Thymocyte composition and method for preparing it

Abstract

This disclosure provides thymocytes, thymic organoids, and animal models comprising such thymocytes and thymic organoids. It also provides methods for inducing the differentiation of pluripotent stem cells into endoderm (DE), anterior foregut endoderm (AFE), ventral pharyngeal endoderm (VPE), thymic epithelial precursor (TEP) cells, thymic epithelial cells (TEC), and thymic organoids in vitro and in vivo.

Description

[Technical Field]

[0001] Related applications This application claims priority under Section 119 of the United States Patent Act to U.S. Provisional Application No. 63 / 457,314, filed on April 5, 2023. The contents of this application are incorporated herein by reference in their entirety.

[0002] Government subsidies This invention was made with government support under Contract No. AY2AX000004, granted by the Advanced Research Projects Agency for Health (ARPA-H). The government has certain rights in this invention.

[0003] Field of Invention This disclosure relates, in general, to the biotechnology of pluripotent stem cells, and more specifically, to biotechnology-treated thymocytes, thymic organoids, humanized animal models comprising these thymocytes and thymic organoids, and methods of using them. [Background technology]

[0004] The thymus is a major lymphoid organ that plays a central role in the immune system. The thymic microenvironment provides a unique training ground for the maturation and development of effector cells, such as lymphocytes (e.g., T cells). Complex interactions between thymocytes and effector cells can determine the phenotype and functionality of effector cells. Some thymocyte-effector cell interactions are modulated so that the recognition of factors expressed by thymocytes promotes the survival of effector cells. Conversely, other thymocyte-effector cell interactions can lead to the death of effector cells. By regulating such interactions, the thymus plays a crucial role in establishing a repertoire of effector cells that can initiate an activated immune response against foreign invaders while establishing resistance to self.

[0005] Thymocyte-effector cell interactions occur within the intricate three-dimensional network of the thymus, creating a complex microenvironment for the development of effector cells. Thymocyte-based therapies that replicate and mimic the natural environment of the thymus remain in need. [Overview of the Initiative]

[0006] This disclosure provides thymocytes, thymic organoids, and animal models comprising such thymocytes and thymic organoids. It also provides methods for producing and maintaining thymocytes and thymic organoids in vitro and in vivo.

[0007] In particular, this disclosure provides a method for inducing the differentiation of pluripotent stem cells into thymocytes. Such a method may include the steps of encapsulating pluripotent stem cells in a polymer and differentiating the pluripotent stem cells into embryonic endoderm (DE) cells. DE cells can be cultured and differentiated into anterior foregut endoderm (AFE) cells by contacting or incubating them with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, and / or a combination thereof. AFE cells can be cultured and differentiated into ventral pharyngeal endoderm (VPE) cells by culturing them in a first VPE medium and / or a second VPE medium. The first VPE medium may contain ascorbic acid, retinoic acid, FGF, and / or a TGFβ inhibitor. In some embodiments, the first VPE medium may further contain a WNT inhibitor. The second VPE medium may contain nogging, a WNT activator, FGF, retinoic acid, and / or ascorbic acid. In some embodiments, the second VPE medium may further contain a BMP inhibitor, an SHH inhibitor, or a combination thereof. VPE cells can be further differentiated into thymocytes such as thymic epithelial precursor cells (TEPs) by contacting or incubating the VPE cells with ascorbic acid, FGF, BMP, and / or WNT activators.

[0008] In one embodiment, the polymer is an alginate. In another embodiment, the polymer comprises an alginate and a gelatin A hydrogel.

[0009] In one embodiment, a method for inducing the differentiation of pluripotent stem cells into thymocytes includes a step of differentiating pluripotent stem cells into endoderm (DE) cells, a step of culturing DE cells and differentiating DE cells into anterior foregut endoderm (AFE) cells by contacting or incubating DE cells with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, or a combination thereof, and a step of culturing AFE cells and (i) contacting or incubating AFE cells in a first VPE medium containing ascorbic acid, retinoic acid, FGF, a TGFβ inhibitor, or a combination thereof. The method comprises (ii) differentiating anterior foregut cells into ventral pharyngeal endoderm (VPE) cells by (ii) contacting or incubating AFE cells in a second VPE medium containing noggin, a WNT activator, FGF, retinoic acid, ascorbic acid, or a combination thereof, and differentiating VPE cells into thymocytes by culturing VPE cells and contacting or incubating VPE cells with ascorbic acid, FGF, BMP, a WNT activator, or a combination thereof, wherein the thymocytes are thymic epithelial precursor (TEP) and / or thymic epithelial cells (TEC).

[0010] In some embodiments, thymocytes may be thymic epithelial precursors (TEPs) or thymic epithelial cells (TECs). TEPs can be further differentiated into TECs by culturing them with interleukins, WNT activators, RANKL, FGF, BMPs, and / or ascorbic acid.

[0011] In some embodiments, pluripotent stem cells can be differentiated into DE cells by contacting or culturing them in a first growth medium containing activin A, PI-103, and / or CHIR99021. Differentiation into DE cells may further include culturing the cells in a second growth medium containing activin A, a BMP inhibitor, PI-103, and / or CHIR99021.

[0012] In some embodiments, the BMP inhibitor may be LDN193189. In some embodiments, the TGFβ inhibitor may be SB431542. In some embodiments, the FGF may be FGF8b, FGF7, FGF10, FGF1, bFGF, or a combination thereof. In some embodiments, the WNT activator may be CHIR99021. In some embodiments, the BMP may be BMP2, BMP4, or a combination thereof. In some embodiments, the interleukin may be IL22. In some embodiments, the WNT inhibitor may be IWR-1. In some embodiments, the BMP inhibitor may be LDN193189. In some embodiments, the SHH inhibitor may be SANT-1.

[0013] The pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes of this disclosure can be cultured in suspension. In some embodiments, pluripotent stem cells, DE cells, AFE cells, VPE cells, and thymocytes can be cultured as aggregates in suspension. In other embodiments, pluripotent stem cells, DE cells, AFE cells, VPE cells, and thymocytes can be cultured as single cells in suspension. In some additional embodiments, one or more of the following cell types: pluripotent stem cells, DE cells, AFE cells, VPE cells, and thymocytes can be cultured as single cells in suspension.

[0014] In some embodiments, pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes can be attached to a solid substrate.

[0015] In some other embodiments, pluripotent stem cells and DE cells can be cultured as aggregates in suspension, and the resulting DE aggregates can then be attached to a solid substrate containing an extracellular matrix-based medium and further differentiated into AFE, VPE, and TEP cells via two-dimensional (2D) adhesion culture. In some additional embodiments, the pluripotent stem cells are human induced pluripotent stem cells. In some other additional embodiments, the human induced pluripotent stem cells are encapsulated. In yet another additional embodiment, the human induced pluripotent stem cells are not encapsulated.

[0016] The method of this disclosure can be carried out for approximately 15 to 30 days. In some embodiments, the method can be carried out for approximately 18 to 25 days. In some embodiments, pluripotent stem cells can be differentiated into endoderm for approximately 5 days. In some embodiments, DE cells can be differentiated into AFE cells for approximately 2 to 3 days. In some embodiments, AFE cells can be differentiated into VPE cells for approximately 2 to 4 days in a first VPE medium and for approximately 2 to 3 days in a second VPE medium. In some embodiments, VPE cells can be differentiated into thymocytes for approximately 3 to 12 days.

[0017] This disclosure also provides thymocytes, for example, TEP and TEC produced by the methods described herein.

[0018] Furthermore, methods for culturing thymocytes in vitro are provided herein. Such methods may include culturing or incubating thymocytes in a thymocyte medium comprising FGF10, BMP4, FGF8b, CHIR99021, and / or ascorbic acid. In some embodiments, the thymocyte medium may further comprise FGF7 and RANKL. The methods may also include culturing thymocytes in a suspension. In a non-limiting example, thymocytes may be cultured as aggregates in a suspension.

[0019] In some embodiments, the present disclosure provides a method for increasing FOXN1 expression in a population of thymocytes. Such a method may include freezing a population of thymocytes, thawing a population of thymocytes, measuring and comparing the expression of FOXN1 in the population of thymocytes before freezing with the expression of FOXN1 after thawing the population of thymocytes. In some embodiments, FOXN1 expression can be increased by about 10 to 100 times. In some embodiments, FOXN1 expression can be increased by about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, FOXN1 expression can be increased by about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times. In some embodiments, a population of thymocytes can be cultured as aggregates in a suspension.

[0020] Furthermore, this specification provides a pharmaceutical composition comprising a population of thymocytes prepared by the method described herein. This disclosure also provides a method for treating or preventing a condition in a subject by administering the pharmaceutical composition of this disclosure. In some embodiments, the condition is related to a deficiency, reduction, or abnormal function of the thymus in the subject. The condition may be immunodeficiency, cancer, autoimmune disease, infection, or graft-versus-host disease (GvHD). In some embodiments, the pharmaceutical composition is administered parenterally. For example, the composition may be implanted or injected into one or more lymph nodes of the subject.

[0021] This disclosure provides compositions that may comprise a population of thymocytes. In some embodiments, the population of thymocytes may comprise one or more cell types. The cell types may be selected from one or more of the following: iTEC, mTEC, keratinocyte-like mTEC, cTEC-high, cTEC-low, and mTEC-low. The compositions may also comprise a thymic support system (TSS).

[0022] In some embodiments, a population of thymocytes may be prepared by differentiating iPS cells into thymocytes.

[0023] In some embodiments, TSS may include polymers. The polymers may be bio-based or synthetic polymers. Bio-based polymers may be polypeptide-based, polynucleotide-based, polysaccharide-based polymers, or combinations thereof. Non-limiting examples of polypeptide-based polymers include collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin, and actin. Non-limiting examples of polysaccharide-based polymers include alginates, chitin, chitosan, hyaluronic acid, cellulose, agarose, starch, cellulose, dextran, hyaluronic acid, glycogen, and glycosaminoglycans.

[0024] In some embodiments, the polymer may be a synthetic polymer. Non-limiting examples of synthetic polymers include polycaprolactone, polyglycolic acid, polylactic acid, polylactic acid-coglycolic acid, poly(ethylene oxide)polyethylene glycol, polyurethane, poly(siloxane), poly(ethylene), poly(vinylpyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), and poly (Ethylene glycol), poly(methacrylic acid), polyhydroxybutyrate (PHB), polypropylene fumarate (PPF), polyvinyl alcohol (PVA), polypropylene carbonate, polyanhydride, polyphosphazene, polygermane, polyorthoester, polyester, polyamide, polyolefin, polycarbonate, polyaramid, polyimide, chitosan, poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), hydroxy Ethoxyethyl methacrylate (HEEMA), hydroxydiethoxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethoxyethyl methacrylate (MEEMA), methoxy-diethoxyethyl methacrylate (MDEEMA), ethylene glycol dimethacrylate (EGDMA), N-vinyl-2-pyrrolidone (NVP), N-isopropyl AAm (NIPAAm), vinyl acetate (VAc), acrylic acid (AA), N-(2-hydroxypropyl Examples include methacrylamide (HPMA), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, and / or poly(N-isopropylacrylamide) (PNIPAM).

[0025] The polymers of this disclosure can form hydrogels. In some embodiments, the polymers may be crosslinked.

[0026] The TSS of this disclosure may further comprise extracellular matrix components and / or drugs. In some embodiments, the extracellular matrix component may be an extracellular matrix protein, or a region or portion thereof. Non-limiting examples of extracellular matrix proteins include fibronectin, laminin, vitronectin, tenacin, entactin, thrombospondin, elastin, gelatin, collagen, fibrin, merosine, ancarin, chondronectin, link protein, bone sialocate protein, osteocalcin, osteopontin, epinectin, hyaluronectin, undurin, epiligrin, or kalinin. In some embodiments, the extracellular matrix component may be a peptide derived from an extracellular matrix protein. Non-limiting examples of peptides include any of the amino acid sequences of SEQ ID NOs: 9-18.

[0027] The TSS of this disclosure may include biological or chemical agents. In some embodiments, the biological agent may be a ligand, an immunomodulator, or a hormone.

[0028] The compositions of this disclosure may further comprise supporting cells, stem cells, and / or effector cells. Non-limiting examples of supporting cells include myelin cells, myosoidal cells, neuroendocrine cells, tuft cells, ionocytes, endothelial cells, mesenchymal stem cells, or fibroblasts.

[0029] This specification also provides a method for treating or preventing a condition in a subject by administering the compositions described herein.

[0030] The aforementioned and other objectives, features, and advantages will be apparent from the following description of specific embodiments of this disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to exact scale, and instead are focused on illustrating the principles of various embodiments of this disclosure. [Brief explanation of the drawing]

[0031] [Figure 1] This graph shows the expression of TEP markers in cells differentiated by encapsulation of iPSC cells, compared to cells differentiated in two-dimensional culture, and cells subjected to freezing and thawing in two-dimensional culture. [Figure 2A] This is a schematic diagram and bar graph illustrating the timing, process, and results of the differentiation of chitosan-coated alginate-encapsulated hiPSC aggregates into thymic epithelial cells. Figure 2A is a schematic diagram illustrating the process for the differentiation of chitosan-coated alginate-encapsulated hiPSC aggregates into thymic epithelial cells (TECs, also called "THY-100") using 3D suspension culture. Cells were encapsulated in alginate (via extrusion or emulsification) and cultured in 3D suspension at 37°C in 5% CO2 at 45 revolutions per minute (RPM). Figure 2A also shows the differentiation stages that take place from day 0 to day 21, represented through stage 0 to stage 4 where the thymic epithelial precursor (TEP) is frozen (indicated by the dashed line on the right), and it also shows the final differentiation stage (stage 5) that takes place after the TEP cells have been thawed. The oval photographs at the bottom in Figure 2A show the encapsulated hiPSC cells (leftmost image) and each stage from stage 0 to stage 5 (rightmost image). All images were taken at a magnification of 10x, and the scale bar in each image represents 100um. [Figure 2B]Figure 2B is a schematic diagram and bar graph illustrating the timing, process, and results of the differentiation of chitosan-coated alginate-encapsulated hiPSC aggregates into thymic epithelial cells. Figure 2B is a bar graph showing the amount of FOXN1 expressed by stage 5 TEC cells after they were thawed and cultured in TEC freeze / thaw medium for 7 days under 3D suspension. This expression data was obtained using qRT-PCR, and FOXN1 expression levels were normalized against GAPDH expression and cell concentration at 200,000 cells / mL. FOXN1 expression levels were approximately 5-fold higher than those measured on day 23 (before TEC FT medium was added to the cells and before they were frozen in liquid nitrogen and stored at -80°C). [Figure 3A] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3B] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3C] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3D] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3E] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3F] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3G] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3H] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3I] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3J] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3K] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3L] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3M] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 3N] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 3A to 3N present the results for the first of four staining methods. [Figure 4A] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4B] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4C] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4D]The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4E] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4F] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4G] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4H] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4I] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4J] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4K] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4L] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 4M] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 4A to 4M present the results for the second of the four staining methods. [Figure 5A]The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5B] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5C] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5D] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5E] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5F] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5G] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5H] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5I] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5J] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5K]The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5L] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5M] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5N] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 5O] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 5A to 5O present the results for the third of five staining methods. [Figure 6A] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 6B] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 6C] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 6D] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 6E] The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 6F]The results of flow cytometry characterizing hiPSC-derived TECs are shown. Figures 6A to 6F present the results for the fourth of the four staining methods. [Figure 7] This bar graph shows the expression levels of various genes in alginate-encapsulated hiPSC-derived TEC cells on day 23 (before freezing) (see the leftmost column "AEMF-200K-090123 TEC"). Expression levels were also evaluated on day G (after freezing, thawing, and growing in TEC FT medium for 7 days) for the same cell samples, and these results are shown in the rightmost column ("AEMF-200K-090123-FT1"). Both the TEC and FT1 samples had a cell concentration of 200,000 cells / mL. These results indicate that differentiated TECs exhibit thymic lymphocyte formation ability to support T cell development in vitro. [Figure 8A] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8B] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8C] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8D] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8E] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8F] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8G]This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8H] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8I] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8J] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8K] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8L] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8M] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 8N] This series of graphs and flow cytometry contour maps demonstrates that human iPS-derived TEP can efficiently induce human T cell development in vivo. [Figure 9A] These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A–9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in a 3D suspension as described in Example 5 (Figures 9A–9C). Successful growth and differentiation into thymic epithelial precursor cells were achieved, qualitatively shown in the representative micrograph of Figure 9A (micrograph taken at 10× magnification, scale bar = 100 μm). [Figure 9B] These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A–9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in a 3D suspension as described in Example 5 (Figures 9A–9C). Successful growth and differentiation into thymic epithelial precursor cells were achieved, qualitatively demonstrated in the representative micrograph of Figure 9B (micrograph taken at 10× magnification showing thymocytes at stage 5, scale bar = 100 μm). [Figure 9C] These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A–9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in a 3D suspension as described in Example 5 (Figures 9A–9C). Figure 9C is a bar graph showing in vitro FOXN1 expression by thymocytes produced as described in Example 5 (iPSC-derived TEP cells were frozen, thawed, and grown in TEC FT medium for 7 days). The level of FOXN1 expression (ranging from 0.0019 to 0.0022) was determined using FOXN1 expression normalized to GADPH expression by using qRT-PCR on the sample at a concentration of approximately 200,000 thymocytes per 1 mL. These results indicate that thymocytes differentiated as single cells in a 3D suspension produced higher levels of FOXN1 in vitro compared to thymocytes produced using other methods described herein. [Figure 9D]These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A-9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in 3D in a bioreactor (Figures 9D-9F) with the same buffer as described in Example 5. The results obtained when hiPSCs were differentiated in 3D in a bioreactor as single cells according to the process illustrated in Figure 9D also exceeded the 0.0001 threshold required to obtain sufficient T cell production in vivo when thymocytes were transplanted into mice, as in Example 6. [Figure 9E] These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A-9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in 3D in a bioreactor (Figures 9D-9F) with the same buffer described in Example 5. [Figure 9F] These are a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in a 3D suspension. Figures 9A-9G show representative hiPSC-to-TEC differentiation experiments using unencapsulated single cells cultured in 3D in a bioreactor (Figures 9D-9F) with the same buffer described in Example 5. [Figure 9G] This is a series of schematic diagrams and graphs illustrating the differentiation of hiPSCs to TECs using unencapsulated single cells cultured in 3D suspension. Figures 9A–9G show representative hiPSC to TEC differentiation experiments. Figure 9G is a schematic diagram showing that multiple attempts were made to obtain single cells and differentiate them in 3D suspension culture without encapsulation at various differentiation stages (ranging from iPS to TEP). All experiments in Figure 9G were performed under standard culture medium conditions. Aggregated or dissociated single cells were cultured in 3D suspension at the indicated differentiation stages. Only in experiment 78B was B27 added to the cell culture medium at the DE stage. Successful growth and differentiation are indicated by dotted lines. Cell death is indicated by diagonal lines. [Figure 10A] Figure 10A is a series of two schematic diagrams and six graphs illustrating the process, timing, and results of differentiating unencapsulated hiPSC aggregates into TECs using a hybrid (2D / 3D) differentiation protocol and transplanting them into humanized NSG MHC-I / II double knockout mice. Figure 10A is a schematic diagram illustrating the hybrid (2D / 3D) process for differentiating unencapsulated hiPSC aggregates into thymic epithelial cells (TECs, also known as "THY-100"). In this hybrid process, unencapsulated hiPSCs were grown in 3D suspension culture at 45 turns per minute (RPM) at 37°C in 5% CO2 through the DE stage, and the DE aggregates were then seeded into Matrigel-coated 24-well culture plates. All remaining differentiation stages (AFE, VPE, and TEP) were performed in 2D adherent cultures. Figure 10A also shows the differentiation stages that occur from day 0 to day 21, representing from stage 0 through stage 4 where the thymic epithelial precursor (TEP) is frozen (indicated by the blue dashed line on the right), and it also shows the final differentiation stage (stage 5) that occurs after the TEP cells are frozen and thawed at -80°C. The lower oval images in Figure 10A show hiPSC cells at each stage from pre-stage 0 (far left) to stage 0 (second image from the left) to stage 5 (far right image). All images were taken at 10× magnification, and the scale bar in all images represents 100 μm. [Figure 10B] Figure 10B is a schematic diagram showing the same hybrid (2D / 3D) differentiation process, with markers for specific differentiation stages indicated within the oval-shaped lower frame. [Figure 10C]Figures 10C-10D are graphs illustrating T cell development in humanized NSG MHC-I / II double knockout mice (Jax) transplanted with hiPS-derived TEP differentiated using the hybrid (2D / 3D) differentiation process described in Figures 10A and 10B. In Figure 10C, the vertical axis of the three graphs represents TCRαβ-positive T cells (leftmost graph), CD4+ T cells (center graph), and CD8+ T cells (rightmost graph), expressed as percentages of the total number of human CD45-positive cells (hCD45+). The horizontal axis represents the time of sample collection (weeks after transplantation). The results shown in Figure 10C represent the results obtained when TEP expressed FOXN1 in vitro at a level higher than the 0.0001 threshold found to be necessary for obtaining sufficient T cells in vivo. Specifically, TEP in Figure 10C expressed FOXN1 at an 8-fold higher level than the threshold (0.0001). Each line (180-184 in Figure 10C, and 190-194 in Figure 10D) represents a different mouse. [Figure 10D] Figures 10C–10D are graphs illustrating T cell development in humanized NSG MHC-I / II dual knockout mice (Jax) transplanted with hiPS-derived TEPs differentiated using the hybrid (2D / 3D) differentiation process described in Figures 10A and 10B. The results shown in Figure 10D represent the results obtained when the transplanted TEPs expressed FOXN1 in vitro at a level below the 0.0001 threshold (0.00009) required to obtain sufficient T cells in vivo. In vitro FOXN1 expression levels were determined using qRT-PCR normalized to GADPH expression. Each line (180–184 in Figure 10C and 190–194 in Figure 10D) represents a different mouse. [Modes for carrying out the invention]

[0032] Introduction Thymic epithelial cells are important in T cell differentiation. Thymic cells prepared as described herein may enable the utilization of thymic tissue and thymic cell properties, such as thymus-associated immune function, for therapeutic applications. For example, age-related decline in immune function is known to be caused by changes in the composition and functional capacity of thymic cells. In addition, changes in sex hormones, including androgens and estrogens, can cause atrophy or aging of the thymus itself. The onset of thymic atrophy can begin as early as puberty. Therefore, the regeneration of thymic epithelial cells may provide compositions and methods for mitigating age-related decline in immune function.

[0033] The complexity and compartmentalization of thymic tissue allow for the definition of distinct microenvironments with specific cues that direct the development of effector cells. Fetal thymic organ culture (FTOC) or re-aggregated thymic organ culture (RTOC) has been developed as a platform for reconstructing niche environments that attempt to replicate the thymic environment. In FTOC, the entire thymus is placed on a membrane on the upper surface of a sponge incorporating culture medium (G. Anderson, et al., Cold Spring Harb. Protoc. 2007, 2007, the contents of which are incorporated herein by reference in their entirety). In RTOC, after thymic dissection, the cells of interest are selected by cell sorting techniques and placed as concentrated droplets on the upper surface of the membrane in a gas-liquid interface phase (E.J. Jenkinson, et al., J. Exp. Med. 1992, 176, 845, the contents of which are incorporated herein by reference in their entirety). While these methodologies reflect the development of effector cells during fetal ontogeny, there is still a need, particularly in adults, for compositions that can mimic natural environments and enable the generation of effector cells. This disclosure provides thymocyte-based compositions that combine thymocytes with a thymic support system to construct a thymic-mimicking microenvironment. The inventors have found that encapsulating iPS cells before differentiation using the differentiation protocol described herein can result in thymocytes that express FOXN1 among other markers. Applying the thymocyte differentiation protocol in suspension provides an opportunity to scale up the production of thymocytes for therapeutic applications.

[0034] composition cell The cells of this disclosure may include, but are not limited to, thymocytes, effector cells, pluripotent stem cells, populations thereof, and cells derived therefrom.

[0035] In some embodiments, the cells of the Disclosure may be autologous, allogeneic, syngeneic, or heterologous with respect to a particular individual or subject. In some embodiments, thymocytes may be autologous, allogeneic, syngeneic, or heterologous with respect to the subjects who will ultimately benefit from their clinical application. In some embodiments, the cells of the Disclosure may be mammalian cells, particularly human cells. The cells may be primary cells or immortalized cell lines. In some embodiments, the cells of the Disclosure may be prepared or drawn from syngeneic cell sources. Any of the cells described herein may feature markers known in the art for their cell type.

[0036] One or more cells of this disclosure can be engineered to ectopically express a polynucleotide. The polynucleotide may encode a polypeptide of interest. In some embodiments, the polypeptide of interest may be operably linked to an inductive element and / or an inductive promoter such that the expression of the polypeptide of interest is controlled by the inductive element and / or promoter. In some embodiments, the inductive element may include a ligand-binding domain, e.g., the ligand-binding domain of FKBP, a cyclophylline receptor, a steroid receptor, a cyclophylline receptor, and / or a tetracycline receptor. Any of the compositions or methods for manipulating cells described in International Patent Publication WO2021 / 150837 may be useful in this disclosure (their contents are incorporated herein by reference in their entirety). In some embodiments, the polypeptide of interest may be a cell death-inducing polypeptide such that the engineered cells are eliminated or killed when induced. Examples of cell death-inducing polypeptides include, but are not limited to, Casp2, Casp3, Casp8, Casp9, Casp10, p53, BAX, DFF40, HSV-TK, and / or cytosine deaminase proteins. Any inducible promoter known in the art may also be useful in this disclosure.

