregates. AFE medium was prepared to contain the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), NEAA (1:400), ITS36 (1:1000), and KSR (0.05%).

[0275] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) 24-well plates were coated with Geltrex (1:100) which was left at room temperature for 1 hour. On day 9, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed in PBS. 250 μl of VPE1 medium was added to each Geltrex-coated well. VPE1 medium was prepared to contain the following: basal medium: DMEM-F12, SB431542 (10 uM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 10, an equal amount of freshly made VPE1 medium (500 ul) was gently added without disturbing the aggregates. On day 11, 50% of the supernatant was removed and freshly prepared VPE1 medium (500 ul) was added per well.

[0276] On days 12-13, VPE2 medium was added. VPE2 medium was prepared to contain the following: basal medium: DMEM-F12, Noggin (50ng / ml), CHIR99021 (2μM), FGF8b (50ng / ml), retinoic acid (0.1μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100). On day 13, an equal volume of freshly made VPE2 medium (500ul) was gently added without disturbing the aggregates.

[0277] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On days 14 to 17, cells were cultured in TEP medium. TEP medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0278] On day 15, an equal volume of freshly made TEP medium (500 μl) was gently added without disturbing the aggregates. On days 16 and 17, 50% of the supernatant was removed and freshly prepared TEP medium (500 μl) was added per well.

[0279] To further differentiate the cells into thymic epithelial cells (TEC), the supernatant was removed and the cells were cultured in basal medium: TEC1 medium containing DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), and BME (100X). On days 18 and 19, 50% of the supernatant was removed and freshly prepared TEC1 medium (500 ul) was added per well. On days 20-22, TEC2 medium containing RANKL was added. TEC2 medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50 ng / ml), FGF7 / KGF (50 ng / ml), RANKL / TRANCE (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), and BME (100X).

[0280] On day 23, cells were treated with Accutase and frozen using Hypothermosol-FRS medium.

[0281] Example 27 Differentiation of iPSC cells into thymocytes (Experiment 33) Expansion of iPSC lines in suspension culture On day 0, mature Fuji iPS-106 cells (3-4 days in suspension, 300-400 micrometers in diameter) were treated with Accutase to convert aggregates into single cells. 2.5 million single iPSCs were placed in suspension in 6-well ultra-low attachment plates and cultured using Stem scale medium with the rock inhibitor Y27632 (10 μM). On day 1, 1 ml of supernatant was removed from the plate and 1 ml of pre-warmed stem scale medium was added. On day 2, the supernatant was transferred together with the spheroids into a 15 ml conical tube. The spheroids were centrifuged at 250G for 5 min at room temperature and the supernatant was aspirated. Spheroids were washed with PBS and resuspended in medium A, which was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400).

[0282] Differentiation of iPSCs into definitive endoderm (DE) On days 1-2, cells were cultured in medium A containing basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400). On day 2, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium A.

[0283] On day 3, cells were washed and resuspended in medium B' prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), CHIRR9901 (2 μM), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400). On days 4–5, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium B'. Medium B' was prepared containing basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200) and NEAA (1:400).

[0284] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, 24-well plates were coated with Geltrex (1:100) that had been left at room temperature for 1 hour. The supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature, and the supernatant was aspirated. The spheroids were washed with PBS, resuspended in AFE medium, and transferred to the Geltrex-coated plate. AFE medium was prepared to contain the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), and NEAA (1:400).

[0285] On day 7, an equal volume of freshly prepared AFE medium (500 ul) was added per well. On day 8, 50% of the supernatant was removed and freshly prepared AFE medium (500 ul) was added per well.

[0286] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On days 9-10, cells were resuspended in VPE1 medium containing the following: basal medium: DMEM-F12, SB431542 (10 uM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400). On day 9, an equal volume of freshly prepared VPE1 medium (500 ul) was gently replaced without disturbing the aggregates. On day 10, an equal volume of freshly prepared VPE1 medium (500 ul) was added per well.

