Compositions and methods for promoting thymocyte production from pluripotent stem cells - Patent Application 20070122999

Abstract

Embodiments of the present disclosure relate to novel compositions and methods for generating thymocytes. In some embodiments, thymocytes can be differentiated from pluripotent stem cells (PSCs), anterior primitive streak (APS) cells, definitive endoderm (DE) cells, anterior foregut endoderm (AFE) cells, pharyngeal endoderm (PE) cells, ventral pharyngeal endoderm (VPE) cells, and third pharyngeal pouch endoderm (TPPE) cells using the compositions and methods disclosed herein. In certain embodiments, thymocytes generated by the compositions, systems, and methods disclosed herein can be used to treat health conditions.

Description

[Technical Field]

[0001]

[0002] Embodiments of the present disclosure relate to novel compositions and methods for generating thymocytes (thymic progenitor cells (TEP) and thymic epithelial cells (TEC)) from pluripotent stem cells. In some embodiments, thymocytes can be differentiated from pluripotent stem cells (PSCs), anterior primitive streak (APS) cells, definitive endoderm (DE) cells, anterior foregut endoderm (AFE) cells, pharyngeal endoderm (PE) cells, ventral pharyngeal endoderm (VPE) cells, and third pharyngeal pouch endoderm (TPPE) cells using the compositions and methods disclosed herein. [Background technology]

[0002]

[0003] The thymus, an essential organ of the immune system, is the primary site of T lymphopoiesis and adaptive immune regulation. The thymus is most active during childhood and involutes around adolescence, resulting in severely reduced or absent naive T cell production, often resulting in immune system impairment in the elderly and in other health conditions. Thymic involution can be accelerated by certain clinical treatments, such as chemotherapy. Therefore, reconstitution of adaptive immunity through the mass production of distinct T cell types is therapeutically necessary in immunocompromised populations and for health conditions requiring adaptive immunotherapy. While pluripotent stem cells are a source of T cells for various clinical applications, current methods of T cell differentiation result in the generation of cells with aberrant phenotypes and / or immature T cells that are not therapeutically useful. Furthermore, negative selection, the process of removing autoreactive T cells during thymic T cell education, is currently not feasible. Other methods for generating thymocytes known in the art are inefficient. Therefore, improved methods for generating thymocytes from renewable and readily available cell sources are needed for the development of immune cell therapy and replacement therapies. Summary of the Invention

[0003]

[0004] Embodiments of the present disclosure relate to novel compositions and methods for generating thymocytes in vitro with improved efficiency. In certain embodiments, compositions for generating thymocytes at various stages of differentiation are disclosed. According to these embodiments, compositions are described that include a cell culture medium and further include, but are not limited to, at least one Wingless-associated integration site (Wnt) activator, at least one bone morphogenetic protein (BMP) inhibitor, and at least one activin A activator or activin A inhibitor. In some embodiments, the compositions disclosed herein may include the medium provided herein, which may further include at least one of mammalian pluripotent stem cells (PSCs), anterior primitive streak (APS) cells, definitive endoderm (DE) cells, or a combination thereof.

[0004]

[0005] In certain embodiments, in addition to the above paragraph

[0004] , the compositions disclosed herein may be media having a basal medium with a low protein concentration of about 1.0% (w / v) or less. In some embodiments, the compositions disclosed herein may have a protein-enriched cell culture medium with a protein concentration of at least 0.5% (w / v) to a maximum of 30% (w / v) protein. According to certain embodiments herein, the compositions disclosed herein having a protein-enriched cell culture medium may further comprise at least one of definitive endoderm (DE) cells; anterior foregut endoderm (AFE), ventral pharyngeal endoderm (VPE), pharyngeal endoderm (PE), and third pharyngeal pouch endoderm (TPPE) cells.

[0005]

[0006] In certain embodiments, in addition to the above paragraphs

[0004] and

[0005] , the compositions disclosed herein may include a protein-free medium. In other embodiments, the compositions disclosed herein may include a protein-enriched cell medium. In other embodiments, the medium may include, but is not limited to, MEM alpha. In certain embodiments, the culture medium may include Minimum Essential Medium (MEM), Eagle's Medium, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM / F12), F10 Nutrient Mix, Ham's F10 Nutrient Mix, Ham's F12 Nutrient Mix, Medium 199, RPMI, RPMI 1640, Reduced Serum Medium, Basal Medium (BME), DMEM / F12 (1:1), Ames' Medium, BGJb Medium (Fitton-Jackson Modification), Crick's Medium, CMRL-1066 Medium, Fisher's Medium, Grass Bovine Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15 Medium (Leibovitz), McCoy's 5A Modification, NCTC Medium, Swim's S-77 Medium, Weymouth's Medium, Williams' Medium E, and the like, and combinations thereof. According to these embodiments, the medium (e.g., MEM alpha medium) may further comprise ribonucleosides, deoxyribonucleosides, and / or L-glutamine. In certain embodiments, the medium (e.g., MEM alpha medium) may comprise, but is not limited to, one or more of phenol red, polyvinyl alcohol, methylcellulose, non-essential amino acids, lipid concentrate, insulin / transferrin / selenium / ethanolamine (ITSE, ITS-X), and / or penicillin-streptomycin. In certain embodiments, the medium (e.g., MEM alpha medium) may comprise, but is not limited to, insulin / transferrin / selenium / ethanolamine (ITSE), or insulin / transferrin / selenium (ITS), ITSX, etc.According to these embodiments, the compositions disclosed herein may comprise a medium supplemented in the absence of protein, or may be a protein-enriched culture medium (e.g., MEM alpha medium), and may further comprise at least one cell population, including at least a population of definitive endoderm (DE) cells; a population of anterior foregut endoderm (AFE) cells, a population of ventral pharyngeal endoderm (VPE) cells, a population of pharyngeal endoderm (PE) cells, and a population of third pharyngeal pouch endoderm (TPPE) cells. According to these embodiments, at least one cell population further comprises a protein-enriched medium and one or more agents described herein. In other embodiments, at least one cell population further comprises a medium containing little or no protein and one or more agents described herein.

[0006]

[0007] In some embodiments, to facilitate the above paragraphs

[0004] -

[0006] , the compositions disclosed herein may comprise a medium (e.g., MEM alpha medium) having at least one activin A activator, where the at least one activin A activator may include, but is not limited to, one or more of recombinant mammalian activin A, alantolactone, or a combination thereof. In some embodiments, the compositions disclosed herein may comprise a medium having at least one activin A inhibitor, where the at least one activin A inhibitor may be one or more of A83-01, RepSox, D4476, Ly364947, R268712, SD208, SB505124, SM16, galunisertib, SB525334, SB431542, or a combination thereof. In certain embodiments, the compositions disclosed herein may comprise a culture medium having at least one BMP inhibitor, which may include, but is not limited to, one or more of LDN193189, LDN214117, LDN212854, DMH2, K02288, ML347, SGC AAK1-1, PD407824, UK383367, A01, recombinant mammalian noggin and dorsomorphin, non-mammalian noggin and dorsomorphin, or combinations thereof.

[0007]

[0008] In certain embodiments, in addition to the above paragraphs

[0004] to

[0007] , the compositions disclosed herein may include, but are not limited to, at least one BMP signaling activator, at least one fibroblast growth factor receptor (FGFR) 3 activator, at least one retinoic acid signaling activator, and a protein-enriched cell culture medium, wherein the protein concentration may be from about 0.5% (w / v) or more protein up to about 30% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v)), and the composition does not contain a transforming growth factor-β (TGF-β) inhibitor. In other embodiments, the compositions disclosed herein do not contain a transforming growth factor-β (TGF-β) inhibitor or a transforming growth factor-β (TGF-β) activator. According to these embodiments, the compositions disclosed herein having a protein-enriched culture medium (e.g., MEM alpha medium) may further comprise at least one cell population, including at least a population of definitive endoderm (DE) cells; a population of anterior foregut endoderm (AFE) cells, a population of ventral pharyngeal endoderm (VPE) cells, a population of pharyngeal endoderm (PE) cells, and a population of third pharyngeal pouch endoderm (TPPE) cells. According to these embodiments, the at least one cell population further comprises the protein-enriched medium disclosed herein and one or more agents. In some embodiments, the compositions disclosed herein may further comprise at least one Wnt activator.

[0008]

[0009] In certain embodiments, in addition to the above paragraphs

[0004] to

[0008] , the compositions disclosed herein may include a medium, a protein-enriched medium, and may include, but are not limited to, at least one of a BMP signaling activator, at least one fibroblast growth factor receptor (FGFR) 3 activator, at least one fibroblast growth factor receptor (FGFR) 2 activator, at least one NOTCH signaling activator, and a protein-enriched cell culture medium, wherein the protein concentration may be greater than about 0.5% (w / v) protein and up to about 30% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v) protein). In some embodiments, the compositions disclosed herein may further include at least one retinoic acid signaling activator and / or at least one sonic hedgehog signaling activator. According to these embodiments, the compositions disclosed herein having a protein-enriched culture medium (e.g., MEM alpha medium) may further comprise at least one cell population, including at least a population of definitive endoderm (DE) cells; a population of anterior foregut endoderm (AFE) cells, a population of ventral pharyngeal endoderm (VPE) cells, a population of pharyngeal endoderm (PE) cells, and a population of third pharyngeal pouch endoderm (TPPE) cells. In some embodiments, in addition to the disclosure herein, the compositions disclosed herein may further comprise at least one Wnt activator.

[0009]

[0010] In certain embodiments, in addition to the above paragraphs

[0004] to

[0009] , the compositions disclosed herein may further comprise, but are not limited to, at least one of a BMP signaling activator, at least one retinoic acid receptor signaling activator, at least one inhibitor of a TGF-β / activin A signaling activator, at least one activator of sonic hedgehog signaling, and a protein-enriched cell culture medium, wherein the protein concentration may be from about 0.5% (w / v) or more protein up to about 30% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v) protein). According to these embodiments, the compositions disclosed herein having a protein-enriched culture medium (e.g., MEM alpha medium) may further comprise at least one cell population, including at least a population of definitive endoderm (DE) cells; a population of anterior foregut endoderm (AFE) cells, a population of ventral pharyngeal endoderm (VPE) cells, a population of pharyngeal endoderm (PE) cells, and a population of third pharyngeal pouch endoderm (TPPE) cells. In some embodiments, the compositions disclosed herein may further comprise at least one vascular endothelial growth factor (VEGF) receptor signaling activator.

[0010]

[0011] In some embodiments, in addition to the above paragraphs

[0004] to

[0010] , the compositions disclosed herein having at least one BMP signaling activator may include at least one of BMP4 or SB4. In some embodiments, the compositions disclosed herein may include at least one FGFR3 activator. According to these embodiments, the at least one FGFR3 activator may include at least one of FGF8, FGF1, FGF2, FGF9, heparin, or a combination thereof. In some embodiments, the compositions may further include at least one retinoic acid signaling activator. According to these embodiments, the compositions having at least one retinoic acid signaling activator may include one or more of retinoic acid, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, Ch55, or a combination thereof. In some embodiments, the compositions disclosed herein may include at least one VEGF receptor signaling activator. According to these embodiments, the compositions disclosed herein having at least one VEGF receptor signaling activator may include at least one of VEGF, VEGF165, multimeric VEGF, VEGF and a cross-linking agent, an anti-VEGF receptor antibody and an antibody cross-linking agent, or combinations thereof.

[0011]

[0012] In certain embodiments, further to the above paragraphs

[0004] to

[0011] , the compositions disclosed herein may comprise a composition or culture medium having, in a protein-enriched medium, at least one FGF receptor 1 and at least one FGF receptor 2 signaling activator, at least one activator of retinoic acid signaling, at least one inhibitor of BMP signaling, at least one inhibitor of Sonic Hedgehog signaling, and at least one activator of Activin A signaling, wherein the protein concentration may be from about 0.5% (w / v) or more protein up to about 30% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v) protein), and the composition does not comprise an activator of BMP signaling. In other embodiments, these compositions in protein-enriched medium may further comprise at least one cell population, which may include definitive endoderm (DE) cells, anterior foregut endoderm (AFE) cells, pharyngeal endoderm (PE) cells, ventral pharyngeal endoderm (VPE) cells, third pharyngeal pouch endoderm (TPPE) cells, and / or thymocytes (e.g., thymic epithelial cells (TECs)). In some embodiments, the compositions disclosed herein may further comprise at least one of a Wnt signaling activator, at least one NOTCH pathway signaling activator, at least one CD40 pathway signaling activator, at least one RANK signaling pathway activator, and / or at least one ghrelin receptor signaling activator.

[0012]

[0013] In some embodiments, in addition to the above paragraphs

[0004] to

[0012] , the compositions disclosed herein may further comprise at least one Wnt activator. According to these embodiments, the at least one Wnt activator may include, but is not limited to, at least one of Wnt3a, Wnt4, CHIR98014, AMBMP hydrochloride, or a combination thereof. In certain embodiments, the compositions disclosed herein may include, or further comprise, at least one NOTCH pathway activator. According to these embodiments, the compositions disclosed herein having at least one NOTCH pathway activator may include, but are not limited to, Yhhu3792, DLL1, DLL3, DLL4, Jagged1, and Jagged2. In certain embodiments, the compositions disclosed herein may include, or further comprise, at least one CD40 pathway activator. According to these embodiments, the at least one CD40 pathway activator may include, but is not limited to, CD40 ligand, multimeric CD40 ligand, CD40 ligand and a crosslinking agent, and an anti-CD40 antibody with an antibody crosslinking agent. In some embodiments, the compositions disclosed herein may include, or further include, at least one RANK pathway activator. According to these embodiments, the at least one RANK pathway activator may include, but is not limited to, a soluble RANK ligand, a RANK ligand, multimeric RANK ligand, a RANK ligand and a crosslinking agent, and an anti-RANK antibody with an antibody crosslinking agent. In other embodiments, the compositions disclosed herein may further include at least one ghrelin receptor pathway activator. According to embodiments, the at least one ghrelin receptor pathway activator may include, but is not limited to, ghrelin, MK0677, tabimorelin hemifumarate, and L-692,585. In certain embodiments, the compositions disclosed herein may comprise at least one NOTCH pathway activator and at least one of at least one CD40 pathway activator and at least one RANK pathway activator.According to these embodiments, the compositions disclosed herein comprise a protein-enriched culture medium (e.g., MEM alpha medium) and one or more agents identified herein. In other embodiments, these compositions may further comprise at least one cell population, including at least a population of definitive endoderm (DE) cells; a population of anterior foregut endoderm (AFE) cells, a population of ventral pharyngeal endoderm (VPE) cells, a population of pharyngeal endoderm (PE) cells, and a population of third pharyngeal pouch endoderm (TPPE) cells. According to these embodiments, the at least one cell population further comprises a protein-enriched medium and two or more, three or more, or four or more agents disclosed herein.

[0013]

[0014] In some embodiments, in addition to the above paragraphs

[0004] through

[0013] , the compositions disclosed herein having at least one FGF receptor 1 and FGF receptor 2 signaling activator may include, but are not limited to, at least one of FGF10, FGF3, FGF4, FGF7, FGF22, or a combination thereof. In some embodiments, the compositions disclosed herein including at least one retinoic acid signaling activator may include, but are not limited to, at least one of retinoic acid, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, Ch55, or a combination thereof. In some embodiments, the compositions disclosed herein having at least one BMP inhibitor may include, but are not limited to, at least one of LDN193189, LDN214117, LDN212854, DMH2, K02288, ML347, SGC AAK11, PD407824, UK383367, A01, recombinant mammalian noggin and dorsomorphin, non-mammalian noggin and dorsomorphin, or combinations thereof. In some embodiments, the compositions disclosed herein having at least one inhibitor of sonic hedgehog signaling may include, but are not limited to, at least one of SANT1, SANT2, U1866A, dynapyrazole-A, dynalestin, cyclopamine, HIP1, GANT58, AY9944 dihydrochloride, RU-SKI43 hydrochloride, or combinations thereof. In some embodiments, the compositions disclosed herein comprising at least one activin A activator may include, but are not limited to, at least one of recombinant mammalian activin A, SB4, alantolactone, or a combination thereof. In some embodiments, the compositions disclosed herein comprising at least one NOTCH signaling activator may include, but are not limited to, at least one of Yhhu3792, DLL1, DLL3, DLL4, Jagged1, and Jagged2, or a combination thereof.In some embodiments, the compositions disclosed herein having at least one Wn signaling activator may include, but are not limited to, Wnt3a, Wnt4, CHIR98014, and AMBMP hydrochloride, or combinations thereof.

[0014]

[0015] In certain embodiments, in addition to paragraphs

[0004] -

[0014] above, activators supplemented into basal media (e.g., protein-free) or protein-rich media having low to moderate levels of protein disclosed herein may have a concentration of about 1 nM to 10 mM, depending on what is being activated, the agent of interest, and the agent inducing activation. Those skilled in the art will understand these distinctions for activating a desired target or system (e.g., NOTCH, Wnt, RA, or other molecules, or another pathway) disclosed herein. In certain embodiments, activators supplemented into protein-free media disclosed herein may have a concentration of about 1 nM to 10 mM, depending on what is being activated, the agent of interest, and the agent inducing activation. In some embodiments, the basal media disclosed herein may further include a protein supplement, or a protein supplement in the form of, for example, serum (e.g., fetal bovine serum (FBS), bovine serum albumin (BSA), human serum, knockout serum, etc.).