[0037] In some embodiments, the cells of the Disclosure may be engineered to ectopically express proteins that activate one or more checkpoint pathways to induce host immune cell depletion and host cell immune unresponsiveness to the cells of the Disclosure. For example, cells may be engineered to express immune checkpoint proteins according to the method described in European Patent Publication EP3886759A1, the contents thereof being incorporated herein in whole by reference. The immune checkpoint proteins may be PD-1, PD-L1, PDL-2, CD47, CD39, CD73, CD200, HVEC, CEACAM1, CD155, TIM-3, LAG-3, CTLA-4, A2AR, B7-H3, B7-H4, HLA-E, BTLA, IDO, KIR, VISTA, or combinations thereof.

[0038] The cells of this disclosure may be organized into structures similar to cylinders, rods, strings, filaments, or networks, as described in U.S. Patent Publication No. 2021 / 0213171 (the contents of which are incorporated herein by reference in their entirety). Such architectures may result in enhanced integration of the compositions of this disclosure into a host organism, as demonstrated by improved blood supply and / or improved vascular systems. The cells of this disclosure may be organized into clusters and / or islands that are incorporated into ECM components.

[0039] In one embodiment, the composition comprises one or more supporting cells, such as thymocytes, effector cells, and mesenchymal stem cells. The composition is maintained or cultured in the presence of one or more agents, such as but not limited to chemokines. Non-limiting examples of chemokines include CXCL12 and / or CCL2.

[0040] thymocyte The compositions described herein may comprise a population of thymocytes. Thymocytes may be cells derived from the thymus, or cells having one or more phenotypic or genotype markers associated with cells destined to become thymic cells. In some embodiments, the population of thymocytes may be derived by differentiation of pluripotent stem cells. In some embodiments, the pluripotent stem cells may be iPSCs.

[0041] In some embodiments, thymocytes may be prepared from the differentiation of pluripotent stem cells that differentiate into thymic stem cells via one or more of the following steps: PSCs can differentiate and / or be induced to differentiate into cells similar to the endoderm (DE) of the embryo. Endoderm cells can differentiate and / or be induced to differentiate into cells similar to the third pharyngeal endoderm (PPE), also known as the ventral pharyngeal endoderm (VPE). Endoderm cells can differentiate and / or be induced to differentiate into cells similar to the anterior foregut endoderm (AFE). AFE can differentiate and / or be induced to differentiate into cells similar to the third pharyngeal endoderm (PPE), also known as the ventral pharyngeal endoderm (VPE). Thymic epithelial precursor cells (TEPCs) can be generated from PPE cells. TECs can be derived from TEPCS. Each of the cell types described herein may be characterized by one or more markers. In some embodiments, pluripotent stem cells may be associated with increased expression of markers such as, but not limited to, OCT4, SOX2, and / or NANOG. In some embodiments, endoderm cells of the embryo may be associated with increased expression of markers such as, but not limited to, SOX17, FOXA2, CXCR4, and / or CER1. In some embodiments, anterior foregut cells (AFE) may be associated with increased expression of markers such as, but not limited to, FOXA2, SOX2, and / or PAX9. In some embodiments, third pharyngeal pouch endoderm cells may be associated with increased expression of markers such as, but not limited to, HOXA3, TBX1, PAX9, EYA1, SIX1, PBX1, and / or PAX1. In some embodiments, thymic epithelial precursor cells may be associated with increased expression of markers such as, but not limited to, FOXN1, K5, K8, and / or HOXA3. In some embodiments, thymocytes may be derived from DE cells, third PPE cells, AFE cells, TEPC cells, and / or TEC cells.

[0042] For the preparation of the thymocyte population of this disclosure, pluripotent stem cells may be cultured and differentiated into endoderm cells. Endoderm cells may be further cultured and differentiated into anterior foregut cells. In some embodiments, anterior foregut cells may be cultured and differentiated into pharyngeal endoderm cells. In some embodiments, pharyngeal endoderm cells may be cultured and differentiated into thymocytes, such as thymic epithelial cells. In some embodiments, differentiation may take place over approximately 14 to 21 days.

[0043] In some embodiments, thymocytes may be prepared from PSCs. In this regard, the method may include culturing pluripotent stem cells for a period and under conditions sufficient to differentiate them into thymocytes. For example, the method may include culturing pluripotent stem cells in the presence of factors and / or inhibitors that drive the differentiation of PSCs into thymocytes. In some embodiments, these factors and / or inhibitors may include, or consist of, pluripotent stem cell-specific inhibitors that selectively exclude human pluripotent stem cells. In some embodiments, the pluripotent stem cell-specific inhibitor includes, or consists of, an oleic acid synthesis inhibitor. In some embodiments, the pluripotent stem cell-specific inhibitor includes, or consists of, a stearoyl-coA desaturase (SCD1) inhibitor that inhibits the activity of SCD1 in human pluripotent stem cells. In some embodiments, the pluripotent stem cell-specific inhibitor includes, or consists of, a derivative of N-acylphenylhydrazine containing a phenylhydrazine (Ph-N[H,C]-NH) moiety. In some embodiments, the pluripotent stem cell-specific inhibitor comprises or consists of N'-phenylpyridine-4-carbohydrazide (NSC14613, also known as PluriSIn #1).

[0044] In some embodiments, the method for differentiating PSCs into thymocytes may be any method known to those skilled in the art. The method for differentiating PSCs into thymocytes may involve the use of one or more parameters known in the art for differentiation, or a combination thereof. Parameters include, but are not limited to, (i) factors that promote differentiation, (ii) inhibitors that promote differentiation, (iii) duration for promoting differentiation, (iv) temperature, (v) substrate, and / or (vi) supporting cells that promote differentiation. Any method or parameter for differentiating PSCs into thymocytes described in the following references may be used herein: Parent et al. Cell Stem Cell. 2013 Aug 1;13(2):219-29, Soh et al. Stem Cell Rep. 2014 Vol.2 j 925-937, Sun et al. Cell Stem Cell. 2013 Aug 1;13(2):230-6, Okabe et al. Cell.Reprog. 2015 Vol 17,No.5, Su et al. Sci.Rep. 2015 5,9882, Otsuka et al. Sci Rep 2020. 10:224, International Patent Publications WO2019 / 060336, WO2020 / 205859, WO2020 / 220040, WO2014 / 134213, WO2010 / 143529, WO2011 / 139628, WO2022 / 076751, WO2014 / 134213, WO2021 / 222297, and Chinese Patent Publication CN2011 / 10121243, the contents of each of these are incorporated herein by reference in their entirety.

[0045] Thymocytes can be from the embryo, fetus, or adult thymus.

[0046] In some embodiments, the population of thymocytes may comprise one or more cell types. The cell types may be iTEC cells, mTEC cells, keratinocyte-like mTEC cells, cTEC-high cells, cTEC-low cells, and mTEC-low cells.

[0047] In some embodiments, the population of thymocytes may include thymic epithelial cells (TECs). In some embodiments, TECs may be derived by differentiation of iPSCs. During embryonic development, TECs may originate from non-hematopoietic cells that are negative for CD45 expression and positive for the epithelial marker EpCAM. TECs may be thymic cortical epithelial cells (cTECs) and / or thymic medullary epithelial cells (mTECs). mTECs are characterized by cytokeratin 5 (KRT5 or K5) and cytokeratin 14 (KRT14 or K14) expression and low levels of cytokeratin 8 (KRT8 or K8) expression, while cTECs express K8 and K18. In some embodiments, thymocytes may originate from (K5+K8+)TECs that express both K5 and K8, which are typical of TEC cells found at the corticomedullary junction. In some embodiments, K5+K8+ cells may be precursors to mTECs and / or cTECs. mTECs may also be positive for the expression of Ulex europaeus agglutinin 1 (UEA-1) on the cell surface but not for Ly51 (e.g., UEA-1+Ly51-), while cTECs may be UEA-1-Ly51+.

[0048] In some embodiments, the population of thymocytes may include thymic medullary epithelial cells (mTECs). In some embodiments, mTECs may be derived from iPSC differentiation. In some embodiments, the thymocytes may be or be derived from mTECs. In some embodiments, mTECs may have high expression of markers such as, but not limited to, cytokeratin 5, cytokeratin 14, UEA-1, CD80, cathepsin L, and / or cathepsin S.

[0049] In some embodiments, thymocytes may be cTECs expressing high levels of markers such as, but not limited to, cytokeratin 8, cytokeratin 18, Ly51, CD205, cathepsin L, and / or thymus-specific serine proteases, or may be derived therefrom. In some embodiments, the population of thymocytes may include thymic cortical epithelial cells (cTECs). cTECs are involved in commitment to T cell lineages and positive selection of early thymocytes. In some embodiments, cTECs may be induced by differentiation of iPSCs. As a non-limiting example, thymocytes may be cTECs expressing markers such as CCL25 and / or K5, or may be derived therefrom.

[0050] In some embodiments, thymocytes may be or may be derived from TECs expressing one or more markers such as FOXN1, PAX9, PAX1, DLIA, ISL1, EYA1, SIX1, IL7, K5, K8, and AIRE.

[0051] Thymocytes may be or be derived from any of the cell types reported by Park et al. 2020 Science Vol.367, Issue 6480 (the contents of which are incorporated herein by reference in their entirety). For example, thymocytes may be derived from myoid cells, e.g., MYOD1 and MYOG-expressing myoid cells (referred herein to as TEC(myo)), and / or NEUROD1, SYP, CHGA-expressing TECs (referred herein to as TEC(neuro)).

[0052] In some embodiments, the population of thymocytes may include any cell type reported by Bautista et al. 2021 Nat Commun 12,1096 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the population of thymocytes drawn by the differentiation of iPSCs may include any cell type reported by Bautista et al. 2021 as "cTEC". lo"These cells may contain lower levels of functional genes (HLA class II) and may feature more KI67 + It may contain proliferating cells. Thymocytes, as reported by Bautista et al. 2021, are characterized by the expression of CLDN4, lower levels of HLA class II, PSMB11, PRSS16, CCL25, and high levels of the chemokine CCL21, and are referred to as "mTEC". lo "It may originate from cells."

[0053] In some embodiments, the population of thymocytes derived by the differentiation of hiPSCs or iPSCs may include any cell type described in US2023 / 0159887 (the contents of which are incorporated herein by reference in their entirety).

[0054] In some embodiments, the thymocyte population induced by iPSC differentiation is described by Bautista et al. 2021 as "mTEC," characterized by higher levels of expression of SPIB, AIRE, FEZF2, and HLA class II. hi "Cells may be included. Thymocytes may be keratinocyte-like mTECs, as reported by Bautista et al. 2021, characterized by the expression of KRT1 and / or IVL, or may be derived therefrom.

[0055] In some embodiments, the population of thymocytes derived by iPSC differentiation may include immature TECs (iTECs) as reported by Bautista et al. 2021, expressing standard TEC identity genes, such as FOXN1, PAX9, and SIX1.

[0056] In some embodiments, the population of thymocytes derived by iPSC differentiation may be characterized by the expression of cell surface markers such as, but not limited to, CTSV, SLC46A2, HLA-DMA, CXCL12, THY1, ENO1, CALR, ALCAM, ATPIF1, and / or HSPA5, and cTEC hi May contain (or high cTEC) cells.

[0057] In some embodiments, the population of thymocytes derived by iPSC differentiation may include AIRE+mTEC hypercellular cells that are characterized by the expression of one or more markers, including but not limited to LTF, HLA-DRA, CD74, HLA DRB1, HLA-DPA, HLA-DPB1, IL2RG, and / or FCER2.

[0058] In some embodiments, the population of thymocytes derived by the differentiation of iPSCs may include TECs expressing one or more markers such as, but not limited to, KRT5, KRT8, AIRE, PSMB11, and / or PRSS16.

[0059] In some embodiments, the population of thymocytes derived by iPSC differentiation may include TECs expressing one or more markers such as, but not limited to, AIRE, CK5, CK8, CXCL12, CCL25, DLL4, and / or HLA-DR.

[0060] In some embodiments, the population of thymocytes derived by the differentiation of iPSCs may include keratinocyte-like mTECs. These cells are called keratinocyte-like because they express genes such as keratinocyte cytoskeleton 1 (KRT1), KRT10, and SPINKS, which are also expressed in skin keratinocytes (terminally differentiated keratinocytes). Keratinocyte-like cells also express transcripts that overlap with mTECs, such as AIRE. Therefore, they are referred to as keratinocyte-like mTECs. They are likely precursors to Hassall's bodies, which are cells unique to the human thymus. It is also possible that these cells originate from mTEC precursor cells (Noam Kadouri et al. Nature Review Immunology 2020 v20:239, the content of which is incorporated herein by reference in its entirety).

[0061] Pluripotent stem cells (PSC) In some embodiments, the cells of this disclosure may be derived from pluripotent stem cells.

[0062] Pluripotent stem cells have the ability to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent stem cells may include, for example, stem cells, such as embryonic stem cells, nuclear transfer-derived embryonic stem cells, and induced pluripotent stem cells (iPSCs). Pluripotent stem cells may have a stem cell phenotype that includes (i) regenerative capacity and (ii) pluripotency. Pluripotency-associated genes may include, but are not limited to, Oct-3 / 4, Sox2, Nanog, GDF3, REXI, FGF4, ESGI, DPPA2, DPPA4, hTERT, and SSEAI.

[0063] The cells described herein may be derived from embryonic stem cells. ES cells may include cells that (a) self-renew, (b) differentiate to produce all cell types within an organism, and / or (c) originate from a developing organism. ES cells may originate from the inner cell mass of a blastocyst of a developing organism. ES cells may also originate from blastomeres generated by a single blastomere biopsy (SBB), which involves the extraction of a single blastomere from an 8-cell stage of a developing organism. ES cells may be characterized by the expression of markers such as, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and / or alkaline phosphatases. Methods for generating and characterizing ES cells are known in the art and can be found, for example, in U.S. Patents 7,029,913, 5,843,780, and 6,200,806 (the contents of each of these are incorporated herein by reference in their entirety).

[0064] Furthermore, the cells of this disclosure may be generated using induced pluripotent stem cells (iPSCs). iPSCs may include cells having one or more characteristics, such as (a) self-renewal, (b) the ability to differentiate to produce all cell types in an organism, and / or (c) somatic cell origin. iPSCs may express markers, such as, but not limited to, SSEA3, SSEA4, SOX2, OCT3 / 4, Nanog, TRA160, TRA1818, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and Zpf42. Methods for generating and characterizing iPS cells can be found, for example, in U.S. Patent Publications 2009 / 0047263, 2009 / 0068742, 2009 / 191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646 (the contents of each of these are incorporated herein by reference in their entirety). In some embodiments, iPSCs may be derived from T cells or non-T cells from peripheral blood mononuclear cells, B cells, or any other cells, hematopoietic precursor cells, or any other somatic cell type.

[0065] In some embodiments, pluripotent stem cells may be derived from adult stem cells. Adult stem cells may be obtained from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood of an object such as a subject. PSCs may also include embryonic stem cells derived from the placenta or umbilical cord, precursor cells (e.g., precursor cells derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood).

[0066] Effector Cells “Effector cell” refers to any cell or cell type that, upon contact with or proximity to a thymocyte, acquires the ability to perform, initiate, or propagate a signal or cell death trigger. “Contact or proximity” may refer to a spatiotemporal approach sufficient to enable intracellular or extracellular (e.g., intercellular) signaling or other transmission or interaction.

[0067] The effector cells described herein may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or precursor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, or thymus, or the stem cells or precursor cells may be differentiated in vitro from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The stem cells or precursor cells or ESCs or iPSCs from primary tissues may originate from humans or non-human animals (e.g., mice).

[0068] In some embodiments, the effector cells may be hematopoietic cells. In some embodiments, the effector cells may be lymphocytes. In some embodiments, the lymphocytes may be CD45-positive lymphocytes.

[0069] Effector cells include CD4+CD8-T cells, CD4-CD8+T cells, CD34+CD7+CDla+ cells, CD3+TCRab+ cells, CD3+TCRgd+ cells, CD3+TCRab+CD4+CD8- cells, C D3+TCRab+CD8+CD4- cells, CD3+TCRab+CD4+CD8-CD45RO-CD45RA+ cells, CD3+TCRab+CD8+CD4-CD45RO-CD45RA+ cells, CD3+TCRab +CD4+CD8-CD45RO-30CD45RA+CCR7+ cells, CD3+TCRab+CD8+CD4-CD45RO-CD45RA+CCR7+ cells, CD3+TCRab+CD4+CD8-CD45RO-C D45RA+CD27+ cells, CD3+TCRab+CD8+CD4-CD45ROCD45RA+ cells, CD27+, CD34+CD7+CD1a+ cells, CD34+CD5+CD7+ cells, CD34+CD5+CD7- Cells, natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or CD45+, CDIlb+, CDIlb-, CD15+, CD15-, CD24+, CD24-, CDI14+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CDlla+, CDlla-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, F Cells that are positive for oxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD3l+, CD3l-, CD30+, CD30-, CD38+, and / or CD38-, as well as / or combinations thereof.

[0070] In some embodiments, effector cells may be T cells. The T cells may be cultured T cells, e.g., primary T cells, or cultured T cell lines, e.g., Jurcut cells, SupTl cells, or T cells derived from mammals. If derived from mammals, effector cells may be obtained from a multitude of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or fluids. Effector cells may also be concentrated or purified. The T cells may be any type of T cell, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells (TILs), memory T cells, and naive T cells, and may be at any developmental stage.

[0071] In some embodiments, the effector cells may be CCRXA-, CD3+, CD69-, MHC-1+, CD62L+, and / or CCR7+.

[0072] Effector cells may have a naive T cell (TN) phenotype, a central memory T cell (TcM) phenotype, or an effector memory T cell (TEM) phenotype. The phenotypes of TN, TcM, and TEM cells are known in the art. For example, CCR7 and CD62L are expressed by TN and TcM cells but not by TEM cells. The transcription factors LEFl, FOXPl, and KLF7 are expressed by TN and TcM cells but not by TEM cells. CD45RO and KLRGl are not expressed by TN cells but are expressed by TEM cells (Gattinoni et al., Nat. Rev. Cancer, 12:671-84 (2012)). Alternatively or additionally, TN and TcM cells may be characterized by longer telomeres compared to TEM cells.

[0073] In some embodiments, effector cells may be TCRα+TCRβ+ cells. TCRα+TCRβ+ effector cells may be T cells expressing receptors that express alpha (α) chains and / or beta (β) chains. TCR alpha and beta chains are known in the art.

[0074] Effector cells can be further modified. In further embodiments, stem cells or precursor cells can be genetically modified. For example, stem cells or precursor cells may express an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR), or both. In further embodiments, stem cells or precursor cells may express an exogenous invariant natural killer T cell (iNKT)-associated TCR. In further embodiments, stem cells or precursor cells may express an exogenous antigen-specific TCR or have exogenous genetic modifications to genes that regulate T cell differentiation, expansion, or function.

[0075] In some embodiments, effector cells may be FOXP3+ Tregs. Tregs can be generated via clonal transmutation of mTECs, thereby resulting in the expression of tissue-specific antigens on the surface (i.e., on antigen-presenting cells (APCs)) of Aire in the mTECs. Autoreactive T cells that recognize tissue-specific antigens give rise to FOXP3+ Tregs that can mediate peripheral tolerance (see Husebye, Eystein S., Mark S. Anderson, and Olle Kaempe. "Autoimmune polyendocrine syndromes." New England Journal of Medicine 378.12 (2018):1132-1141 (the whole article is incorporated herein by reference)).

[0076] supporting cells In some embodiments, the cells of the present disclosure may include, or may be cultured with, supporting cells that assist in the generation and / or maintenance of thymocytes in culture. Non-limiting examples of supporting cells include hematopoietic non-T cell precursors such as macrophages and dendritic cells (DCs); non-hematopoietic cells such as epithelial cells and fibroblasts; and stromal cells such as skeletal tissue precursors, components such as bone, cartilage, hematopoietic supporting stroma, and adipocytes. In some embodiments, supporting cells promote the proliferation, survival, maturation, or function of thymocytes. In some embodiments, supporting cells may be of mesenchymal origin.

[0077] Supporting cells may be non-immune cells that can be present in the thymic microenvironment. For example, supporting cells may be fibroblasts, vascular smooth muscle cells (VSMCs), endothelial cells, and / or lymphoid endothelial cells. Supporting cells may also include mesenchymal stem cells (MSCs) from different sources, including bone marrow-derived MSCs, adipose tissue-derived MSCs, thymic-derived MSCs, or iPS-derived MSCs (Tong Ming Liu et al Stem Cell Reports 2020 14:210, Samsonraj et al Stem Cells Translational Medicine 2017 6:2173; the contents of each of these are incorporated herein by reference in their entirety).

[0078] In some embodiments, supporting cells may be neuroendocrine cells (expressing BEX1, NEUROD1), myosomal cells (expressing MYOD1, DES), and myelin-positive epithelial cells (also referred to herein as myelin cells) (expressing SOX10, MPZ), as reported by Bautista et al. 2021 Nat Commun 12, 1096 (2021) (the contents of which are incorporated herein by reference in their entirety). In some embodiments, mesenchymal cells may be associated with markers such as, but not limited to, LAMA2, LAMA4, PDGFRA, PDGFRB, LUM, CSPG4, COL1A2, COL3A1, IGF1, FGF7, FGF10, FST, BMP4, SFRP2, WNT5A, etc. Mesenchymal cells may be positive or negative for one or more of these markers. In some embodiments, mesenchymal cells may be positive for some of the marks described herein but negative for others.

[0079] In some embodiments, endothelial cells may be associated with one or more markers, including but not limited to VEGFC, PECAM1, APLNR, PROX1, LYVE1, ACKR1, SELE, SELP, FN1, and / or TGFB1. Endothelial cells may be positive or negative for one or more of these markers. In some embodiments, endothelial cells are adult venous endothelial cells, adult arterial endothelial cells, embryonic stem cell-derived endothelial cells, iPS cell-derived endothelial cells, umbilical vein endothelial cells, umbilical artery endothelial cells, bone marrow-derived endothelial precursor cells, umbilical cord blood-derived endothelial precursor cells, peripheral blood-derived endothelial precursor cells, adipose tissue-derived endothelial precursor cells, adult skin-derived endothelial cells, or a combination thereof. In some embodiments, umbilical vein endothelial cells are human umbilical vein endothelial cells (HUVECs).

[0080] In some embodiments, the supporting cells of this disclosure may be fibroblasts and / or fibroblast-like cells. In some embodiments, the fibroblasts are human foreskin fibroblasts, human embryonic fibroblasts, mouse embryonic fibroblasts, cutaneous fibroblasts, angiofibroblasts, myofibroblasts, smooth muscle cells, mesenchymal stem cell (MSC)-derived fibroblasts, or a combination thereof. In some embodiments, the fibroblasts are normal human dermal fibroblasts (NHDF).

[0081] In some embodiments, supporting cells may be ciliated cells positive for ATOH1, GFI1, LHX3, and / or FOXJ1. In some embodiments, supporting cells may be myelin cells closely resembling Schwann cells, which may be positive for SOX10, MPZ, MBP, and / or S100A1. In some embodiments, supporting cells may be tuft cells, which may be positive for the markers GNB3, TRPM5, GNAT3, PLCB2, OVOL3, and / or POU2F3.

[0082] In some embodiments, the supporting cells may be a population of ionocytes that may be positive for FOXI1, ASCL3, CFTR, and / or CLCNKB.

[0083] Other non-limiting examples of supporting cells include hepatocytes, pancreatic exocrine cells, muscle cells, pancreatic endocrine cells, neurons, intestinal cells, adipocytes, spleen cells, renal cells, cholangiocarcinoma cells, Kupffer cells, astrocytes, cardiomyocytes, alveolar cells, bronchocytes, club cells, urothelial cells, mucosal cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gallbladder cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, or corneal epithelial cells.

[0084] Thymic organoids In some embodiments, the compositions of this disclosure may include thymic organoids. Organoids are in vitro, three-dimensional, reduced-scale reproductions of organs. Thymic organoids may be in vitro, three-dimensional, reduced-scale versions of thymic organs that can mimic the physiology and function of the human thymus. A method for preparing thymic organoids is described in International Patent Publication WO2019 / 060336, the contents of which are incorporated herein by reference in their entirety.

[0085] In some embodiments, effector cells may be prepared by differentiating pluripotent stem cells or precursor cells into lymphocytes by culturing PSCs or precursor cells with thymocytes. In some embodiments, thymocytes may express Notch ligand. In some embodiments, Notch ligand may be Delta-like 1 (DLL1). In some embodiments, Notch ligand may be Delta-like 4 (DLL4). In some embodiments, Notch ligand may be one described herein or one of the art, such as U.S. Patent No. 7,795,404 (which is incorporated herein by reference in its entirety). Effector cells of this disclosure may be prepared using a thymic organoid cell culture system. In some embodiments, the method further comprises contacting co-cultured stem cells or precursor cells and stromal cells with Flt-3 ligand and / or IL-7 and / or stem cell factor / Kit ligand and / or thrombopoietin. In some embodiments, differentiating stem cells or precursor cells into T cells involves culturing a three-dimensional (3D) cell aggregate comprising a) a selected population of supporting cells endogenously or exogenously expressing Notch ligand, and b) a selected population of stem cells or precursor cells, in a serum-free medium comprising B-27® supplement, xeno-free B-27® supplement, GS2l™ supplement, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem cells or precursor cells described in International Patent Publication WO2017 / 075389 may be useful to this disclosure (their contents are incorporated herein by reference in their entirety).