[0287] On day 12, an equal volume of freshly prepared VPE2 medium (500ul) was gently replaced without disturbing the aggregates. VPE2 medium was prepared to contain the following: basal medium: DMEM-F12, Noggin (50ng / ml), CHIR99021 (2μM), FGF8b (50ng / ml), retinoic acid (0.1μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100). On day 13, an equal volume of freshly prepared VPE2 medium (500ul) was added per well.

[0288] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On day 14, an equal volume of freshly made TEP medium (500 ul) was gently added without disturbing the aggregates. TEP medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0289] On day 15, an equal volume of freshly prepared TEP medium (500 μl) was added per well. On days 16–18, 50% of the supernatant was removed and freshly prepared TEP medium (500 ul) was added per well.

[0290] TEC medium was added on days 19 to 22. TEC medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X) and BME (100X).

[0291] On day 22, cells were treated with Accutase and frozen using Hypothermosol-FRS medium. Cell populations were divided according to several experimental conditions. For 33FT1 (7.5M / plate), cells were thawed in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). Different concentrations and pulses of survivin inhibitor (YM155) were evaluated. For 33FT2 (7.8M / plate), cells were thawed in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). Different concentrations and pulses of survivin inhibitor (YM155) were evaluated. On day 6, cells from groups 3 and 4 were selected and transplanted into 5 animals. For 33FT3 (5M / plate), cells were thawed in 2D (24-well plate coated with Geltrex) and maintained in TEC medium for 5 days (Y27-10uM on day 1). Various concentrations and pulses of Plurisin#1 were evaluated. On day 5, they were resuspended in 3D and maintained in TEC medium for 5 days. On day 5, they were treated with 20 μM Plurisin#1. For 33FT4 (7.6M / plate), cells were thawed in 3D and maintained in TEC medium for 5 days (Y27-10uM on day 1). On day 5, one group was treated with 20nM YM155 and another group was treated with Plurisin#1. For 33FT6 (8M / plate), cells were thawed in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). On day 6, they were treated with 20 μM Plurisin#1 (36 hours) and transplanted into 5 animals on day 7. For 33FT7 (7.8M / plate), cells were thawed in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). On day 6, they were treated with 20μM Plurisin#1 (24h) and transplanted into two animals on day 7.

[0292] Example 28 Differentiation of iPSC cells into thymocytes (Experiment 37) Expansion of iPSC lines in suspension culture On day 0, mature iPSC line 18945 (3-4 days in suspension, 300-400 micrometers in diameter) was treated with Accutase to prepare single cells from aggregates. Three million single iPSCs were placed in suspension in 6-well ultra-low attachment plates in Stem scale medium along with the ROCK inhibitor Y27632 (10 μM). On day 1, 1 ml of supernatant was removed from the plate and 1 ml of pre-warmed Stem scale medium was added. On day 2, the supernatant was transferred together with the spheroids into a 15 ml conical tube and resuspended in medium A in 6-well low attachment plates.

[0293] Differentiation of iPSCs into definitive endoderm (DE) On day 1, cells were washed with PBS and resuspended in medium A. Medium A was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIR99021, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400). On day 2, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium A.

[0294] On day 3, the cells were washed with PBS and resuspended in medium B. Medium B was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), CHIRR9901 (2 μM), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400).

[0295] On days 4–5, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium B. Medium B was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400).

[0296] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) A 24-well plate was coated with Geltrex (1:100) that had been left at room temperature for 1 hour. On day 6, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature, and the supernatant was aspirated. The spheroids were washed with PBS, resuspended in AFE medium, and cultured from days 6 to 8. AFE medium was prepared to contain the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), and NEAA (1:400).

[0297] On day 7, an equal volume of freshly prepared AFE medium (500 μl) was added per well. On day 8, 50% of the supernatant was removed and freshly prepared AFE medium (500 μl) was added per well.