[0015]

[0016] In certain embodiments, further to paragraphs

[0004] through

[0015] above, a method for differentiating mammalian pluripotent stem cells (PSCs) is disclosed. In some embodiments, the present disclosure provides a method for differentiating mammalian PSCs into anterior primitive streak cells and subsequently into definitive endoderm (DE) cells. In certain embodiments, the method for differentiating mammalian PSCs into anterior primitive streak cells and subsequently into DE cells may include incubating mammalian PSCs or anterior primitive streak cells or DE cells with a composition disclosed herein, generating DE cells that may have the ability to differentiate into at least one of AFE, VPE, TPPE, PE, and / or thymocytes.

[0016]

[0017] In some embodiments, further to paragraphs

[0004] through

[0016] above, the initial incubation period may include a cell culture composition having a protein concentration of less than 0.5%, and after a predetermined period, the enriched protein condition may be switched to a cell culture composition having a protein concentration of at least 0.5% (w / v) or more up to about 30.0% (w / v) (e.g., about 0.5% to about 20.0% (w / v)). In some embodiments, the method may include incubating mammalian PSCs, anterior primitive streak cells, or DE cells in a low-protein or protein-free medium for about 12 hours to up to about 6 days, and the low-protein medium may have a protein concentration of less than 0.5% (w / v) in the medium. In certain embodiments, the methods disclosed herein may further include incubating PSC or APS cells in a medium having a protein concentration of less than 0.5% (w / v) protein for about 12 hours to up to about 4 days to generate DE cells capable of efficiently differentiating into AFE cells, VPE cells, TPPE cells, PE cells, and / or thymocytes, and the medium may further include, but is not limited to, at least one BMP inhibitor and at least one activin inhibitor. In other embodiments, the culture composition for the methods disclosed herein may include at least one BMP inhibitor and at least one activin inhibitor to generate DE cells from PSC or APS cells capable of efficiently differentiating into AFE cells, VPE cells, TPPE cells, PE cells, and / or thymocytes (TEP or TEC cells). In other embodiments, the cell culture compositions used in the methods disclosed herein may comprise a medium and at least one activin activator to generate DE cells from PSCs or APS cells that have the ability to efficiently differentiate into AFE cells, VPE cells, TPPE cells, PE cells, and / or thymocytes (TEP or TEC cells).

[0017]

[0018] In certain embodiments, further to the above paragraphs

[0004] through

[0017] , the methods and compositions disclosed herein may include incubating mammalian PSCs, or APS cells, or DE cells, or AFE cells, or VPE cells, or PE cells, or TPPE cells, or thymocytes in any of the compositions disclosed herein and supplementing the composition with ITS (insulin, transferrin, and selenium), ITSX (insulin, transferrin, selenium, and ethanolamine), ITSE, or the like, at a dilution of about 1:50 to about 1:5000, from about day 0 up to 24 hours of incubation. In some embodiments, the methods disclosed herein may include supplementing the composition with ITS or ITS-X, which may be about 1:2000 or may be increased to about 1:2000. In certain embodiments, the methods disclosed herein may further include incubating the mammalian PSCs or anterior primitive streak cells or DE cells in a medium that may include one or more of epidermal growth factor (EGF), hydrocortisone, vitamin C, vitamin E, non-essential amino acids, sodium pyruvate, glutamine, trace elements, lipids, and / or beta-mercaptoethanol, or any combination thereof.

[0018]

[0019] In certain embodiments, further to the above paragraphs

[0004] to

[0018] , the methods disclosed herein may include incubating DE cells or AFE cells in a replacement composition or replacement medium having a protein concentration of at least about 0.5% (w / v) protein or more and up to about 30.0% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v)) protein, for about 12 hours to up to about 4 days, wherein the replacement composition includes at least one of at least one activator of FGF receptor 3 signaling, at least one activator of retinoic acid signaling, and at least one activator of BMP signaling, for about 12 hours to up to about 14 days to produce at least one AFE cell and / or VPE cell capable of efficiently differentiating into TEP cells and / or TEC cells. According to these embodiments, the methods disclosed herein may further include replacing the culture medium with medium having a protein concentration that can range from about 0.5% (w / v) or more protein to about 30.0% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v) protein) for about 12 hours to up to about 60 days or more, and the medium may further include at least one of at least one FGF receptor 1 and FGF receptor 2 signaling activator, at least one retinoic acid signaling activator, at least one inhibitor of BMP signaling, at least one inhibitor of sonic hedgehog signaling, at least one activin A activator, and at least one Wnt pathway signaling activator. According to these embodiments, the methods disclosed herein may obtain a final population of cells comprising thymocytes, and the thymocytes may be at least one of TEP cells and TEC cells.

[0019]

[0020] In certain embodiments, in addition to the above paragraphs

[0004] to

[0019] , the methods disclosed herein may further include replacing the composition with a replacement composition having from about 0.5% (w / v) or more protein up to about 30.0% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v)), where the replacement composition contains at least one of at least one FGF receptor 1 and FGF receptor 2 signaling activator, at least one retinoic acid signaling activator, at least one inhibitor of BMP signaling, at least one inhibitor of sonic hedgehog signaling, and at least one activin A activator; and incubating the DE, AFE, and / or VPE cells for from about 12 hours up to about 60 days or more to generate thymocytes (e.g., TEC and / or TEP cells).

[0020]

[0021] In some embodiments, further to the above paragraphs

[0004] -

[0020] , the methods and compositions disclosed herein may include differentiating at least about 70% to up to about 100% of mammalian PSCs, anterior primitive streak cells, and / or DE cells into AFE cells that are SOX2+, FOXA2+, and SOX17-, which can further differentiate into thymocytes. In some embodiments, the methods disclosed herein may include differentiating at least about 60% to up to about 100% of mammalian AFE cells into VPE cells that are HOXA3+HOXB1-NKX2.1-. In some embodiments, the compositions and methods disclosed herein may include at least about 5% or more, 10% or more, 15% or more, or 20% or more of the mammalian AFE or VPE cells differentiated into thymocytes (e.g., TEPs and TECs). Alternatively, 30% or 40% or more of the mammalian AFE or VPE cells differentiated into thymocytes (e.g., TEPs and TECs). According to these embodiments, thymocytes generated or differentiated using the compositions and methods disclosed herein include cells that are EPCAM+, CD104+, CD205+, and MHC-II+.

[0021]

[0022] In certain embodiments, in addition to paragraphs

[0004] through

[0021] above, the present disclosure provides kits containing one or more compositions disclosed herein and at least one container. In some embodiments, the kits may include mammalian PSCs or components needed to harvest mammalian PSCs. In some embodiments, the kits disclosed herein may include one or more agents for supplementing the compositions or media disclosed herein, and, optionally, media or compositions or replacement compositions disclosed herein. In certain embodiments, the present disclosure provides kits for differentiating cells into functional thymocytes disclosed herein for use in treating or preventing a health condition. In certain embodiments, the kits may include kits for storage, transportation, and use in improving, preventing, and / or treating a health condition in a subject.

[0022]

[0023] In certain embodiments, further to paragraphs

[0004] through

[0022] above, the present disclosure provides pharmaceutical compositions and methods for treating a condition in a subject using the disclosed pharmaceutically acceptable compositions. In certain embodiments, the pharmaceutical compositions include one or more cell populations produced by the compositions and methods of the present disclosure. In some embodiments, the methods disclosed herein include treating an immune-related condition in a subject by administering to a subject in need thereof a cell population produced by any one of the compositions and methods disclosed herein. In some embodiments, the methods disclosed herein may treat a condition in a subject by administering to a subject in need thereof thymocytes produced by any one of the methods disclosed herein.

[0023]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0024] [Figure 1]

[0025] FIG. 1 shows an exemplary schematic of the methods and compositions disclosed herein for generating intermediate cells and thymocytes from pluripotent stem cells, according to certain embodiments of the present disclosure. [Figure 2]

[0026] FIG. 2 shows a decoding chart of the drugs, regulators, additives, and basal media used in the compositions and methods disclosed herein, according to certain embodiments of the present disclosure. [Figure 3A]

[0027] 3A-3C show compositions and methods for AFE and VPE induction from various cells and conditions according to certain embodiments of the present disclosure (3A), as well as representative flow plots and comparative quantification of SOX17-SOX2+ cell production under various growth conditions (3B), and flow plots and FOXA2+SOX2+ cell production under various growth conditions (3C). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4A]

[0028] 4A-4E show compositions and methods for AFE, VPE, and thymic epithelial progenitor (TEP) cell derivation from various cells and conditions (4A), according to certain embodiments of the present disclosure, as well as representative comparative quantification of CD205+EPCAM+ cells under conditions on selected days (4B), and analysis of HOXA3 (4C), KRT8 (4D), and FOXN1 (4E) comparative gene expression under various differentiation protocols. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 5A]

[0029] 5A-5C show compositions and methods for AFE and VPE production according to certain embodiments of the present disclosure (5A), as well as representative flow plots and comparative quantification of SOX17-SOX2+ cells under various conditions at selected times (5B), and representative flow plots and comparative quantification of FOXA2+SOX2+ cells under various conditions at selected times according to various differentiation protocols (5C). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A]

[0030] 6A-6G show compositions and methods for AFE, VPE, and thymic epithelial progenitor (TEP) cell induction according to certain embodiments of the present disclosure (6A), as well as representative flow plots (6B), and quantification of EPCAM+ / CD104hi cells (6C), and comparative gene expression analysis of FOXN1 (6D), KRT5 (6E), NKX2 (6F), and NKX2-1 (6G) gene expression from each differentiation protocol. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 7]

[0031] FIG. 7 illustrates an exemplary experimental setup, in accordance with certain embodiments of the present disclosure. [Figure 8]

[0032] 8A-8B show additional stimulatory agents used to enhance thymocyte production with improved marker presentation. 8A and 8B show representative marker percentage results under various conditions according to certain embodiments of the present disclosure. [Figure 9]

[0033] 9A-9B show exemplary results of qPCR detection of representative markers under control and experimental conditions according to certain embodiments of the present disclosure. [Figure 10]

[0034] FIG. 10 is a schematic diagram of the derivation of stem cell-derived thymic organoids (sTO) according to certain embodiments of the present disclosure. [Figure 11AB]

[0035] 11A-11C show exemplary results of an experimental thymocyte induction protocol under various conditions: 11A shows exemplary flow cytometry results; 11B shows the production of sTO compared to the production of various T cell populations; and 11C depicts TEP versus sTO under certain experimental conditions according to certain embodiments of the present disclosure. [Figure 11C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition

[0036] Unless defined herein, terms have the meaning commonly understood or applicable by a person of ordinary skill in the art relevant to a particular embodiment disclosed herein.

[0026]

[0037] As used herein, unless otherwise indicated, "about" applies to all numbers expressing quantities, properties (e.g., molecular weight, reaction conditions) of agents and / or compounds, and is intended to be modified in all instances by this term as disclosed herein. Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that may vary from about 10% to about 15% plus and / or minus, depending on the desired properties sought as disclosed herein. Numerical values ​​expressed herein inherently contain standard deviations necessarily resulting from errors found in numerical testing measurements.

[0027]

[0038] As used herein, "individual," "subject," "host," and "patient" may be used interchangeably herein and refer to any mammalian subject (e.g., human, pet, livestock, horse, or other animal) for whom diagnosis, treatment, prevention, or therapy is desired.

[0028]

[0039] As used herein, "treat," "treating," or "treatment" can mean reversing, ameliorating, or inhibiting the onset or progression of a condition or disease or symptoms of a condition or disease. In other embodiments, a condition may be prevented or the risk of onset may be ameliorated.

[0029]

[0040] As used herein, "pluripotent stem cells" or "pluripotent cells" can refer to cells that, under appropriate conditions, can produce progeny of several different cell types, which are derivatives of or reprogrammed from all three germ layers (i.e., endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells (PSCs) include, but are not limited to, embryonic stem (ES) cells, embryonic germ stem (EG) cells, induced pluripotent stem (iPSC) cells, adult stem cells, and the like. PSC cells can be derived from any organism of interest, including, but not limited to, primates (e.g., humans), dogs, cats, mice, horses, pigs, birds, camels, cows, sheep, and the like.

[0030]

[0041] As used herein, "marker" can refer to any molecule that can be measured or detected, for example. In certain embodiments disclosed herein, markers can include, but are not limited to, nucleic acids (e.g., transcripts of a gene), polypeptide products of a gene, polypeptides, proteins, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, carbohydrates, and / or small molecules. As used herein, "expression" and its grammatical equivalents, in the context of a marker, can refer to the production or transcription or translation of the marker. Furthermore, the level or amount of a marker can be assessed and compared to a control to evaluate a process.

[0031] Detailed Description

[0042] In the following sections, certain exemplary compositions and methods are described to detail certain embodiments of the present invention.It is clear to those skilled in the art that implementing certain embodiments does not require the use of all or even some of the specific details outlined herein, and that concentrations, times, and other specific details can be modified through routine experimentation.In some cases, well-known methods or components are not included in the description.

[0032]

[0043] Embodiments of the present disclosure relate to novel compositions and methods for generating thymocytes from pluripotent stem cells and their intermediate cells. A key challenge in accelerating research and treatment efforts on thymic function and T cell development has been the lack of effective and efficient differentiation protocols for pluripotent stem cell lines. Current differentiation protocols produce a small number of mature thymocytes with a high proportion of immature T cells with aberrant phenotypes that are not useful for research or therapeutic use. Furthermore, negative selection, the process of removing autoreactive T cells during thymic T cell education, is not currently feasible. Therefore, there is a need in the art for improved compositions, conditions, and methods for generating thymocytes from pluripotent stem cells. In certain embodiments, novel and efficient compositions and methods for generating thymocytes (e.g., TEC and TEP cells) and intermediate cells (e.g., DE, AFE, and VPE cells) from pluripotent stem cells (PSCs) are disclosed herein, where the functional thymocytes can generate conventional T cells suitable for clinical applications, including, but not limited to, adoptive cell therapy, immunotherapy, and cancer therapy.

[0033]

[0044] Appendix A, submitted with the priority application, is incorporated herein by reference in its entirety for all purposes. Those skilled in the art understand that there are several causes for the decline of thymocytes and naive T cells. For example, thymic atrophy (e.g., involution, immunosenescence), chemotherapy side effects, graft-versus-host disease (GvHD), and human immunodeficiency virus (HIV) can cause these declines. State-of-the-art thymic epithelial cell differentiation protocols lack functional TECs after extended periods in vivo, low numbers of FOXN1-positive cells in vitro, and low expression levels of TEP / TEC markers in vivo and in vitro, providing a need for improved methods for generating these cells for therapeutic use and recovery. The compositions and methods disclosed herein provide improved methods for producing functional thymocytes, for example, using novel supplemented media. In certain embodiments, early thymic progenitor signaling can enhance FOXN1 expression during differentiation of thymic epithelial cells (TECs), e.g., CD40 ligand, RANK ligand, and NOTCH ligand. In some embodiments disclosed herein, it is demonstrated that ETP-derived signals (e.g., CD40L, RANKL, and NOTCH) can enhance iPSC-derived TEP expression of FOXN1 and MHC-II. In other embodiments, the compositions and methods disclosed herein can be used to form stem cell-derived thymic organoids, produce several single-positive T cells, and provide further TEP / TEC maturation. In other embodiments, the optimized TEP / TEC protocols disclosed herein can be combined with stem cell-derived thymic organoid systems to improve outcomes, such as thymic functional recovery.

[0034]

[0045] In certain embodiments, in addition to paragraphs

[0043] -

[0044] above, the compositions disclosed herein can be used to provide culture conditions for generating thymocytes (e.g., TEC cells and TEP cells) and intermediate cells (e.g., DE cells, AFE cells, VPE cells) from PSCs according to the methods of the present disclosure. In some embodiments, the compositions disclosed herein can comprise a culture medium. As used herein, the terms "medium" or "media" are used in the context of cell culture, i.e., the phrases "cell culture medium" or "cell medium," "replacement composition," or "composition" can refer to a cell growth medium suitable for culturing the cells of the present disclosure, as appropriate. A "basal medium" referred to herein for use as a low- or enriched-protein-containing medium can be a cell culture medium that has not been modified by the addition of one or more additives / supplements. Non-limiting examples of basal media suitable for use herein include MEM alpha medium, Minimum Essential Medium (MEM), Eagle's Medium, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM / F12), F10 Nutrient Mix, Ham's F10 Nutrient Mix, Ham's F12 Nutrient Mix, Medium 199, RPMI, RPMI 1640, Reduced Serum Medium, Basal Medium (BME), DMEM / F12 (1:1), Ames' Medium, BGJb Medium (Fitton-Jackson Modification), Crick's Medium, CMRL-1066 Medium, Fischer's Medium, Grass Bovine Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15 Medium (Leibovitz), McCoy's 5A Modification, NCTC Medium, Swim's S-77 Medium, Weymouth's Medium, Williams' Medium E, and the like, and combinations thereof. In some embodiments, the compositions disclosed herein may comprise a basal medium containing one or more additives as disclosed herein.