[0086] In some embodiments, the thymic organoid may be based on the artificial thymic organoid described by Seet CS, et al. Nat Methods. 2017;14(5):521-530 (the contents of which are incorporated herein by reference in their entirety). To prepare thymic organoids, thymocytes may be isolated by trypsin treatment and resuspended in serum-free culture medium ("RB27") which may contain RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 μM L-ascorbic acid 2-phosphate sesquimagnium salt hydrate reconstituted in PBS (Sigma-Aldrich, St. Louis, MO), 1% penicillin / streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng / ml rhFLT3L, and 5 ng / ml rhIL-7 (Peprotech, Rocky Hill, NJ). Different ratios of thymocytes to effector cells were prepared in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 minutes at 4°C in a swing-bucket centrifuge. The supernatant was carefully removed and the cell pellet was resuspended by vortexing for a short time. For each organoid, a 0.4 μm Millicell Transwell insert (EMD Millipore, Billerica, MA, catalog PICM0RG50) could be placed in a 6-well plate containing 1 ml of RB27 per well. To seed the organoids, the insert was removed and placed at the edge of the plate to drain excess medium. Cell slurry was prepared to 5 μl per organoid and added to the plate by drawing it up with a 20 μl pipette tip and forming a droplet at the end of the pipette tip, and gently deposited onto the cell insert. The cell insert could be returned to the wells containing 1 mL of RB27. The culture medium can be completely replaced every 3-4 days by aspirating from around the cell insert, followed by replacement with 1 ml containing fresh RB27 / cytokines.In some embodiments, organoids can be cultured by this means for up to 10, 15, 20, 25, or 30 weeks.

[0087] During the indicated period, organoid cells were collected by adding FACS buffer (PBS / 0.5% bovine serum album / 2 mM EDTA) to each well, pipetting with a 1 ml "P1000" pipette to briefly deagglomerate the organoids, and then passing them through a 50 μm nylon strainer. In some experiments, single-cell suspensions of MS5-hDLL1 cells were irradiated with gamma rays at the indicated dose before being used as organoids.

[0088] Thymic organoid effector cell co-cultures can be prepared as reported in Seet CS, et al. Nat Methods. 2017;14(5):521-530 (the contents of which are incorporated herein by reference in their entirety). Thymic cells can be seeded in 12-well plates coated with 0.1% gelatin 1-2 days before use to achieve a 70-80% density. The medium can be aspirated from a monolayer, 1.5 × 10⁶ 4 FACS-purified effector cells (CD34+CD3-hematopoietic cells) can be seeded with thymic organoids in 2 ml of culture medium consisting of MEMα, 20% FBS, 30 μM L-ascorbic acid, 5 ng / ml rhFLT3L, and 5 ng / ml rhIL-7. In some embodiments, effector cells can be transferred to thymic organoids every 4-5 days by harvesting the cells, filtering them through a 50 μm nylon strainer, and reseeding them in fresh culture medium. Upon culling, the cells were divided into multiple wells containing fresh stroma.

[0089] In some embodiments, the thymocytes of this disclosure may be combined with double-negative 14-day T cells to form cell clusters, which may then be deposited as “organoids” on a transwell and maintained under gas-liquid interface culture conditions. In some embodiments, cells may be harvested from the culture medium every few days to assess T cell maturation.

[0090] Thymic support system (TSS) In some embodiments, the compositions of the Disclosure may include a thymic support system. As used herein, a thymic support system refers to a system of one or more non-viable components that interact with the cells of the Disclosure to provide structural, supportive, and / or essential cues for the viability, function, proliferation, and / or differentiation of the cells of the Disclosure. In one embodiment, the TSS promotes the differentiation of iPS cells, AFE cells, VPE cells, DE cells, and / or TEP cells. In some embodiments, the TSS may mimic the thymus or thymic tissue structure in a three-dimensional manner.

[0091] The thymic support system may include one or more components such as polymers, matrices, and matrix components, and optionally one or more drugs.

[0092] In some embodiments, the TSS may (i) provide a structure for thymocyte adhesion, proliferation, and differentiation, (ii) create a suitable biomechanical environment, and / or (iii) allow for the diffusion of nutrients and oxygen.

[0093] In some embodiments, the TSS can mimic the tensegrity, viscoelasticity, and / or rigidity of the thymus (Engler AJ, et al. Cell. 2006;126(4):677-89, the contents of which are incorporated herein by reference in their entirety).

[0094] In some embodiments, the TSS may have an architecture that enables T cell migration toward and from the TSS and / or cells of the present disclosure.

[0095] In some embodiments, TSS may be “bioinactive,” “biocompatible,” “bioactive,” or “reabsorbable,” depending on their biological response in vivo. As used herein, “biocompatible” means a substance or object that, when introduced into an organism, performs its desired function without inducing an inflammatory response, immunogenicity, or cytotoxicity in the cells, tissues, or organs, or substances or objects into which it is introduced.

[0096] In some embodiments, the TSS may include a porous network capable of exchanging oxygen, nutrients, and metabolites. With respect to porosity, the TSS may be biphasic, comprising regions of the TSS having a highly porous morphology to mimic the thymic medulla and other regions of the TSS having a less porous morphology to mimic the thymic cortex.

[0097] Other desirable properties of TSS include, but are not limited to, the ability to deliver a high number of cells to a desired site without disrupting their properties (e.g., pre-formed cell structures), reduced ethical concerns, and / or reduced batch-to-batch variability.

[0098] In some embodiments, TSS may comprise decellularized thymic tissue. This approach provides an opportunity to grow thymocytes in their native environment while retaining biomechanical and / or biochemical clues. Decellularization can be achieved by any method known in the art, such as whole-organ perfusion followed by detergent washing. Decellularized thymic tissue may also be homogenized and crosslinked to produce the TSS of the Disclosure. In some embodiments, the composition of the Disclosure may comprise decellularized extracellular matrix extracted from thymic tissue. The thymic tissue may be fetal, neonatal, juvenile, or adult. The thymic tissue may be autologous, allogeneic, or heterogeneous.

[0099] Cells may be patterned within or on the TSS of the Disclosure by selective polymerization of the polymers of the Disclosure, by patterning of cells using an electric field, or both. Cells may be patterned by placing them within specific regions of a relatively homogeneous preparation of polymers (resolution up to approximately 5 microns), or by constructing a patterned polymer scaffold of a defined pattern in which living cells are contained (resolution up to approximately 100 microns). Patterning may be performed without direct mechanical manipulation or physical contact and without relying on active cell processes such as cell adhesion.

[0100] The methods described herein can be used to produce any of a number of patterns in monolayers or multilayers, comprising characteristic geometric shapes or repeating sequences of dots in various sizes. Alternatively, multilayer biopolymer gels can be produced using a single mask rotated in various directions. The formation of high-resolution patterned cells in three dimensions can be achieved by methods other than photopolymerization, such that the limitations of the method are overcome.

[0101] The TSSs of this disclosure may be organized into structures similar to cylinders, rods, strings, filaments, or networks. Such architectures may result in enhanced integration of the compositions of this disclosure into a host organism, as demonstrated by improved blood supply and / or an improved vascular system. The TSSs may be patterned to allow for organization into clusters or islands of cells. Patterning may be carried out according to the process shown in Figure 1A or Figure 5A of U.S. Patent Publication 2021 / 0213171, the contents thereof incorporated herein in their entirety by reference. The TSSs may also be assembled using custom 3D printing technology.

[0102] polymer In some embodiments, the TSS may comprise polymers. Any substance or blend of natural or synthetic sources may be used in the thymic support system. In some embodiments, the TSS may be a biopolymer, a synthetic polymer, or a compound thereof.

[0103] In some embodiments, the polymers may be hydrophilic polymers. Hydrophilic polymers contain polar or charged functional groups that make them soluble in water. They can interact with or dissolve in water or other polar substances. In some embodiments, one or more polymers (or their monomers) or at least one of the one or more polymers has one or more hydrophilic groups. In some embodiments, each of the one or more hydrophilic groups may be individually selected from -NH2, -COOH, -OH, -CONH2, -CONH-, and -SO3H. Each of the one or more polymers may be individually selected from bio-derived polymers and synthetic polymers. In some embodiments, all polymers in the hydrogel are bio-derived polymers. In some embodiments, all polymers in the hydrogel are synthetic polymers. In some embodiments, if two or more polymers are present, at least one polymer is a bio-derived polymer and at least one polymer is a synthetic polymer.

[0104] In some embodiments, the TSS may be a temperature-responsive polymer (e.g., pNIPAM, PVME) that transitions between a hydrophobic and hydrophilic state at a specific temperature, enabling control of cell culture and growth, as well as subsequent ECM deposition and the formation of cell sheets that adhere to biological surfaces.

[0105] The molecular weight of the polymer can be any preferred molecular weight. In some embodiments, each polymer present in the hydrogel has an average molecular weight independently selected from about 100 to about 1000 Da, about 100 to about 900 Da, about 100 to about 800 Da, about 100 Da to about 700 Da, about 200 Da to about 600 Da, about 300 Da to about 600 Da, about 400 Da to about 600 Da, about 500 Da to about 600 Da, about 525 Da to about 600 Da, about 550 Da to about 600 Da, and 575 Da to about 600 Da.

[0106] In one embodiment, cells of the Disclosure, including but not limited to iPS cells, AFE cells, VPE cells, DE cells, TEP cells, and / or TECs, are encapsulated in the polymer described herein. The 3D arrangement of TECs in the thymic microenvironment is important for maintaining their thymic epithelial genetic signature. Thymocytes prepared as aggregates in biocompatible hydrogels can maintain their molecular properties and extend their survival in vitro for up to 7 days. Encapsulating the cells of the Disclosure is also advantageous when scaling up the production of thymocytes for clinical application. In one embodiment, the polymer of the Disclosure provides 3-D matrix support, which is important for the survival of iPSC-derived thymocytes.

[0107] In some embodiments, the concentration of the polymer is about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, 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%, or more. Approximately 0.1%~0.5%, approximately 0.5%~1%, approximately 1%~1.5%, approximately 1.5%~2%, approximately 2%~2.5%, approximately 2.5%~3%, approximately 3%~3.5%, approximately 3.5%~4%, approximately 4%~4.5%, approximately 4.5%~5%, approximately 5%~5.5%, approximately 5.5%~6%, approximately 6%~6.5%, approximately 6.5%~7%, approximately 7%~7.5%, approximately 7.5%~8%, approximately 8%~8.5%, approximately 8.5% ~9%, approximately 9%~9.5%, approximately 9.5%~10%, approximately 10%~15%, approximately 15%~20%, approximately 20%~25%, approximately 25%~30%, approximately 30%~35%, approximately 35%~40%, approximately 40%~45%, approximately 45%~50%, approximately 50%~55%, approximately 55%~60%, approximately 60%~65%, approximately 65%~70%, approximately 70%~75%, approximately 75%~80%, or more.

[0108] Bio-derived polymers Any polymer derived from natural sources or living organisms may be defined as a bio-based polymer and may be used in the thymic support system.

[0109] Examples of bio-derived polymers include, but are not limited to, one or more of the following: proteins, polypeptides, polysaccharides, lipids, nucleic acids, and glycosaminoglycans. Examples of bio-derived polymers include, but are not limited to, one or more of the following: silk, fibroin, sericin, keratin, alpha-keratin, beta-keratin, alginate, elastin, fibrillin, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, fibrinogen, fibrin, fibronectin, laminin, collagen, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, vimentin, nerve filaments, light chain nerve filaments (NF-L), medium chain nerve filaments (NF-M), heavy chain nerve filaments (NF-H), amyloid, alpha-amyloid, beta-amyloid, actin, myosin, chitin, gelatin, chitin, hyaluronic acid, D-glucuronic acid, regumin, bicillin, and / or DN-acetylglucosamine.

[0110] Based on their monomer units and structure, bio-based polymers can be classified as polypeptide-based bio-based polymers, polysaccharide-based bio-based polymers, and polynucleotide-based bio-based polymers.

[0111] In some embodiments, the bio-based polymer may be a polypeptide-based bio-based material. Non-limiting examples of polypeptide-based bio-based polymers include collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin, and / or actin. In some embodiments, the bio-based polymer may be collagen, which is a primary structural element of the extracellular matrix (ECM) and has several functional features that help it bind to cells, such as ECM proliferation, differentiation, and secretion. In some embodiments, collagen may consist of three identical chains (homotrimers), e.g., types II, III, VII, VIII, and / or X. Collagen may also consist of two or more different chains (heterotrimers), e.g., types I, IV, V, VI, IX, and XI. Collagen compositions may be modified by combining them with other molecules such as hyaluronic acid (HA), chitosan, and chondroitin sulfate (CS) to achieve improved biological activity and mechanical properties of the final scaffold. In some embodiments, the bio-based polymer may be gelatin, which is the result of degradation derived from insoluble collagen by disintegration or denaturation. In some embodiments, the bio-based polymer may be silk, which is a natural protein-based polymer derived from various Lepidoptera larvae such as spiders, as well as silkworms.

[0112] In some embodiments, the biopolymer may be a polysaccharide-based biopolymer. Non-limiting examples of polysaccharide-based biopolymers include chitin, chitosan, alginates, hyaluronic acid, cellulose, agarose, dextran, and / or glycosaminoglycans. Polysaccharide-based biopolymers can be made from different units of monosaccharides or disaccharide chains (e.g., starch, cellulose). The effect is that a very large number of structurally diverse polysaccharides are mixed as numerous different sugar isomers, utilizing the range of chemical bonding. In some embodiments, the biopolymer may be based on structural polysaccharides, e.g., cellulose in plants and chitin in crustacean shells. Chitin is commonly found in crustacean shells, and its derivative, chitosan, is obtained by deacetylation of chitin. These are glycosaminoglycans, such as naturally occurring cationic polysaccharides. In one embodiment, the polymer may be dextran sulfate.

[0113] In some embodiments, the bio-derived polymers may be starch and glycogen.

[0114] In some embodiments, the bio-derived polymer may be hyaluronic acid (HA), a linear polysaccharide that is ubiquitous in the extracellular matrix (ECM) of mammals and can be highly biocompatible. HA contains functional groups such as carboxylic acids and alcohols, which can be used to implement functional domains or develop hydrogels by linking them together.

[0115] In some embodiments, the bio-based polymer may be a polynucleotide-based bio-based polymer. Non-limiting examples of polynucleotide-based bio-based polymers include DNA, linear plasmid DNA, and / or RNA.

[0116] In some embodiments, the polymers of the present disclosure may be a combination of two bio-derived polymers described in International Patent Publication WO2022 / 015902, for example, heparin and dextran, the contents of which are incorporated herein by reference in their entirety.

[0117] Synthetic polymers In some embodiments, the TSS may include synthetic polymers. As used herein, the term “synthetic” means produced, prepared, and / or manufactured by human hands. The synthesis of the polymers or other molecules of this disclosure may include chemical or enzymatic synthetic methods.

[0118] In some embodiments, the synthetic polymer may be poly(α-hydroxyesters) including PCL, PGA, PLA, and their copolymer PLGA, as well as poly(ethers) including PEO and PEG, PVA, and PU.

[0119] In some embodiments, the synthetic polymer may be polylactic acid (PLA), a semi-crystalline polymer that gradually crystallizes. PLA can be prepared from lactic acid (LA) monomers through fermentation processes of natural resources such as wheat and grains, or by various polymerization routes as petrochemical derivatives. In some embodiments, PLA may be poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLA), and / or poly(D,L-lactic acid) (PDLLA). In some embodiments, the synthetic polymer may be polyglycolic acid (PGA).

[0120] In some embodiments, the synthetic polymer may be polylactic acid-coglycolic acid (PLGA), a randomly ring-opened copolymer of PLA and PGA. In some embodiments, the synthetic polymer may be polycaprolactone (PCL), a semicrystalline and aliphatic polymer. In some embodiments, the synthetic polymer may be poly(ethylene oxide) (PEO), an inert, hydrophilic polymer with minimal antigenicity, immunogenicity, cell adhesion, and protein-binding properties. In some embodiments, the synthetic polymer may be polyurethane (PU), which contains a urethane moiety in its repeating units. Reactions of diisocyanates with polyols typically produce these polymers.

[0121] Examples of synthetic polymers include, but are not limited to, one or more of the following: poly(urethane), poly(siloxane), poly(ethylene), poly(vinylpyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), PLA, PGA, PLGA, polyhydroxybutyrate (PHB), polypropylene fumarate (PPF), polyvinyl alcohol (PVA), polypropylene carbonate, polyanhydride, polyphosphazene, polygermane, polyorthoester, polyester, polyamide, polyolefin, polycarbonate, polyaramid, polyimide, PCL, and their copolymers, derivatives, or combinations.

[0122] In some embodiments, one or more polymers are individually polyethylene glycol (PEG), chitosan, poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), hydroxyethoxyethyl methacrylate (HEEMA), hydroxydiethoxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethoxyethyl methacrylate (MEEMA), methoxydiethoxyethyl methacrylate (MDEEMA), ethylene glycol dimethacrylate (EGDMA), N-vinyl-2-pyrrolidone (NVP), N-isopropyl AAm (NIPAAm), vinyl acetate (VAc), acrylic acid (AA), N -Selected from (2-hydroxypropyl)methacrylamide (HPMA), ethylene glycol (EG), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, poly(N-isopropylacrylamide) (PNIPAM), self-assembling peptides, acrylate-modified PEG and acrylate-modified hyaluronic acid, heparin, amine-terminated functionalized 4-arm star-shaped PEG, or any combination thereof. In some embodiments, at least one of the polymers is PEGDA.

[0123] In some embodiments, the Disclosure provides synthetic polymers comprising polysaccharides modified by converting one or more groups present in the polysaccharide to negatively charged functional groups, the negatively charged groups providing the synthetic polymer with a sufficient amount of negative charge to promote one or more of the following: growth factor binding, growth factor activity, and angiogenesis. In some embodiments, the functional group in the polysaccharide converted to a negatively charged group may be a hydroxyl group. In some embodiments, the polysaccharide of the synthetic polymer may be dextran, alginate, agarose, chondroitin sulfate, chitin / chitosan, cellulose, starch, hyaluronic acid, galactogen, inulin, pectin, or glycogen. As a non-limiting example, the synthetic polymer may be a heparin mimic described in International Patent Publication WO2022 / 015902 (the contents of which are incorporated herein by reference in their entirety).

[0124] Composite polymers By combining bio-derived polymers and synthetic polymers, composite polymers can be produced that combine the characteristics of both polymer types, thereby producing composite polymers with improved properties. For example, the thymocytes of this disclosure can be cultured with a collagen-PCL composite polymer (see DJ Choi, et al. J. Biotechnol. 2015, 205, 47, the contents of which are incorporated herein by reference in their entirety). Thymocytes and / or thymocyte organoids can also be prepared using a gelatin-and-PEG composite polymer (see ABSuraiya, ACS Biomater. Sci. Eng. 2020, 6, 2198, the contents of which are incorporated herein by reference in their entirety).

[0125] Hydrogel In some embodiments, the polymer may be a hydrogel. Hydrogels are generally prepared by converting a hydrophilic polymer solution into a 3D network structure via physical or chemical crosslinking. During this process, the hydrogel can uniformly encapsulate cells and provide them with a 3D microenvironment similar to the innate extracellular matrix (ECM). The behavior and function of cells in vivo are influenced by stimuli generated by the surrounding ECM. Similarly, the structure and physicochemical properties of the hydrogel provide important clues for controlling the function of the embedded cells. The structure and physicochemical properties of the hydrogel can be designed and controlled by selecting different polymers, crosslinking methods, and assembly strategies.

[0126] Hydrogels can be prepared from polypeptide-based polymers (such as gelatin, collagen, fibrin, and silk fibroin) and polysaccharide-based biopolymers (such as hyaluronic acid (HA), chondroitin sulfate (CS), alginate, and chitosan), but are not limited to these. Collagen can be used in hydrogel preparations as a major ECM component in various tissues. In some embodiments, gelatin, a collagen derivative with higher solubility, can also be used to prepare hydrogels in this disclosure. Hyaluronic acid (HA), a glycosaminoglycan, is commonly found in body fluids, and its ECM can be used in hydrogel preparations. Other polysaccharide-based biopolymers such as alginate (obtained from bacteria and brown algae) and chitosan (derived from chitin produced from crab and shrimp shells) can also be used in this disclosure.

[0127] The hydrogels of this disclosure may also be prepared using synthetic polymers such as, but not limited to, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAM), and polyacrylamide (PAM) (Darnell et al., 2013).

[0128] The hydrogels described herein may include EAK16-II / EAKIIH6 self-assembling hydrogels as reported by A. Tajima et al. Clin.Immunol.2015,160,82 (the contents of which are incorporated herein by reference in their entirety).

[0129] In some embodiments, hydrogels may be prepared from polymers of hydrophilic monomers. As used herein, a hydrophilic monomer refers to any monomer that, when polymerized, produces a hydrophilic polymer that can form a hydrogel when in contact with an aqueous medium such as water. Examples of hydrophilic monomers include hydroxyl-containing monomers such as 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylamide, 2-hydroxyethyl acrylamide, N-2-hydroxyethyl vinylcarbamate, 2-hydroxyethyl vinyl carbonate, 2-hydroxypropyl methacrylate, hydroxyhexyl methacrylate, and hydroxyoctyl methacrylate; carboxyl-containing monomers such as acrylic acid, methacrylic acid, itaconic acid, fumaric acid, crotonic acid, maleic acid, and their salts, monomethyl maleic acid esters, monoethyl maleic acid esters Examples include, but are not limited to, free carboxyl group-containing esters such as ster, monomethyl fumarate, monoethyl fumarate, and their salts; amide-containing monomers such as (meth)acrylamide, crotonamide, cinnamic acid amide, maleic acid diamide, and fumaric acid diamide; thiol-containing monomers such as methanethiol, ethanethiol, 1-propanethiol, butanethiol, tert-butyl mercaptan, and pentanethiol; and sulfonic acid-containing monomers such as p-styrene sulfonic acid, vinyl sulfonic acid, pa-methylstyrene sulfonic acid, isoprene sulfonide, and their salts.

[0130] In some embodiments, hydrogels may be formed by the self-assembly of one or more types of hydrophilic polymers in an aqueous medium. The term “self-assembly” refers to the process of spontaneous organization of higher-order structural components by relying on mutual incentives between components, without the formation of chemical bonds between components. For example, polymer chains may interact with each other via hydrophobic forces, hydrogen bonds, van der Waals interactions, electrostatic forces, or polymer chain entanglement induced on the polymer chains, so as to aggregate or solidify in an aqueous medium, which can form a three-dimensional network, thereby trapping water molecules and forming a hydrogel.

[0131] Complexes of bio-derived and synthetic polymers may also be used. For example, PAM, PVA, PNIPAM, and PEG hydrogels can be blended with gelatin. Cell diffusion and proliferation in these hybrid hydrogels may be enhanced compared to cells cultured in 3D in synthetic polymer-based hydrogels.

[0132] Crosslinked polymers Crosslinking, as used herein, may be defined as the induction of chemical or physical bonding between polymer chains. The polymers of the Disclosure may be crosslinked to improve their mechanical, biological, and / or degradation properties.

[0133] In some embodiments, hydrophilic polymers can be crosslinked to form hydrogels.

[0134] Various methods for crosslinking polymer chains are known in the art and may be used herein. The method may be selected depending on the materials chemistry and the desired function. In some embodiments, crosslinking may be formed by covalent or ionic bonding.

[0135] Polymers can be physically crosslinked under very mild conditions without the use of crosslinking agents that can often cause toxicity to cells or affect the activity of biological molecules. Physical crosslinking methods include, but are not limited to, ionic interactions, guest-host interactions, and thermal gelation.

[0136] In some embodiments, the polymers described herein may be chemically crosslinked. In some embodiments, some or all of the crosslinks are formed via thiol-Michael addition reactions, such as thiol-Michael addition click reactions. Chemically crosslinked hydrogels may have stronger bond energy and improved flexibility depending on the nature of the crosslinking reaction. Hydrophilic polymers have many functional groups, such as OH, COOH, and NH2. 3D networks can be established by covalent bonding between these functional groups using glutaraldehyde and EDC / NHS. In some embodiments, chemical crosslinking may be achieved by photopolymerization (using photoreactive moieties such as methacrylate or acrylate groups), enzymatic crosslinking (using enzymes such as transglutaminase and horseradish peroxidase), click chemistry, and / or Schiff base reactions (via coupling between aldehyde and amine groups in the polymer chain).