[0298] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) On day 9, an equal volume of freshly prepared VPE1 medium (500 ul) was gently replaced without disturbing the aggregates. VPE1 was prepared to contain the following basal medium: DMEM-F12, SB431542 (10 uM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400). On day 10, an equal volume of freshly prepared VPE1 medium (500 μl) was added per well.

[0299] On day 12, an equal volume of freshly made VPE2 medium (500ul) was gently replaced without disturbing the aggregates. VPE2 medium was prepared to contain the following: basal medium: DMEM-F12, Noggin (50ng / ml), CHIR99021 (2μM), FGF8b (50ng / ml), retinoic acid (0.1μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100).

[0300] On day 13, an equal volume of freshly prepared VPE2 medium (500 μl) was added per well.

[0301] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) On day 14, an equal amount of freshly made TEP medium (500 μl) was gently replaced without disturbing the aggregates. Cells were cultured in TEP medium from days 14 to 18. TEP medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIRR99 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without Vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 15, an equal volume of freshly prepared TEP medium (500 ul) was added per well. On days 16–18, 50% of the supernatant was removed and freshly prepared TEP medium (500 ul) was added per well.

[0302] Cells were cultured in TEC medium from days 19 to 22. On day 19, an equal volume of freshly made TEC medium (500 μl) was gently replaced without disturbing the aggregates. TEC medium was prepared containing the following: basal medium: DMEM-F12, FGF10 (50ng / ml), IL-22 (20ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), and BME (100X). On day 20, an equal volume of freshly prepared TEC medium (500ul) was added together with Plurisin#1 (20μM) (untreated and treated groups). On day 21, an equal volume of freshly made TEC medium (500ul) was gently replaced without disturbing the aggregates. On day 22, cells were treated with Accutase and frozen using Hypothermosol-FRS medium. Cells were divided into the following groups depending on the experiment: Group 37FT1 (8.85M treated and 9.5M untreated groups) were thawed in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). On day 6, cells were treated with 20μM Plurisin#1 (24h) and transplanted into 4 animals on day 7. Group 37FT2 (8.85M / plate) were frozen in 3D and maintained in TEC medium for 6 days (Y27-10uM on day 1). On day 6, cells were treated with 20 μM Plurisin#1 (24 h) and transplanted into 8 animals on day 7.

[0303] Example 29 Differentiation of iPSC cells into thymocytes (Experiments 40A, 40B and 41) iPSCs were differentiated into thymocytes as described below.

[0304] Expansion of iPS18945 lines in suspension culture On day 0, mature iPSC cells (3-4 days in suspension, 300-400 micrometers in diameter) were treated with Accutase to convert aggregates into single cells. Three million single iPSC cells were placed in suspension in a 6-well ultra-low attachment plate in Stem Scale medium along with the ROCK inhibitor Y27632 (10 μM). The plate was placed on a shaker at 70 RPM and 37°C. On day 1, 1 ml of supernatant was removed from the plate and 1 ml of pre-warmed Stem Scale medium was added. On day 2, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS, centrifuged at 250G for 5 minutes and the PBS was aspirated. Medium A was prepared containing basal medium: DMEM-F12, activin A (100 ng / mL), CHIR99021 (2 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400). 1 ml of medium A was added to each well of a 6-well low attachment plate.

[0305] Differentiation of iPSCs into definitive endoderm (DE) For differentiation of iPSCs into DE, small molecules and growth factors need to be introduced at the appropriate time. On days 1-2, medium A was added (medium A was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), CHIR99021 (2 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400)). On day 3, medium B was added (medium B was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), 2 μM CHIRR99021, LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:500), KSR (0.05%), Pen strep (1:200), and NEAA (1:400)). On days 4-5, medium B' was added (medium B' was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:500), KSR (0.05%), Pen strep (1:200), and NEAA (1:400)).