[0035]

[0046] In certain embodiments, in addition to the above paragraphs

[0043] through

[0045] , the compositions disclosed herein can be used in one or more stages of differentiation to produce thymocytes from PSCs. As illustrated in one exemplary schematic diagram shown in FIG. 1 , producing thymocytes from PSCs according to the present disclosure can involve several stages of differentiation. In this example, five exemplary differentiation stages are shown to aid in understanding the compositions and methods disclosed herein: (1) culturing PSCs to generate anterior primitive streak (APS) cells; (2) culturing APS cells to generate definitive endoderm (DE) cells; (3) culturing DE cells to generate anterior foregut endoderm (AFE) cells; (4) culturing AFE cells to generate ventral pharyngeal endoderm (VPE) cells; and (5) culturing VPE cells to generate thymocytes (e.g., thymic epithelial progenitor (TEP) cells, thymic epithelial cells (TECs), or a combination thereof).

[0036] Stage 1 - Culturing PSCs to generate APS cells

[0047] In certain embodiments, the present disclosure provides compositions for use in the in vitro differentiation of pluripotent stem cells (PSCs) into thymocytes and their intermediate cells. In some embodiments, the PSCs used in the present disclosure may include, but are not limited to, embryonic stem cells, embryonic germ cells, or induced pluripotent stem cells (iPSCs). In some embodiments, the PSCs may be mammalian PSCs. In other embodiments, the PSCs may be human PSCs. In other embodiments, the PSCs used in the present disclosure may be isolated from an autologous source. As used herein, the term "autologous" refers to PSCs obtained from the same subject who will be treated and / or monitored with the thymocytes generated as disclosed herein. In some embodiments, the PSCs used in the embodiments of the present disclosure may be isolated from an allogeneic source. As used herein, the term "allogeneic" refers to PSCs obtained from a subject different from the subject who will be treated and / or monitored with the thymocytes generated as disclosed herein. In some embodiments, the PSCs used in the present disclosure may be harvested, generated, cultured, and / or characterized using standard methods in the art.

[0037]

[0048] In certain embodiments, the compositions disclosed herein can be used to provide conditions for culturing PSCs to generate APS cells according to the methods of the present disclosure. According to these embodiments, the compositions disclosed herein can include a basal medium having a low concentration of protein. In certain embodiments, the protein used in the basal medium disclosed herein can be serum or other protein supplements. In some embodiments, the protein used in the basal medium disclosed herein can be serum from a mammalian source (e.g., bovine, human) or a protein from any source.

[0038]

[0049] In some embodiments, the compositions disclosed herein used to culture PSCs to generate APS cells according to the methods of the present disclosure can be basal media having a low protein concentration of about 0.5% (w / v) or less. In some embodiments, basal media having a low protein concentration for use herein can have a protein concentration of less than about 0.01% (w / v), less than about 0.05% (w / v), less than about 0.1% (w / v), less than about 0.2% (w / v), less than about 0.3% (w / v), less than about 0.4% (w / v), or less than about 0.5% (w / v).

[0039]

[0050] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure may comprise a basal medium having at least one additive capable of modulating Wnt and / or the Wnt signaling pathway. As used herein, the term "modulate" refers to the activation, propagation, inhibition, upregulation of expression, downregulation of expression, and / or other alteration of the activity of a signaling pathway or a component of a signaling pathway. According to these embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure may be a basal medium having at least one Wnt-modulating agent. As used herein, "Wnt-modulating agent" may refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that modulates Wnt and / or the Wnt signaling pathway. In some embodiments, the compositions disclosed herein for culturing PSCs to generate anterior primitive streak (APS) cells according to the methods of the present disclosure can be a basal medium having at least one Wnt modulating agent (e.g., a "Wnt activator") capable of activating Wnt and / or the Wnt signaling pathway. In certain embodiments, the agent capable of modulating Wnt can be an activating agent. Non-limiting examples of Wnt activators suitable for use as disclosed herein include DNA encoding β-catenin (e.g., naked DNA encoding β-catenin, a plasmid expression vector encoding β-catenin, a viral expression vector encoding β-catenin), a β-catenin polypeptide, one or more Wnt / β-catenin pathway agonists (e.g., Wnt ligands, DSH / DVL-1, -2, -3, LRP6N, WNT3A, WNT5A, and WNT3A, 5A), one or more glycogen synthase kinase 3β (GSK3(3)) inhibitors (e.g., lithium chloride (LiCl), purvalanol A, olomoucine, alsterpaullone, kenpaullone, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), α-4-dibromoacetophenone (i.e., tau protein kinase I (TPK) inhibitor) I) Inhibitors), 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanol, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), indirubin-5-sulfonamide; indirubin-5-sulfonic acid (2-hydroxyethyl)-amide indirubin-3'-monoxime; 5-iodo-indirubin-3'-monoxime; 5-fluoroindirubin; 5,5'-dibromoindirubin; 5-nitroindirubin; 5-chloroindirubin indirubin; 5-methylindirubin; 5-bromoindirubin, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), a-4-dibromoacetophenone (i.e., tau protein kinase I (TPK) inhibitor) (I) inhibitors), 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi) N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazole-2-aurea (AR-A014418), H-KEAPPAPPQSpP-NH2 (L803) and Myr-N-GKEAPPAPPQSpPNH2 (L803-mts)), one or more antisense RNAs or siRNAs that specifically bind to GSK3β mRNA, one or more casein kinase 1 (CK1) inhibitors (e.g., antisense RNAs or siRNAs that specifically bind to CK1 mRNA), and the like.

[0040]

[0051] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be, for example, a basal medium having at least one Wnt activator at a concentration of at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one Wnt activator at a concentration of about 5 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 150 ng / ml, about 15 ng / ml to about 125 ng / ml, or about 15 ng / ml to about 100 ng / ml, or about 15 ng / ml to about 50 ng / ml). In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be comprised of a basal medium having at least one Wnt activator at a concentration of about 5 ng / ml, about 10 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, or about 200 ng / ml.

[0041]

[0052] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one activin A modulating agent (and, optionally, at least one Wnt activator and / or at least one BMP inhibitor). As used herein, "activin A modulating agent" can refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that modulates activin A and / or the activin A signaling pathway. In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods disclosed herein can include a basal medium having at least one activin A activator. According to these embodiments, the activin A activator can be activin A, and / or a variant or functional analog thereof. Non-limiting examples of activin A analogs suitable for use as disclosed herein include IDE1 (2-[6-carboxy-hexanoyl)-hydrazonomethyl]-benzoic acid), IDE2 (7-(2-cyclopentylidenehydrazino)-7-oxoheptanoic acid), and the like. In some embodiments, the compositions disclosed herein may have at least one activin A activator, which may include at least one of recombinant mammalian activin A, SB4, alantolactone, or a combination thereof.

[0042]

[0053] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one activin A activator (and optionally at least one Wnt activator and / or at least one BMP inhibitor) at a concentration of, for example, at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one activin A activator (and, optionally, at least one Wnt activator and / or at least one BMP inhibitor) at a concentration of about 5 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 75 ng / ml or about 15 ng / ml to about 50 ng / ml). In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can include a basal medium having at least one activin A activator (and, optionally, at least one Wnt activator and / or at least one BMP inhibitor) at a concentration of about 5 ng / ml, about 10 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, or about 200 ng / ml. In certain embodiments, the basal medium is a low protein content (eg, 1% (w / v) protein or less) medium.

[0043]

[0054] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one phosphoinositide 3-kinase (PI3K) modulator (and, optionally, at least one activin A inhibitor, at least one Wnt activator, and / or at least one BMP inhibitor). As used herein, "PI3K modulator" can refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that modulates PI3K and / or the PI3K signaling pathway. In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having at least one PI3K inhibitor. Non-limiting examples of PI3K inhibitors suitable for use in the compositions and methods disclosed herein include BKM120, BEX235, BGT226, idelalisib, GDC-0941, IPI-145 (INK1197), GSK2636771, PI-103, BEZ235, BGT226, VS-5584m, (SB2343), PI-103, ZSTK474, GSK1059615, gedatolisib, HS-173, alperi sib (BYL719), PIK-75, A66, YM201636, TGX-221, GSK2636771, CZC24832, AS-252424, AS-604850, CAY10505, CAL-101 (idelalisib, GS-1101), PIK-294, PI-3065, PIK-293, IC-87114, AS-605240, PIK-90, other P3K inhibitors, and the like, and combinations thereof.

[0044]

[0055] In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having, for example, a concentration of at least one PI3K inhibitor (and optionally at least one activin A inhibitor, at least one Wnt activator, and / or at least one BMP inhibitor) of at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure may comprise a basal medium having at least one PI3K inhibitor at a concentration of about 5 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 75 ng / ml or about 15 ng / ml to about 50 ng / ml). In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells according to the methods disclosed herein may comprise a basal medium having at least one PI3K inhibitor at a concentration of about 5 ng / ml, about 10 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, or about 200 ng / ml.

[0045]

[0056] In certain embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having a low concentration of protein (e.g., 0.5% (w / v) or less protein) and at least one Wnt modulating agent, at least one activin A modulating agent, at least one PI3K modulating agent, or a combination thereof. In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can include a basal medium having a low concentration of protein and at least one Wnt activator, at least one activin A activator, at least one PI3K inhibitor, or a combination thereof. In some embodiments, the compositions disclosed herein for culturing PSCs to generate APS cells according to the methods of the present disclosure can be a basal medium having a low concentration of protein, at least one Wnt activator, at least one activin A activator, and at least one PI3K inhibitor, and optionally other culture agents.

[0046]

[0057] In certain embodiments, compositions disclosed herein for culturing PSCs to generate APS cells can be provided to cell culture and incubated for a suitable period of time for the PSC cells to differentiate into APS cells. In some embodiments, the culture conditions disclosed herein can be from about 12 hours to about 48 hours, or about 72 hours or more. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells can be provided to cell culture and incubated for a suitable period of time for at least about 70% to at least about 99% up to 100% of the PSC cells to differentiate into APS cells. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells can be provided to cell culture and incubated for a suitable period of time for about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the PSC cells to differentiate into APS cells. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells may be provided to cell cultures and incubated for a period of about 12 hours to about 5 days. In some embodiments, compositions disclosed herein for culturing PSCs to generate APS cells may be provided to cell cultures and incubated for a period of 12 hours or less, or less than about 1 day, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, or any time in between.

[0047] Step 2 - Culturing APS cells to generate DE cells

[0058] In certain embodiments, the compositions disclosed herein can be used to provide conditions for culturing APS cells to generate DE cells according to the methods of the present disclosure. According to these embodiments, the compositions disclosed herein can be a basal medium having a low concentration of protein (e.g., a "low-protein medium") or a basal medium having a high concentration of protein (e.g., a "protein-rich medium"). In some embodiments, the compositions disclosed herein can be used to provide conditions for culturing APS cells to generate DE cells according to the methods of the present disclosure, and can be a basal medium having a high protein concentration, from about 0.5% (w / v) or more protein up to about 30% (w / v) protein (e.g., about 0.5% to about 20.0% (w / v)). In some embodiments, a high protein concentration basal medium for use as a culture medium disclosed herein can have a protein concentration of greater than about 0.5% (w / v), greater than about 5.0% (w / v), greater than about 10% (w / v), greater than about 15% (w / v), greater than about 20% (w / v), greater than about 25% (w / v), or up to about 30.0% (w / v). In some embodiments, a high protein concentration basal medium for use as a culture medium disclosed herein can have a protein concentration of at least about 0.5% (w / v) and up to 30% (w / v) protein. In some embodiments, a basal medium having a high protein concentration for use herein may have a protein concentration in the range of about 0.5% (w / v) to about 30.0% (w / v) (e.g., about 0.5% (w / v), about 5.0% (w / v), about 10% (w / v), about 15% (w / v), about 20% (w / v), about 25% (w / v), about 30% (w / v)).

[0048]

[0059] In certain embodiments, compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may comprise a basal medium having at least one bone morphogenetic protein (BMP) modulating agent. As used herein, "BMP modulating agent" may refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that modulates BMP and / or the BMP signaling pathway. In some embodiments, compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may be a basal medium having at least one BMP inhibitor. Non-limiting examples of BMP inhibitors suitable for use in and as supplements for the enriched protein media disclosed herein include, but are not limited to, LDN193189, LDN214117, LDN212854, DMH2, K02288, ML347, SGC AAK11, PD407824, UK383367, and A01, recombinant mammalian noggin and dorsomorphin, non-mammalian noggin and dorsomorphin, etc. Any BMP inhibitor is contemplated for use herein to reduce or inhibit the BMP pathway. In some embodiments, the composition disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure can be a basal medium containing at least one BMP inhibitor. In some embodiments, the compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure can be a basal medium having at least one BMP inhibitor at a concentration of at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least 400 ng / ml, at least 500 ng / ml, or at least about 1000 ng / ml.In some embodiments, the compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may be a basal medium having a high protein concentration of at least 1.0% (w / v) or more, and may further comprise at least one BMP inhibitor at a concentration of about 5 ng / ml to about 350 ng / ml (e.g., about 10 ng / ml to about 150 ng / ml, about 15 ng / ml to about 100 ng / ml, or about 15 ng / ml to about 50 ng / ml).

[0049]

[0060] In certain embodiments, compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may include a basal medium having at least one activin A modulating agent (and at least one BMP modulating agent). In some embodiments, compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may include a basal medium having at least one activin A inhibitor (and at least one BMP modulating agent), and may optionally include low protein concentration levels as disclosed herein. Non-limiting examples of activin A inhibitors include A83-01, RepSox, D4476, Ly364947, R268712, SD208, SB505124, SM16, galunisertib, SB525334, and SB431542, among others. In some embodiments, compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may comprise a basal medium having at least one activin A inhibitor (and at least one BMP modulating agent) at a concentration of at least about 5 ng / ml to at least about 2 μg / ml, e.g., at least about 10 ng / ml, at least 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, at least 1 μg / ml, at least about 1.5 μg / ml, or at least about 2 μg / ml. In some embodiments, the compositions disclosed herein for culturing APS cells to generate DE cells according to the methods of the present disclosure may comprise a basal medium having at least one activin A inhibitor at a concentration of about 0.5 μg / ml to about 2 μg / ml (e.g., about 0.5 μg / ml to about 2.0 μg / ml or about 1 μg / ml).

[0050]

[0061] In certain embodiments, the compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure may be a basal medium having an enriched concentration of a protein as disclosed herein and at least one BMP modulating agent, at least one activin A modulating agent, or a combination thereof. In some embodiments, the compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure may comprise a basal medium having an enriched concentration of a protein and at least one BMP inhibitor, at least one activin A inhibitor, or a combination thereof. In some embodiments, the compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure may comprise a basal medium having an enriched concentration of a protein, at least one BMP inhibitor, and at least one activin A inhibitor.

[0051]

[0062] In some embodiments, compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure can include a basal medium having at least one activin A activator. In some embodiments, compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure can be a basal medium having at least one activin A activator at a concentration of, for example, at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure can be a basal medium having at least one activin A activator at a concentration of about 5 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 75 ng / ml or about 15 ng / ml to about 50 ng / ml). In some embodiments, compositions disclosed herein for culturing APS cells to produce DE cells according to the methods of the present disclosure can be a basal medium having at least one activin A activator at a concentration of about 5 ng / ml, about 10 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, or about 200 ng / ml.

[0052]

[0063] In certain embodiments, the compositions disclosed herein for culturing APS cells to produce DE cells may be provided to a cell culture after the previous culture medium has been removed or washed away, as needed. According to these embodiments, the compositions disclosed herein for culturing APS cells to produce DE cells may be provided to an APS cell culture after the previous culture medium has been removed, with or without a wash step before the introduction of fresh medium.

[0053]

[0064] In certain embodiments, a composition disclosed herein for culturing APS cells to produce DE cells can be provided to an APS cell culture and incubated for a suitable period of time for the APS cells to differentiate into DE cells. In some embodiments, a composition disclosed herein for culturing APS cells to produce DE cells can be provided to an APS cell culture and incubated for a suitable period of time for at least about 70% to at least about 99% or 100% of the APS cells to differentiate into DE cells. In some embodiments, a composition disclosed herein for culturing APS cells to produce DE cells can be provided to an APS cell culture and incubated for a suitable period of time for about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or 100% of the APS cells to differentiate into DE cells. In some embodiments, a composition disclosed herein for culturing APS cells to produce DE cells can be provided to an APS cell culture and incubated for a period of time for about 12 hours to about 5 days. In some embodiments, the compositions disclosed herein for culturing APS cells to generate DE cells may be provided to a cell culture and incubated for a period of less than about 1 day, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days.

[0054] Stage 3 and 4—DE cells are cultured to generate AFE cells and AFE cells are cultured to generate VPE cells, or DE cells are cultured to generate PE cells and PE cells are cultured to generate VPE cells.