[0137] Examples of compounds that act as crosslinking agents include dextrandialdehyde, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), vinylamine, 2-aminoethyl methacrylate, 3-aminopropyl methacrylamide, ethylenediamine, ethylene glycol dimethacrylate, methyl methacrylate, N,N'-methylene-bisacylamide, N,N'-methylenebis-methacrylamide, diallyl tartrate diamide, allyl (meth)acrylate, and lower alkylene glycol di( Examples include, but are not limited to, meth)acrylates, polylower alkylene glycol di(meth)acrylates, lower alkylene di(meth)acrylates, divinyl ethers, divinyl sulfones, di- or trivinylbenzenes, trimethylolpropane tri(meth)acrylates, pentaerythritol tetra(meth)acrylates, bisphenol A di(meth)acrylates, methylene bis(meth)acrylamides, triallyl phthalates, diallyl phthalates, transglutaminases, or mixtures thereof.

[0138] In some embodiments, the crosslinking may be biodegradable or non-biodegradable.

[0139] In some embodiments, the crosslinking agent may be a peptide crosslinking agent. In non-limiting examples, the crosslinking agent may be a protease-degradable crosslinking agent, such as the VPM peptide GCRDVPMSMRGGDRCG (SEQ ID NO: 1), which is rapidly cleaved by matrix metalloproteinase (MMP)-1 and MMP-2 proteases. In some embodiments, the crosslinking agent may be a matrix metalloproteinase (MMP)-cleavable peptide. In some embodiments, the peptide may include amino acid sequences CGPQGIAGQGCR (SEQ ID NO: 2), GPQGIAGQ (SEQ ID NO: 3), GPQGIWGQ (SEQ ID NO: 4), VPMSMRGG (SEQ ID NO: 5), QPQGLAK (SEQ ID NO: 6), GPLGLSLGK (SEQ ID NO: 7), or GPLGMHGK (SEQ ID NO: 8). Additional MMP-cleavable peptides can be found in Tu, Y. et al. Smart Pharmaceutical Nanocarriers; 83-116 (2016), the full contents of which are incorporated herein by reference.

[0140] In some embodiments, the polymers of the present disclosure may be crosslinked PEG polymers. In non-limiting examples, PEG polymers may be crosslinked with maleimide functional groups. In one embodiment, the crosslinked PEG polymer may be 4-armed PEG-maleimide (PEG-4MAL).

[0141] Extracellular matrix components (ECMC) The compositions of the Disclosure may comprise one or more components of the extracellular matrix. In some embodiments, the ECMC may be tethered to or decorated with the polymer of the Disclosure for presentation to the cells of the Disclosure.

[0142] In some embodiments, ECMC may be an extracellular matrix protein. In some embodiments, ECMC may be a biopolymer. Non-limiting examples of extracellular matrix proteins include, but are not limited to, fibronectin, laminin, vitronectin, tenacin, entactin, thrombospondin, elastin, gelatin, collagen, fibrin, merosin, ancarin, chondronectin, link proteins, bone sialosate proteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undurin, epiligrin, and kalinin.

[0143] Extracellular matrix proteins useful in this disclosure may be of biological origin or may be purified from human or animal tissue. Alternatively, ECM proteins may be genetically engineered recombinant proteins or intrinsic synthetic products. ECM proteins may be whole proteins or peptide fragments. Examples of potentially useful ECM proteins include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin.

[0144] In some embodiments, the extracellular matrix component may be Matrigel (or a similar commercially available product such as Geltrex), which is a laminin-111-rich basement membrane extracted from Engelblesse-Holm-Swarm mouse sarcoma. In some embodiments, the ECMC may be produced from naturally occurring materials such as fibrin, collagen, or hyaluronic acid, or from synthetic hydrogels.

[0145] In some embodiments, ECMC may comprise peptides that match the short key amino acid sequences of the ECM protein. ECMC may comprise peptides that bind to integrin receptor subunits α5, α6, αv, β1, and β5. In some embodiments, ECMC may be the fibronectin-derived three-amino acid peptide Arg-Gly-Asp (RGD), which binds to both αvβ3 and αvβ5 integrins. The cyclic form of RGD has been identified as the most effective peptide for hPSC culture, and the cyclic RGD peptide is conveniently comparable to other peptides derived from laminin, fibronectin, and vitronectin and may be used in this disclosure (Lambshead JW et al. Sci. Rep 8, 701 (2018), the contents of which are incorporated herein in their entirety by reference). In some embodiments, the peptide may be CGRGDS (SEQ ID NO: 9).

[0146] Other examples of ECMC include peptides corresponding to the collagen motif GFXGER (SEQ ID NO: 10), the laminin motifs IKVAV (SEQ ID NO: 11) and YIGSR (SEQ ID NO: 12), MNYYSNS (SEQ ID NO: 13) or CNYYSNS (SEQ ID NO: 14), DAPS (SEQ ID NO: 15), AELDPP (SEQ ID NO: 16), VALDEP (SEQ ID NO: 17), NGRAHA (SEQ ID NO: 18), peptides derived from vitronectin, and / or bone sialoproteins.

[0147] In some embodiments, ECMC may be a synthetic matrix component.

[0148] In some embodiments, ECMC may include growth factors such as transforming growth factor (TGF) family peptides (e.g., TGF-β), fibroblast growth factor (FGF), and integrins, as well as enzymes such as matrix metalloproteinases (MMPs).

[0149] medication TSS may be prepared using one or more agents. One or more agents may be released from the TSS (either continuously or in a controlled manner) so that they are available for interaction with the cells of this disclosure. The agents may be biological or chemical agents. The agents may promote the survival, growth, expansion, differentiation, and / or one or more functions of the cells of this disclosure.

[0150] In some embodiments, the agent may be a biological agent. As used herein, “biological agent” means any compound, composition, biopolymer, molecule, etc., produced by a living organism, and includes, but is not limited to, polynucleotides (e.g., DNA, RNA), peptides and polypeptides, as well as chemical compounds.

[0151] In one embodiment, the biological agent may be a protein tag, such as a biotin molecule.

[0152] In some embodiments, the biological agent may be an antibody or a fragment thereof. In a non-limiting example, the antibody may be an EpCAM antibody that conjugates to the polymer of the Disclosure via an adapter complex. Such an antibody may enable the sequestration of thymocytes of the Disclosure within or on the TSS of the Disclosure (see A. Tajima, et al. Fan, Clin. Immunol. 2015, 160, 82, the contents of which are incorporated herein by reference in their entirety).

[0153] One or more drugs may be individually selected from peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit the translation or transcription of essential proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory drugs, antihistamines, anti-infective drugs, radiosensitizers, drug sensitizers, contrast agents, chemokines, chemokines, cytokines, anti-migration compounds that can reduce cell entry by inhibiting chemokine receptors, or any combination thereof.

[0154] In some embodiments, a biological agent may interact with a counterpart receptor that is ligand-specific and specific to that ligand, thereby jointly transmitting a message or signal to assume a specific function or phenotype. For example, the cells described herein may have an exogenous nucleotide sequence encoding a ligand that can be introduced into the cell by transfection or transduction (or has been introduced previously). In a non-limiting example, the ligand may be a Notch ligand. As used herein, the term “Notch ligand” includes intact (full-length), partial (cleaved form), or modified (including one or more mutations such as conservative mutations) Notch ligands, as well as Notch ligands from any species or fragment thereof that retain at least one activity or function of a full-length Notch ligand. Peptides that mimic Notch ligands are also included. A Notch ligand may be a “standard Notch ligand” or a “non-standard Notch ligand.” A standard Notch ligand typically features an N-terminal (NT) domain followed by a Delta / Serrate / LAG-2 (DSL) domain and an extracellular domain containing multiple tandem sequences of epidermal growth factor (EGF)-like repeats. The DSL domain, along with the adjacent NT domain, and the first two EGF repeats containing Delta and OSM-11-like protein (DOS) motifs are typically required for standard ligands to bind to Notch. The intracellular domains of some standard ligands contain a carboxy-terminal PSD-95 / Dlg / ZO-l-ligand (PDZL) motif, which plays a role independent of Notch signaling. While the C. elegans DSL ligand lacks the DOS motif, it has been proposed that it cooperates with ligands containing only DOS to activate Notch signaling. Exemplary standard Notch ligands include, but are not limited to, Delta-like ligand 4 (DLL4), Delta-like ligand 1 (DLL1), Jagged 1 (JAG1), Jagged 2 (JAG2), Delta-like ligand 3 (DLL3), and X-delta2, and other similar exemplary standard ligands are intended in additional embodiments.Non-standard Notch ligands lacking the DSL domain (Delta / Serrate / LAG-2) are structurally diverse and include integral- and GPI-binding membrane proteins as well as various secreted proteins. Where “Notch ligand fragment” or “standard Notch ligand fragment” is referred to herein, the fragment is intended to be a fragment that binds to Notch. Examples of non-standard Notch ligands include, but are not limited to, contactin-1, NOV / CCN3, contactin-6, periostin / OSF-2, DLK2 / EGFL9, Pref-1 / DLK1 / FAl, DNER, thrombospondin-2, MAGP-1 / MF AP2, thrombospondin-3, MAGP-2 / MF AP5, thrombospondin-4, and netrin-1. In some embodiments, the ligand may be JAG1, JAG2, and / or Delta-like 1. In some embodiments, the ligand may be VCAM1. Shukla S et al. and Michaels et al. have shown that both DLL4 and vascular cell adhesion molecule (VCAM1) play important roles in the differentiation of effector cells (e.g., T cells) (see Shukla et al. Nature Methods. 2017;14(5):531-8 and Michaels YS, et al. bioRxiv. 2021, the contents of each of these are incorporated herein by reference in their entirety). In some embodiments, the ligand may be an apoptosis regulator such as Fas ligand (FasL), and the Fas receptor / Fas ligand pathway has been shown to play an important role in tolerance to autoantigens, with Fas-L expression at the transplantation site inducing tolerance of allogeneic tissue (see Ji Lei et al. Sci Adv, 8, 2022; Lau et al Science 1996 273:109). In some embodiments, the agent may be a growth factor.Examples of growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), bone morphogenetic protein(s) (BMP), transforming growth factors (TGF) such as transforming growth factor beta, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), insulin-like growth factor (e.g., insulin-like growth factor I), dispersion factor / hepatocyte growth factor (HGF), granulocyte / macrophage colony-stimulating factor (GMCSF), glial growth factor (GGF), and fibroblast growth factor (FGF), GCSF, erythropoietin, TPO, GDF, and New Examples include rotrophin, MSF, SGF, GDF, activin, CTGF, epigen, galectin, KGF, leptin, MMIF, MIA (melanoma inhibitory activity), myostatin, noggin, NOV, omentin, oncostatin-M, osteopontin, OPG, periostin, placental growth factor, placental lactogen, prolactin, RANK ligand, retinol-binding protein (RBP), stem cell factor, amphiregulin, lymphocyte function-associated antigen 3, bone marrow-derived growth factor, osteoclast-stimulating factor, progranulin, colony-stimulating factor, and combinations thereof.Growth factors include stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-stimulating factor (GM-CSF), stromal cell-derived factor-1, hematopoietic stem cell factor, VEGF, TGFb, platelet-derived growth factor (PDGF), angiopoietin (Ang), epidermal growth factor (EGF), bFGF, HNF, NGF, fibroblast growth factor (FGF), hepatocyte growth factor, hepatic growth factor (LGF), insulin-like growth factor (IGF-1), colony-stimulating factor, thrombopoietin, erythropoietin, fit3-ligand, tumor necrosis factor α (TNFα), and bone morphogenesis factor. It may be one or more of the following: growth factors of the protein (BMP) family (e.g., BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15), growth factors that function in the Wnt signaling pathway (e.g., WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16), and other growth factors.

[0155] In some embodiments, ligands may promote the long-term survival of thymocytes of this disclosure. For example, ligands that promote the long-term survival of thymocytes may be FGF2, FGF7, and RANKL (see Lee HW et al. Expt & Mol. Med. 2008;40(1):59-70, the contents of which are incorporated herein by reference in their entirety). In some embodiments, ligands, such as VEGF, may promote angiogenesis (see de Barros SC. The Journal of Immunology. 2020;205(9):2423-36, and Chung B, et al. Stem Cells. 2014;32(9):2386-96, the contents of each of these are incorporated herein by reference in their entirety).

[0156] In some embodiments, the drug may be an immunomodulator. Preferred immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, IL-6, IL-3, IL-a, IL-Ib, IL-6, IL-7, IL-8, IL-11, IL-13, IL-12), cytokines, chemokines, cytosine phosphate guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers. In some embodiments, one or more of the drugs are chemokines. Examples of exemplary chemokines include, but are not limited to, CCL3, CCL26, CXCL13, CXCL14, CCL6, CCL27, CXCL16, CXCL17, CXCL6, CXCL5, eotaxin, CCL2, CX3CL1, CXCL1, 2, 3, CCL14, CCL1, CXCL8, CXCL11, CC3L1, XCL1, CCL2, 7, 8, 12, 13, CCL22, CCL28, CXCL9, CCL3, 4, 9, 15, CXCL7, CCL4, CXCL4, CXCL12, CCL17, CCL21, CCL25, CCL16, FAM19A5, CXCL15, and any combination thereof. In some embodiments, one or more of the drugs are cytokines.Examples of cytokines include, but are not limited to, interferons (e.g., IFN-α, IFN-J3, IFN-S, IFN-K, IFN-CO, and IFN-Y), granulocyte-colony-stimulating factor, imiquimod, 4-1BB, adiponectin, AITR, AIFl, B-cell activator, beta-defensin, beta-cerulin, BMP, BST1, type B natriuretic peptide, cardiotrophin, CTLA4, EBB, endoglin, epiregulin, FAS, Flt3 ligand, follistatin, hedgehog protein, interferons (e.g., interferon-alpha, interferon-gamma, interferon-tau, interferon-beta, interferon regulators), and interleukins. (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-8, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-27, IL-28A, IL-29, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37), Otraprin, Resistin, Leukemia Suppressor, Serum Amyloid A, TPO, Trefoil Factor, Thymic Interstitial Lymhopoietin, Tumor Necrosis Factor, Utegrobin, Visfatin, Wingless MMTV Nitration Site Family, AIMP1, CLCF1, CYTL1, EMAP Examples include II, TAFA2, buspin, and any combination thereof. In some embodiments, the immunomodulator may be a drug such as CXCL12 that promotes the migration of dendritic cells toward and away from the cells and / or TSS of the present disclosure (see Ramos SA, et al. Journal of Allergy and Clin. Imm. 2021, the contents of which are incorporated herein by reference in their entirety).In some embodiments, the immunomodulator may be ligands for CCR7 and / or CCR9 that can promote the recruitment of effector cells, such as T cells, to thymocytes or TSSs of the present disclosure (Gameiro J et al. Cell adhesion & migration. 2010;4(3):382-90, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the immunomodulator may be thymic-interstitial lymphopoietin (TSLP) and / or IL10 that can promote the development of effector T cells, such as regulatory T cells (see Alawam AS, et al. Frontiers in immunology. 2020;11:858, the contents of which are incorporated herein by reference in their entirety).

[0157] In some embodiments, the agent may be a hormone. Preferred hormones include, but are not limited to, amino acid-derived hormones (e.g., melatonin and thyroxine), low molecular weight peptide hormones and protein hormones (e.g., thyroid-stimulating hormone-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxin, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone-cortisol). Examples of hormones include, but are not limited to, endothelin, exendin, follicle-stimulating hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, ipamorelin, glucagon, glucagon-like peptide, insulin, chorionic gonadotropin, inhibin-beta C chain, inhibin alpha, inhibin alpha chain, luteinizing hormone, luteinizing hormone-releasing hormone, peptide hormones (e.g., adrenocorticotropic hormone, ararelin, antide, atosiban, buserelin, cetrorelix, desmopressin, deslorerin, elcatonin, ganirelix, ghrelin, goserelin, hexarelin, distrelin, lanreotide, leuprolide, repressin) Examples include melanotan-I and -II, nafarelin, octreotide, plumrintide, secretin, cincalid, somatostatin, terlipressin, thymopentin, triptorelin, vasopressin, neuropeptide Y, cholecystokinin), procalcitonin, prolactin, oxytocin, parathyroid hormone, estrogen, testosterone, staniocalcin-1 and -2, thymosine, tyrostimulin, thyroid-stimulating hormone, agouti-related protein, calcitonin, corticotropin-releasing hormone-binding protein, prouroguaniline, oxytomodulin, thyroid-stimulating hormone-releasing hormone, and any combination thereof.

[0158] In some embodiments, the agents of this disclosure may promote angiogenesis.

[0159] The drug may be a gene modifier. Exemplary gene modifiers include, but are not limited to, RNA-inducible or programmable nuclease systems such as CRISPR-Cas systems, meganucleases, zinc finger nucleases, and / or similar systems. Such systems are generally known in the art.

[0160] As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. RNA may be in the form of tRNA (transfer RNA), snRNA (micronuclear RNA), rRNA (ribosomal RNA), antisense RNA, RNAi (RNA interference construct), siRNA (short interference RNA), microRNA (miRNA), or non-coding RNA such as ribozymes, aptamers, guide RNA (gRNA), or coding mRNA (messenger RNA).

[0161] In some embodiments, the agent is a chemical agent. As used herein, “chemical agent” means a chemical substance, molecule, or composition. Exemplary chemical agents include those suitable for use as a pharmaceutical in animals, and those that are not. In some embodiments, the chemical agent is a hazardous chemical agent. In some embodiments, the chemical agent is not hazardous. In some embodiments, the chemical agent may be a carcinogen. In some embodiments, the chemical agent is biocompatible. As used herein, “biocompatible” means a substance or object that, when introduced into an organism, performs its desired function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in the cells, tissues, or organs of nature, or in the cells, tissues, or organs into which it is introduced along with the substance or object. For example, a biocompatible product is a product that, when introduced into an organism, performs its desired function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in the cells, tissues, or organs of nature.

[0162] device The cells and / or TSS of this disclosure may be incorporated into a device. Mechanical parameters such as flow rate, shear stress, pressure, and motion can influence the culture, differentiation, and tissue homeostasis of thymocytes. For example, hemodynamically induced shear stress in cells can trigger final maturation. Similarly, a variety of physical forces are involved in the normal and pathological function of cells, tissues, organs, and / or organisms. Incorporation of the compositions of this disclosure into a device that enables the generation of forces and motion allows the compositions to more closely mimic the thymic environment in vivo. Organ chip technology introduces many techniques for generating and maintaining forces and may be useful in this disclosure. Organ chip-like devices also enable the growth of epithelium on a 2D membrane having microfluidic channels on both the apical and basal sides, providing bilateral accessibility and the ability to apply fluid flow. Such microfabrication-based devices offer an opportunity to combine biophysical and biochemical stimuli and thus increase the physiological relevance of in vitro models and the robustness of their generation protocols.

[0163] Microfluidic devices can be used to integrate access channels for waste removal and nutrient delivery within cell populations, enabling independent control over experimental conditions.

[0164] Any of the microfluidic devices described in U.S. Patent Publication No. 2021 / 0115369 and U.S. Patent No. 10,829,727 may be useful to this disclosure. The contents of each of these applications are incorporated herein by reference in their entirety.

[0165] method This disclosure provides a method for differentiating pluripotent stem cells into thymocytes. In some embodiments, this disclosure provides a method for differentiating induced pluripotent stem cells into thymocytes.

[0166] In some embodiments, the steps involved in the differentiation of iPSCs into thymocytes may include the encapsulation of a single iPSC. In some other embodiments, the steps involved in the differentiation of iPSCs into thymocytes do not include the encapsulation of a single iPSC.

[0167] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymocytes may include activation of WNT signaling. In a non-limiting example, the activator of WNT signaling may be CHIR99021.

[0168] In some embodiments, one or more steps involved in the differentiation of iPSCs into thymocytes may include the inhibition of WNT signaling. In a non-limiting example, the WNT signaling inhibitor may be IWR1 (or IWR-1).

[0169] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymocytes may include the inhibition of BMP signaling. In some embodiments, the inhibition of BMP signaling can be achieved using LDN193189, a BMP pathway inhibitor.

[0170] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymocytes may include the inhibition of SHH signaling. In some embodiments, inhibition of SHH is achieved by using SANT-1, an SHH antagonist.

[0171] In some embodiments, one or more steps involved in the differentiation of iPSCs into thymocytes may include the inhibition of TGFβ signaling. In some embodiments, the inhibition of TGFβ signaling is achieved by using the TGFβ inhibitor SB431542.

[0172] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymocytes may include a cell culture medium containing insulin transferrin selenium (ITS), knockout replacement serum (KSR), penicillin streptomycin (also referred to herein as "Pen Strep"), and non-essential amino acids (NEAAs).

[0173] In one embodiment, the cells of the Disclosure are cultured in the presence of one or more polymers described herein. In one embodiment, the cells of the Disclosure are encapsulated in the polymer of the Disclosure. In one embodiment, the polymer is an alginate.

[0174] In one embodiment, iPS cells are cultured in a suspension and / or encapsulated in alginate. In one embodiment, DE cells are cultured in a suspension and / or encapsulated in alginate. In one embodiment, AFE cells are cultured in a suspension and / or encapsulated in alginate. In one embodiment, VPE cells are cultured in a suspension and / or encapsulated in alginate. In one embodiment, TEP cells are cultured in a suspension and / or encapsulated in alginate. In one embodiment, TEC cells are cultured in a suspension and / or encapsulated in alginate.

[0175] In one embodiment, iPS cells are cultured in suspension and not encapsulated in alginate. In one embodiment, DE cells are cultured in suspension and not encapsulated in alginate. In one embodiment, AFE cells are cultured in suspension and not encapsulated in alginate. In one embodiment, VPE cells are cultured in suspension and not encapsulated in alginate. In one embodiment, TEP cells are cultured in suspension and not encapsulated in alginate. In one embodiment, TEC cells are cultured in suspension and not encapsulated in alginate.

[0176] In one embodiment, iPS cells are cultured in suspension and are not encapsulated. In one embodiment, DE cells are cultured in suspension and are not encapsulated. In one embodiment, AFE cells are cultured in suspension and are not encapsulated. In one embodiment, VPE cells are cultured in suspension and are not encapsulated. In one embodiment, TEP cells are cultured in suspension and are not encapsulated. In one embodiment, TEC cells are cultured in suspension and are not encapsulated.

[0177] In one embodiment, iPS cells are differentiated in suspension and / or encapsulated in alginate. In one embodiment, DE cells are differentiated in suspension and / or encapsulated in alginate. In one embodiment, AFE cells are differentiated in suspension and / or encapsulated in alginate. In one embodiment, VPE cells are differentiated in suspension and / or encapsulated in alginate. In one embodiment, TEP cells are differentiated in suspension and / or encapsulated in alginate. In one embodiment, TEC cells are differentiated in suspension and / or encapsulated in alginate.

[0178] In one embodiment, iPS cells are differentiated in suspension and not encapsulated in alginate. In one embodiment, DE cells are differentiated in suspension and not encapsulated in alginate. In one embodiment, AFE cells are differentiated in suspension and not encapsulated in alginate. In one embodiment, VPE cells are differentiated in suspension and not encapsulated in alginate. In one embodiment, TEP cells are differentiated in suspension and not encapsulated in alginate. In one embodiment, TEC cells are differentiated in suspension and not encapsulated in alginate.

[0179] In one embodiment, iPS cells are differentiated in suspension and not encapsulated. In one embodiment, DE cells are differentiated in suspension and not encapsulated. In one embodiment, AFE cells are differentiated in suspension and not encapsulated. In one embodiment, VPE cells are differentiated in suspension and not encapsulated. In one embodiment, TEP cells are differentiated in suspension and not encapsulated. In one embodiment, TEC cells are differentiated in suspension and not encapsulated.

[0180] In one embodiment, alginate-encapsulated iPS cells are differentiated into DE cells in suspension. In one embodiment, alginate-encapsulated iPS cells are differentiated into DE and AFE cells in suspension. In one embodiment, alginate-encapsulated iPS cells are differentiated into DE or AFE cells in suspension. In one embodiment, alginate-encapsulated iPS cells are differentiated into DE, AFE, VPE, and thymocytes in suspension. In one embodiment, alginate-encapsulated iPS cells are differentiated into DE, AFE, VPE, or thymocytes in suspension.

[0181] In one embodiment, alginate-encapsulated DE and AFE cells are differentiated into VPE cells in suspension. In one embodiment, alginate-encapsulated DE and AFE cells are differentiated into VPE cells and thymocytes in suspension. In one embodiment, alginate-encapsulated DE or AFE cells are differentiated into VPE or thymocytes in suspension. In one embodiment, alginate-encapsulated AFE and VPE cells are differentiated into thymocytes in suspension. In one embodiment, alginate-encapsulated AFE or VPE cells are differentiated into thymocytes in suspension. In one embodiment, alginate-encapsulated VPE cells are differentiated into thymocytes in suspension.

[0182] In one embodiment, unencapsulated iPS cells are differentiated into DE cells in suspension. In one embodiment, unencapsulated iPS cells are differentiated into DE cells and AFE cells in suspension. In one embodiment, unencapsulated iPS cells are differentiated into DE or AFE cells in suspension. In one embodiment, unencapsulated iPS cells are differentiated into DE, AFE, VPE, and thymocytes in suspension. In one embodiment, unencapsulated iPS cells are differentiated into DE, AFE, VPE, or thymocytes in suspension.

[0183] In one embodiment, unencapsulated DE and AFE cells are differentiated into VPE cells in suspension. In one embodiment, unencapsulated DE and AFE cells are differentiated into VPE cells and thymocytes in suspension. In one embodiment, unencapsulated DE or AFE cells are differentiated into VPE or thymocytes in suspension. In one embodiment, unencapsulated AFE and VPE cells are differentiated into thymocytes in suspension. In one embodiment, unencapsulated AFE or VPE cells are differentiated into thymocytes in suspension. In one embodiment, unencapsulated VPE cells are differentiated into thymocytes in suspension.