[0306] Differentiation of definitive endoderm (DE) into anterior foregut endoderm (AFE) On day 6, the supernatant was transferred into a 15 ml conical tube along with the spheroids. The spheroids were centrifuged at 250G for 5 minutes at room temperature and the supernatant was aspirated. The spheroids were washed with PBS, centrifuged at 250G for 5 minutes and the PBS was aspirated. 250 ul of AFE medium was added to each well of a Geltrex (1:100) coated 24-well plate that had been left at room temperature for 1 hour. AFE medium was prepared containing the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:500), KSR (0.05%), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), and NEAA (1:400). 6 ml of AFE medium was added to the DE-aggregates. The aggregates were gently mixed and 250 ul was added to each well of a 24-well plate containing AFE medium. On day 6, the medium was replaced with fresh AFE medium. On day 7, 500 μL of AFE' medium was added. AFE' medium was prepared to contain the following: basal medium: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:500), KSR (0.05%), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), and NEAA (1:400). In experiments 40A and 40B, cells were cultured in AFE' medium for 2 days, whereas in experiment 40B, cells were cultured in AFE medium for 3 days.

[0307] Differentiation of anterior foregut endoderm (AFE) into ventral pharyngeal endoderm (VPE) The cells were then cultured in VPE1 medium as shown in Table 4.

[0308] Table 4 VPE1 medium for experiment 40A and experiment 40B TIFF2024536496000005.tif81128

[0309] On day 10, for Experiment 40A and Experiment 40B, an equal volume of freshly made VPE1 medium (500 μl) was gently added without disturbing the aggregates.

[0310] In experiment 41, on day 10, 500 μl of freshly prepared VPE1 medium was added to the aggregates.

[0311] For experiment 41, VPE1 medium was prepared containing the following: basal medium: DMEM-F12, all-trans retinoic acid (0.1 μM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:500), KSR (0.05%), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), NEAA (1:400).

[0312] VPE2 medium was added to the cells in each experiment as shown in Table 5.

[0313] Table 5 VPE2 medium for experiments 40A, 40B and 41 TIFF2024536496000006.tif85152

[0314] HOXA3 and PAX9 expression was measured at the end of the VPE stage. Figure 15 shows that experiments 40A and 40B resulted in higher levels of HOXA3 and Pax9 compared to experiment 41.

[0315] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial progenitor cells (TEP) For experiment 41, TEP medium shown in Table 6 was added on days 13–17. On day 14, an equal volume of freshly made TEP medium (500 μl) was gently added without disturbing the aggregates. On days 15–17, 50% of the supernatant was removed and freshly prepared TEP medium (500 μl) was added per well.

[0316] Table 6: TEP medium for experiments 40A, 40B and 41 TIFF2024536496000007.tif97144

[0317] On day 16, an equal volume of freshly prepared TEP medium (500 μl) was gently added without disturbing the aggregates. On day 17, 50% of the supernatant was removed and freshly prepared TEP medium (500 μl) was added per well.

[0318] On day 18, the aggregates were transferred to TEC medium. TEC medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50ng / ml), FGF7 / KGF (50ng / ml), RANKL / TRANCE (50ng / ml), BMP4 (50ng / ml), FGF8b (50ng / ml), CHIRR99021 (2μM), IL-22 (20nM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100X), BME (100X).

[0319] On day 19, 50% of the supernatant was removed and freshly prepared TEC medium (500 μl) was added per well. On day 20, 20 μM PluriSIn-1 (a stearoyl-CoA desaturase inhibitor) was added to the freshly prepared medium. On day 21, PluriSIn-1-free TEC medium was added. On day 22, cells were treated with the enzyme Accutase to generate single cells that were frozen before further testing and experimentation.

[0320] Example 30 Reproducibility of Thymocyte Differentiation Protocol In experiment 37, new iPSC lines were tested using the differentiation protocol. For each experiment, FOXN1 expression in iPSC-derived TEPs was measured before cell freezing at the end of differentiation and 5 days after thawing the cells into 3D aggregates. A significant increase in FOXN1 expression was observed in thawed cells compared to FOXN1 expression before freezing of iPSC-derived thymocytes. This experiment demonstrates that the differentiation protocol can be easily transferred across iPSC cell lines and consistently upregulates FOXN1 expression through the freeze / thaw process.