[0055]

[0065] In certain embodiments, the compositions disclosed herein can be used to provide conditions for culturing DE cells to produce PE and / or AFE cells, and / or conditions for culturing PE and / or AFE cells to produce VPE cells, according to the methods of the present disclosure. According to these embodiments, the compositions disclosed herein can comprise a basal medium having a high concentration of protein (e.g., a "protein-rich medium"). In some embodiments, the compositions disclosed herein can be used to provide conditions for culturing DE cells to produce AFE cells and / or conditions for culturing AFE cells to produce VPE cells, according to the methods of the present disclosure, and can comprise a basal medium having a high protein concentration of at least about 1.0% (w / v) or more. In some embodiments, a basal medium with a high protein concentration for use herein may have a protein concentration of at least 0.5% (w / v) or more, or about 1.0% (w / v) or more, or about 2.5% (w / v) or more, or about 5.0% (w / v) or more, or about 7.5% (w / v) or more, or about 10.0% (w / v) or more, or about 15.0% (w / v) or more, or about 20% (w / v) or more, or about 25% or more, up to about 30% (w / v). In some embodiments, a basal medium with a high protein concentration for use herein may have a protein concentration ranging from at least about 1.0% (w / v) to about 30% (w / v) protein. According to these embodiments, the protein used to supplement the media disclosed herein may be derived from any source. In certain embodiments, proteins may be supplemented using serum, such as human serum albumin (HSA), bovine serum albumin (BSA), fetal bovine serum (FBS), or other serum, or other protein sources used in culture media.

[0056]

[0066] In some embodiments, the culture medium may be washed off the differentiated cells disclosed herein, and the washed differentiated cells may be used in pharmaceutical compositions. In certain embodiments, the cells may be harvested and stored in a buffer solution for long-term storage and transportation at low temperatures for later use. In other embodiments, the differentiated cells disclosed herein may be quickly frozen and stored for later use in therapy or for culture.

[0057]

[0067] In certain embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a basal medium having at least one bone morphogenetic protein (BMP) modulating agent. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a basal medium having at least one BMP activator. In certain embodiments, these culture compositions may no longer contain a BMP pathway inhibitor. Non-limiting examples of BMP activators include, but are not limited to, BMP4, SB4, and the like. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a protein-rich medium having at least about 0.5% (w / v) protein or more. In some embodiments, a basal medium with a high protein concentration for use herein may have a protein concentration of at least about 0.5% (w / v) or more, or about 1.0% (w / v) or more, or about 2.5% (w / v) or more, or about 5.0% (w / v) or more, or about 7.5% (w / v) or more, or about 10.0% (w / v) or more, or about 15.0% (w / v) or more, or about 20% (w / v) or more, or 25% or more, up to about 30% (w / v) of protein and at least one BMP activator. The concentration of the BMP activator can be at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml.In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a protein-enriched medium having at least one BMP activator at a concentration of about 1 ng / ml to about 100 ng / ml (e.g., about 10 ng / ml to about 75 ng / ml or about 15 ng / ml to about 50 ng / ml). In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or culturing AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a protein-enriched medium having at least one BMP activator at a concentration of about 1.0 ng / ml to about 150 ng / ml, or about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml.

[0058]

[0068] In certain embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or AFE cells to generate VPE cells according to the methods of the present disclosure may comprise a medium having an enriched protein concentration of at least about 0.5% (w / v) to about 30% (w / v), or about 0.5% to about 20% (w / v), and at least one fibroblast growth factor receptor (FGFR) activator (and at least one BMP activator). As used herein, "FGFR activator" may refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that activates FGFR and / or the FGFR signaling pathway. In some embodiments, the compositions disclosed herein for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells according to the methods of the present disclosure can be a medium having an enriched protein concentration of at least 0.5% (w / v) and at least one FGFR activator (and at least one BMP activator). In some embodiments, the compositions disclosed herein for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells according to the methods of the present disclosure can be a basal medium having at least one FGFR3 activator. Non-limiting examples of FGFR3 activators suitable for use herein can include fibroblast growth factors (FGFs), such as FGF8, FGF1, FGF2, FGF9, or variants thereof, heparin, etc.

[0059]

[0069] In some embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or culturing AFE cells to generate VPE cells according to the methods of the present disclosure may be a medium having a high protein concentration of at least 0.5% (w / v) and at least one FGFR3 activator (e.g., FGF8, FGF1, FGF2, FGF9, or a variant thereof). According to these embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or culturing AFE cells to generate VPE cells according to the methods of the present disclosure can be media having a high protein concentration of at least 0.5% (w / v) and at least one FGFR3 activator (e.g., FGF8, FGF1, FGF2, FGF9, or a variant thereof) (and at least one BMP activator) at a concentration of at least about 5.0 ng / ml to about 150 ng / ml, 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, FGF may be present in cell culture medium at a concentration ranging from about 10 ng / ml to about 100 ng / ml (e.g., about 20 ng / ml to about 100 ng / ml, or about 30 ng / ml to about 100 ng / ml). In some embodiments, FGF may be present in cell culture medium at a concentration of about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml, or about 150 ng / ml.

[0060]

[0070] In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells according to the methods of the present disclosure may comprise a medium having a high protein concentration of at least 0.5% (w / v) and including heparin (and optionally, at least one of at least one BMP activator and at least one FGFR3 activator). According to these embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells according to the methods of the present disclosure may include a high protein medium including heparin at a concentration ranging from about 1.0 μg / ml to about 100 μg / ml. In certain embodiments, the concentration of heparin in the culture medium is about 1 μg / ml, about 2 μg / ml, about 4 μg / ml, about 6 μg / ml, about 8 μg / ml, about 10 μg / ml, about 12 μg / ml, about 14 μg / ml, about 16 μg / ml, about 18 μg / ml, or about 20 μg / ml.

[0061]

[0071] In certain embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or culturing AFE cells to generate VPE cells according to the methods of the present disclosure may comprise a medium having an enriched protein concentration of at least 0.5% (w / v) protein and at least one retinoic acid signaling modulator (and optionally, heparin and at least one BMP activator and at least one FGFR3 activator). As used herein, "retinoic acid signaling modulator" may refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that activates retinoic acid, a retinoic acid receptor, and / or a signaling pathway. In some embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or AFE cells to generate VPE cells according to the methods of the present disclosure can include a medium having a high protein concentration of at least 0.5% (w / v) protein and at least one retinoic acid signaling activator (and optionally heparin and at least one BMP activator and at least one FGFR3 activator). Non-limiting examples of retinoic acid signaling activators include, but are not limited to, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, Ch55, and the like.In some embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or AFE cells to generate VPE cells according to the methods of the present disclosure may comprise a medium having a high protein concentration of at least 0.5% (w / v) protein and at least one retinoic acid signal activator (and optionally heparin and at least one BMP activator and at least one FGFR3 activator), wherein the retinoic acid signal activator may be present in the compositions disclosed herein at a concentration ranging from about 0.1 nM / ml to about 3,000 nM / ml; from about 1.0 nM / ml to about 2,500 nM / ml, from about 1.5 nM / ml to about 2,000 nM / ml, or any concentration therebetween, to obtain the desired signaling response. In some embodiments, the retinoic acid signaling activator is present in the compositions disclosed herein at a concentration of about 0.1 nM, about 0.5 nM, about 1.0 nM, about 2.5 nM, about 5.0 nM, about 10 nM, about 15 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about It may be present at a concentration of 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1.0 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2.0 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, or about 3.0 μM.

[0062]

[0072] In certain embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or PE cells and / or for culturing AFE cells and / or PE cells to generate VPE cells according to the methods of the present disclosure may comprise a medium having a high concentration of protein and at least one of at least one BMP activator, at least one FGFR3 activator, at least one FGFR2 activator, at least one NOTCH activator, or a combination thereof. In certain embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or PE cells to generate VPE cells according to the methods herein may comprise a medium having a high concentration of protein and at least one of at least one BMP activator, at least one FGFR3 activator, at least one FGFR2 activator, at least one NOTCH activator, at least one agent that activates retinoic acid receptor signaling, or a combination thereof. In certain embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or PE cells and / or for culturing AFE cells and / or PE cells to generate VPE cells according to the methods of the present disclosure can be a medium having a high concentration of protein and at least one BMP activator, at least one FGFR3 activator, at least one FGFR2 activator, at least one NOTCH activator, at least one agent that activates retinoic acid receptor signaling, one sonic hedgehog signaling activator, or a combination thereof.In certain embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or PE cells and / or for culturing AFE cells and / or PE cells to generate VPE cells according to the methods of the present disclosure can be a medium having a high concentration of protein and at least one BMP activator, at least one agent that activates retinoic acid receptor signaling, at least one inhibitor of TGF-β receptor signaling, and at least one sonic hedgehog signaling activator, at least one VEGF receptor activator, or a combination thereof.

[0063]

[0073] In certain embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or culturing AFE cells to generate VPE cells according to the methods of the present disclosure can be media having a high concentration of protein and at least one BMP modulating agent, at least one FGFR3 activator, at least one retinoic acid signaling activator, or a combination thereof. In some embodiments, the compositions disclosed herein for culturing DE cells to generate AFE cells and / or culturing AFE cells to generate VPE cells according to the methods of the present disclosure can be compositions or media having a high concentration of protein and at least one BMP activator, at least one FGFR3 activator, at least one retinoic acid signaling activator, or a combination thereof. In some embodiments, compositions disclosed herein for culturing DE cells to generate AFE cells and / or AFE cells to generate VPE cells according to the methods of the present disclosure may include compositions or media having high concentrations of protein (e.g., enriched concentrations) including at least one of at least one BMP activator, at least one FGFR3 activator, at least one retinoic acid signaling activator, and heparin. In certain embodiments, these media compositions may further include at least one Wnt pathway signaling activator and / or NOTCH pathway signaling activator.

[0064]

[0074] In certain embodiments, the compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells do not comprise a transforming growth factor-β (TGF-β) modulator, hi certain embodiments, the compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells do not comprise a TGF-β inhibitor.

[0065]

[0075] In certain embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells may replace cell culture medium after the previous culture medium has been removed and, if necessary, washed to remove residual agents. According to these embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells may be provided to a cell culture after the previous culture medium has been removed, with or without a washing step before application.

[0066]

[0076] In certain embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a suitable period of time to differentiate the DE cells into AFE cells. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a suitable period of time to differentiate at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% to about 99%, or about 100% of the DE cells into AFE cells. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a period of time suitable for about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, or about 100% of the DE cells to differentiate into AFE cells.

[0067]

[0077] In certain embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a suitable period of time to differentiate the AFE cells into VPE cells. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a suitable period of time to differentiate at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, or about 100% of the AFE cells into VPE cells. In some embodiments, compositions disclosed herein for culturing DE cells to produce AFE cells and / or compositions disclosed herein for culturing AFE cells to produce VPE cells can be provided to a cell culture and incubated for a period of time suitable for about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, or 100% of the AFE cells to differentiate into VPE cells.

[0068]

[0078] In some embodiments, culturing DE cells to produce AFE cells and / or culturing AFE cells to produce VPE cells can include culturing the cells in a medium having a supplement as described herein for a period of about 1 day to about 9 days. In some embodiments, compositions for culturing DE cells to produce AFE cells and / or AFE cells to produce VPE cells can include, but are not limited to, culturing the cells in a composition disclosed herein for a period of about 12 hours to about 1 day, to about 2 days, to about 3 days, to about 4 days, or about 5 days to about 9 days or more.

[0069]

[0079] In some embodiments, culturing DE cells to produce AFE cells and / or culturing AFE cells to produce VPE cells can include culturing the cells for a period of about 1 day to about 9 days in a medium having a supplement as provided herein. In some embodiments, a composition for culturing DE cells to produce AFE cells and / or culturing AFE cells to produce VPE cells can include, but is not limited to, culturing the cells in a composition disclosed herein for a period of about 12 hours to about 1 day, to about 2 days, to about 3 days, to about 4 days, or about 5 days to about 9 days or more. In certain embodiments, a composition disclosed herein for culturing DE cells to produce AFE cells and / or culturing AFE cells to produce VPE cells according to the methods of the present disclosure can be a medium having a high concentration of protein and at least one NOTCH pathway signaling activator, at least one CD40 pathway signaling activator, and / or at least one RANK pathway signaling activator. According to these embodiments, the compositions disclosed herein comprising at least one NOTCH pathway activator may include, but are not limited to, Yhhu3792, DLL1, DLL3, DLL4, Jagged1, and Jagged2. In certain embodiments, the compositions disclosed herein may include, or further comprise, at least one CD40 pathway activator. According to these embodiments, the at least one CD40 pathway activator may include, but is not limited to, CD40 ligand, multimeric CD40 ligand, CD40 ligand and a crosslinking agent, and an anti-CD40 antibody with an antibody crosslinking agent. In some embodiments, the compositions disclosed herein may include, or further comprise, at least one RANK pathway activator. According to these embodiments, the at least one RANK pathway activator may include, but is not limited to, a soluble RANK ligand, a RANK ligand, a multimeric RANK ligand, a RANK ligand and a crosslinking agent, and an anti-RANK antibody with an antibody crosslinking agent.In other embodiments, the compositions disclosed herein may further comprise at least one ghrelin receptor pathway activator. According to embodiments, the at least one ghrelin receptor pathway activator may include, but is not limited to, ghrelin, MK0677, tabimorelin hemifumarate, and L-692,585. In certain embodiments, the compositions disclosed herein may comprise at least one NOTCH pathway activator and at least one of at least one CD40 pathway activator and at least one RANK pathway activator. According to these embodiments, the compositions disclosed herein may comprise a protein-enriched culture medium (e.g., MEM alpha medium).

[0070] Stage 5 - Culturing VPE or TPPE cells to generate thymocytes

[0080] In certain embodiments, the compositions disclosed herein can be used to provide conditions for culturing VPE cells and / or TPPE cells to generate thymocytes (TEC and / or TEP cells) according to the methods of the present disclosure. According to these embodiments, the compositions disclosed herein can comprise a basal medium having a high or enriched concentration of protein (e.g., a "protein-rich medium"). In some embodiments, the high protein concentration can comprise at least about 0.5% (w / v) to about 30% (w / v). In some embodiments, a basal medium having a high protein concentration for use herein can comprise a protein concentration of at least 0.5% (w / v) to about 1.0% (w / v), or about 2.0% (w / v), or about 3.0% (w / v), or about 4.0% (w / v), or about 5.0% (w / v), or about 10% (w / v), or about 20% (w / v), or about 30% (w / v) protein, or any concentration between at least 0.5% (w / v) and 30.0% (w / v). In some embodiments, a basal medium having an enriched protein concentration for use in the compositions disclosed herein may contain a protein concentration in the range of at least about 0.5% (w / v) to about 30% (w / v) (e.g., about 1.0% (w / v), about 5.0% (w / v), about 10% (w / v), about 15% (w / v), about 20% (w / v), about 25% (w / v), about 30% (w / v)).

[0071]

[0081] In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium and at least one BMP modulating agent. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium and at least one BMP inhibitor. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium and at least one BMP inhibitor. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium and at least about 5 ng / ml to about 1,000 ng / ml of a BMP modulating agent. The composition may comprise at least one BMP inhibitor at a concentration of at least about 1000 ng / ml, or at least about 1000 ng / ml, or any concentration between 10 ng / ml and 1,000 ng / ml, e.g., at least 10 ng / ml, at least 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium and at least one BMP inhibitor at a concentration of about 5 ng / ml to about 300 ng / ml (e.g., about 10 ng / ml to about 250 ng / ml or about 15 ng / ml to about 100 ng / ml).

[0072]

[0082] In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the disclosure may comprise a protein-enriched medium having at least one activin A modulator. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the disclosure may comprise a protein-enriched medium having at least one activin A activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein and up to about 30% (w / v) protein, and at least one activin A activator at a concentration of, for example, at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium having at least 1% (w / v) protein and up to 30% (w / v) protein, and at least one activin A activator at a concentration of about 5 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 100 ng / ml or about 15 ng / ml to about 50 ng / ml).

[0073]

[0083] In certain embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30% (w / v) protein, and at least one retinoic acid signaling modulator (and optionally at least one activin A activator and a BMP inhibitor). In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30% (w / v) protein, and at least one retinoic acid signaling activator. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium having at least 0.5% (w / v) protein up to about 30% (w / v) protein, and at least one retinoic acid signaling activator at a concentration ranging from about 0.1 nM / ml to about 3,000 nM / ml, about 1.0 nM / ml to about 2,500 nM / ml, about 1.5 nM / ml to about 2,000 nM / ml, or any concentration therebetween, to obtain a desired signaling response.In some embodiments, the retinoic acid signaling activator is present in the compositions disclosed herein at a concentration of about 0.1 nM, about 0.5 nM, about 1.0 nM, about 2.5 nM, about 5.0 nM, about 10 nM, about 15 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about It may be present at a concentration of 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1.0 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2.0 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, or about 3.0 μM. In some embodiments, the retinoic acid signaling activator may be present in the cell culture medium at a concentration ranging from about 0.1 nM to about 3.0 μM (e.g., about 0.5 nM to about 2.5 μM or about 1 nM to about 2 μM), and may further include at least one activin A activator and a BMP inhibitor.

[0074]

[0084] In certain embodiments, in addition to the above paragraphs

[0038] to

[0077] , the concentration of an agent disclosed herein used to supplement a medium disclosed herein (e.g., a low-protein medium or a protein-rich medium) can be about 1 nM to about 10 μM at a final concentration. In some embodiments, the concentration of an agent disclosed herein used to supplement a medium disclosed herein can be about 5 to about 15 (e.g., about 9 nM) for small molecules and about 0.5 μM to about 5 μM (e.g., about 2 μM) for biomolecules or other agents.