[0184] Method of alginate encapsulation In some embodiments, an oil / water emulsion method is used for alginate encapsulation. Emulsion encapsulation allows for a uniform cellular spatial distribution within small microcapsules. An alginate solution containing cells is stirred with non-toxic paraffin oil and then crosslinked to obtain cell-filled microcapsules. Using this technique, parameters such as stirring speed can be adjusted to optimize the capsule size for differentiation. In some embodiments, an extrusion (droplet) method can be used for alginate encapsulation.

[0185] In one embodiment, the cells of this disclosure are encapsulated by extrusion. The autoclaved alginate solution is prepared with alginate, gelatin, HEPES, and / or NaCl. The cells can be resuspended in the alginate solution at various concentrations, e.g., 200,000 cells / mL. A 1 mL syringe equipped with a 27 gauge needle tip is filled with the solution. The syringe is then placed in a syringe pump and extruded into a CaCl2 gelling tank at a predetermined rate. The alginate capsules can be maintained in the tank for an additional time. The capsules are then washed with PBS and suspended in Stem Scale medium containing Y-27632. The cells are cultured for 1 to 5 days before differentiation.

[0186] In one embodiment, the cells of the present disclosure are encapsulated by an emulsion method using an autoclaved solution of alginate, gelatin, HEPES, NaCl, and / or CaCO3. Mineral oil can be added to a sterile container including a 35 mm magnetic stirring rod. The cells are resuspended in alginate solutions of varying concentrations. The cell alginate solution is added to the oil with stirring and emulsified. After emulsification, mineral oil containing acetic acid is added, and gelation is allowed to continue with stirring. After gelation is complete, culture medium can be added to neutralize the pH. Oil separation and capsule collection are achieved by centrifugation. The capsules are washed and resuspended in cell medium for differentiation.

[0187] In some embodiments, the cells of this disclosure are encapsulated in alginate. In one embodiment, single cells are obtained by pretreatment of a dense cell population with Y-27632 dihydrochloride (R&D Systems) before dissociation with Accutase (StemPro). In some embodiments, the cells are encapsulated in a predetermined density (e.g., 5 × 10⁶ cells per ml of alginate). 5The cells are suspended in a 1.1 w / v% low-viscosity alginate (Sigma). In some embodiments, the single-cell alginate mixture is polymerized by dropwise addition with a stirred solution of CaCl2 (e.g., 100 mM CaCl2) containing HEPES (e.g., 10 mM HEPES) to form capsules. In one embodiment, the capsules are spherical. In some embodiments, the capsules are washed three times with DMEM / F12 (Gibco), and then cultured for 4-6 days in mTeSR1 (StemCell Technologies) supplemented with 10 μM Y-27632 dihydrochloride before differentiation is initiated.

[0188] Method for decapsulation of alginates At any step of differentiation, the encapsulated cells of this disclosure can be deencapsulated, i.e., the alginate encapsulation is removed. In one embodiment, iPS cells are encapsulated in alginate and then differentiated into thymocytes using the method described herein. The differentiated thymocytes are then deencapsulated before in vivo transplantation.

[0189] In one embodiment, the cells of the present disclosure are decapsulated in a solution containing sodium citrate, HEPES, and / or NaCl. The capsules are suspended in the decapsulation solution while being vibrated to facilitate decapsulation. The aggregates are collected by centrifugation and then treated with an acutase to dissociate into single cells. The single cells are collected by centrifugation and used for further testing.

[0190] In one embodiment, the cells of this disclosure are decapsulated with EDTA. In one embodiment, the cells are placed on a Ficoll gradient to remove both capsule debris and dead cells. In some embodiments, the live cell layer is pipetted off the gradient and dissociated for analysis.

[0191] Preparation and maintenance of thymocytes This specification provides a method for differentiating pluripotent stem cells into thymocytes. Such a method may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-targeted P13K inhibitor). In some embodiments, the first growth medium comprises DMEM-F12, activin A, CHIR99021, insulin transferrin selenium (ITS), and knockout serum replacement (KSR). In some embodiments, the second growth medium comprises DMEM-F12, bFGF, activin A, LDN193189, ITS, and KSR. In some embodiments, the cells are cultured in the presence of PI-103. The concentration of PI-103 may be about 1 nM to 1000 nM. In one embodiment, the concentration of PI-103 may be 50 nM.

[0192] Embryonic endoderm cells can be further cultured and differentiated into anterior foregut cells by contacting or incubating them with at least one of SB431542, LDN-193189, and KSR. In some embodiments, anterior foregut cells can be cultured and differentiated into pharyngeal endoderm cells by contacting or incubating them with at least one of EGF, retinoic acid, FGF8B, and SHH. In some embodiments, pharyngeal endoderm cells can be cultured and differentiated into thymic epithelial cells by contacting or incubating them with at least one of BMP4, FGF8b, EGF, SANT, CHIR99021, ascorbic acid, or a combination thereof. In some embodiments, differentiation takes place over approximately 14 to 25 days. For example, differentiation may take place over approximately 18, 19, 20, 21, 22, 23, 24, or 25 days.

[0193] In some embodiments, this disclosure provides methods for preparing one or more cells or cell types as described herein. In some embodiments, the cells may be thymocytes.

[0194] The accumulation of data in publicly available databases provides single-cell transcriptomes of primary human and mouse thymus (see Bautista et al. 2021 Nat Commun 12, 1096, Kernfeld, et al. Immunity. 2018 Jun 19; 48(6): 1258-1270.e6, Zeng et al. Immunity. 2019 Nov 19; 51(5): 930-948.e6, the contents of each of these are incorporated herein in their entirety by reference). This provides a rich source of material for identifying factors that promote the differentiation and / or maturation of cells of the present disclosure into thymocytes. By analysis of scRNA sequencing data, the present disclosure identifies potential factors and / or supporting cells that can promote and / or maintain the thymocyte phenotype.

[0195] In some embodiments, the cells of the Disclosure may be isolated from an organism. In some embodiments, the organism may be a mammal. Mammalian cells may be isolated from human, rodent, pig, and / or bovine sources. The human source of the cells of the Disclosure may be autologous or allogeneic. In some embodiments, tissue containing the cells of the Disclosure may be collected and used as for the applications described herein. The cells of the Disclosure may be obtained from embryos, fetuses, or adult organisms. In some embodiments, the organism may be living or a dead organism.

[0196] The cells described herein may be derived from other cell types. In a non-limiting example, the cells of this disclosure may be derived from pluripotent stem cells (PSCs). In some embodiments, the cells of this disclosure may be derived from precursor cells. In some embodiments, the cells of this disclosure may be derived from the differentiation of PSCs and / or precursor cells.

[0197] In some embodiments, thymocytes can be prepared from PSCs. In this regard, the method may include culturing pluripotent stem cells for a sufficient time and under conditions to differentiate them into thymocytes. For example, the method may include culturing pluripotent stem cells in the presence of factors and / or inhibitors that drive the differentiation of PSCs into thymocytes. Methods for differentiating PSCs into thymocytes are known in the art. Methods for differentiating PSCs into thymocytes may include the use of one or more parameters known in the art for differentiation or combination thereof. Parameters include, but are not limited to, (i) factors that promote differentiation, (ii) inhibitors that promote differentiation, (iii) duration for promoting differentiation, (iv) temperature, (v) substrate, and / or (vi) supporting cells that promote differentiation. Any method or parameter for differentiating PSCs into thymocytes described in the following references may be used herein: Parent et al. Cell Stem Cell. 2013 Aug 1;13(2):219-29, Soh et al. Stem Cell Rep. 2014 Vol.2 j 925-937, Sun et al. Cell Stem Cell. 2013 Aug 1;13(2):230-6, Okabe et al. Cell.Reprog. 2015 Vol 17,No.5, Su et al. Sci.Rep. 2015 5,9882, Otsuka et al. Sci Rep 2020 10:224, International Patent Publications WO2019 / 060336, WO2020 / 205859, WO2020 / 220040, WO2014 / 134213, WO2010 / 143529, WO2011 / 139628, and Chinese Patent Publication CN2011 / 10121243, the contents of each of these are incorporated herein by reference in their entirety.

[0198] In one embodiment, iPS cells are encapsulated in a polymer such as an alginate, as described herein, before their differentiation into thymocytes.

[0199] Preparation and maintenance of endoderm (DE) cells This disclosure provides a method for preparing endoderm cells of an embryo, which can then be differentiated into thymocytes. In some embodiments, endoderm can be prepared by culturing cells in a two-dimensional or three-dimensional culture. Such a method may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-targeted P13K inhibitor). In some embodiments, the first growth medium may include activin A, CHIR99021, insulin transferrin selenium (ITS), and / or knockout serum substitution (KSR). In one embodiment, iPS cells are encapsulated in a polymer such as an alginate, as described herein, before their differentiation. The encapsulated cells are grown in a three-dimensional culture.

[0200] In some embodiments, the second growth medium comprises basic fibroblast growth factor (bFGF), activin A, LDN193189, ITS, and KSR. In some embodiments, the second growth medium comprises CHIR99021.

[0201] In some embodiments, the concentration of CHIR99021 is approximately 0.1 μM to 100 μM. In some embodiments, the concentration of CHIR99021 is approximately 2 μM to 3 μM.

[0202] In some embodiments, cells are cultured in the presence of PI-103. The concentration of PI-103 can be about 1 nM to 1000 nM. In one embodiment, the concentration of PI-103 can be 50 nM. In some embodiments, the concentration of PI-103 can be 25 nM. In some embodiments, pluripotent stem cells can be cultured for about 3 to 5 days. PI-103 can be added for 1 to 2 days. The pluripotent stem cells can be embryonic stem cells or induced pluripotent stem cells.

[0203] In some embodiments, the pluripotent stem cells can be cultured for about 3 to 5 days. The stem cells can be cultured for about 1 to 2 days in a first growth medium and for about 2 to 3 days in a second growth medium. The pluripotent stem cells can be cultured for 2 days in the first growth medium and for 3 days in the second growth medium. In some embodiments, the concentration of activin A can be about 100 ng / ml. In some embodiments, the concentration of CHIR99021 can be 2 μM. In some embodiments, the concentration of bFGF can be 10 ng / ml. In some embodiments, the concentration of LDN193189 can be 200 nM. In some embodiments, CHIR99021 can be added to the second growth medium for about 1 day.

[0204] Provided herein is a method of differentiating pluripotent stem cells into thymocytes. Such method can include culturing the pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium can include PI-103 (a multi-targeted P13K inhibitor). In some embodiments, the first growth medium includes DMEM-F12, activin A, CHIR99021, insulin transferrin selenium (ITS), and knockout serum replacement (KSR). In some aspects, the second growth medium includes DMEM-F12, bFGF, activin A, LDN193189, ITS, and KSR. In some embodiments, the second growth medium includes CHIR99021. In some embodiments, the concentration of CHIR99021 is about 0.1 μM to 1,00 μM. In some embodiments, the concentration of CHIR99021 is 2 μM. In some embodiments, the cells are cultured in the presence of PI-103. The concentration of PI-103 can be about 1 nM to 1,000 nM. In one embodiment, the concentration of PI-103 can be 50 nM.

[0205] Preparation of anterior foregut endoderm (AFE) cells The embryonic endoderm cells can be further cultured and differentiated into anterior foregut cells. In some embodiments, the AFE cells can be prepared by culturing the cells in two-dimensional or three-dimensional culture. The embryonic endoderm cells can be differentiated into AFE cells by contacting the DE cells with a BMP inhibitor, a TGFβ inhibitor, at least one FGF, and / or ascorbic acid.

[0206] In one embodiment, the cells are encapsulated in a polymer as described herein, such as alginate, prior to their differentiation. The encapsulated cells are grown in a three-dimensional culture.

[0207] In some embodiments, the cell culture medium used for the differentiation of DE cells into AFE cells can include N2-supplement (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement, non-essential amino acids, KSR, and / or ITS.

[0208] In some embodiments, the cell culture medium used for the differentiation of DE cells into AFE cells does not include the B27™ serum-free supplement.

[0209] In some embodiments, the BMP inhibitor can be LDN193189. In some embodiments, the concentration of LDN193189 is from about 0.1 nM to about 1000 nM. In some aspects, the concentration of LDN193189 is from about 100 to 200 nM.

[0210] In some embodiments, the TGFβ inhibitor can be SB431542. In some embodiments, the concentration of SB431542 is from about 1 μM to about 100 μM. By way of non-limiting example, the concentration of SB431542 is 10 μM.

[0211] In some embodiments, FGF can be FGF8. In some embodiments, the concentration of FGF8 is about 1 ng / ml to about 100 ng / ml. In a non-limiting example, the concentration of FGF8b is about 25 to 50 ng / ml.

[0212] DE cells can be differentiated into AFE cells in approximately 1, 2, 3, 4, or 5 days.

[0213] Preparation of ventral pharyngeal endoderm (VPE) cells Differentiation from AFE to VPE cells is carried out as a single-step process or as a multi-step process. The multi-step process can be a two-step process. In the first step, AFE is cultured in VPE1 medium, and in the second step, the cells are cultured in VPE2 medium. In one embodiment, the cells are encapsulated in a polymer such as alginate, as described herein, before their differentiation. The encapsulated cells are grown in a three-dimensional culture.

[0214] In some embodiments, VPE cells can be prepared by culturing cells in a two-dimensional or three-dimensional culture. Step VPE1 may include culturing cells for approximately 1, 2, 3, 4, or 5 days. Step VPE2 may include culturing cells for approximately 2, 3, 4, 5, or 6 days.

[0215] In some embodiments, the VPE1 medium may contain retinoic acid, at least one FGF, a WNT inhibitor, a TGFβ inhibitor, and / or ascorbic acid.

[0216] In some embodiments, the VPE2 medium may contain noggin, a BMP inhibitor, a WNT activator (e.g., CHIR99021), at least one FGF, retinoic acid, an SHH antagonist, and / or ascorbic acid.

[0217] In some embodiments, FGF can be FGF8, FGF7, and / or FGF10. In some embodiments, the concentration of FGF8 is about 1 ng / ml to about 100 ng / ml. In a non-limiting example, the concentration of FGF8b is about 25 to 50 ng / ml.

[0218] In some embodiments, the WNT inhibitor is IWR1. The concentration of IWR1 can be about 0.01 to 10 μM. In a non-limiting example, the concentration of IWR1 is 2.5 μM.

[0219] In some embodiments, the TGFβ inhibitor can be SB431542. In some embodiments, the concentration of SB431542 is about 1 μM to about 100 μM. In a non-limiting example, the concentration of SB431542 is 10 μM.

[0220] In some embodiments, the concentration of ascorbic acid is approximately 0.1 to 30 μM. In a non-limiting example, the concentration of ascorbic acid can be 10 μM.

[0221] In some embodiments, the BMP inhibitor can be LDN193189. In some embodiments, the concentration of LDN193189 is about 0.1 nM to about 1000 nM. In some embodiments, the concentration of LDN193189 is about 100 to 200 nM.

[0222] In some embodiments, the SHH inhibitor can be SANT-1. In some embodiments, the concentration of SANT-1 is about 0.01 μM to about 10 μM. In a non-limiting example, the concentration of SANT-1 is 0.25 μM.

[0223] In some embodiments, anterior foregut cells can be cultured and differentiated into pharyngeal endoderm cells by contacting or incubating them with at least one of EGF, retinoic acid, FGF8B, and / or SHH.

[0224] In some embodiments, the VPE1 and / or VPE2 medium can include N2 - Supplement (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ Serum - Free Supplement (with or without added vitamin A), non - essential amino acids, KSR, and / or ITS.

[0225] Preparation of thymic epithelial progenitor (TEP) cells Differentiation of VPE cells into TEP cells can be performed by culturing the cells in TEP medium. In some embodiments, TEP cells can be prepared by culturing the cells in two - dimensional or three - dimensional culture. The TEP step can include culturing the cells for about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0226] In one embodiment, the cells are encapsulated in a polymer as described herein, such as alginate, prior to their differentiation. The encapsulated cells are grown in a three - dimensional culture.

[0227] In some embodiments, VPE cells can be differentiated into TEP cells using BMP (e.g., BMP4, BMP2), a WNT agonist, e.g., CHIR99021, at least one FGF, and / or ascorbic acid.

[0228] In some embodiments, the TEP medium can include N2 - Supplement (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ Serum - Free Supplement (with or without added vitamin A), non - essential amino acids, KSR, and / or ITS.

[0229] In some embodiments, BMP can be BMP2 or BMP4. The concentration of BMP can be 1 ng / ml to about 100 ng / ml. In some embodiments, the concentration of BMP can be 50 ng / ml.

[0230] In some embodiments, FGF can be FGF8, FGF7, FGF1, and / or FGF10. In some embodiments, the concentration of FGF is about 1 ng / ml to about 100 ng / ml. In non-limiting examples, the concentration of FGF is about 25 to 50 ng / ml.

[0231] In some embodiments, pharyngeal endoderm cells can be cultured and differentiated into thymic epithelial cells by contacting or incubating them with at least one of BMP4, FGF8b, EGF, SANT-1 (SHH antagonist), CHIR99021, ascorbic acid, or a combination thereof.

[0232] Preparation of thymic epithelial cells (TECs) TEP cells can be further differentiated into TECs in vitro. Differentiation into TECs can be carried out in 2D or 3D culture. In some embodiments, TEP differentiation can be carried out for approximately 2, 3, 4, 5, 6, or 7 days.

[0233] In one embodiment, cells are encapsulated in a polymer such as an alginate, as described herein, before their differentiation. The encapsulated cells are grown in a three-dimensional culture.

[0234] In some embodiments, differentiation from TEP to TEC is carried out in TEC medium.

[0235] The TEC medium may contain RANKL, interleukins, e.g., (IL22), at least one FGF, at least one BMP (e.g., BMP4), a WNT activator, and / or ascorbic acid.

[0236] In some embodiments, the concentration of RANKL can be about 1 ng / ml to about 100 ng / ml. In some embodiments, the concentration of RANKL can be about 20 ng / ml to about 50 ng / ml.

[0237] In some embodiments, FGF can be FGF8, FGF7, FGF1, and / or FGF10. In some embodiments, the concentration of FGF is about 1 ng / ml to about 100 ng / ml. In non-limiting examples, the concentration of FGF is about 25 to 50 ng / ml.

[0238] In some embodiments, the interleukin concentration is approximately 1 ng / ml to approximately 100 ng / ml. In a non-limiting example, the IL22 concentration is approximately 20 ng / ml.

[0239] TEC medium may contain N2-supplements (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27® serum-free supplements (with or without added vitamin A), non-essential amino acids, KSR, and / or ITS.

[0240] In some embodiments, differentiation takes place over approximately 14 to 17 days. In some embodiments, the cells of this disclosure can be cultured as aggregates. In some embodiments, the cells of this disclosure can be cultured in an extracellular matrix-based medium, such as Geltrex.

[0241] Preparation of effector cells Furthermore, methods for preparing effector cells are provided herein. In some embodiments, the effector cells may be lymphocytes. Effector cells may be obtained from primary cells from mammals or from established cell lines. When obtained from mammals, effector cells may be obtained from a multitude of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or fluids. Effector cells may also be concentrated or purified. In some embodiments, the effector cells may be T cells. The T cells may be any type of T cell, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells (TILs), memory T cells, and naive T cells, and may be at any developmental stage. Methods for isolating and / or enriching lymphocytes are known in the art. Methods for enriching a population of lymphocytes obtained from a mammal or donor can be achieved by any suitable separation method, including, but not limited to, separation media (e.g., FICOLL-PAQUE®, ROSETTESEP® HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog number 0850494X)), cell size, shape, or density separation by filtration or elution, immunomagnetic separation (e.g., magnetically activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence-activated cell sorting system, FACS), and / or bead-based column separation.

[0242] In some embodiments, the effector cells described herein may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or precursor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, or thymus, or the stem cells or precursor cells may be differentiated in vitro from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The stem cells or precursor cells or ESCs or iPSCs from primary tissues may be of human or non-human animal origin (e.g., mouse).

[0243] In some embodiments, effector cells can be prepared and cultured with supporting cells ectopically expressing Notch ligand to enable differentiation of pluripotent stem cells or precursor cells into lymphocytes. In some embodiments, the supporting cells may be OP9 cells. In some embodiments, the Notch ligand is Delta-like 1 (DLL1). In some embodiments, the Notch ligand is Delta-like 4 (DLL4). In some embodiments, the Notch ligand is one described herein or one of the art, such as U.S. Patent No. 7,795,404 (which is incorporated herein by reference in its entirety). The effector cells of this disclosure can be prepared using an artificial thymic organoid (ATO) cell culture system utilizing supporting cells ectopically expressing OP9-DLL1. In some embodiments, the method further comprises contacting co-cultured stem cells or precursor cells and stromal cells with Flt-3 ligand and / or IL-7 and / or stem cell factor / Kit ligand and / or thrombopoietin. In some embodiments, differentiating stem cells or precursor cells into T cells involves culturing a three-dimensional (3D) cell aggregate comprising a) a selected population of supporting cells expressing an exogenous Notch ligand, and b) a selected population of stem cells or precursor cells, in a serum-free medium comprising B-27® supplement, xeno-free B-27® supplement, GS2l™ supplement, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem cells or precursor cells described in International Patent Publication WO2017 / 075389 may be useful in this disclosure (their contents are incorporated herein by reference in their entirety).

[0244] In some embodiments, effector cells may be or may be derived from hematopoietic cells. Methods for preparing hematopoietic cells from pluripotent stem cells are known in the art, for example, as described in U.S. Patent No. 9,834,754, and may include one or more of the following steps: (i) an induction step of inducing hematopoietic differentiation in a population of human pluripotent stem cells, wherein activin / lymph node signaling is inhibited between days 1 and 4 of differentiation; (ii) a selection step of sorting the induced population based on the expression of CD34 and CD43, and / or (iii) a selection step of selecting a fraction of cell populations that are CD34-positive and / or CD43-negative, wherein the sorting and cell fraction selection are performed around days 6 to 11 of differentiation (the contents of U.S. Patent No. 9,834,754 are incorporated herein by reference in their entirety).

[0245] In some embodiments, thymocytes and / or effector cells can be cultured in the presence of extracellular vesicles (e.g., exosomes) derived from thymocytes and / or effector cells. A method for preparing thymocyte exosomes is described in U.S. Patent Publication 2020 / 299641, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the exosomes can be derived from thymocytes engineered to ectopically express DLL1.

[0246] In some embodiments, effector cells such as T cells can be derived by differentiation of other cell types. T cell differentiation may include four stages: 1) mesoderm induction (approximately days 1–4), 2) hematopoietic specification (approximately days 4–8), 3) hematopoietic commitment and expansion (approximately days 8–10), and / or 4) T lymphocyte differentiation. PSCs (iPSCs or ESCs) can be used as a starting cell population for mesoderm differentiation. These cells can be differentiated into mesoderm cells. Mesoderm cells can be further differentiated into hematopoietic cells, which can be expanded in number. Cell culture systems for use in this disclosure include, but are not limited to, a first cell culture medium for mesoderm induction, a second cell culture medium for hematopoietic specification and expansion, and a third cell culture medium for T lymphocyte differentiation. The first cell culture medium may include BMP4 (e.g., human BMP4) and bFGF (e.g., human bFGF). PSCs or ESCs can be used as a starting cell population. Undifferentiated PSCs or ESCs can be transferred to low-adhesion plates to allow embryoid body (EB) formation. EB formation during the first stage can be promoted by overnight incubation in the presence of hBMP4. EBs can then be cultured with BMP4 and bFGF until day 4 to allow mesoderm induction. Successful mesoderm induction can be tested, for example, by measuring the percentage of KDR+PDGFR- cells. A second cell culture medium can contain VEGF (e.g., hVEGF) and a cocktail of hematopoietic cytokines. The hematopoietic cytokine cocktail can contain SCF (e.g., hSCF), Flt3L (e.g., hFlt3L), at least one cytokine, and bFGF for hematopoietic specification. The cytokines can be Th1 cytokines, including but not limited to IL3, IL15, IL7, IL12, and IL21. EBs can be cultured in the second cell culture medium for hematopoietic specification until approximately day 10. EB can be analyzed immunophenotypically by FACS for the expression of CD34, CD31, CD43, CD45, CD41a, c-kit, Notch 1, and IL7Ra.In some embodiments, CD34+ cells from day 10 of EB express major transcription factors in lymphocyte differentiation, e.g., CD127 (IL7Ra) and Notch 1, at the highest levels. A third cell culture medium may include feeder cells as well as SCF, Flt3L, and at least one cytokine. The cytokine may include, but is not limited to, Th1 cytokines such as IL3, IL15, IL7, IL12, and IL21. In some embodiments, around day 10, EB can be dissociated, and the hematopoietic precursor can be transferred to feeder cells to induce T lymphocyte differentiation in an established co-culture system in the presence of SCF, Flt3L, and Th1 cytokines (multiple) (e.g., IL-7). In some embodiments, the co-culture system may include thymocytes and / or feeder cultures, e.g., OP9-DL11 feeder cells.