[0321] Example 31 Removal of undifferentiated pluripotent stem cells from the TEP population The potential for residual undifferentiated pluripotent cells to ultimately remain is a major safety issue to be addressed in iPSC-derived cell therapy products. Such iPSCs in the product may cause teratomas. To eradicate the residual iPSCs, small molecules that have been shown to selectively target pluripotent stem cells were utilized. The survivin inhibitor YM155 and the stearoyl-CoA desaturase inhibitor PluriSIn-1 were tested. TEP populations treated with YM155 prior to transplantation caused smaller teratomas in vivo compared to animals transplanted with TEPs that were not treated with YM155.

[0322] Treatment with PluriSIn-1 (one 24-hour pulse before freezing and one 24-hour pulse after freezing in aggregates) resulted in lower Oct4 and Nanog expression in the TEP population compared to the population treated with one pulse of PluriSIn-1, but treatment with only one dose of PluriSIn-1 was sufficient to reduce Oct4 and Nanog expression when another iPSC cell line was used to generate TEPs.

[0323] Frozen cells from experiment 29B were thawed into aggregates over 5 days and treated with YM155 for 24 hours before transplantation under the kidney capsule of NSG dKO mice that had been transplanted with huCD34+ cells 2 weeks previously. Animals were followed, blood was collected every 3 weeks, and peripheral blood mononuclear cells were analyzed by flow cytometry for human hematopoietic cells (CD45), B cells (CD19), myeloid cells (CD19), T cells (CD3 / CD4 and CD3 / CD8) and mature T cell receptors (TCRα and β). All animals showed robust repopulation of PBMCs with human CD45+ hematopoietic cells by 5 weeks post-transplant. Double staining for CD3 and CD4 or CD8 was used to measure single positive T cells in the peripheral blood circulation. One of the two animals transplanted with fetal thymus developed CD4 and CD8 T cells by cells at 8-9 weeks. One of six animals transplanted with iPSC-derived TEPs developed T cells by week 9, with slightly higher levels of CD4 than CD8. T cells in these experiments also expressed mature α and β T cell receptors. Control sham-transplanted animals continued to show basal levels of T cells below 1% at week 13.

[0324] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not limited to the above description, but is as set forth in the appended claims.

[0325] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless specifically stated to the contrary or clear from the context. A claim or description containing "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, employed in, or relevant to a given product or process, unless specifically stated to the contrary or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or relevant to a given product or process. The present disclosure includes embodiments in which a plurality, or the entire group member, is present in, employed in, or relevant to a given product or process.

[0326] It should also be noted that the term "comprising" is intended to be open, allowing for, but not requiring, additional elements or steps to be included. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0327] When ranges are specified, the endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood as contemplated in different embodiments of the present disclosure to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0328] Furthermore, it is understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims, regardless of the reason, whether related to the existence of prior art or not.

[0329] The words that have been used are words of description rather than of limitation, and it is understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0330] While the present disclosure has been described in some detail and with respect to certain described embodiments, the present disclosure should not be limited to such details or embodiments, or to specific embodiments, but should be interpreted against the appended claims in a manner that provides the broadest possible interpretation of such claims in view of the prior art, thereby effectively encompassing the intended scope of the present disclosure.

Claims

1. A method for differentiating pluripotent stem cells into thymocytes, comprising: a. differentiating the pluripotent stem cells into definitive endoderm (DE) cells; b. culturing the DE cells and differentiating the DE cells into anterior foregut endoderm (AFE) cells by contacting or incubating the DE cells with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, or a combination thereof; c. Culturing the AFE cells and differentiating the anterior foregut cells into ventral pharyngeal endoderm (VPE) cells, i. contacting or incubating the AFE cells in a first VPE medium containing ascorbic acid, retinoic acid, FGF, a TGFβ inhibitor, or a combination thereof; ii. contacting or incubating the AFE cells in a second VPE medium containing noggin, a WNT activator, FGF, retinoic acid, ascorbic acid, or a combination thereof; Including, the steps; d. Culturing the VPE cells and differentiating the VPE cells into thymocytes by contacting or incubating the VPE cells with ascorbic acid, FGF, BMP, a WNT activator, or a combination thereof; Including, the thymocytes are thymic epithelial progenitor (TEP) cells; The method.