[0075]

[0085] In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure can include a protein-rich medium having at least 0.5% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising heparin (and optionally at least one of at least one activin A activator, a BMP inhibitor, and at least one retinoic acid receptor activator). According to these embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure can include a protein-rich medium having at least 0.5% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising heparin at a concentration ranging from about 1 μg / ml to about 100 μg / ml. In certain embodiments, the concentration of heparin in the culture medium is about 1 μg / ml, about 2 μg / ml, about 4 μg / ml, about 6 μg / ml, about 8 μg / ml, about 10 μg / ml, about 12 μg / ml, about 14 μg / ml, about 16 μg / ml, about 18 μg / ml, or about 20 μg / ml.

[0076]

[0086] In certain embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30.0% (w / v) protein, and further comprising at least one fibroblast growth factor receptor (FGFR) modulating agent. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30.0% (w / v) protein, and further comprising at least one FGFR activator (and optionally at least one of at least one activin A activator, a BMP inhibitor, at least one retinoic acid receptor activator, and heparin). In some embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising at least one FGFR activator (and optionally at least one of at least one activin A activator, a BMP inhibitor, and at least one retinoic acid receptor activator, and heparin), including, but not limited to, at least one FGFR1 activator, at least one FGFR2 activator, or a combination thereof. Non-limiting examples of FGFR1 and FGFR2 activators suitable for use herein include fibroblast growth factors (FGFs), such as FGF10, FGF3, FGF4, FGF7, and FGF22, or variants thereof.In some embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein and up to about 30% (w / v) protein, and further comprising at least one FGFR activator, including, but not limited to, FGF (e.g., FGF10, FGF3, FGF4, FGF7, and FGF22, or variants thereof) (and optionally at least one of at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, and heparin). According to these embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30% (w / v) protein, and may be at least about 0.5 ng / ml, at least about 1.0 ng / ml, at least about 1.5 ng / ml, at least about 2.0 ng / ml, at least about 2.5 ng / ml, at least about 3.0 ng / ml, at least about 3.5 ng / ml, at least about 4.0 ng / ml, at least about 4.5 ng / ml, at least about 5.0 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 30 ... and further comprising at least one FGFR activator (and optionally at least one of at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, and heparin), including but not limited to FGF (e.g., FGF10, FGF3, FGF4, FGF7, and FGF22, or variants thereof) at a concentration of at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml.In some embodiments, FGF may be present in the cell culture medium at a concentration ranging from about 0.5 ng / ml to about 100 ng / ml (e.g., from about 1.0 ng / ml to about 100 ng / ml, or from about 2.0 ng / ml to about 100 ng / ml).

[0077]

[0087] In certain embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a basal medium having at least one hedgehog signaling modulator, including sonic hedgehog (Shh). As used herein, "Shh-modulating agent" may refer to any chemical (e.g., a small molecule or other chemical agent), compound, polypeptide, protein, or fragment thereof, polynucleotide (DNA or RNA), or other agent that modulates the hedgehog and / or Shh and / or Shh signaling pathway. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 1.0% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising at least one Shh inhibitor (and optionally at least one of at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, at least one FGFR2 activator, and heparin). Non-limiting Shh inhibitors suitable for use as disclosed herein may include SANT1, SANT2, U1866A, dynapyrazole a, dynalestin, cyclopamine, HIP1, GANT58, AY9944 dihydrochloride, RU-SKI43 hydrochloride, and the like.In some embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the disclosure may comprise a protein-rich medium having at least 1.0% (w / v) protein and up to about 30.0% (w / v) protein, and may include at least about 0.5 nM, about 1.0 nM, about 2.0 nM, about 3.0 nM, about 4.0 nM, about 5.0 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, about 250 nM, about 275 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 460 nM, about 470 nM, about 480 nM, about 490 nM, about 510 nM, about 520 nM, about 530 nM, about 540 nM, about 550 nM, about 560 nM, about 570 nM, about 580 nM, about 590 nM, about 600 nM, about 610 nM, about 620 nM, about 630 nM, about 640 nM, about 650 nM, about 660 nM, about 670 nM, about 680 nM, about 690 nM, about 700 nM, about 710 nM, about 720 nM, about 730 nM, about 740 nM, about 750 nM, about 760 n at least one Shh inhibitor (and optionally at least one of at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, at least one FGFR2 activator, and heparin) at a concentration of about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 725 nM, about 750 nM, about 775 nM, about 800 nM, about 825 nM, about 850 nM, about 875 nM, about 900 nM, about 925 nM, about 950 nM, about 975 nM, or about 1000 nM.

[0078]

[0088] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a basal medium having an enriched protein concentration and at least one NOTCH pathway signaling activator, at least one CD40 pathway signaling activator, and / or at least one RANK pathway signaling activator. According to these embodiments, compositions disclosed herein having at least one NOTCH pathway activator may include, but are not limited to, Yhhu3792, DLL1, DLL3, DLL4, Jagged1, and Jagged2. In certain embodiments, compositions disclosed herein may include, or further include, at least one CD40 pathway activator. According to these embodiments, at least one CD40 pathway activator may include, but is not limited to, CD40 ligand, multimeric CD40 ligand, CD40 ligand and crosslinker, and anti-CD40 antibody with antibody crosslinker. In some embodiments, compositions disclosed herein may include, or further include, at least one RANK pathway activator. According to these embodiments, the at least one RANK pathway activator may include, but is not limited to, a soluble RANK ligand, a RANK ligand, a multimeric RANK ligand, a RANK ligand and a crosslinker, and an anti-RANK antibody with an antibody crosslinker. In other embodiments, the compositions disclosed herein may further include at least one ghrelin receptor pathway activator. According to embodiments, the at least one ghrelin receptor pathway activator may include, but is not limited to, ghrelin, MK0677, tabimorelin hemifumarate, and L-692,585. In certain embodiments, the compositions disclosed herein may include at least one NOTCH pathway activator and at least one of at least one CD40 pathway activator and at least one RANK pathway activator. According to these embodiments, the compositions disclosed herein may include a protein-enriched culture medium (e.g., MEM alpha medium).

[0079]

[0089] In certain embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising at least one epidermal growth factor (EGF) (and optionally at least one of at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, at least one FGFR2 activator, at least one Shh inhibitor, and heparin). In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein and up to about 30.0% (w / v) protein, and further comprising at least one epidermal growth factor (EGF) (and optionally at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, at least one FGFR2 activator, at least one Shh inhibitor, and heparin), wherein the EGF is present at a concentration ranging from about 0.1 ng / ml to about 30.0 ng / ml.In some embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium having at least 0.5% (w / v) protein up to about 30.0% (w / v) protein, and further comprising at least one epidermal growth factor (EGF) (and optionally at least one activin A activator, at least one BMP inhibitor, and at least one retinoic acid receptor activator, at least one FGFR2 activator, at least one Shh inhibitor, and heparin), and about 0.1 ng / ml, about 0.5ng / ml, about 1.0ng / ml, about 5.0ng / ml, about 10ng / ml, about 15ng / ml, about 20ng / ml, about 25ng / ml, about 30ng / ml, about 40ng / ml, about 50ng / ml, about 75ng / ml, about 100ng / ml, about 125ng / ml, about 150ng / ml, about 175ng / ml, about 200ng / ml, about 225ng / ml, about 250ng / ml, about 275ng / ml, about 300ng / ml, about 325ng / ml, about 350ng / ml, about 375ng / ml, about 400ng / ml, about 425ng / ml, about 450ng / ml, about 475ng / ml, or about 500ng / ml.

[0080]

[0090] In certain embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one Wnt modulating agent. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may be in a protein-rich medium, optionally including at least one Wnt activator. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may be a basal medium, optionally including at least one Wnt activator at a concentration ranging from about 5 ng / ml to about 200 ng / ml (e.g., from about 10 ng / ml to about 150 ng / ml, or from about 15 ng / ml to about 100 ng / ml).

[0081]

[0091] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may optionally comprise a protein-enriched medium containing at least one NOTCH activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may optionally comprise a protein-enriched medium containing at least one NOTCH activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-enriched medium, optionally containing at least one NOTCH activator at a concentration ranging from about 5 ng / ml to about 100 ng / ml (e.g., from about 10 ng / ml to about 75 ng / ml, or from about 15 ng / ml to about 50 ng / ml).

[0082]

[0092] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may optionally include a protein-rich medium containing at least one RANK activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may optionally include a protein-rich medium containing at least one RANK activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may include a protein-rich medium, optionally containing at least one RANK activator at a concentration ranging from about 5 ng / ml to about 100 ng / ml (e.g., from about 10 ng / ml to about 75 ng / ml, or from about 15 ng / ml to about 50 ng / ml).

[0083]

[0093] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one CD40 activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one CD40 activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one CD40 activator at a concentration ranging from about 5 ng / ml to about 100 ng / ml (e.g., from about 10 ng / ml to about 75 ng / ml, or from about 15 ng / ml to about 50 ng / ml).

[0084]

[0094] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one ghrelin receptor signaling activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one ghrelin receptor signaling activator. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-rich medium, optionally including at least one ghrelin receptor signaling activator at a concentration ranging from about 5 ng / ml to about 100 ng / ml (e.g., from about 10 ng / ml to about 75 ng / ml, or from about 10 ng / ml to about 30 ng / ml).

[0085]

[0095] In certain embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes according to the methods of the present disclosure may comprise a protein-enriched basal medium and at least one activin A modulator, at least one BMP modulator, at least one FGFR modulator, at least one retinoic acid signaling modulator, at least one Shh modulator, and optionally a Wnt activator and / or a NOTCH activator, or a combination thereof.

[0086]

[0096] In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure can include a base having an enriched protein content and at least one activin A activator, at least one BMP inhibitor, at least one FGFR activator, at least one retinoic acid signaling activator, at least one Shh inhibitor, or a combination thereof. In some embodiments, compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes according to the methods of the present disclosure can be a basal medium with a high concentration of protein and includes at least one activin A activator, at least one BMP inhibitor, at least one FGFR1 activator / FGFR2 activator, at least one retinoic acid signaling activator, at least one Shh inhibitor, heparin, and EGF.

[0087]

[0097] In certain embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes do not comprise a transforming growth factor-β (TGF-β) modulating agent. In certain embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes do not comprise a (TGF-β) inhibitor.

[0088]

[0098] In certain embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes may be provided to the cell culture after the previous culture medium has been removed. According to these embodiments, the compositions disclosed herein for culturing VPE and / or TPPE cells to generate thymocytes may be provided to the cell culture after the previous culture medium has been removed, without a washing step before application.

[0089]

[0099] In certain embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes can be provided to a cell culture for a period suitable for differentiating the VPE cells and / or TPPE cells into thymocytes. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes can be provided to a cell culture for a period suitable for at least about 70% to at least about 99% of the VPE cells and / or TPPE cells to differentiate into thymocytes. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes can be provided to a cell culture for a period suitable for about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the VPE cells and / or TPPE cells to differentiate into thymocytes. In some embodiments, compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes can be introduced into a cell culture and incubated for a period of about 1 day to about 5 days. In some embodiments, the compositions disclosed herein for culturing VPE cells and / or TPPE cells to generate thymocytes can be provided to a cell culture and incubated for a period of less than about 1 day, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days.

[0090]

[0100] In certain embodiments, one or more additional supplements may be added to any of the compositions described herein to provide cells with trace elements for improved proliferation and expansion. In certain embodiments, supplemental trace elements may include, but are not limited to, iron (Fe), copper (Cu), zinc (Zn), rubidium (Rb), selenium (Se), strontium (Sr), molybdenum (Mo), manganese (Mn), lead (Pb), arsenic (As), chromium (Cr), cobalt (Co), vanadium (V), and cadmium (Cd). In certain embodiments, supplemental trace elements may include, but are not limited to, iron (Fe) (hemoglobin), copper (Cu), cobalt (Co) (vitamin B12), iodine, manganese (Mn), and zinc (Zn), or combinations thereof. In other embodiments, additional supplements may be used in compositions used in one or more stages of differentiation to produce thymocytes from PSCs. Such supplements may include, but are not limited to, insulin, transferrin, sodium selenium, and combinations thereof. These ingredients may be included in any known acceptable form. In some embodiments, these agents may be present in a salt solution, including, but not limited to, Hank's Balanced Salt Solution™ (HBSS), Earle's Salt Solution™, antioxidant supplements, MCDB-201™ supplements, phosphate-buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid, and / or ascorbic acid-2-phosphate, and additional amino acids. In some examples, amino acids used herein may include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. In other embodiments,

[0091]

[0101] In other embodiments, antibiotics can be used in cell cultures as needed to reduce or eliminate bacterial, mycoplasmal, and fungal contamination. Typically, the antibiotic or antifungal compound used is a penicillin / streptomycin mixture, but may also include, but is not limited to, amphotericin (Fungizone®), ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tyrosine, and zeocin.

[0092]

[0102] Hormones may also be used in cell culture, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. β-mercaptoethanol and other hormones are contemplated herein.

[0093]

[0103] Lipids and lipid carriers can also be used to supplement cell culture media, depending on the type of cell and the fate of differentiated cells. Such lipids and carriers can include, but are not limited to, cyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic acid-oleic acid-arachidonic acid conjugated to albumin, oleic acid not conjugated to albumin, and oleic acid conjugated to albumin.

[0094]

[0104] In some embodiments, the compositions herein may comprise a basal medium supplemented with an insulin-transferrin-selenium (ITS) supplement. According to these embodiments, the ITS may be provided in the medium at a concentration ranging from about 1:10 (v / v) to about 1:10,000 (v / v), or at a concentration greater than about 1:200 (v / v). In some embodiments, the concentration of ITS in the medium may be about 1:1000 (v / v), about 1:900 (v / v), about 1:800 (v / v), about 1:700 (v / v), about 1:600 ​​(v / v), about 1:500 (v / v), about 1:400 (v / v), about 1:300 (v / v), about 1:200 (v / v), about 1:100 (v / v), or about 1:50 (v / v).

[0095]

[0105] In certain embodiments, further to the preceding paragraph, the compositions herein may include a basal medium supplemented with cortisol, e.g., hydrocortisone. According to these embodiments, cortisol may be provided in the medium at a concentration ranging from about 0.05 μg / ml to about 5 μg / ml. In some embodiments, for example, the concentration of hydrocortisone in the culture medium is about 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, or about 1.0 μg / ml. In certain embodiments, the basal medium disclosed herein may further include serum or other protein supplements or other agents contemplated herein.

[0096]

[0106] In some embodiments, cells of the present disclosure in culture can be maintained either in suspension or attached to a solid support (e.g., a coated plate or where extracellular matrix components and synthetic or biopolymers are included). Cells can also be supplemented with additional factors that promote their attachment to the solid support, including, but not limited to, types I, II, and IV collagen, concanavalin A, chondroitin sulfate, fibronectin, "superfibronectin" and / or fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, Matrigel™, thrombospondin, and / or vitronectin. In certain embodiments, multiwell plates can be used (e.g., G-Rex culture plates).

[0097]

[0107] In certain embodiments, cell populations cultured according to the methods disclosed herein can be monitored to assess cellular changes imparted by culturing (e.g., during certain stages of the culture methods disclosed herein) to characterize the cell populations produced. In some embodiments, the production of APS cells, DE cells, AFE cells, VPE cells, pharyngeal endoderm (PE) cells, third pharyngeal pouch endoderm (TPPE) cells, TEP cells, and / or TECs, including various subpopulations of TECs, can be assessed by determining the expression of markers characteristic of these cell populations. In some embodiments, DE cell identification markers may include protein markers and may include SOX17+ and / or FOXA2. In other embodiments, AFE cell identification protein markers expressed herein may include SOX2+, FOXA2+, and / or SOX17-. In certain embodiments, VPE cell identification markers may include HOXA3+, HOXB1-, and / or NKX2.1-. In some embodiments, thymic (e.g., TEP / TEC) cell identification markers may include SOX17-, SOX2+ and / or FOXA2+; EPCAM+CD104+; EPCAM+CD205+, HLA-class II+; or any combination thereof. In certain embodiments, thymic (e.g., TEP / TEC) cell identification markers may include EPCAM+CD104+, FOXN1, HLA-class II+, KRT5+, and / or KRT8+, and more than about 5%, more than about 8%, more than about 10%, more than about 11%, more than about 12%, more than about 13%, more than about 14%, or more than about 15% of the cells may be identified as EPCAM+CD104+KRT5+ and / or KRT8+. In some embodiments, thymic (e.g., TEP / TEC) cell identification markers may include EPCAM+CD205+, and greater than about 55%, greater than about 56%, greater than about 57%, greater than about 58%, greater than about 59%, greater than about 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, or greater than 65% of the cells may be identified as EPCAM+CD104+.In some embodiments, thymic (e.g., TEP / TEC) cell identification markers may include SOX17-, SOX2+ and / or FOXA2+; EPCAM+CD104+; CD205+, HLA-class II+, FOXN1+, KRT5+, KRT8+; or any combination thereof, and greater than about 55%, greater than about 56%, greater than about 57%, greater than about 58%, greater than about 59%, greater than about 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, or greater than 65% of the cells may be identified as SOX17-, SOX2+ and / or FOXA2+; CD104+; EPCAM+CD205+, HLA-class II+, FOXN1+, KRT5+, KRT8+; or any combination thereof. In certain embodiments, thymic (e.g., TEP / TEC) cell identification markers include HLA-class II+ molecules, FOXN1+, KRT5+, KRT8+; or a combination thereof, and more than about 55%, more than about 56%, more than about 57%, more than about 58%, more than about 59%, more than about 60%, more than 61%, more than 62%, more than 63%, more than 64%, or more than 65% of cells can be identified as expressing these protein markers. According to these embodiments, the expression of cellular markers can be determined by detecting the presence or absence of a marker (e.g., detecting protein expression) and / or by analyzing the expression of a particular marker by measuring the level at which the marker is present in cells of a cell culture or cell population (e.g., detecting protein concentration). Other methods known in the art can also be used to detect and / or quantify marker gene expression. Non-limiting examples of methods suitable for use herein include PCR, RT-PCR, qRT-PCR, immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and the like.