[0247] In some embodiments, co-culture can be performed using a co-culture medium. In some embodiments, the co-culture medium may contain StemSpan SFEM II and StemSpan® T cell precursor cell maturation supplement. In some embodiments, the co-culture medium may contain αMEM, 4% B27 supplement, 30 μM ascorbic acid, 50 ng / ml IL7, 50 ng / ml FLT3L, 50 ng / ml TPO, 50 ng / ml SCF, and / or 1 × Pen Strep. In some embodiments, the co-culture medium may include DMEM / F12, a vitamin A-free 1% B27 supplement, 50 μM ascorbic acid, 50 ng / ml FGF8b, 50 ng / ml BMP, 50 ng / ml FGF10, 2 μM CHIR99021, 0.1% ITS, 0.0025% KSR, 0.5 × Pen Strep, 1 × NEAA, 1% N2, 1% Glutamax, 1% β-ME, 50 ng / ml IL7, 50 ng / ml FLT3L, 50 ng / ml TPO, and / or 50 ng / ml SCF.

[0248] In some embodiments, effector cell precursors are co-cultured with thymocytes of the present disclosure to promote their differentiation. The co-culture may further comprise TSS and one or more supporting cells.

[0249] aggregate size In some embodiments, the cells of the present disclosure can be cultured in a three-dimensional culture. In some embodiments, the cells of the present disclosure may be in the form of aggregates or spheroids. The term “spheroid” refers to a cluster of cells and / or cell colonies. Spheroids can be formed from various cell types, e.g., thymocytes, pluripotent cells, effector cells, stem cells, and / or supporting cells. Spheroids may have a spherical or irregular shape. Spheroids may include heterogeneous populations of cells, cell types, and cells in different states, such as proliferating cells, quiescent cells, and necrotic cells.

[0250] In some embodiments, the spheroid / aggregate size can be adjusted. For example, aggregate size in pluripotent stem cells can be deterministic during the expansion period, as the aggregate size can determine the oxygen distribution within the cellular spheroid, resulting in distinct zones consisting of outer, intermediate, and inner spheroid regions along high to low oxygen supply, which exhibit the core characteristics of proliferation, quiescent survival, and apoptosis, respectively (Langan et al. Plos One. 2016; 11(2), the contents of which are incorporated herein in their entirety by reference). In some embodiments, aggregates can be about 50 μm to 500 μm, about 100 μm to 1000 μm, about 200 μm to 2000 μm, about 250 μm to 2500 μm, about 300 μm to 3000 μm, and about 400 μm to 4000 μm. In embodiments, the spheroid / aggregate size can be 250 μm.

[0251] This disclosure provides a method for treating or preventing a condition in a subject. The method may include administering to a mammal an amount effective in treating or preventing a condition in a subject, of any of the cell populations described herein, or a pharmaceutical composition comprising any of the cell populations described herein. The condition may be cancer, immunodeficiency, autoimmune condition, infection, or hematological condition.

[0252] Indications for thymus The thymocytes, effector cells, and / or compositions described herein may be used to treat or prevent one or more diseases or indications related to the absence, reduction, or abnormal function of the thymus of a subject. In some embodiments, the thymocytes, effector cells, and / or compositions described herein may be used to treat athymia, a condition in which the subject may be born with severe thymic agenesis or in which the thymus is completely absent in the subject. Conditions related to athymia include, but are not limited to, complete or partial DiGeorge syndrome, complete or partial CHARGE syndrome, thymoma, thymic carcinoma, type A thymoma, type B thymoma, thymic atrophy, age-related thymic atrophy, thymic cyst, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, thymic radiation, myasthenia gravis, thymic carcinoma, thymic hyperplasia, thymic radiation, age-related or infection-related thymic function reduction. Thymocytes, effector cells, and / or compositions may be used to treat athymia associated with mutations, deletions, or deficiencies in genes involved in the development of thymic tissue. Thymocytes, effector cells, and / or compositions may be used to treat athymia associated with mutations, deletions, or deficiencies in the PAX1 gene (referred to herein as PAX1 deficiency). Thymocytes, effector cells, and / or compositions may be used to treat athymia associated with mutations, deletions, or deficiencies in the TBX1 gene (referred to herein as TBX1 deficiency). Thymocytes, effector cells, and / or compositions may be used to treat athymia associated with mutations, deletions, or deficiencies in the FOXN1 gene (referred to herein as FOXN1 deficiency).

[0253] In some embodiments, the thymocytes, effector cells, and / or compositions described herein may be used to treat thymic insufficiency associated with aging, chemotherapy, radiotherapy, immunosuppressive drug therapy, graft-versus-host disease, T-cell depletion hematopoietic stem cell transplantation, and / or HIV infection.

[0254] surgery In certain embodiments, the thymocytes, effector cells, and / or compositions described herein may be used on a subject to treat a subject who may undergo thymectomy. In certain embodiments, the subject may have a congenital cardiac defect and may have undergone or have undergone open cardiac surgery. The subject may undergo thymectomy for the treatment of one or more indications related to the thymus, such as myasthenia gravis or thymoma.

[0255] immunodeficiency In some embodiments, the compositions of the present disclosure may be used to treat immunodeficiency. The term immunodeficiency can refer to any condition in which the immune system in question is impaired and / or requires reconstruction, for example, after radiation or chemotherapy. Immunodeficiency may be primary immunodeficiency, which is inherited or caused by genetic factors, or secondary immunodeficiency, which is caused by environmental factors. In some embodiments, the compositions of the present disclosure may be used to treat primary immunodeficiency, including but not limited to Wiscott-Aldrich syndrome, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia telangiectasia, chronic granulomatous disease, transient infantile hypogammaglobulinemia, agammaglobulinemia, complement deficiency, T-cell lymphopenia, and / or selective IgA deficiency. In some embodiments, the compositions of the present disclosure may be used to treat secondary immunodeficiency caused by diseases such as AIDS and / or hepatitis.

[0256] As used herein, lymphopenia refers to a condition in which there is a lower-than-normal number of lymphocytes (a type of white blood cell) in the blood. When lymphopenia is associated with a decrease in the number of T cells, it may be referred to as T-cell lymphopenia. Compositions of this disclosure may be used to treat intrinsic or acquired lymphopenia that may be caused by hematopoietic stem cell therapy, bone marrow transplantation, radiotherapy, chemotherapy, surgery, immunosenescence, and / or aging.

[0257] cancer Various cancers can be treated with the pharmaceutical compositions of this disclosure. As used herein, the term “cancer” refers to any of the various malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new sites of the body, and refers to the pathological condition characterized by such malignant neoplasm growth. Cancer can be a tumor or hematological malignancy and includes, but is not limited to, all types of lymphoma / leukemia, carcinomas, and sarcomas, such as cancers or tumors of the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testis, thyroid, and uterus.

[0258] The types of carcinomas that can be treated with the compositions of this disclosure include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell anaplastic carcinoma, basal cell carcinoma, and anaplastic sinus carcinoma.

[0259] The types of cancers that can be treated with the compositions of this disclosure include, but are not limited to, soft tissue sarcomas such as hydatidiform soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, tendonoid sarcoma, fibroplastic round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangioepoid cytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, as well as Askin tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0260] As a non-exclusive example, treatable cancers include acute granulocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, anaplastic astrocytoma, angiosarcoma, appendiceal carcinoma, astrocytoma, basal cell carcinoma, B-cell lymphoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, intestinal carcinoma, brain carcinoma, brainstem glioma, brain tumor, breast carcinoid tumor, cervical carcinoma, cholangiocarcinoma, chondrosarcoma, and chronic lymphocytic leukemia. Chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, systemic germ cell tumor, glioblastoma multiforme, glioma, pilocytic cell leukemia, head and neck cancer, hemangioendothelioma, Dikin's lymphoma, Hodgkin's disease, Hodgkin's lymphoma, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, colon cancer, intrahepatic cholangiocarcinoma, invasive / invasive breast cancer, islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leukemia, lip cancer, liposarcoma, liver cancer, non-invasive lobular carcinoma, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, medulloblastoma Breast cancer, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymous tumor, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cell carcinoma of the neck, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-small cell lung cancer, ophthalmic cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancerCancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, primary peritoneal cancer of the ovary, ovarian cord-stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal gland tumor, pineoblastoma, pituitary cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma It may be tumor, soft tissue sarcoma, uterine sarcoma, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular cancer, ureteral cancer, ureteral cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0261] autoimmune disease In some embodiments, the thymocytes, effector cells, or any composition described herein may be useful for the treatment of autoimmune diseases. Autoimmune diseases can occur in a subject when autoantigens are recognized by effector cells in extrathymic tissue and / or when such recognition triggers an activated immune response in the subject. This disclosure provides a method for preparing effector cells that can induce an immune tolerance response in a subject. Effector cells and pharmaceutical compositions containing them may be useful in training the immune system of a subject. In some embodiments, the engineered thymocytes of this disclosure may also be administered to a subject for the treatment of autoimmune diseases.

[0262] In some embodiments, compositions of the present disclosure may be used to treat type 1 polyglandular autoimmune syndrome (APS-1) or autoimmune polyglandular endocrine deficiency-candidiasis-ectodermal dystrophy (APECED) syndrome. More than 60 mutations in the autoimmune regulator (AIRE) gene are associated with the development of type 1 polyglandular autoimmune syndrome (APS-1). AIRE plays a crucial role in shaping the T cell repertoire through its role in the elimination of T cells that are reactive to autoantigens in the thymus. Clinical manifestations associated with APS-1 are classically associated with mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, chronic mucocutaneous candidiasis, hypoparathyroidism, hypergonadotropic hypogonadism, ovarian failure, and / or autoimmune hepatitis. In some embodiments, the compositions of the present disclosure may be administered in conjunction with current standard care therapies for APS-1, including but not limited to lifelong antifungal agents, calcium regulators, endocrine hormone replacements, corticosteroids with or without 5-azacitidine, 5-azacitidine with mycophenolic acid (autoimmune hepatitis), and / or 5-azacitidine with or without rituximab (autoimmune pneumonia).

[0263] Autoimmune diseases may include rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and allergic encephalomyelitis (EAE), systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, autoimmune pneumonia, autoimmune encephalomyelitis, Guillain-Barré syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, Crohn's disease, scleroderma, psoriasis, Sjögren's syndrome, autoimmune inflammatory eye disease, primary biliary cirrhosis, Sjögren's syndrome, temporal arteritis, ulcerative colitis, vasculitis, Wegener's granulomatosis, mixed connective tissue disease, myasthenia gravis, narcolepsy, Guillain-Barré syndrome, celiac disease, alopecia areata, polymyalgia, asthma, and Hashimoto's disease.

[0264] In some embodiments, the autoimmune disease may be an immunodysregulation multiple endocrine disorder X-linked syndrome (IPEX).

[0265] In some embodiments, the compositions of the present disclosure may be used in combination with a second therapeutic agent for the treatment of autoimmune diseases. The second therapeutic agent may be an immunosuppressant or anti-inflammatory agent and may include, but are not limited to, alkylating agents, antimetabolites, cytotoxic antibiotics, folate analogs, purine analogs, antibodies, TNF-binding proteins, interferons, opioids, mycophenolic acids, and calcineurin inhibitors. The compositions of the present disclosure may also be used in combination with peripheral lymphatic depletion.

[0266] In some embodiments, thymocytes may be engineered to express MHC molecules, and their expression may be associated with a disease or disorder. In some embodiments, the expression of MHC molecules may be protective against a particular disease or disorder in the subject. In some embodiments, the disease or disorder may be an autoimmune disease.

[0267] In some embodiments, the MHC haplotypes expressed by the thymocytes of this disclosure may be associated with a disease or disorder in the subject. In some embodiments, the expression of an MHC haplotype may be protective against a particular disease or disorder. As a non-limiting example, thymocytes may be engineered to express the DR1501-DQ6 haplotype as a therapeutic strategy for the treatment and / or prevention of type 1 diabetes in a subject (see Wen et al., Science Immunology 5.44 (2020), the contents of which are incorporated herein by reference in their entirety).

[0268] Cell, tissue, and organ transplantation In some embodiments, the cells, compositions, and pharmaceutical compositions of this disclosure may be used to improve the acceptance of cell, tissue, or organ transplants and / or prevent rejection of cell, tissue, or organ transplants. In some embodiments, the cells, compositions, and / or pharmaceutical compositions of this disclosure may be used to treat or prevent graft-versus-host disease (GvHD). In some embodiments, the thymocytes of this disclosure may be administered to a subject prior to cell, tissue, or organ transplantation. Pre-transplantation of thymocytes may result in an immune tolerance response in the subject. In some embodiments, the compositions may be administered in combination with therapeutic agents known to suppress the immune system. Non-limiting examples of immunosuppressive therapeutic agents include calcineurin inhibitors, e.g., tacrolimus, cyclosporine; antiproliferative agents, e.g., mycophenolate mofetil, mycophenolate sodium, and azathioprine; mTOR inhibitors, e.g., sirolimus, and / or steroids, e.g., prednisone.

[0269] In some embodiments, the cells of this disclosure, for example, thymocytes, may be administered concurrently with or after cell, tissue, or organ transplantation.

[0270] In some embodiments, organ transplantation may be solid organ transplantation (SOT) representing a therapeutic modality for end-stage organ failure of the kidney, liver, pancreas, heart, and lung. SOT may also include small intestine and vascularized composite allografts.

[0271] In some embodiments, organ transplantation may be hematopoietic stem cell transplantation (HSCT). Non-limiting indications requiring HSCT include aplastic anemia, Fanconi anemia, Diamond-Blackfan syndrome, sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, Chediak-Higashi syndrome, chronic granulomatous diseases, Glanzmann thrombasthenia, osteoporosis, lysosome storage disorders, Gaucher disease, Niemann-Pick disease, mucopolysaccharidosis, glycoproteinosis, immunodeficiency, ataxia telangiectasia, DiGeorge syndrome, severe combined immunodeficiency (SCID), Wiscott-Aldrich syndrome, Costman syndrome, and Schwakman-Diamond syndrome. Non-limiting examples of indications requiring HSCT include, but are not limited to, leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, and myelodysplasia; lymphomas such as Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, myeloproliferative neoplasms, myelofibrosis, and chronic myeloid leukemia; and malignant indications such as solid tumors such as neuroblastoma, fibrinogenic round cell tumor, Ewing's sarcoma, and / or choriocarcinoma.

[0272] In some embodiments, the tissue transplant may be a bone marrow transplant (BMT).

[0273] Compositions and cells, such as the thymocytes of this disclosure, may be used to treat T-cell lymphopenia observed after hematopoietic stem cell transplantation or bone marrow transplantation.

[0274] infectious disease The cells, compositions, and pharmaceutical compositions of this disclosure may be used to treat infectious diseases. Organisms that cause infectious diseases include, but are not limited to, any bacterial species (spp.), such as Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), and Corynebacterium. spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenzae), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp.(e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Porphyromonas spp. (e.g., P. Gingavalis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus Examples of microorganisms that cause infection include saprophyticus, coagulase-negative staphylococcus (e.g., U.S. Patent No. 7,473,762), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). Additional microorganisms that cause infection include, for example, one or more parasites (spp.) (e.g., parasite targets), such as Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., and Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmaniaspp., Loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium), Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T.Examples include *Trichobilharzia gondii*, *Trichobilharzia regenti*, *Trichinella spiralis*, *Trichuris trichiura*, *Trombiculidae* spp., *Trypanosoma* spp., *Tunga penetrans*, and / or *Wuchereria bancrofti*.

[0275] Pharmaceutical composition The pharmaceutical compositions of this disclosure may comprise one or more cell-containing compositions described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the compositions of this disclosure are prepared for intravenous administration.

[0276] In some embodiments, the pharmaceutical composition may include any isotonic carrier, such as physiological saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate solution. In some embodiments, the pharmaceutically acceptable carrier may be supplemented with human serum albumin.

[0277] In some embodiments, the pharmaceutical composition may not substantially contain contaminants selected from, for example, endotoxins, mycoplasmas, reproducible lentiviruses (RCLs), p24, VSV-G nucleic acids, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packing cells or plasmid components, bacteria, and fungi, and may not be present at, for example, detectable levels. In some embodiments, the bacteria are at least one selected from Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0278] buffer solution In some embodiments, the pharmaceutical compositions of this disclosure are prepared with one or more buffering agents.

[0279] Examples of buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, hydroxyapatite (calcium hydroxide phosphate), potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0280] Non-limiting examples include aqueous formulations such as pH 7.4 phosphate-buffered formulations or pH 6.2 citrate-buffered formulations; formulations for lyophilization such as pH 6.2 citrate-buffered formulations with 3% mannitol or pH 6.2 citrate-buffered formulations with 4% mannitol / 1% sucrose; or formulations prepared by the process disclosed in U.S. Patent No. 8,883,737 by Reddy et al. (the contents of which are incorporated herein by reference in their entirety).

[0281] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated into parenteral administration forms. Parenteral formulations may be aqueous solutions containing carriers or excipients such as salts, carbohydrates, and buffers (e.g., at pH 3–9), or sterile non-aqueous solutions, or dry forms that can be used in conjunction with a suitable vehicle such as sterile pyrogen-free water. For example, aqueous solutions of the therapeutic agents of the present disclosure may contain isotonic saline, 5% glucose, or other pharmaceutically acceptable liquid carriers such as liquid alcohols, glycols, esters, and amides, as disclosed, for example, in U.S. Patent No. 7,910,594 by Vlahov et al. (Endocyte), the entirety of which is incorporated herein by reference. In another example, aqueous solutions of the therapeutic agents of the present disclosure may contain a phosphate-buffered formulation (pH 7.4) for intravenous administration, as disclosed in Example 23 of WO2011 / 014821 by Leamon et al. (the entirety of which is incorporated herein by reference). Parenteral administration can be in the form of a reconstituteable lyophilized product containing a dose of the therapeutic agent of the present disclosure. Any long-release administration form known in the art can utilize, for example, the biodegradable carbohydrate matrix described in U.S. Patents No. 4,713,249, No. 5,266,333, and No. 5,417,982 (their disclosures are incorporated herein by reference), or alternatively, a slow pump (e.g., an osmotic pump) can be used.

[0282] Nutrients In some embodiments, the pharmaceutical compositions of this disclosure comprise one or more nutrients that promote the health, survival, and / or proliferation of the cells described herein.

[0283] In some embodiments, the pharmaceutical preparation contains vitamins. In some embodiments, the pharmaceutical composition contains (and any derivable range thereof) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelf, or thirteen of the following: biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12, or the pharmaceutical composition contains a combination thereof or salts thereof. In some embodiments, the pharmaceutical composition contains, or essentially consists of, biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamins include or are essentially biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof.

[0284] In some embodiments, the pharmaceutical composition further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the pharmaceutical composition comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or a combination thereof.

[0285] In some embodiments, the pharmaceutical composition comprises amino acids, inorganic ions, and / or monosaccharides. In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations thereof or salts thereof. In some embodiments, the pharmaceutical composition further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof; in some embodiments, the pharmaceutical composition further comprises: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, amino acids (arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0286] Preservatives Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluend (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®.

[0287] Medication and administration The cells and compositions of this disclosure described above may be administered by any delivery route, systemic delivery, or local delivery, and may result in therapeutically beneficial outcomes.

[0288] In some embodiments, thymocytes and effector cells may be co-delivered to the same anatomical site in the subject. In some embodiments, thymocytes and effector cells may be delivered to different anatomical sites in the subject.

[0289] In some embodiments, thymocytes and effector cells may be delivered to the target simultaneously, either via the same delivery pathway or via different delivery pathways.

[0290] In some embodiments, thymocytes may be administered to the subject before the administration of effector cells.

[0291] In some embodiments, thymocytes may be administered to the subject after administration of effector cells.

[0292] Non-limiting examples of delivery routes include enteral (into the intestines), gastrointestinal, epidural (into the dura mater), oral (through the mouth), percutaneous, intracerebral (into the cerebrum), intraventricular (into the cerebral ventricles), supercutaneous (applied on the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous infusion, intraarterial (into an artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous injection (into the bone marrow), intramedullary (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (injection into the peritoneum), intravesical injection, intravitreous (through the eye), intracavitary injection (into pathological cavities), intracavitary (into the penis) (Into the base), vaginal administration, intrauterine, extra-amniotic fluid administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, inhalation (inhalation through the nose), sublingual, sublabial, enema, eye drops (on the conjunctiva), or ear drops, ear (into the ear or via the ear route), cheek (towards the cheek), conjunctiva, skin, teeth (into one or more teeth), electroosmosis, cervix, sinus, trachea, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, amniotic fluid, intraarticular, biliary tract, bronchial, bursa, intracartilage (within the range of cartilage), caudal (within the range of the cauda equina), cisterna magna (within the range of the cisterna magna of the medulla oblongata), cornea (within the range of the cornea), dental intracornal, coronary artery (within the range of the coronary arteries), intracavernosum (intracorpus cavernosum) (corpus cavernosum of the penis)Within the expansive space of the cavernosa, within the intervertebral disc (within the intervertebral disc), within the tubule (within the glandular tubule), within the duodenum (within the duodenum), within the dura mater (within or directly beneath the dura mater), within the epidermis (in the epidermis), within the esophagus (in the esophagus), within the stomach (within the stomach), within the gingiva (within the gingiva), within the ileum (within the distal part of the small intestine), within the lesion (within the local lesion, or directly introduced therein), within the lumen (within the lumen of the tubule), within the lymph (within the lymph), within the medullary cavity (within the medullary cavity of the bone), within the meninges (within the meninges), within the myocardium (within the myocardium), Intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardial), intrapleural (within the pleural), intraprostate (within the prostate), intrapulmonary (within the lung or its bronchi), sinus (within the nasal sinuses or perioboribular sinuses), intraspinal cord (within the spinal column), synovial sac (within the synovial space of a joint), intratendinous (within the tendon), intratesticular (within the testis), intramedullary cavity (within the cerebrospinal fluid at any level of the brain-spinal axis), intrathoracic (within the thoracic cavity), intratubular (within the tubular of an organ), intratumoral (within the tumor), intratympanic cavity (middle ear (aurus) (within the media), intravascular (within the range of one or more blood vessels), intraventricular (within the range of the ventricles), iontophoresis (using electric current to move ions of a soluble salt into the body's tissues), irrigation (immersing or flushing an open wound or body cavity), larynx (directly into the larynx), transnasogastrostomy (into the stomach through the nose), occlusive dressing technique (local route administration, which is then covered with a bandage that occludes the area), ocular (in the external eye), oropharynx (directly into the mouth and pharynx), parenteral, percutaneous, joint These include the surrounding, epidural, perineurial, periodontal, rectal, respiratory (within the airway by oral or nasal inhalation for local or systemic effects), retrobulbar (after the pons or after the eyeball), soft tissue, subarachnoid, subconjunctival, submucosal, local, transplacental (through or beyond the placenta), transtracheal (through the tracheal wall), transtympanic (beyond or through the tympanic cavity), ureter (into the ureter), urethra (into the urethra), vagina, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and spinal cord.

[0293] In some embodiments, compositions containing the cells of the present disclosure may be delivered intrathymally (into the thymus).

[0294] In some embodiments, compositions containing the cells of this disclosure may be surgically placed in a subject. In non-limiting examples, the cells may be surgically placed within the kidney capsule or within the quadriceps femoris muscle in the thigh.

[0295] In some embodiments, thymocytes and / or compositions containing the cells of the present disclosure may be administered intrahepatically, via intrasplenic injection, or via intraportal injection.

[0296] The thymocytes and / or compositions described herein may be provided to a subject by direct injection into the bone marrow (referred to herein as intraosseous injection). The bone may be a long bone such as the tibia, fibula, femur, metatarsal, phalanges of the lower limb, humerus, radius, ulna, metacarpal, and / or phalanges of the upper limb.

[0297] Parenteral and injectable administration In some embodiments, the cells and compositions described herein may be administered parenterally.

[0298] Preparations for injection, such as sterile aqueous or oily suspensions for injection, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile preparations for injection may be, for example, solutions in 1,3-butanediol, sterile solutions, suspensions, and / or emulsions in non-toxic, parenterally acceptable diluents and / or solvents. Acceptable vehicles and solvents that may be used include, among others, water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixatives have conventionally been used as solvents or suspension media. For this purpose, any non-irritating fixative containing synthetic monoglycerides or diglycerides can be used. Fatty acids, such as oleic acid, can be used in the preparation of injections.

[0299] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0300] To extend the effect of the active ingredient, it is often desirable to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The absorption rate of the active ingredient may depend on the dissolution rate, and then on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot formulations are prepared by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Depot injection formulations are prepared by capturing the drug in liposomes or microemulsions that are compatible with body tissues. Lymph node administration

[0301] In certain embodiments, the thymocytes and / or compositions of the Disclosure may be delivered to a subject for transplantation into lymph nodes. In some embodiments, the thymocytes and / or compositions of the Disclosure may be delivered to a subject in an amount effective to form ectopic thymic tissue in the lymph nodes. In certain embodiments, the thymocytes and / or compositions of the Disclosure may be delivered to the lymph nodes of a subject using the methods and compositions described herein, enabling the thymocytes to engraft and produce ectopic thymus in the lymph nodes. In certain embodiments, the ectopic thymus may restore thymic function in the subject, for example, by supplementing or enhancing one or more functions that a normal, healthy thymic organ can perform. For example, but not limited to, the ectopic thymus may be involved in the body's immunomodulation by promoting T cell growth, development, maturation, and selection.