2. 2. The method of claim 1, wherein the TEPs are further differentiated into thymic epithelial cells (TECs) by contacting or incubating the TEPs with an interleukin, a WNT activator, RANKL, FGF, BMP, ascorbic acid, or a combination thereof, and optionally the TEPs are differentiated into TECs over a period of about 4 days.

3. i. the first VPE medium in step c.i. further comprises a WNT inhibitory substance, and optionally, the WNT inhibitory substance is IWR-1; and / or ii. The second VPE medium in step c.ii. further comprises a BMP inhibitor, an SHH inhibitor, or a combination thereof, and optionally, the BMP inhibitor is LDN193189 and / or the SHH inhibitor is SANT-1; The method of claim 1.

4. Differentiation of pluripotent stem cells into DE cells a. contacting or culturing the pluripotent stem cells in a first growth medium, wherein the first growth medium comprises activin A, PI-103, CHIR99021, or a combination thereof; b. culturing the pluripotent stem cells in a second growth medium to generate definitive endoderm cells, wherein the second growth medium comprises activin A, a BMP inhibitor, PI-103, CHIR99021, or a combination thereof; The method of claim 1 , comprising:

5. i. The BMP inhibitor is LDN193189; ii. The TGFβ inhibitor is SB431542; iii. The FGF is FGF8b, FGF7, FGF10, FGF1, bFGF, or a combination thereof; iv. the WNT activator is CHIR99021, and / or v. The BMP is BMP2, BMP4, or a combination thereof; The method of claim 1.

6. The method of claim 2, wherein the interleukin is IL22.

7. 2. The method of claim 1, wherein the pluripotent stem cells, the DE cells, the AFE cells, the VPE cells, or the thymocytes are cultured in a 3D culture, optionally cultured as aggregates in suspension.

8. 10. The method of claim 1, wherein the method is carried out for about 15 days to 30 days, or about 18 days to 25 days.

9. i. the pluripotent stem cells differentiate into definitive endoderm cells over a period of about 5 days; ii. The DE cells differentiate into AFE cells within about 2 to 3 days; iii. The AFE cells are cultured in the first VPE medium for about 2 to 4 days; iv. the AFE cells are cultured in the second VPE medium for about 2-3 days; and / or v. The VPE cells are differentiated into thymocytes for about 3 to 6 days; The method of claim 1.

10. A population of thymocytes prepared according to the method of any one of claims 1 to 9.

11. A pharmaceutical composition comprising the population of thymocytes according to claim 10 and at least one excipient.

12. 12. The pharmaceutical composition of claim 11 for use in a method for treating or preventing a condition in a subject.

13. i. the condition is associated with lack, reduction, or abnormal thymic function, immunodeficiency, cancer, an autoimmune disease, an infectious disease, or graft-versus-host disease (GvHD) in the subject; ii. the pharmaceutical composition is administered to the subject via a parenteral route, and / or iii. The pharmaceutical composition is implanted or injected into one or more lymph nodes of the subject; The pharmaceutical composition of claim 12.

14. 1. A method for increasing FOXN1 expression in a population of thymocytes, comprising: a. freezing the population of thymocytes; b. thawing the population of thymocytes; c. measuring FOXN1 expression in the population of thymocytes before freezing and comparing it to FOXN1 expression after thawing the population of thymocytes; The method comprising:

15. i. the FOXN1 expression is increased by about 10- to 100-fold; ii. the population of thymocytes is cultured in suspension after thawing, optionally as aggregates in turbidity; and / or iii. The thymocytes comprise thymic epithelial progenitor cells (TEPs), thymic epithelial cells (TECs), or a combination thereof; 15. The method of claim 14.

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