[0098]

[0108] In certain embodiments, the methods disclosed herein provide a method of preventing, reducing the occurrence of, and / or treating one or more immune-mediated diseases or conditions in a subject by administering a composition, including, but not limited to, thymocytes prepared by the compositions and methods disclosed herein. Non-limiting examples of such immune-mediated diseases or conditions include graft-versus-host disease (GvHD), DiGeorge syndrome, inflammatory bowel disease (IBD), Crohn's disease, type 1 diabetes, kidney disease or kidney condition or injury, psoriasis, asthma, allergies, rheumatoid arthritis, ankylosing spondylitis, heart conditions or cardiovascular disease, psoriasis, psoriatic arthritis, Behcet's disease, arthritis, viral infections (e.g., DNA viruses (e.g., adenoviruses, herpes viruses (e.g., herpes simplex type 1, herpes simplex type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8)), papillomaviruses (e.g., human papillomavirus), poxviruses (e.g., smallpox), parvoviruses (e.g., human Examples of viruses that may be used include, but are not limited to, tobocavirus, parvovirus B19), hepadnaviridae (e.g., hepatitis B virus) and / or reovirus (e.g., rotavirus), RNA viruses (picornavirus (e.g., coxsackievirus, hepatitis A virus, poliovirus, rhinovirus), togavirus (rubella virus), orthomyxovirus (e.g., influenza virus), and / or rhabdovirus (e.g., rabies virus)), or reverse-transcribed viruses (including, but not limited to, retroviruses and hepadnaviruses, Retroviridae (human immunodeficiency virus (HIV)), Metaviridae, Pseudoviridae, Caulimoviridae, and Hepadnaviridae). In some embodiments, the condition requiring treatment in the subject is GvHD, type 1 diabetes, IBD, cardiovascular disease, renal injury, renal disease, etc. The present disclosure also contemplates methods for treating one or more immune-mediated conditions that occur in certain types of cancer.Such cancers include, but are not limited to, carcinoma (e.g., breast cancer, prostate cancer, lung cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), or colon cancer), sarcoma (e.g., cancer of bone, cartilage, neuron, or fat (e.g., liposarcoma)), lymphoma, leukemia (blood type cancer), blastoma (e.g., hepatoblastoma). In some embodiments, the condition in need of treatment in a subject may be, for example, restoration of immune function in a subject after a thymectomy. In some embodiments, the condition in need of treatment in a subject may be restoration of immune function in a subject after cancer treatment (e.g., chemotherapy, radiation therapy). In some embodiments, the condition in need of treatment in a subject may be restoration of immune function in a subject after one or more conditioning regimens (e.g., chemotherapy, radiation therapy) as part of a hematopoietic cell transplant (HCT). In some embodiments, the condition in need of treatment in a subject may be reversing age-related thymic involution in a subject. In some embodiments, the condition in need of treatment in a subject may be boosting thymic function in a subject. According to these embodiments, boosted thymic function in a subject (e.g., complementary and / or replacement therapy) can be used as a boost to the subject's response to a vaccine, anti-cancer therapy, and / or immunotherapy.

[0099]

[0109] As used herein, the term "subject" may refer to any mammal, including, but not limited to, a non-human primate (e.g., a monkey or ape), a livestock or pet, e.g., a cow, pig, cat, dog, rat, mouse, horse, goat, rabbit, sheep, hamster, guinea pig), or other subject. In some embodiments, the mammalian subject is a human, such as an adult, young child, adolescent, toddler, infant, or fetus. In some embodiments, the mammalian subject is a human, e.g., an elderly person, e.g., about 50 years of age or older, about 60 years of age or older, about 70 years of age or older, about 80 years of age or older, about 90 years of age or older, or about 100 years of age or older.

[0100]

[0110] In certain embodiments, thymic epithelial progenitor (TEP) cells and / or thymic epithelial cells (TECs) produced using the methods disclosed herein can be used to generate functional thymic epithelium (TECs) in a subject in need thereof. In certain embodiments, the methods disclosed herein can include transplanting TEP cells generated according to the methods disclosed herein into a subject. According to these embodiments, the TEP cells generated according to the methods disclosed herein can generate TECs after transplantation into a subject. In some embodiments, at least about 5% of the TEP cells generated according to the methods disclosed herein can differentiate into TEC cells after transplantation into a subject. In some embodiments, about 5% to about 99% of the TEP cells generated according to the methods disclosed herein can differentiate into TECs after transplantation into a subject. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the TEP cells generated according to the methods disclosed herein can differentiate into TECs after transplantation into a subject. In some embodiments, about 100% of the TEP cells generated according to the methods disclosed herein can differentiate into TECs or TEs after transplantation into a subject.

[0101]

[0111] In certain embodiments, the methods disclosed herein may include transplanting one or more subpopulations of TECs generated according to the compositions and methods disclosed herein into a subject. Examples of subpopulations of TECs generated according to the methods disclosed herein include, but are not limited to, cortical thymic epithelial cell (cTEC) lineage cells, unipotent, bipotent, and / or pluripotent TEP cells, committed medullary thymic epithelial cell (mTEC) precursors, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, and / or myoid cells. According to these embodiments, one or more subpopulations of TECs generated according to the methods disclosed herein may generate TEs after transplantation into a subject. In some embodiments, at least about 5% of TECs generated according to the methods disclosed herein may differentiate into TEs after transplantation into a subject. In some embodiments, between about 5% and about 99% of TECs generated according to the methods disclosed herein may differentiate into TEs after transplantation into a subject. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the TECs generated according to the methods disclosed herein can differentiate into TE cells after transplantation into a subject. In some embodiments, about 100% of the TECs generated according to the methods disclosed herein can differentiate into TE cells after transplantation into a subject.

[0102]

[0112] In certain embodiments, the methods disclosed herein may include transplanting thymocytes produced according to the methods disclosed herein into a subject, where the thymocytes may have a mixed population of cell types. In some embodiments, the thymocytes produced according to the methods disclosed herein transplanted into a subject may be a mixture of TEP, TEC, and / or TE cells. In some embodiments, the thymocytes produced according to the methods disclosed herein transplanted into a subject may be a mixture of TEP cells and one or more subpopulations of TECs. In some embodiments, the thymocytes produced according to the methods disclosed herein transplanted into a subject may be a mixture of TEP cells and TECs, where about 1.0% to about 99.0% (e.g., about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99% to 100%) of the mixture are TEP cells. In some embodiments, the thymocytes generated according to the methods disclosed herein that are transplanted into a subject may be a mixture of TEP cells and TECs, with about 1.0% to about 99.0% (e.g., about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%) of the mixture being TECs.

[0103]

[0113] In certain embodiments, the methods disclosed herein may include transplanting thymocytes produced according to the methods disclosed herein into a subject in combination with one or more non-thymocyte cells. According to these embodiments, thymocytes produced according to the methods disclosed herein may be transplanted into a subject in combination with lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, red blood cells, or any combination thereof. In some embodiments, the methods disclosed herein may include transplanting thymocytes produced according to the methods disclosed herein into a subject in combination with one or more cell-based immunotherapies. In some embodiments, the methods disclosed herein may include transplanting thymocytes produced according to the methods disclosed herein into a subject in combination with one or more adoptive cell therapies. Non-limiting examples of adoptive cell therapies suitable for use herein include tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, natural killer (NK) cell therapy, etc.

[0104]

[0114] For the purposes described herein, either autologous, allogeneic, or xenogeneic thymocytes prepared according to the methods of the present disclosure may be administered to a subject. According to these embodiments, the thymocytes may be in an undifferentiated, partially differentiated, or fully differentiated form, may be genetically modified or unmodified, and may be introduced by direct injection into a tissue site, by infusion via the portal vein, in a bolus delivered to an organ, or may be administered systemically on or around the surface of an acceptable matrix, encapsulated, or combined with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions disclosed herein may be formulated for parenteral administration (e.g., intravenous or intravascular, bolus injection, intrarenal introduction, intraventricular injection, intracisternal injection, intraparenchymal injection, or any combination thereof). In some embodiments, the pharmaceutical compositions disclosed herein may be formulated for intramuscular implantation and / or intramuscular injection. In some embodiments, the pharmaceutical compositions disclosed herein may be formulated for orthotopic implantation.

[0105]

[0115] In some embodiments, thymocytes produced by the compositions and methods disclosed herein can be prepared for administration to a subject by any suitable method known in the art. In some embodiments, the cells can be administered to a subject by local or systemic injection. In some embodiments, the thymocyte preparation can be administered by methods comparable to bone marrow transplantation (e.g., through the renal artery or similar). In other embodiments, the thymocyte preparation can be introduced directly to the site of interest, such as an infection or other area requiring such treatment. In other embodiments, the thymocyte preparation disclosed herein can be introduced intramuscularly into a subject by transplantation and / or injection. In still other embodiments, in the process of transplanting at least one solid organ (e.g., kidney, lung, heart, liver, etc.) and / or cell transplant (e.g., bone marrow transplant) into a subject, the thymocytes produced herein can be co-administered to the subject receiving such transplant. In accordance with these embodiments, thymocytes produced by the compositions and methods disclosed herein can be placed, for example, under the kidney capsule of a subject in need thereof for further transplantation in the subject. Methods for cell transplantation, pharmaceutically acceptable vehicles for delivery, and improved efficacy of cell transplantation to improve the integrity of the transplanted cell population are known in the art.

[0106]

[0116] In some embodiments, the number of cells transplanted into a subject can be a therapeutically effective number or amount. As used herein, "therapeutically effective amount" can refer to the number of transplanted cells that has a therapeutic effect on the specific injury, disease, or condition for which treatment is sought. For example, if the treatment is for tissue injury, transplantation of a therapeutically effective number of cells can typically result in a reduction in the severity of symptoms associated with the injury, and in certain cases, can eliminate the injury. Those skilled in the art or medical professionals will understand how to determine the appropriate cell dosage or concentration of the cell population used herein.

[0107]

[0117] In some embodiments, the amount of thymocytes of the present disclosure administered can be optimized to achieve optimal effects in the subject. Different scenarios may require optimization of the number of cells injected into the target tissue. For example, the amount of cells administered can vary depending on the subject being treated. In some embodiments, approximately 10 thymocytes produced by the compositions and methods disclosed herein are administered. 4 pieces ~ about 10 10 pieces, or about 10 6 pieces ~ about 10 8 pieces, or about 10 9 More than one thymocyte may be administered in a single bolus or multiple boluses for optimal effect. However, the precise determination of what is considered an effective dose may be based on factors individual to each patient, including the patient's size, age, extent of tissue injury / damage, and the length of time since the injury occurred. Dosages can be readily ascertained by those skilled in the art from this disclosure and knowledge in the art, and taking into account transplant doses or other cell transplant doses.

[0108]

[0118] In some embodiments, thymocytes prepared according to the methods disclosed herein can be administered to a subject in need thereof alone, in combination with other therapeutic agents and / or treatments, and / or in combination with other transplant organs and / or cells, over the course of a day, for several hours, daily, every other day, twice a week, weekly, every other week, monthly, or other suitable treatment regimen. According to these embodiments, thymocytes transplanted by the methods disclosed herein can be included in the kidney subcapsule alone or in a transplant or combination of transplants. Additionally, according to embodiments further disclosed herein, the survival period of the cells after administration to a subject can be a few hours (e.g., about 2 hours, about 6 hours, about 12 hours, about 24 hours), a few days (e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days), a few weeks (e.g., about 2 weeks, about 4 weeks, about 6 weeks, about 12 weeks, about 40 weeks, about 52 weeks), to several years (e.g., about 2 years, about 5 years), or even the lifespan of the subject, i.e., long-term engraftment.

[0109]

[0119] In some embodiments, pharmaceutical formulations suitable for injection disclosed herein may include sterile aqueous solutions and dispersions or buffers for preserving the cell populations of the present invention. The carrier or pharmaceutical excipient may be, for example, a dispersion medium containing water, saline, phosphate-buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0110]

[0120] In some embodiments, certain additives that enhance the stability, sterility, and isotonicity of the thymocyte compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added to the compositions contemplated herein. In some embodiments, antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) may be added to reduce contamination of cultures for administration.

[0111]

[0121] Sterile injectable solutions can be prepared by incorporating the thymocytes utilized in practicing the present disclosure into the required amount of an appropriate solvent, along with a certain amount of other ingredients, as needed. Examples of compositions containing the thymocytes of the present disclosure can include liquid preparations for administration, including suspensions. Such compositions can be mixed with a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, etc.).

[0112]

[0122] The pharmaceutical compositions of the present disclosure may be provided as liquid preparations (e.g., isotonic aqueous solutions, suspensions, emulsions, or viscous compositions) that may be buffered to a selected pH. The selection of suitable carriers and other additives may depend on the route of administration and the particular dosage form, for example, the nature of the liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, e.g., a sustained-release form or a liquid-filled form). Solutions, suspensions, and gels typically contain large amounts of water (e.g., purified, sterile water) in addition to cells. Small amounts of other components, such as pH adjusters (e.g., bases such as NaOH), emulsifiers or dispersants, buffers, preservatives, wetting agents, and gelifying agents (e.g., methylcellulose), may also be present. In some embodiments, the pharmaceutical compositions contemplated herein may be isotonic, i.e., have the same osmotic pressure as blood and tears. In other embodiments, agents to reduce cell lysis or other adverse effects on cells may be provided for delivery to a subject.

[0113]

[0123] In some embodiments, the desired isotonicity of the cell compositions of the present disclosure can be achieved using sodium chloride or other pharmaceutically acceptable agents (e.g., dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes). The viscosity of the composition can be maintained at a selected level, if desired, using a pharmaceutically acceptable thickening agent. Methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent depends on the agent selected. The key is to use an amount that achieves the selected viscosity. Viscous compositions are typically prepared from solutions by adding such thickening agents.

[0114]

[0124] Pharmaceutically acceptable preservatives or cell stabilizers may be used to extend the shelf life of the composition. When preservatives are used, it is well within the skill of the art to select a composition that does not affect the viability or efficacy of the cells as described herein.

[0115]

[0125] The pharmaceutical compositions of the present disclosure can be administered in dosages and by techniques well known to those skilled in the medical and veterinary fields, taking into account factors such as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals can be determined without undue experimentation by those skilled in the art from this disclosure and knowledge in the art.

[0116]

[0126] In some embodiments, kits for use in producing the thymocyte populations disclosed herein or for storing or transporting final and intermediate populations of cells for expansion or use are contemplated. In certain embodiments, kits for use in treating or alleviating a targeted disease or condition treatable by the use of thymocytes (e.g., an immune-mediated or immunocompromised condition or disease) are disclosed herein. In some embodiments, the kit may include instructions for use according to any of the methods described herein. The instructions found on the kit may include instructions for administering a thymocyte-containing composition, and optionally a second therapeutic agent, to treat, delay the onset of, or alleviate a target disease as described herein. The kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the individual has the target disease or condition, e.g., applying a diagnostic method as described herein and / or identifying symptoms in the subject. In some embodiments, the instructions may include instructions for administering an antibody to a subject at risk for developing a disease or condition as disclosed herein.

[0117]

[0127] In some embodiments, instructions for use of the thymocyte-containing product may generally include information including, but not limited to, dosage, such as the number of cells, administration schedule, and route of administration for the intended treatment. The containers of the kit may contain unit doses or bulk packages (e.g., multi-dose packages) or sub-unit doses. The kit may further include a delivery device, such as a syringe, implant device, or cell delivery device. The instructions provided with the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0118]

[0128] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating the disease, such as cancer or an immune disorder (e.g., an autoimmune disease). Instructions can be provided for practicing any of the methods described herein.

[0119]

[0129] In some embodiments, the kit may be in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar® or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices such as minipumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle).

[0120] Example

[0130] The following examples are included to illustrate certain embodiments. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered to function well in the practice of the claimed methods, compositions, and devices. However, those of skill in the art should, in light of this disclosure, understand that changes can be made in some of the disclosed embodiments and still obtain like or similar results without departing from the spirit and scope of the invention.