[0302] In certain embodiments, thymocytes may be delivered to the subject as a liquid suspension.

[0303] Non-limiting examples of lymph nodes that can deliver thymocytes include abdominal lymph nodes, celiac lymph nodes, paraaortic lymph nodes, hilar lymph nodes, hepatic lymph nodes, left gastric lymph nodes, right gastric lymph nodes, left gastroomental lymph nodes, right gastroomental lymph nodes, retroperitoneal lymph nodes, pyloric lymph nodes (e.g., suprapyloric lymph nodes, subpyloric lymph nodes, postpyloric lymph nodes), pancreatic lymph nodes (e.g., upper pancreatic lymph nodes, lower pancreatic lymph nodes, lymph nodes of the line of the spleen), splenic lymph nodes, hepatic lymph nodes (e.g., gallbladder lymph nodes, omental lymph nodes, foramen of Winslow), pancreaticoduodenal lymph nodes (e.g., upper pancreaticoduodenal lymph nodes, lower pancreaticoduodenal lymph nodes), and superior mesenteric lymph nodes. Lymph nodes include the ileocolic lymph nodes, prececal lymph nodes, postcecal lymph nodes, appendiceal lymph nodes, mesenteric lymph nodes (e.g., paracolic lymph nodes, left colonic lymph nodes, middle colonic lymph nodes, right colonic lymph nodes, inferior mesenteric lymph nodes, sigmoid colonic lymph nodes, superior rectal lymph nodes), common iliac lymph nodes (e.g., medial common iliac lymph nodes, intermediate common iliac lymph nodes, lateral common iliac lymph nodes, subaortic common iliac lymph nodes, common iliac lymph nodes at the promontory), and external iliac lymph nodes (e.g., medial external iliac lymph nodes, intermediate external iliac lymph nodes, lateral external iliac lymph nodes, medial intracavitary femoral lymph nodes, intermediate intracavitary femoral lymph nodes, lateral intracavitary femoral lymph nodes, internal and external iliac lymph nodes, and external iliac occlusive lymph nodes).

[0304] As a non-limiting example, any of the methods for transplanting thymic tissue into lymph nodes described in International Patent Publication WO2021 / 026195 may be useful in this disclosure (its contents are incorporated herein by reference in their entirety).

[0305] Depot administration As described herein, in some embodiments, compositions including the cells and pharmaceutical compositions of this disclosure are formulated into depots for sustained release. Generally, a specific organ or tissue ("target tissue") is targeted for administration. In some embodiments, local release is achieved through the use of biocompatible devices. For example, biocompatible devices can restrict the diffusion of cells in the target.

[0306] In some aspects of this disclosure, cells, compositions, and pharmaceutical compositions are spatially retained within or near a target tissue. A method is provided for providing a pharmaceutical composition to a target tissue of a mammalian subject by bringing the target tissue (including one or more target cells) into contact with the pharmaceutical composition under conditions such that they are substantially retained within the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99.99% of the composition is retained within the target tissue. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more than 99.99% of the pharmaceutical composition administered to the subject is present during the period following administration.

[0307] Dosage and regimen The present disclosure provides a method of administering cells, compositions, and pharmaceutical compositions according to the present disclosure to a subject that needs them. The pharmaceutical compositions containing the described cells can be administered to the subject using any amount and any route of administration effective to prevent, treat, manage, or diagnose a disease, disorder, and / or condition. The exact amount required will vary for each subject depending on factors such as the species, age, and general condition of the subject, the severity of the disease, the specific composition, its mode of administration, its mode of activity, etc. The subject can be a human, mammal, or animal. The specific therapeutically effective, prophylactically effective, or appropriate diagnostic dosage level for any particular individual will depend on various factors including the disorder being treated and the severity of the disorder, the activity of the specific payload being used, the specific composition being used, the age, weight, general health, gender, and diet of the patient, the duration of administration, as well as the route of administration.

[0308] In certain embodiments, the cells, compositions according to the present disclosure, and pharmaceutical compositions described herein are administered at a dosage level sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg of the subject's body weight per day, one or more times per day, to obtain the desired therapeutic, diagnostic, or prophylactic effect.

[0309] In some embodiments, the dosage of the cells, compositions, and / or pharmaceutical compositions described herein is 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×107 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , or 5×10 8 It may be cells / kg. In some embodiments, the dose of the cells, compositions, and / or pharmaceutical compositions described herein is at least about 1 × 10⁻⁶ 6 , 1.1 × 10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1 x 10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , or 5×10 8 It may be cells / kg. In some embodiments, the dose of the cells, compositions, and / or pharmaceutical compositions described herein is up to about 1 × 10⁶ 6 , 1.1 × 10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1 x 10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , or 5×10 8 It may be cells / kg. In some embodiments, the dose of the cells, compositions, and / or pharmaceutical compositions described herein is about 1.1 × 10⁻⁶ 6 ~1.8×10 7 It may be cells / kg. In some embodiments, the dose of the cells, compositions, and / or pharmaceutical compositions described herein is about 1 × 10⁻⁶ 7 , 2×10 7 , 5×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 5×10 8 , 1 x 10 9 , 2×109 or 5×10 9 cells / kg. In some embodiments, the dosage of the cells, compositions, and / or pharmaceutical compositions described herein is about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 2×10 9 or 5×10 9 cells / kg. In some embodiments, the dosage of the cells, compositions, and / or pharmaceutical compositions described herein is about 1×10 7、 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 2×10 9 or 5×10 9 cells / kg. In some embodiments, the dosage of the cells, compositions, and / or pharmaceutical compositions described herein is about 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 5×10 7 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 8 , 5×10 8 , 1×10 9 , 2×10 9 or 5×10 9 cells / kg. In some embodiments, the dosage of the cells, compositions, and / or pharmaceutical compositions described herein is about 1 - 3×10 7From 1 to 3 x 10 8 It can be cells / kg.

[0310] In certain embodiments, the cells described herein or the pharmaceutical compositions according to this disclosure may be administered in amounts of about 10 to about 600 μl / site, 50 to about 500 μl / site, 100 to about 400 μl / site, 120 to about 300 μl / site, 140 to about 200 μl / site, and about 160 μl / site.

[0311] The desired dose may be delivered at least once, three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose may be delivered using multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses).

[0312] The desired dose of the cells of this disclosure may be administered once or multiple times. The cells, compositions, and pharmaceutical formulations may be administered regularly at a set frequency over a period of time, or continuously as a “continuous flow.” The total daily dose, which is the amount administered or prescribed over a 24-hour period, may be administered by any of these methods, or as a combination of these methods.

[0313] In some embodiments, the delivery of cells(s) to a target provides a therapeutic effect lasting at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer than 10 years.

[0314] The cells of this disclosure may be used sequentially or concurrently in combination with one or more other therapeutic, prophylactic, research, or diagnostic agents, or medical procedures. Generally, each agent will be administered in a dose and / or time schedule determined for that agent. In some embodiments, this disclosure includes the delivery of pharmaceutical, prophylactic, research, or diagnostic compositions in combination with agents that can improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution in the body.

[0315] For example, the cells of the Disclosure may be administered as a biocompatible device that restricts diffusion in a target area and increases its bioavailability in a therapeutically targeted area. The cells(s) of the Disclosure may also be administered by local delivery.

[0316] The term "conditioning regimen" refers to the therapeutic process a patient undergoes before stem cell transplantation. For example, before hematopoietic stem cell transplantation, a patient may undergo myeloablative therapy, non-myeloablative therapy, or reduced-intensity conditioning to prevent rejection of the stem cell transplant, even if the stem cells originate from the same patient. Conditioning regimens may include the administration of cytotoxic agents. Conditioning regimens may also include immunosuppression, antibodies, and radiation therapy. Other possible conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007), Palchaudari et al., 34(7) Nature Biotechnology 738-745 (2016), Chhabra et al., 10:8(351) Science Translational Medicine 351ra105 (2016)) and CAR-T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2018), each of which is incorporated herein by reference in its entirety). Conditioning is used to create space in the brain for microglia derived from manipulated HSCs to migrate and deliver the target protein (recent gene therapy trials for ALD and MLD). A conditioning regimen is also designed to create a niche “space” within the body that allows transplanted cells to have a place to engraft and proliferate. In hematopoietic stem cell transplantation, for example, the conditioning regimen creates a niche space in the bone marrow for the transplanted hematopoietic stem cells to engraft. Without the conditioning regimen, the transplanted hematopoietic stem cells cannot engraft. In some embodiments, the subject may be administered the cells, compositions, and / or pharmaceutical formulations of this disclosure after treatment with a conditioning regimen.

[0317] The use of the cells described in this disclosure for the treatment of a disease, disorder, or condition is also included in this disclosure.

[0318] Certain embodiments provide a disease, disorder, or condition selected from cancer, Parkinson's disease, graft-versus-host disease (GvHD), autoimmune conditions, hyperproliferative disorders or conditions, malignant transformation, hepatic conditions, hereditary conditions including hereditary gene defects, juvenile-onset diabetes, and ocular compartment conditions.

[0319] In certain embodiments, a disease, disorder, or condition affects at least one system of the body selected from the muscular system, skeletal system, circulatory system, nervous system, lymphatic system, respiratory endocrine system, digestive system, excretory system, and reproductive system.

[0320] Effector cell: As used herein, “effector cell” means any cell or cell type that acquires the ability to perform, initiate, or propagate a signal or cell death trigger when in contact with or in close proximity to a thymocyte. “Contact or proximity” means a spatiotemporal approach sufficient to enable intracellular or extracellular (e.g., intercellular) signaling or other transmission or interaction.

[0321] Immune tolerance response: As used herein, “immune tolerance response” refers to a class of immune responses characterized by a tendency for the immune system to reduce or eliminate the activation of an immune response to at least one antigen.

[0322] Infectious pathogen antigens: As used herein, the term “infectious pathogen antigens” refers to a class of antigens derived from pathogens or microorganisms that cause infectious diseases. Non-exclusive examples of infectious pathogen antigens include viral antigens, bacterial antigens, protozoan antigens, prion antigens, and / or fungal antigens.

[0323] Lymphocytes: As used herein, “lymphocytes” means, in addition to the meaning and use that a person skilled in the art would understand the term to encompass, a type of immune cell of bone marrow origin that is present in lymphoid tissue or blood. In some embodiments, lymphocytes mature in the thymus.

[0324] Negative: As used herein, the term "negative" (which may be abbreviated as "-") means that a cell does not express the indicated cell marker at a detectable level, when used herein in relation to the expression of the indicated cell marker.

[0325] Neoantigen: As used herein, the term “neoantigen” refers to a class of tumor antigens arising from tumor-specific mutations in expressed proteins, in addition to the meanings and uses that a person skilled in the art would understand to encompass the term.

[0326] Positive: As used herein, the term “positive” (which may be abbreviated as “+”) means that a cell expresses the indicated cell marker at any detectable level in relation to the expression of the indicated cell marker, which may include, for example, low (but detectable) levels of expression as well as high (hi) levels of expression.

[0327] Pre-T cells: As used herein, “pre-T cells” refers to lymphocytes that can mature or differentiate into T cells.

[0328] Autoantigen: As used herein, the term “autoantigen” means a class of antigens derived from one or more cells or cell types of a parent organism that induce an immune response in another organism but not in the healthy parent organism from which it originates, in addition to the meaning and use that a person skilled in the art would understand the term to encompass.

[0329] Thymus origin or lineage: As used herein, “thymus origin or lineage” means cells of thymic origin or cells having one or more phenotypic or genotype markers associated with cells destined to become thymic cells. As used herein, the thymus may be an embryo, fetus, or adult thymus.

[0330] Tumor antigen: As used herein, the term “tumor antigen” refers to a class of antigens derived from cancer cells, in addition to the meaning and use that a person skilled in the art would understand to encompass the term.

[0331] Tumor-associated antigens (TAAs): As used herein, “tumor-associated antigens” refers to a class of tumor antigens derived from tumor cells, which may also be derived from one or more non-tumor cells or cell types.

[0332] Tumor-specific antigen (TSA): As used herein, “tumor-specific antigen” refers to a class of tumor antigens characterized by being specific to a particular tumor or cancer cell.

[0333] Variant: When used in relation to biomolecules, the term "variant" refers to a biomolecule that is related to or derived from a parent molecule. A variant may be, for example, a modified form, cleaved form, mutated form, homologous form, or other altered form of a parent molecule. The term variant can be used to describe either a polynucleotide or a polypeptide.

[0334] Details of one or more embodiments of this disclosure are given in the following supplementary descriptions. Any materials and methods similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred materials and methods are described herein. Other features, purposes, and advantages of this disclosure will become apparent from the descriptions. In the descriptions, singular forms also include plural forms unless the context expressly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. In case of inconsistency, this description shall prevail.

[0335] Equivalents and range Those skilled in the art will recognize, or can confirm by using only ordinary experiments, many equivalents to specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather as set forth in the appended claims.

[0336] In these claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or evident from the context. Any claim or description containing “or” between one or more members of a group is deemed satisfied if one, two or more, or all of the group members are present in, used in, or otherwise related to a given product or process, unless otherwise indicated or evident from the context. This disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which two or more, or all, group members are present in, used in, or otherwise related to a given product or process.

[0337] It should also be noted that the term “contains” is intended and permitted to be open, but does not require the inclusion of additional elements or steps. Wherever the term “contains” is used herein, the term “consisting of” is also encompassed and disclosed.

[0338] Where a range is indicated, it includes the endpoint. Furthermore, unless otherwise indicated or otherwise obvious from the context and the understanding of those skilled in the art, values ​​expressed as a range should be understood to mean any specific value or subrange within the range described in different embodiments of this disclosure, up to one-tenth of the lower limit unit of the range, unless the context clearly indicates otherwise.

[0339] In addition, any particular embodiment of the present disclosure that constitutes prior art should be understood to be expressly excluded from any one or more of the claims. Since such embodiments are considered to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly stated herein. Any particular embodiment of the compositions of the present disclosure may be excluded from any one or more claims for any reason, whether or not it relates to the existence of prior art.

[0340] The words used are descriptive, not restrictive, and should be understood to be modified within the scope of the attached claims without departing from the true scope and intent of this disclosure in its broadest form.

[0341] While this disclosure describes some described embodiments with a certain length and some specificity, it is not intended to be limited to such details or embodiments or any particular embodiment, but rather should be interpreted in relation to the appended claims to provide the broadest possible interpretation of such claims in consideration of the prior art, and thus effectively encompass the intended scope of this disclosure. [Examples]

[0342] Example 1. Preparation of thymocytes by alginate encapsulation. Expansion of iPSC stocks On day 0, iPSC strains (300-400 microns in diameter, suspended for 3-4 days) were treated with Accutase to prepare single cells from aggregates. The cells were then resuspended in Stemscale medium (Thermo Fisher) containing 1% pen strep and 10 μM Y-27632, and left on ice for 30 minutes before encapsulation.

[0343] Encapsulation of iPSCs by extrusion Autoclaved alginate solutions were prepared with 1.1% alginate, 0.2% gelatin, 10 mM HEPES, and 0.9% NaCl. Cells were resuspended in the alginate solution at a concentration of 200,000 cells / mL. The solution was filled into a 1 mL syringe equipped with a 27 gauge needle tip. The syringe was then placed in a syringe pump and extruded at a rate of 0.25 mL / min into a 100 mM CaCl2 gelation tank. The alginate capsules were maintained in the tank for a further 5 minutes. The capsules were then washed with PBS and suspended in Stem Scale medium containing 1% pen strep and 10 μM Y-27632. Cells were cultured in this medium for 5 days before differentiation. Differentiation was performed using 3 mL of each differentiation medium per mL capsule. Complete medium changes were performed daily.

[0344] Differentiation of iPSCs into endoderm (DE) On day 1, the cells were washed with PBS and resuspended in medium A. Medium A was prepared to contain basal medium: DMEM-F12, activin A (100 ng / mL), CHIR99021 2 μM, insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25 nM), and NEAA (1:400). On day 2, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium A.

[0345] On day 3, the cells were washed with PBS and resuspended in medium B. Medium B was prepared to contain the following: basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), and NEAA (1:400).

[0346] On days 4-5, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared medium B. Medium B was prepared to contain the following: basal medium: DMEM-F12, activin A (100 ng / mL), LDN193189 (200 nM), PI-103 (25 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), and NEAA (1:400).

[0347] Differentiation of endoderm (DE) into anterior foregut endoderm (AFE) 24-well plates were coated with Geltrex (1:100) and left at room temperature for 1 hour. On day 6, the supernatant along with the alginate capsules was transferred to a 15 ml conical tube. The alginate capsules were centrifuged at 250 G for 5 minutes at room temperature, and the supernatant was aspirated. The alginate capsules were washed with PBS, resuspended in AFE medium, and cultured from day 6 to 8. The AFE medium was prepared to contain the following basal mediums: DMEM-F12, LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / ml), ascorbic acid (10 μM), Pen Strep (1:200), B27 (RA-free) (1:200), N2 (1:100), Glutamax (1:100), BME (1:100), and NEAA (1:400).

[0348] On day 7, an equal volume of freshly prepared AFE medium (500 μl) was added to each well. On day 8, 50% of the supernatant was removed, and freshly prepared AFE medium (500 μl) was added to each well.

[0349] Differentiation of the anterior foregut endoderm (AFE) into the ventral pharyngeal endoderm (VPE) On day 9, an equal volume of freshly prepared VPE1 medium (500 μl) was gently replaced without disturbing the alginate capsules. VPE1 was prepared to contain the following basal medium: DMEM-F12, SB431542 (10 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 10, an equal volume of freshly prepared VPE1 medium (500 μl) was added to each well.

[0350] On day 12, the alginate capsules were gently replaced with an equal volume of freshly prepared VPE2 medium (500 μl) without disturbing them. The VPE2 medium was prepared to contain the following: basal medium: DMEM-F12, noggin (50 ng / ml), CHIR99021 (2 μM), FGF8b (50 ng / ml), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100).

[0351] On day 13, an equal volume of freshly prepared VPE2 medium (500 μl) was added to each well.

[0352] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial precursor (TEP) On day 14, the alginate capsules were gently replaced with an equal volume of freshly prepared TEP medium (500 μl) without disturbing them. Cells were cultured in TEP medium from day 14 to 18. The TEP medium was prepared to contain the following: basal medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 15, an equal volume of freshly prepared TEP medium (500 μl) was added to each well. From days 16 to 18, 50% of the supernatant was removed, and freshly prepared TEP medium (500 μl) was added to each well.

[0353] Cells were cultured in TEC medium from day 19 to day 22. On day 19, an equal volume of freshly prepared TEC medium (500 μl) was gently replaced without disturbing the alginate capsules. The TEC medium was prepared to contain the following basal mediums: DMEM-F12, FGF10 (50 ng / ml), IL-22 (20 ng / ml), FGF7 / KGF (50 ng / ml), RANKL / TRANCE (50 ng / ml), BMP4 (50 ng / ml), FGF8b (50 ng / ml), CHIR99021 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400), Glutamax (100×), and BME (100×). On day 20, an equal volume of freshly prepared TEC medium (500 μl) was added along with Plurisin #1 (20 μM) (group - untreated and treated). On day 21, an equal volume of freshly prepared TEC medium (500 μl) was gently replaced without disturbing the alginate capsules.

[0354] Removal of alginate for harvesting differentiated cells Alginate capsules were dissociated using a decapsulation solution containing 55 mM sodium citrate, 10 mM HEPES, and 0.9% NaCl. The capsules were suspended in the decapsulation solution for 10 minutes with shaking at 100 rpm. The aggregates were collected by centrifugation (1100 rpm for 4 minutes) and then treated with Accutase to dissociate into single cells. The single cells were collected by centrifugation (1100 rpm for 4 minutes) and used for further testing.

[0355] Example 2. Characterization of TEP extracted by differentiation of alginate-encapsulated iPS cells in 3D suspension. Single-cell iPS cells were encapsulated in low-density alginate microbeads and cultured in a 3D suspension. By day 4, aggregates of different sizes were observed, indicating that iPS cells could proliferate and form colonies within the alginate. Differentiation was induced by the addition of culture media for DE, AFE, VPE, TPE, and TEC, as described in Example 1. At the TEC stage (days 15-16), the aggregates were larger (approximately 600 microns) and fewer in number. TEP marker expression was compared between iPSC aggregates differentiated in alginate capsules using 3D suspension culture and iPS cells differentiated under 2D conditions, as well as after 2D culture, followed by freezing and thawing. FOXN1 expression was observed in alginate-TEC cells at levels comparable to those under 2D culture conditions before freezing and after freezing and thawing to aggregate suspension culture in TEC medium for 5 days. FOXN1 expression was highest after freeze-thawing. HOXA3 and DLL4 expression were higher in alginate-TEC cells, and only alginate-TEC cells showed PAX1 expression. The data demonstrated that iPS cells can differentiate in 3D aggregates throughout the entire pathway from iPS to TEC (Figure 1).

[0356] Example 3. Differentiation of chitosan-coated alginate-encapsulated iPSC aggregates into thymocytes using 3D suspension culture. Expansion of iPSC strains (-4 days or -3 days) iPSC strains were thawed and grown in Stemscale medium (Thermo Fisher) in 6-well plates containing Pen Strep (1:100) and 10 μM Y-27632, in an incubator at 37°C and 5% CO2, on a platform shaken at 70 revolutions per minute (rpm). When aggregates reached an average diameter of 300–400 μm (3–4 days), the expanded iPSCs were left on ice for 30 minutes before encapsulation.

[0357] Preparation of a solution for chitosan-alginate encapsulation. A buffered saline solution containing 10 mM HEPES and 0.9% NaCl was prepared. An encapsulated solution containing 10 mM HEPES, 0.9% NaCl, 0.2% gelatin type A, and 1.1% low-viscosity sodium alginate was prepared. This was mixed at maximum stirring speed for at least 2 hours. CaCO3 was added to bring the solution to a concentration of 24 mM, and the entire solution was autoclaved.

[0358] Sterile glacial acetic acid was filtered using a 22 μm filter and added to sterile mineral oil to prepare a 0.4% glacial acetic acid and mineral oil mixture, which was then stirred at maximum speed for 1-2 minutes and autoclaved.

[0359] A 1.66% low molecular weight chitosan coating solution was prepared using 0.5 M HCl. This solution was dissolved for 2 hours while stirring at 1000 rpm, and then autoclaved.

[0360] A decapsulation solution containing 10 mM HEPES, 95 mM NaCl, and 55 mM sodium citrate was prepared.

[0361] Encapsulation of hiPSC aggregates in chitosan-coated alginate microbeads via emulsification Approximately 200,000 hiPS cells were resuspended in 10 mL of encapsulation solution and vortexed for 20-30 seconds to prepare a homogeneous mixture. All 10 mL of this homogenized mixture was slowly added to 20 mL of mineral oil and stirred at 500 rpm for 12 minutes to prepare an emulsion. Under continuous stirring, 10 mL of a 0.4% glacial acetic acid and mineral oil mixture was added to the emulsion and stirring continued for 8 minutes. After gelation was complete, 40 mL of DMEM / F12 medium was added and stirred for 1 minute to neutralize the pH. The emulsion was divided into two 50 mL conical tubes, and the oil was separated by centrifugation at 700 × g for 4 minutes, and the capsules were collected. The oil layer was removed, and as much medium as possible was removed using a vacuum trap. The alginate beads were aspirated from each conical tube and then resuspended in 40 mL of buffered saline.

[0362] Separately, 9 mL of chitosan-coated solution was combined with 21 mL of buffered saline solution to prepare 30 mL of chitosan-saline solution. 10 mL of this chitosan-saline solution was added to each conical tube to resuspend the hiPSC-containing alginate microbeads. The resuspended alginate microbeads were slowly added to the remaining chitosan-buffered saline solution and stirred at 500 rpm for 1 minute. The solution was then filtered using a 40 μm cell strainer. The filtered chitosan-coated alginate microbeads containing hiPSC aggregates were washed with 10 mL of buffered saline and placed in a shaker flask containing 24 mL of StemScale medium supplemented with 1% PenStrep and 2 mM Y-27632. The shaker flask was placed on a shaker platform at 70 rpm and incubated at 37°C and 5% CO2 for 3–4 days before differentiation began.

[0363] Preparation of cell culture media Medium A was prepared using DMEM-F12 as the basal medium and the following reagents: activin A (100 ng / mL), CHIR99021 (2 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), and NEAA (1:400).

[0364] Medium B was prepared using DMEM-F12 as the basal medium and the following reagents: activin A (100 ng / mL), LDN193189 (200 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), and NEAA (1:400).

[0365] AFE medium was prepared using DMEM-F12 as the basal medium and the following reagents: LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / mL), ascorbic acid (10 μM), PenStrep (1:200), B27 (without retinoic acid) (1:200), N2 (1:100), BME (1:100), KSR (0.05%), NEAA (1:400), and Glutamax (1:100).

[0366] VPE1 medium was prepared using DMEM-F12 as the basal medium and the following reagents: SB431542 (10uM), FGF8b (50ng / mL), retinoic acid (0.1nM), ascorbic acid (10uM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0367] VPE2 medium was prepared using DMEM-F12 as the basal medium and the following reagents: Noggin (50 ng / mL), CHIR99021 (2 μM), FGF8b (50 ng / mL), retinoic acid (0.1 nM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), NEAA (1:400), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100).