[0121] Example 1

[0131] In one exemplary method, AFE induction from iPSCs was evaluated. Specifically, six protocols for iPSC differentiation were tested to evaluate the effects of optimized anterior primitive streak induction (AW vs. AC vs. ACW); BMP4 signaling modulation during days 1 and 2 of direct differentiation on the production of anterior foregut endoderm (AFE); and basal medium. Figure 3A is a schematic diagram showing the timeline and culture conditions for the six protocols performed in this exemplary method. Figure 2 provides a chart of the drugs, regulators, additives, and basal medium used in this and other exemplary methods herein.

[0122]

[0132] In this example, all six protocols followed the same volume schedule as shown in Table 1.

[0123] [Table 1]

[0124]

[0133] SR Condition. For condition "SR" (one of two control conditions), iPSCs were plated on day 1 for differentiation in mTeSR+ supplemented with ROCKi. Briefly, iPSCs were first dissociated with TrypLE as follows: a) Aspirate the medium from each well and wash with 1 mL of 1x PBS; b) Replace with 1 mL of TrypLE and place in the incubator for 7 minutes; c) Gently tape the plate to release the iPSCs, add 1 mL of mTeSR+ to each well, and gently pipette to obtain single cells; d) Transfer to a 15 mL conical containing 4 mL of mTeSR+ (total volume now 5 mL); e) Count and then pellet. The pellet was resuspended, and the cells were plated as follows: a) iPSCs were resuspended at 10e6 / mL in mTeSR+ supplemented with ROCK inhibitor; b) 600,000 iPSCs (60 μL suspension) were plated per well in a total of 0.5 mL of mTeSR+ containing ROCKi; c) The plate was gently tapped back and forth to evenly distribute the iPSCs and placed in the incubator overnight.

[0125]

[0134] On day 0, anterior primitive streak (APS) specification in "Medium AW" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 (e.g., protein-rich medium) supplemented with 1:5000 ITS, 100 ng / mL activin A, and 50 ng / mL Wnt3a (composition replaced). On days 1-3, DE specification in "Medium A" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS and 100 ng / mL activin A. On day 4, DE specification in "medium AR" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 6 nM TTNPB. On days 5-6, AFE specification in "medium BRLySAG" was performed as follows: a) the medium in each well was replaced with X-VIVO10 supplemented with 20 ng / mL BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / mL SAG.

[0126]

[0135] Ctrl Condition. For the condition "Ctrl" (one of the two control conditions), on day 1, iPSCs were plated for differentiation in mTeSR+ supplemented with ROCKi according to the method disclosed above. On day 0, anterior primitive streak specification in "medium AC" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:5000 ITS, 100 ng / mL activin A, and 2 μM CHIR. On days 1-3, DE specification in "medium A" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, and 100 ng / mL activin A. From day 3 onwards, cells were cultured in "medium K", which was RPMI supplemented with 1% N21, 1:1000 ITS, and 50 ng / mL KGF.

[0127]

[0136] Conditions 3-6. For all three exemplary experimental conditions, on day 1, iPSCs were plated for differentiation in mTeSR+ supplemented with ROCKi according to the methods disclosed above. On day 0, anterior primitive streak specification in "medium ACW" for all three exemplary experimental conditions was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:5000 ITS, 100 ng / mL activin A, 2 µM CHIR, and 50 ng / mL Wnt3a. On days 1-2, DE specificity in "medium ALDN" or "medium AB" was performed as follows: a) each well was washed with 1x PBS; b1) for conditions 3 and 5, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, 100 ng / mL activin A, and 250 nM LDN; or b2) for conditions 4 and 6, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, 100 ng / mL activin A, and 20 ng / mL BMP4. On days 3-4, DE specificity in "medium LDNA83" was performed as follows: a) each well was washed with 1x PBS; b1) for conditions 3 and 4, the medium in each well was replaced with DMEM supplemented with 1% N21, 1:2000 ITS, 250 nM LDN, and 1 μM A83; or b2) for conditions 5 and 6, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 250 nM LDM, and 1 μM A83. On days 5-6, AFE characterization in "medium LDNA83" was performed as follows: a1) for conditions 3 and 4, the medium in each well was replaced with DMEM supplemented with 1% N21, 1:2000 ITS, 250 nM LDN, and 1 μM A83; a2) for conditions 5 and 6, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 250 nM LDM, and 1 μM A83.

[0128]

[0137] Cells from each condition were subjected to flow cytometry on day 7 to assess the amount of cell types obtained by measuring marker expression. Endoderm-derived cells were identified by FOXA2 expression. Anterior cells were identified by co-expression of SOX2. Further posteriorly, SOX17 co-expression was observed. AFE cells were marked as FOXA2+, SOX2+, and SOX17-. Figure 3B shows representative flow plots and quantification of SOX17-SOX2+ cells from each differentiation protocol. Figure 3C shows FOXA2 expression from each differentiation protocol. + SOX2 + Representative flow plots and quantification of cells are shown. The data demonstrate that XVIVO10 basal medium with high protein (conditions 5 and 6) significantly increased SOX17 expression compared to DMEM (conditions 3 and 4). - SOX2 + FOXA2 + The data also demonstrated that SOX17 enhanced the production of AFE. -S OX2 + FOXA2 + We also demonstrated that AFE generation is not affected by BMP signaling regulation.

[0129] Example 2

[0138] In another exemplary method, the effects of TGF-β signaling during the generation of TEPs at two distinct stages were evaluated. Figure 4A is a schematic diagram showing the timeline and culture conditions for the nine protocols performed in this exemplary method. Figure 2 provides a chart of the drugs, regulators, additives, and basal media used in this and other exemplary methods herein.

[0130]

[0139] All six protocols followed the same volume schedule as shown in Table 1. For all nine exemplary experimental conditions, on day 1, iPSCs were plated for differentiation in mTeSR+ supplemented with ROCKi according to the method disclosed above. On day 0, anterior primitive streak specification in "medium AW" was performed for all nine exemplary experimental conditions as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:5000 ITS, 100 ng / mL activin A, and 50 ng / mL Wnt3a. On days 1-3, DE specification in "medium A" was performed for all nine exemplary experimental conditions as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS and 100 ng / mL activin A. On day 4, DE specification in "medium AR" for all nine exemplary experimental conditions was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 6 nM TTNPB.

[0131]

[0140] On days 5-6, AFE characterization was performed as follows: a) for conditions 1, 4, and 7, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG; b) for conditions 2, 5, and 8, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, and 100 ng / ml SAG; c) for conditions 3, 6, and 9, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 1 ng / ml TGF-β, and 100 ng / ml SAG.

[0132]

[0141] On days 7-8, VPE characterization was performed as follows: a) for conditions 1-3, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG; b) for conditions 4-6, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, and 100 ng / ml SAG; c) for conditions 7-9, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 1 ng / ml TGF-β, and 100 ng / ml SAG.

[0133]

[0142] On day 9 and thereafter, TEP characterization in "medium LRWF8S1A" for all nine exemplary experimental conditions was performed as follows: a) The medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 250 nM LDN, 6 nM TTNPB, 50 ng / mL Wnt3a, 50 ng / mL FGF8, 250 nM SANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAAs).

[0134]

[0143] Nine protocols testing the effect of TGF-β inhibition (Ly), omission, or activation (T) on TEP induction were tested and analyzed by flow cytometry and qPCR on day 14. Figure 4B shows the CD205 expression levels from each differentiation protocol. + EPCAM + Flow plot quantification of cells is shown, and Figures 4C-4E show qPCR analysis of HOXA3, KRT8, and FOXN1 gene expression in cells from each differentiation protocol.

[0135]

[0144] During the AFE stage (days 5–6), we evaluated the effects of 1) inhibition of TGF-β signaling (Ly-conditions SR, 2, and 3); 2) no modulation of TGF-β signaling (conditions 4, 5, and 6); and 3) activation of TGF-β signaling (T-conditions 7, 8, and 9). During the VPE stage (days 7–8), we also evaluated the effects of 1) inhibition of TGF-β signaling (Ly-conditions SR, 4, and 7); 2) no modulation of TGF-β signaling (conditions 2, 5, and 8); and 3) activation of TGF-β signaling (T-conditions 3, 6, and 9). Overall, the data showed no effect of TGF-β signaling modulation on the expression of TEP / TEC markers CD205 and EPCAM or the expression of VPE marker HOXA3 or thymic epithelial progenitor cell (TEP) markers KRT8 and FOXN1. These results unexpectedly demonstrated that TGF inhibition was not required during AFE and VPE generation to efficiently establish thymocytes using direct differentiation, in contrast to methods currently practiced in the art for thymocyte generation.

[0136] Example 3

[0145] In another exemplary method, the effect of basal medium during the first stage of thymocyte differentiation was evaluated. Figure 5A is a schematic diagram showing the timeline and culture conditions for the four protocols performed in this exemplary method. Figure 2 provides a chart of the drugs, regulators, additives, and basal medium used in this and other exemplary methods herein. All four protocols followed the same volume schedule as shown in Table 1. For all four exemplary experimental conditions, on day 1, iPSCs were plated for differentiation in mTeSR+ supplemented with ROCKi according to the methods disclosed above.

[0137]

[0146] Condition SR. On day 0 of "SR," anterior primitive streak specification in "medium AW" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:5000 ITS, 100 ng / mL activin A, and 50 ng / mL Wnt3a. On days 1-3, DE specification in "medium A" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS and 100 ng / mL activin A. On day 4, DE specification in "medium AR" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 6 nM TTNPB. On days 5-6, AFE specification in "medium BRLySAG" was performed as follows: a) The medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG. On days 7-8, VPE specification in "medium BRLySAG" was performed as follows: a) The medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG.

[0138]

[0147] Ctrl condition. For condition "Ctrl" (one of the two control conditions), anterior primitive streak specification in "medium AC" was performed on day 0 as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:5000 ITS, 100 ng / mL activin A, and 2 μM CHIR. On days 1–3, DE specification in "medium A" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, and 100 ng / mL activin A. From day 3 onward, cells were cultured in "medium K," which was RPMI supplemented with 1% N21, 1:1000 ITS, and 50 ng / mL KGF.

[0139]

[0148] Conditions 3 and 4. For the exemplary experimental conditions, anterior primitive streak specification in "medium ACP" was performed on day 0 as follows: a) each well was washed with 1x PBS; b1) for condition 3, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:5000 ITS, 100 ng / mL activin A, 2 µM CHIR, and 100 nM PIK-90; or b2) for condition 4, the medium in each well was replaced with XVIVO10 supplemented with 1:5000 ITS, 100 ng / mL activin A, 2 µM CHIR, and 100 nM / mL PIK-90.

[0140]

[0149] On days 1 and 2, DE specification in "medium ALDN" was performed as follows: a) each well was washed with 1x PBS; b1) for condition 3, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, 100 ng / mL activin A, and 250 nM LDN; or b2) for condition 4, the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 250 nM LDN. On days 3 and 4, DE specification in "medium LDNA83" was performed as follows: a) each well was washed with 1x PBS; b) for both conditions 3 and 4, the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 250 nM LDN, and 1 µM A83. On days 5–6, AFE specification in “medium LDNA83” was performed as follows: for both conditions 3 and 4, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 250 nM LDN, and 1 μM A83.

[0141]

[0150] The ability of the four protocols to induce AFE was tested and analyzed via flow cytometry on day 7. Figure 5B shows the SOX17 cells from each differentiation protocol. - SOX2 + Representative flow plots and quantification of cells are shown in Figure 5C. FOXA2 cells from each differentiation protocol are shown in Figure 5D. + SOX2+ Representative flow plots and quantification of cells are shown. The data demonstrate that utilizing RPMI basal medium (high protein medium, very low protein compared to XVIVO10) during the early stages of thymocyte differentiation significantly improved the performance of SOX17 compared to XVIVO10. - SOX2 + FOXA2 + These data demonstrated that using the same signaling pathway regulators in conditions 3 or 4 both resulted in AFEs marked by co-expression of FOXA2 and SOX2. However, only cells generated in condition 3 using RPMI as the basal medium during days 0, 1, and 2 did not co-express SOX17 compared to condition 4.

[0142] Example 4

[0151] In another exemplary method, the timing of BMP signaling regulation on TEP / TEC generation was evaluated. Figure 6A is a schematic diagram showing the timeline and culture conditions for the nine protocols implemented in this exemplary method. Figure 2 provides a chart of the drugs, regulators, additives, and basal media used in this and other exemplary methods herein. All nine protocols followed the same volume schedule as shown in Table 1. For all four exemplary experimental conditions, on day 1, iPSCs were plated for differentiation in mTeSR+ supplemented with ROCKi according to the methods disclosed above.

[0143]

[0152] Condition SR. On day 0 of "SR," anterior primitive streak specification in "medium AW" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:5000 ITS, 100 ng / mL activin A, and 50 ng / mL Wnt3a. On days 1-3, DE specification in "medium A" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS and 100 ng / mL activin A. On day 4, DE specification in "medium AR" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 6 nM TTNPB. On days 5-6, AFE specification in "medium BRLySAG" was performed as follows: a) The medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG. On days 7-8, VPE specification in "medium BRLySAG" was performed as follows: a) The medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG. On days 7-8, VPE specification in "medium BRLySAG" was performed as follows: a) The medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG. On day 9 and thereafter, TPE characterization in "medium LRWF8S1A" was performed as follows: the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 250 nM LDN, 6 nM TTNPB, 50 ng / mL Wnt3a, 50 ng / mL FGF8, 250 nM SANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAAs).

[0144]

[0153] Conditions 2-9. For these exemplary conditions, on day 0, anterior primitive streak specification in "medium ACP" was performed as follows: a) each well was washed with 1x PBS; b1) for condition 2, the medium in each well was replaced with XVIVO10 supplemented with 1:5000 ITS, 100 ng / mL activin A, 2 µM CHIR, and 100 nM PIK-90; b2) for conditions 3-9, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:5000 ITS, 100 ng / mL activin A, 2 µM CHIR, and 100 nM / mL PIK-90. On days 1 and 2, DE specification in "medium ALDN" was performed as follows: a) each well was washed with 1x PBS; b1) for condition 2, the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 100 ng / mL activin A, and 250 nM LDN; b2) for conditions 3 to 9, the medium in each well was replaced with RPMI supplemented with 0.2% N21, 1:2000 ITS, 100 ng / mL activin A, and 250 nM LDN. On days 3 and 4, DE specification in "medium LDNA83" was performed as follows: a) each well was washed with 1x PBS; b) the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 250 nM LDN, and 1 μM A83. On days 5-6, AFE characterization was performed as follows: a) for conditions 2 and 3, the medium in each well was replaced with XVIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG; b) for conditions 4 and 5, the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 250 nM LDN, and 1 μM A83. c) for conditions 6 and 7, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 20 ng / ml BMP4, 6 pM TTNPB, and 50 ng / ml FGF8; for conditions 8 and 9, the medium in each well was replaced with X-VIVO10 supplemented with 250 nM LDN, 1 μM A83, 6 pM TTNPB, and 50 ng / ml FGF8.On days 7-8, AFE characterization was performed as follows: a) for conditions 2 and 3, the medium in each well was replaced with X-VIVO10 supplemented with 20 ng / ml BMP4, 6 nM TTNPB, 5 μM Ly, and 100 ng / ml SAG; b) for conditions 4-9, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 20 ng / ml BMP4, 6 pM TTNPB, and 50 ng / ml FGF8. On day 9 and thereafter, TEP characterization was performed as follows: a) for conditions 2 and 3, the medium in each well was replaced with XVIVO10 supplemented with 1:2000 ITS, 250 nM LDN, 6 nM TTNPB, 50 ng / mL Wnt3a, 50 ng / mL FGF8, 250 nM SANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAA); b) for conditions 4, 6, and 8, the medium was replaced with XVIVO10 supplemented with 1:2000 ITS, 20 ng / mL BMP4, 6 pM TTNPB, 2.5 ng / mL FGF10, 250 nM SANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAA). c) For conditions 5, 7, and 9, the medium in each well was replaced with X-VIVO10 supplemented with ANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAA); c) For conditions 5, 7, and 9, the medium in each well was replaced with X-VIVO10 supplemented with 1:2000 ITS, 250 nM LDN, 6 pM TTNPB, 2.5 ng / mL FGF10, 250 nM SANT-1, 20 ng / mL activin A, 10 μg / mL heparin, 500 ng / mL hydrocortisone, 20 ng / mL EGF, and 1× non-essential amino acids (NEAA).

[0145]

[0154] Cells were analyzed by flow cytometry and qPCR on day 30. Figure 6C shows the flow gating strategies and CD104 expression levels for each protocol. hi EPCAM +Quantification of cells is shown. Figures 6D-6G show qPCR analysis of FOXN1, KRT5, NKX2-3, and NKX2-1 expression at day 30 for each protocol. The data showed how the timing of BRF8 introduction at day 7 (conditions 5 and 6) or day 5 (conditions 6 and 7), or the introduction of non-overlapping factors in addition to LDN / A83 at day 5 (conditions 8 and 9), affected TEP / TEC production. The data showed that introduction of BRF8 at day 5 resulted in the highest EPCAM expression. + CD104 hi We demonstrated that TEP / TECs with a population expressed the most cells with the markers: FOXN1, KRT5, and NKX2-3.

[0146]

[0155] The data also demonstrated how activation of BMP-4 signaling (conditions 4, 6, and 8) versus inhibition of BMP-4 signaling (L-conditions 5, 6, and 9) at the TEP stage (day 9 onward) affected TEP / TEC generation. The data demonstrated that inhibition of BMP-4 signaling gave the best target gene expression in condition 7; specifically, NKX2-1 expression (lung and thyroid marker) was absent in condition 7, while condition 6 expressed it much more than any other condition.

[0147] Example 5

[0156] Those skilled in the art understand that there are several causes for the decline of thymocytes and naive T cells. For example, thymic atrophy (e.g., involution, immunosenescence), chemotherapy side effects, graft-versus-host disease (GvHD), and human immunodeficiency virus (HIV) can cause these declines. The development of the mature thymus produces distinct cortical and medullary regions. State-of-the-art thymic epithelial cell differentiation protocols lack functional TECs after extended periods in vivo, produce low numbers of FOXN1-positive cells in vitro, and exhibit low expression levels of TEP / TEC markers in vivo and in vitro, providing a need for improved methods for generating these cells for therapeutic use and recovery. The compositions and methods disclosed herein provide improved methods for producing functional thymocytes, for example, using novel supplemented media. In certain embodiments, early thymic progenitor signaling can enhance FOXN1 expression during differentiation of thymic epithelial cells (TECs), e.g., CD40 ligand, RANK ligand, and NOTCH ligand. In some embodiments disclosed herein, it is demonstrated that ETP-derived signals (e.g., CD40L, RANKL, and NOTCH) can enhance iPSC-derived TEP expression of FOXN1 and MHC-II. In other embodiments, the compositions and methods disclosed herein can be used to form stem cell-derived thymic organoids, produce several single-positive T cells, and provide further TEP / TEC maturation. In other embodiments, the optimized TEP / TEC protocols disclosed herein can be combined with stem cell-derived thymic organoid systems to improve outcomes, such as thymic functional recovery.

[0148]

[0157] 7 shows an exemplary experimental setup according to certain embodiments of the present disclosure, in which RANKL, CD40L, and NOTCH agonists are added in different combinations at stage 4 or 5 of differentiation of progenitor cells for production. Differentiation can be assessed using flow cytometry and qPCR by evaluating specific markers, such as MHC-II and EPCAM markers, that indicate effective differentiation using the compositions and methods disclosed herein.

[0149]

[0158] Figures 8A-8B show additional stimulatory agents used to enhance thymocyte production with improved marker presentation. 8A and 8B show representative marker percentage results under various conditions, according to certain embodiments of the present disclosure. Previous results showed the production of approximately 10% FOXN1+EPCAM+ cells, and while the addition of CD40L and / or RANKL alone in this study did not enhance the production of these cells, the addition of a NOTCH agonist enhanced FOXN1-GFP expression and also enhanced MHC-II expression using the compositions and methods disclosed herein. It was also observed that the use of all supplements positively enhanced the expression of FOXN1+ and EPCAM+ cells (see Figures 8A and 8B).

[0150]

[0159] 9A-9B show exemplary results of qPCR detection of representative markers FOXN1 and MHC-II (e.g., HLA-DR) protein expression corroborated by gene expression under control and experimental conditions, according to certain embodiments of the present disclosure.

[0151]

[0160] FIG. 10 is a schematic diagram of the derivation of stem cell-derived thymic organoids (sTO) according to certain embodiments of the present disclosure.

[0161] Figures 11A-11C show exemplary results under various conditions of an experimental thymocyte induction protocol demonstrating that TEPs can be further matured into sTOs using the compositions and methods disclosed herein. Figure 11A shows exemplary flow cytometry results; Figure 11B shows sTO production compared to the production of various T cell populations; and Figure 11C depicts assays for tissue-restricted antigen levels in TEP cells versus sTOs, demonstrating superior expression of the tested antigens in the sTO population (immunoassays showing expression of AIRE and AIRE + DAPI are available upon request, not shown). As demonstrated herein, ETP-derived signals (CD40L, RANKL, and NOTCH) were identified to enhance iPSC-derived TEP expression of FOXN1 and MHC-II. Stem cell-derived thymic organoids from further TEP / TEC maturation could be formed and produce single-positive T cell populations.

[0152] All compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions and methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. 1. A composition comprising at least one bone morphogenetic protein (BMP) signaling activator, at least one fibroblast growth factor receptor 3 (FGFR3) activator, at least one retinoic acid (RA) signaling activator, and a protein-enriched cell culture medium, wherein the protein concentration is from about 0.5% (w / v) to a maximum of about 30.0% (w / v) protein, and the composition does not contain a transforming growth factor-β (TGF-β) inhibitor.

2. 10. The composition of claim 1, further comprising definitive endoderm (DE) cells or anterior foregut endoderm (AFE) or ventral pharyngeal endoderm (VPE) cells.

3. The composition of claim 1 or 2, wherein the BMP activator comprises one or more of BMP4 or SB4.

4. The composition of any one of claims 1 to 3, wherein the at least one FGFR3 activator comprises at least one of FGF8, FGF1, FGF2, FGF9, and heparin.

5. 5. The composition of any one of claims 1 to 4, wherein the at least one retinoic acid signaling activator comprises at least one of retinoic acid, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, and Ch55.

6. 3. The composition of claim 1 or 2, wherein the BMP activator comprises one or more of BMP4 or SB4, the at least one FGFR3 activator comprises at least one of FGF8, FGF1, FGF2, FGF9, and heparin, and the at least one RA signaling activator comprises at least one of RA, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, and Ch55.

7. A composition comprising at least one FGF receptor 1 and FGF receptor 2 signaling activator, at least one RA signaling activator, at least one inhibitor of BMP signaling, at least one inhibitor of sonic hedgehog signaling, and a protein-enriched cell culture medium, wherein the protein concentration is from about 0.5% (w / v) to about 30.0% (w / v) protein, and the composition does not contain an activator of BMP signaling.

8. 8. The composition of claim 7, further comprising at least one of a Wnt signaling activator and an activin A activator.

9. The composition of claim 7 or 8, further comprising at least one of definitive endoderm (DE) cells, anterior foregut endoderm (AFE) cells, pharyngeal endoderm (PE) cells, ventral pharyngeal endoderm (VPE) cells, third pharyngeal pouch endoderm (TPPE) cells, and thymocytes.

10. 10. The composition of any one of claims 7 to 9, wherein the at least one FGF receptor 1 and FGF receptor 2 signaling activator comprises at least one of FGF10, FGF3, FGF4, FGF7, and FGF22.

11. 11. The composition of any one of claims 7 to 10, wherein the at least one RA signaling activator comprises at least one of RA, vitamin A, TTNPB, AC261066, SR1078, SR221, BMS493, fenretinide, AM580, adapalene, and Ch55.

12. 12. The composition of any one of claims 7 to 11, wherein the at least one BMP inhibitor comprises at least one of LDN193189, LDN214117, LDN212854, DMH2, K02288, ML347, SGC AAK11, PD407824, UK383367, A01, recombinant mammalian noggin and dorsomorphin, and non-mammalian noggin and dorsomorphin.

13. 13. The composition of any one of claims 7 to 12, wherein the inhibitor of sonic hedgehog signaling comprises at least one of SANT1, SANT2, U1866A, dynapyrazole-A, dynalestin, cyclopamine, HIP1, GANT58, AY9944 dihydrochloride, and RU-SKI43 hydrochloride.

14. 14. The composition of claim 13, wherein the at least one activin A activator comprises at least one of recombinant mammalian activin A, SB4, and alantolactone.

15. A composition comprising a protein-enriched medium having a protein concentration of about 0.5% to about 30.0% and at least one agent comprising at least one NOTCH pathway signaling activator, at least one CD40 pathway signaling activator, and at least one RANK signaling pathway activator.

16. 16. The composition of claim 15, comprising at least one NOTCH pathway signaling activator and at least one of a CD40 pathway signaling activator and a RANK signaling pathway activator.

17. 17. The composition of claim 15 or 16, further comprising at least one cell population comprising at least one of pharyngeal endoderm (PE) cells, third pharyngeal pouch endoderm (TPPE) cells, and thymic epithelial progenitor (TEP) cells.

18. 1. A method for differentiating mammalian pluripotent stem cells (PSCs) into anterior primitive streak cells and subsequently into definitive endoderm (DE) cells, comprising: incubating mammalian PSCs or anterior primitive streak cells or DE cells with a composition comprising at least one bone morphogenetic protein (BMP) signaling activator, at least one fibroblast growth factor receptor 3 (FGFR3) activator, at least one retinoic acid (RA) signaling activator, and a protein-enriched cell culture medium, wherein the protein concentration is from about 0.5% (w / v) to up to about 30.0% (w / v) protein, and the composition does not contain a transforming growth factor-β (TGF-β) inhibitor; generating DE cells having the ability to differentiate into at least one of AFE cells, VPE cells, and thymocytes.

19. 20. The method of claim 18, wherein incubating further comprises incubating in low protein or protein-free medium for about 12 hours up to 6 days, wherein low protein comprises a concentration of less than 0.5% (w / v) in said medium.

20. 20. The method of claim 18 or 19, further comprising supplementing the composition or the medium with at least one of insulin, transferrin, selenium; insulin, transferrin, and selenium (ITS); insulin-transferrin-selenium-ethanolamine (ITS-X) at a dilution of about 1:50 to about 1:5000 from about day 0 of incubation up to about 24 hours.

21. 21. The method of claim 20, wherein the ratio of insulin, transferrin, selenium; insulin, transferrin, and selenium (ITS); insulin-transferrin-selenium-ethanolamine (ITS-X) is about 1:2000.

22. 22. The method of any one of claims 18-21, further comprising incubating the cells in a composition further comprising one or more of trace elements, vitamin C, non-essential amino acids, sodium pyruvate, glutamine, lipids, and beta-mercaptomethanol.

23. 23. The method of any one of claims 18-22, further comprising incubating the DE cells with at least one BMP inhibitor and at least one activin inhibitor in a composition having a protein concentration of at least 0.5% (w / v) protein to at most about 30.0% (w / v) protein for a total of about 12 hours to up to about 7 days, or at least 5 days, or up to 7 days to generate DE cells, wherein the DE cells can be further differentiated into at least one of anterior foregut endoderm (AFE) cells, ventral pharyngeal endoderm (VPE), and thymocytes.

24. 24. The method of any one of claims 19 to 23, further comprising incubating DE cells or AFE cells, as applicable, with at least one of at least one activator of FGF receptor 3 signaling, at least one activator of retinoic acid signaling, and at least one activator of BMP signaling, in the composition having a protein concentration of at least 0.5% (w / v) protein to at most about 30% (w / v) protein, for a total time of about 12 hours to up to about 6 days, or at least 5 days, or up to 6 days, to produce at least one of AFE and ventral pharyngeal endoderm (VPE) cells capable of efficiently differentiating into thymocytes.

25. 25. The method of claim 24, further comprising, after a total incubation period of about 12 hours up to about 6 days, or at least 5 days, or up to 6 days, replacing the composition with a composition having a protein concentration of about 0.5% (w / v) protein up to about 30.0% (w / v) protein, further comprising at least one of at least one FGF receptor 1 and FGF receptor 2 signaling activator, at least one RA signaling activator, at least one inhibitor of BMP signaling, at least one inhibitor of Sonic hedgehog signaling, at least one activin A activator, and at least one Wnt pathway signaling activator, for about 12 hours up to about 60 days or more.

26. 26. The method of claim 25, wherein the final population of cells comprises thymocytes, including at least one of TEP cells and TEC cells.

27. 20. The method of claim 18, further comprising replacing the composition with a composition comprising about 0.5% (w / v) to about 30% (w / v) protein, comprising at least one of at least one FGF10 signaling activator, at least one RA signaling activator, at least one inhibitor of BMP signaling, at least one inhibitor of sonic hedgehog signaling, and at least one activin A activator, and incubating the cells for about 12 hours up to about 60 days or more to generate thymocytes.

28. 28. The method of any one of claims 18-27, wherein at least about 50% and up to about 100% of the mammalian PSCs, anterior primitive streak cells, or DE cells differentiate into AFE cells, which comprise SOX2+, FOXA2+, and SOX17-.

29. The method of any one of claims 18 to 28, wherein at least about 40% and up to about 100% of the mammalian AFE cells are differentiated into VPE cells comprising HOXA3+HOXB1-NKX2.1-.

30. 30. The method of claim 28 or 29, wherein at least about 5% or more of the mammalian AFE or mammalian VPE cells differentiate into FOXN1+, HLA class II+, KRT5+, and / or KRT8+ thymocytes.

31. 31. The method of any one of claims 28-30, wherein at least about 5% or more of the mammalian AFE or mammalian VPE cells differentiate into EPCAM+, CD104+, FOXN1+, HLA class II+, KRT5+, and / or KRT8+ thymocytes.

32. 31. The method of any one of claims 28-30, wherein at least about 5% or more of the mammalian AFE or mammalian VPE cells differentiate into thymocytes, and the thymocytes comprise EPCAM+, CD205+, FOXN1+, HLA class II+, KRT5+, and / or KRT8+ thymocytes.

33. The method of claim 18, wherein the cells comprise VPE cells or VPE-like cells having characteristics of VPE, and the VPE cells or VPE-like cells having characteristics of VPE are incubated for at least 12 hours to up to about 21 days, or about 60 days or more to produce a cell population comprising at least one of TEC and TEP cells, wherein the composition for culturing comprises a protein concentration of about 0.5% (w / v) to up to 30.0% (w / v) protein, and further comprises at least one FGF receptor 1 and 2 signal activator, at least one retinoic acid signaling activator; at least one inhibitor of BMP signaling; at least one inhibitor of sonic hedgehog signaling, and at least one activin A activator; and optionally, at least one Wnt pathway signaling activator and at least one NOTCH pathway signaling activator.

34. 19. The method of claim 18, wherein the cells comprise AFE cells or AFE-like cells having characteristics of AFE, and the composition for culturing is replaced with a replacement composition having a protein concentration of at least 0.5% (w / v) to a maximum of 30.0% (w / v) protein, and the replacement composition further comprises at least one of at least one FGF10 signal activator, at least one retinoic acid signaling activator; at least one inhibitor of BMP signaling; at least one inhibitor of sonic hedgehog signaling, and at least one activin A activator; and optionally at least one Wnt pathway signaling activator and at least one NOTCH pathway signaling activator, and the AFE cells or AFE-like cells having characteristics of AFE are incubated for at least 12 hours to a maximum of about 21 days, or about 60 days or more, to produce a cell population comprising at least one of TEC and TEP cells.

35. The method of claim 18, wherein the cells comprise VPE cells or VPE-like cells having characteristics of VPE, the composition for culturing is replaced with a replacement composition comprising a protein concentration of from about 0.5% (w / v) to up to 30.0% (w / v) protein, the replacement composition further comprising at least one of a NOTCH pathway activator, a CD40 pathway activator, a RANK pathway activator, or a combination thereof, and the VPE cells or VPE-like cells having characteristics of VPE are incubated for at least 12 hours to up to about 21 days, or up to about 60 days or more, to produce a cell population comprising at least one of TEC and TEP cells.

36. 19. The method of claim 18, wherein the cells comprise AFE cells or AFE-like cells having characteristics of AFE, the composition for culturing is replaced with a replacement composition comprising a protein concentration of at least 0.5% (w / v) to at most 30% (w / v) protein, the replacement composition further comprising at least one of a NOTCH pathway activator, a CD40 pathway activator, a RANK pathway activator, or a combination thereof, and the AFE cells or AFE-like cells having characteristics of AFE are incubated for at least 12 hours to up to about 21 days, or up to about 60 days or more, to produce a cell population comprising at least one of TEC and TEP cells.

37. The method of claim 18, wherein the cells comprise VPE cells or VPE-like cells having characteristics of VPE, the composition for culturing is replaced with a replacement composition comprising a protein concentration of from about 0.5% (w / v) to up to 30% (w / v) protein, the replacement composition further comprising at least one of a NOTCH pathway activator, a CD40 pathway activator, a RANK pathway activator, or a combination thereof, and the VPE cells or VPE-like cells having characteristics of VPE are incubated for at least 12 hours to up to about 21 days, or up to about 60 days or more to produce at least one of TEC and TEP cells.

38. 19. The method of claim 18, wherein the cells comprise AFE cells or AFE-like cells having characteristics of AFE, the composition for culturing is replaced with a replacement composition comprising a protein concentration of at least 0.5% (w / v) to at most 30% (w / v) protein, the replacement composition further comprising at least one of a NOTCH pathway activator, a CD40 pathway activator, a RANK pathway activator, or a combination thereof, and the AFE cells or AFE-like cells having characteristics of AFE are incubated for at least 12 hours to up to about 21 days, or up to about 60 days or more to produce at least one of TECs and TEP cells.

39. A kit comprising the composition of any one of claims 1 to 17 and at least one container.

40. 39. A method for treating a condition in a subject, the method comprising administering to a subject in need thereof a cell population produced by any one of the methods of any one of claims 18 to 38.

41. 41. The method of claim 40, wherein administering the cell population to the subject comprises administering the cell population to the subject simultaneously with, before, or after treating the subject with a standard of care, or before, simultaneously with, or after organ or cell transplantation.

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