[0368] TEP medium was prepared using DMEM-F12 as the basal medium and the following reagents: FGF10 (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), and NEAA (1:400).

[0369] TEC medium was prepared using DMEM-F12 as the basal medium and the following reagents: IL-22 (20 ng / mL), FGF10 (50 ng / mL), FGF7 / KGF (50 ng / mL), RANKL / TRANCE (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (2 μM), ascorbic acid (10 μM), and insulin-transferrin-selenium (ITS-G) (1:1000).

[0370] TEC freeze / thaw (FT) medium was prepared using DMEM-F12 as the basal medium and the following reagents: IL-22 (20 ng / mL), FGF10 (50 ng / mL), FGF7 / KGF (50 ng / mL), RANKL / TRANCE (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (3 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), vitamin A-free B27 (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0371] Differentiation of chitosan-coated, alginate-encapsulated hiPSCs into endoderm (DE) On day 0, hiPSCs were deaggregated using Accutase. 3–5 million single iPS cells were placed in suspension in a 6-well ultra-low adhesion plate with medium A and ROCK inhibitor Y-27632 and incubated at 37°C and 5% CO2 at 70 rpm. Incubator conditions remained constant throughout the iPS differentiation steps described herein, and unless otherwise specified, seeded cells were returned to the incubator after each differentiation step.

[0372] On day 1, 1 mL of supernatant was removed from each well of each 6-well plate and spun down. The pelleted cells were washed with PBS, resuspended in 1 mL of preheated medium A and Y-27632, and then added back to the 6th well of each plate (well #6).

[0373] On the second day, approximately 1.8 mL of supernatant was removed from each well, and 2 mL of pre-warmed medium A (without Y-27632) was added to each well. The supernatant was spun down, the pelleted cells were washed with PBS, resuspended in 2 mL of medium A (without Y-27632), and then added to the sixth well of each plate.

[0374] On day 3, the cells were collected in 50 mL conical tubes along with the supernatant and allowed to settle. The remaining aggregates were collected by adding 2 mL of PBS to each well and transferring the remaining cells to a new conical tube. 1 mL of fresh medium B was added to each well, and the plate was returned to the incubator. The supernatant (medium A) was aspirated from the settled cells in each conical tube. 20 mL of phosphate-buffered saline (PBS) was added to each tube, the tubes were gently vortexed, and the tubes were spun down at 1100 rpm for 4 minutes. The cells were resuspended in the required amount of medium B (depending on the number of plates) and evenly distributed into the wells of each plate.

[0375] On days 4 and 5, 1 mL of supernatant was removed from each well and replaced with 1 mL of pre-warmed medium B. The collected supernatant was spun down and residual cells were added to the 6th well of each plate (well #6).

[0376] Differentiation of DE cells into anterior foregut endoderm (AFE) cells On day 6, the aggregates were collected along with the supernatant and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared AFE medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of AFE medium and evenly distributed into a 6-well plate.

[0377] On days 7-8, 1 mL of supernatant from each well was replaced with 1 mL of freshly prepared AFE medium. 1 mL of supernatant from each well was removed by spinning down, the remaining cells were resuspended in AFE medium, and added back to the last well.

[0378] Differentiation of anterior foregut endoderm (AFE) cells into ventral pharyngeal endoderm (VPE) cells At the start of day 9, aggregates were collected along with the supernatant in a 50 mL conical tube and allowed to settle. 1–2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE1 medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE1 medium and evenly distributed into a 6-well plate.

[0379] On day 10, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed VPE1 medium. The collected supernatant was removed by spinning down, and the residual cells were resuspended in the required amount of VPE1 medium and added to the 6th well of each plate.

[0380] On day 11, the aggregates were collected along with the supernatant in a 50 mL conical tube and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE2 medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE2 medium and evenly distributed into a 6-well plate.

[0381] On day 12, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed VPE2 medium. The collected supernatant was removed by spinning down, and the remaining cells were resuspended in the required amount of VPE2 medium and added back to the 6th well of each plate.

[0382] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial precursor (TEP) On day 13, the aggregates were collected along with the supernatant in a 50 mL conical tube and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared TEP medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of TEP medium and evenly distributed into a 6-well plate. On days 14, 15, and 16, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed TEP medium. The collected supernatant was removed by spinning down, and the residual cells were resuspended in the required amount of TEP medium and added to the sixth well of each plate.

[0383] On day 17, approximately 1.8 mL of supernatant was collected from each well, and 2 mL of pre-warmed TEP medium was added in its place. The collected supernatant was removed by spinning down, the residual cells were resuspended in TEP medium, and added back to the last well (well #6). In cases of numerous aggregates, the residual cells were added to two or more wells and returned to the wells.

[0384] On day 18, approximately 1 mL of supernatant was collected from each well, and 1 mL of pre-warmed TEP medium was added in its place. The collected supernatant was removed by spinning down, the residual cells were resuspended in pre-warmed TEP medium, and added back to the last well (well #6). This step was repeated on days 19, 20, 21, and 22.

[0385] Removal of chitosan-alginate for harvesting and freezing differentiated cells. On day 23, chitosan-coated alginate capsules were decapsulated by suspending them in a decapsulation solution (55 mM sodium citrate, 10 mM HEPES, and 0.9% NaCl) for 10 minutes while shaking at 100 rpm. TEP aggregates were collected, and the plates were washed with DPBS to collect any remaining TEP aggregates. After allowing the TEP aggregates to settle, the supernatant was aspirated. The TEP aggregates were treated with Accutase to dissociate into single cells, and 0.5–1.0 mL of sample was taken for cell counting and qRT-PCR.

[0386] 1-2 mL of fresh TEP medium and 10 mL of pre-warmed DMEM-F12 medium were added to the cells, and they were spun down at 1110 rpm for 4 minutes. 10 mL of fresh TEP medium was added, and the TEP cells were counted using a cell counter. The remaining cells were spun down, the supernatant was removed, and replaced with cryopreservation medium 1 (500 μL per cryopreservation vial). Cryopreservation medium 2 was added dropwise to the cells (500 μL per vial), and each cryopreservation vial contained 6 million to 10 million cells. The vials were frozen in liquid nitrogen and stored at -80°C.

[0387] Differentiation of thymic epithelial precursors into thymic epithelial cells (TEP to TEC) On day A, the TEP was thawed, and 1 mL of TEC freeze / thaw (FT) medium was added to each vial. The thawed cells were transferred to conical tubes and spun down at 1110 rpm for 4 minutes. The supernatant was removed, and 6 mL of TEC FT medium (with Y27632 added at a ratio of 1:1000) was added to each tube to resuspend the TEP. TEC FT medium containing Y27632 at a ratio of 1:1000 was also added to the wells of each 6-well plate (1 mL per well), and 1 mL of resuspended cells was added to each well. The plates were placed in an incubator (37°C, 5% CO2, shaking at 70 rpm).

[0388] After 24 hours, on day B, 1 mL of supernatant was removed from each well of each plate, and 1 mL of pre-warmed TEC FT medium was added to each well. The supernatant was spun down and aspirated, the residual cells were resuspended in TEC FT medium, and added back to the last well (well #6) of each plate. These steps were repeated for the next three days (days C, D, and E).

[0389] On day F, 1 mL of supernatant was removed from each plate well, and 1 mL of pre-warmed TEC FT medium containing PluriSin (20 μM) was added to each well. The supernatant was spun down and aspirated, and the remaining cells were resuspended in TEC FT medium containing PluriSin (20 μM) and added back to the last well (well #6).

[0390] On day G, TEC aggregates were collected and washed twice with DPBS. The supernatant was then aspirated and the cells were resuspended in 10 mL of DPBS. Four separate samples (0.5–1 mL) were collected and used for cell counting, flow cytometry, qRT-PCR, and CFU analysis. For qPCR analysis, DPBS medium was removed from the corresponding 0.5–1.0 mL samples and 200–400 μL of RLT buffer was added. TEC cells in the qPCR samples were lysed and stored at -80°C for RNA extraction. For flow cytometry analysis, TEC aggregates in 0.5–1.0 mL samples were washed once with DPBS and spun down at 1100 rpm for 4 minutes. The supernatant was aspirated and 1–2 mL of Accutase was added to each tube. After 4 minutes of incubation, the TEC aggregates were dissociated, washed with 1 mL of DPBS, and then spun down. The Accutase / supernatant was aspirated, DPBS was added to the cells, and then they were placed on ice for staining.

[0391] A schematic diagram illustrating the process of differentiation of chitosan-coated alginate-encapsulated hiPSC aggregates using 3D suspension culture is provided in Figure 2A ("3D Differentiation from Aggregates in Alginate"). Typical levels of FOXN1 expression in these TECs on day G (after freezing, thawing, and 7 days of growth in TEC FT medium) are provided in the right-hand column shown in Figure 2B, when measured by GADPH-normalized qRT-PCR at a cell concentration of 200,000 TEC cells / mL (0.0002). This level was approximately 5 times higher than the FOXN1 expression level measured on day 23 (before TEC FT medium was added to the cells and before they were frozen in liquid nitrogen and stored at -80°C).

[0392] Flow cytometry results for TEC aggregates are shown in Figures 3, 4, 5, and 6. The procedure was repeated three times (staining 1, Figures 3A-3N; staining 2, Figures 4A-4M; staining 3, Figures 5A-5O; staining 4, Figures 6A-6F).

[0393] On day 23 prior to freezing, the expression levels of the following genes were evaluated in TEC cells (the leftmost column represents the sample titled "AEMF-200K-090123 TEC"): Oct4, NANOG, HOXA3, TBX1, EYA1, EPCAM, PAX9, PAX1, FOXN1, DLL4, HLA-DRA, AIRE, CK5, CK8, FEZf2, CLDN3, CLDN4, PRSS16, LY75, and JAG2. The expression levels of the following genes were evaluated on day G (after thawing the TEC cells frozen on day 23 and growing them in TEC FT medium for 7 days), and these results are shown in the right-hand column of Figure 7: Oct4, NANOG, HOXA3, EPCAM, PAX9, and FOXN1 ("AEMF-200K-090123-FT1"). These results demonstrate that differentiated TECs exert thymic lymphocyte formation capabilities in vitro to support T cell development.

[0394] Example 4. Intramuscular transplantation of differentiated TEP supports human thymic lymphocyte formation in a humanized animal model. Human iPSC cells encapsulated in alginate and coated with chitosan were grown in a 3D suspension culture and differentiated into TEP cells as described in Example 4 (before the freeze / thaw step). Approximately 830,000 TEP cells were added to 300,000 mesenchymal stem cells, which were then treated with humanized NOD / SCID / IL2Rg Null (NSG) MHC-I / II double knockout mice (Jackson Laboratories) (n=3, identified by numbers 235, 236, and 237) were intramuscularly injected. Humanization was achieved as follows: Mice were sublethally irradiated, followed by injection of human umbilical cord blood-derived CD34+ hematopoietic stem cell precursors. Two weeks later, 830,000 TEP cells were intramuscularly injected into the quadriceps femoris muscle of each mouse (along with 300,000 mesenchymal stem cells). Humanized NSG mice have inefficient human T cell development due to residual thymic tissue. Thymectomy could be performed on the mice to create an athymic background in NSG mice, but this step was avoided by using an NSG variant line in which both MHC class I and MHC class II genes are knocked out. Since MHC is required for thymic function, the double knockout line functionally creates an athymic background, and NSG mice do not produce FOXN1.

[0395] To track the emergence of T cells and their immunophenotypes, the efficiency of thymic lymphocyte formation and T cell generation were assessed every three weeks via flow cytometry. Markers such as CD3 and TCRαβ related to human hematopoietic cells (hCD45) and human T cells were observed as shown in Figures 8A–8N. At week 3, none of the mice expressed any CD3+ (Figures 8G, 8I, 8K). At week 16, CD3-expressing cells were present, and within 13 weeks of TEP intramuscular injection, human CD3+ TCRαβ+ T cells ranged from 2% to 18% of total human blood (CD45+) cells, and by week 16, that range was 15% to 43%. Importantly, pseudo-transplant mice (negative controls – not shown) did not develop detectable T cells in their blood, demonstrating that the mice were effectively athymic and thus proved to be an excellent model for testing the thymic lymphocyte formation ability of iPS-TEP. These results demonstrate that human iPS-TEP can efficiently induce human T cell development in vivo.

[0396] Example 5. Differentiation of unencapsulated single hiPS cells into thymocytes using 3D suspension culture Preparation of cell culture media Medium A was prepared using DMEM-F12 as the basal medium and the following reagents: activin A (100 ng / mL), CHIR99021 (2 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400), and vitamin A-free B27 (1:400).

[0397] Medium B was prepared using DMEM-F12 as the basal medium and the following reagents: activin A (100 ng / mL), LDN193189 (200 nM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400), and vitamin A-free B27 (1:400).

[0398] AFE medium was prepared using DMEM-F12 as the basal medium and the following reagents: LDN193189 (200 nM), SB431542 (10 μM), FGF8b (50 ng / mL), ascorbic acid (10 μM), PenStrep (1:200), B27 (without retinoic acid) (1:200), N2 (1:100), BME (1:100), NEAA (1:400), and Glutamax (1:100).

[0399] VPE1 medium was prepared using DMEM-F12 as the basal medium and the following reagents: SB431542 (10 μM), FGF8b (50 ng / mL), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0400] VPE2 medium was prepared using DMEM-F12 as the basal medium and the following reagents: Noggin (50 ng / mL), CHIR99021 (2 μM), FGF8b (50 ng / mL), retinoic acid (0.1 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), NEAA (1:400), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), and N2 (1:100).

[0401] TEP medium was prepared using DMEM-F12 as the basal medium and the following reagents: FGF10 (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (2 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0402] TEC medium was prepared using DMEM-F12 as the basal medium and the following reagents: IL-22 (20 ng / mL), FGF10 (50 ng / mL), FGF7 / KGF (50 ng / mL), RANKL / TRANCE (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (3 μM), ascorbic acid (10 μM), and insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 (vitamin A-free) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0403] TEC freeze / thaw (FT) medium was prepared using DMEM-F12 as the basal medium and the following reagents: IL-22 (20 ng / mL), FGF10 (50 ng / mL), FGF7 / KGF (50 ng / mL), RANKL / TRANCE (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (3 μM), ascorbic acid (10 μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), vitamin A-free B27 (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400).

[0404] Differentiation of unencapsulated single hiPSCs into endoderm (DE) On day 0, iPSCs were thawed and deaggregated using Accutase. 3–5 million single iPS cells were placed in suspension in a 6-well ultra-low adhesion plate with medium A and ROCK inhibitor Y-27632 and incubated at 37°C and 5% CO2 at 70 rpm. Incubator conditions remained constant throughout the iPS differentiation steps described herein, and unless otherwise specified, seeded cells were returned to the incubator after each differentiation step.

[0405] On day 1, 1 mL of supernatant was removed from each well of each 6-well plate and spun down. The pelleted cells were washed with PBS, resuspended in 1 mL of preheated medium A and Y-27632, and then added back to the 6th well of each plate (well #6).

[0406] On the second day, approximately 1.8 mL of supernatant was removed from each well, and 2 mL of pre-warmed medium A (without Y-27632) was added to each well. The supernatant was spun down, the pelleted cells were washed with PBS, resuspended in 2 mL of medium A (without Y-27632), and then added to the sixth well of each plate.

[0407] On day 3, the cells were collected in 50 mL conical tubes along with the supernatant and allowed to settle. The remaining aggregates were collected by adding 2 mL of PBS to each well and transferring the remaining cells to a new conical tube. 1 mL of fresh medium B was added to each well, and the plate was returned to the incubator. The supernatant (medium A) was aspirated from the settled cells in each conical tube. 20 mL of phosphate-buffered saline (PBS) was added to each tube, the tubes were gently vortexed, and the tubes were spun down at 1100 rpm for 4 minutes. The cells were resuspended in the required amount of medium B (depending on the number of plates) and evenly distributed into the wells of each plate.

[0408] On days 4 and 5, 1 mL of supernatant was removed from each well and replaced with 1 mL of pre-warmed medium B. The collected supernatant was spun down and residual cells were added to the 6th well of each plate (well #6).

[0409] Differentiation of DE cells into anterior foregut endoderm (AFE) On day 6, the aggregates were collected along with the supernatant and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared AFE medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of AFE medium and evenly distributed into a 6-well plate.

[0410] On days 7-8, 1 mL of supernatant from each well was replaced with 1 mL of freshly prepared AFE medium. 1 mL of supernatant from each well was removed by spinning down, the remaining cells were resuspended in AFE medium, and added back to the last well.

[0411] Differentiation from the anterior foregut endoderm (AFE) to the ventral pharyngeal endoderm (VPE) At the start of day 9, aggregates were collected along with the supernatant in a 50 mL conical tube and allowed to settle. 1–2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE1 medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE1 medium and evenly distributed into a 6-well plate.

[0412] On day 10, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed VPE1 medium. The collected supernatant was removed by spinning down, and the residual cells were resuspended in the required amount of VPE1 medium and added to the 6th well of each plate.

[0413] On day 11, the aggregates were collected along with the supernatant in a 50 mL conical tube and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE2 medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE2 medium and evenly distributed into a 6-well plate.

[0414] On day 12, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed VPE2 medium. The collected supernatant was removed by spinning down, and the remaining cells were resuspended in the required amount of VPE2 medium and added back to the 6th well of each plate.

[0415] Differentiation of ventral pharyngeal endoderm (VPE) into thymic epithelial precursor (TEP) On day 13, VPE aggregates were collected in a 50 mL conical tube along with the supernatant and allowed to settle. 1-2 mL of PBS was added to each well, and any remaining aggregates were collected in a new tube. 1 mL of freshly prepared TEP medium was added to each well, and the plate was returned to the incubator. The supernatant was aspirated from the settled cells, 20 mL of PBS was added, and the cells were spun down at 1100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of TEP medium and evenly distributed into a 6-well plate.

[0416] On days 14, 15, and 16, 1 mL of supernatant was collected from each well and replaced with 1 mL of pre-warmed TEP medium. The collected supernatant was removed by spinning down, and the residual cells were resuspended in the required amount of TEP medium and added to the sixth well of each plate.

[0417] On day 17, approximately 1.8 mL of supernatant was collected from each well, and 2 mL of pre-warmed TEC medium was added in its place. The collected supernatant was removed by spinning down, the residual cells were resuspended in TEC medium, and added back to the last well (well #6). In cases of numerous aggregates, the residual cells were added to two or more wells and returned to the wells.

[0418] On day 18, approximately 1 mL of supernatant was collected from each well, and 1 mL of pre-warmed TEC medium was added in its place. The collected supernatant was removed by spinning down, and the remaining cells were resuspended in pre-warmed TEC medium and added back to the last well (well #6). This step was repeated on days 19, 20, 21, and 22.

[0419] Differentiated cells are collected, sampled, and frozen. On day 23, TEP aggregates were collected, and the plates were washed with DPBS to collect any remaining TEP aggregates. After the TEP aggregates settled, the supernatant was aspirated. The TEP aggregates were treated with Accutase to dissociate into single cells, and 0.5–1.0 mL of sample was taken for cell counting and qRT-PCR.

[0420] 1-2 mL of fresh TEC medium and 10 mL of pre-warmed DMEM-F12 medium were added to the cells, and they were spun down at 1110 rpm for 4 minutes. 10 mL of fresh TEC medium was added, and the TEC cells were counted using a cell counter. The remaining cells were spun down, the supernatant was removed, and replaced with cryopreservation medium 1 (500 μL per cryopreservation vial). Cryopreservation medium 2 was added dropwise to the cells (500 μL per vial), and each cryopreservation vial contained 6 million to 10 million cells. The vials were frozen in liquid nitrogen and stored at -80°C.

[0421] Differentiation from thymic epithelial precursor to thymic epithelial cells (TEP to TEC) On day A, the TEC cells were thawed, and 1 mL of TEC freeze / thaw (FT) medium was added to each vial. The thawed cells were transferred to conical tubes and spun down at 1110 rpm for 4 minutes. The supernatant was removed, and 6 mL of TEC FT medium (with Y27632 added at a ratio of 1:1000) was added to each tube to resuspend the TEC cells. TEC FT medium containing Y27632 at a ratio of 1:1000 was also added to the wells of each 6-well plate (1 mL per well), and 1 mL of resuspended cells was added to each well. The plates were placed in an incubator (37°C, 5% CO2, shaking at 70 rpm).

[0422] On days B, C, D, and E, 1 mL of supernatant was removed from each well of each plate, and 1 mL of pre-warmed TEC FT medium was added to each well. The supernatant was spun down and aspirated, the residual cells were resuspended in TEC FT medium, and added back to the last well (well #6) of each plate.

[0423] On day F, 1 mL of supernatant was removed from each plate well, and 1 mL of pre-warmed TEC FT medium containing PluriSin (20 μM) was added to each well. The supernatant was spun down and aspirated, and the remaining cells were resuspended in TEC FT medium containing PluriSin (20 μM) and added back to the last well (well #6).

[0424] On day G, TEC aggregates were collected and washed twice with DPBS. The supernatant was then aspirated and the cells were resuspended in 10 mL of DPBS. Four separate samples (0.5–1 mL) were collected and used for cell counting, flow cytometry, qRT-PCR, and CFU analysis. For qPCR analysis, DPBS medium was removed from the corresponding 0.5–1.0 mL samples and 200–400 μL of RLT buffer was added. TEC cells in the qPCR samples were lysed and stored at -80°C for RNA extraction. For flow cytometry analysis, TEC aggregates in 0.5–1.0 mL samples were washed once with DPBS and spun down at 1100 rpm for 4 minutes. The supernatant was aspirated and 1–2 mL of Accutase was added to each tube. After 4 minutes of incubation, the TEC aggregates were dissociated, washed with 1 mL of DPBS, and then spun down. The Accutase / supernatant was aspirated, DPBS was added to the cells, and then they were placed on ice for staining.

[0425] Figure 9A illustrates the differentiation process from hiPSCs to TECs described in this example, where unencapsulated single cells were cultured in a 3D suspension. Successful growth and differentiation into thymic epithelial precursor cells were achieved, as shown in the representative micrograph at the bottom of Figure 9A and in Figure 9B (a magnified version of the micrograph on the far right in Figure 9A, when the thymocytes were at stage 5 of the differentiation process, scale bar = 100 μm). Thymocytes produced from hiPSCs differentiated as single cells in 3D suspension culture expressed FOXN1 levels (ranging from 0.0019 to 0.0022) in vitro, which were approximately 19 to 22 times higher than the threshold level (0.0001) required to obtain sufficient T cell production in vivo, when the thymocytes were transplanted into mice as in Example 6 (as shown by the data in Figure 10A). The level of FOXN1 expression (shown in Figure 9C) was determined using qRT-PCR on thymocyte samples at a concentration of approximately 200,000 thymocytes per mL, with FOXN1 expression normalized to GADPH expression. As shown in Figure 9G, unencapsulated single-cell differentiation using 3D suspension culture was successful when B27 was added at the DE stage.

[0426] The expression levels of the following genes were also evaluated in TEP cells on day 23 (before freezing) and again on day G (after freezing, thawing, and 7 days of growth in TEC FT medium): Oct4, NANOG, HOXA3, TBX1, EYA1, EPCAM, PAX9, PAX1, FOXN1, DLL4, HLA-DRA, AIRE, CK5, CK8, FEZf2, CLDN3, CLDN4, PRSS16, LY75, and JAG2. These results also demonstrated that differentiated TECs exert thymic lymphocyte formation capabilities to support T cell development in vitro.

[0427] When hiPSCs were differentiated in 3D as single cells in a bioreactor according to the process illustrated in Figure 9D, the in vitro FOXN1 expression level also exceeded the threshold of 0.0001, as shown in Figure 9F.

[0428] Example 6. Intramuscular transplantation of differentiated TEPs demonstrates that in vitro FOXN1 expression levels correlate with in vivo T cell production. Unencapsulated human iPSC cells were differentiated into TECs using the same culture medium as in Example 5, with the hybrid (2D / 3D) protocol illustrated in Figures 10A and 10B.

[0429] In addition to approximately 1 million TEP cells, 300,000 mesenchymal stem cells were used with humanized NOD / SCID / IL2Rg Null Intramuscular injection was administered to (NSG) MHC-I / II dual knockout mice (Jackson Laboratories).

[0430] To track T cell emergence and their immunophenotypes, the efficiency of thymic lymphocyte formation and T cell generation were assessed every three weeks via flow cytometry. Markers for human hematopoietic cells (hCD45) and human T cells (CD4, CD8, and TCRαβ) were observed as shown in Figures 10C and 10D. The results shown in Figure 10C represent results obtained in similar experiments when TEC expressed FOXN1 in vitro at a level higher than the 0.0001 threshold found to be necessary for obtaining high levels of T cells in vivo. Specifically, TEC in Figure 10C expressed FOXN1 at an 8-fold higher level than the threshold (0.0001). When these TECs were transplanted into NSG MCH class I-II knockout mice, the levels of total T cells, CD4+ T cells, and CD8+ T cells observed between weeks 18 and 26 were significantly higher in mice receiving these TECs (see, e.g., 181, 182, and 183 in Figure 10C). In contrast, when TECs expressing FOXN1 at a threshold level of less than 0.0001 in vitro were transplanted, the levels of total T cells, CD4+ T cells, and CD8+ T cells were significantly lower. In vitro FOXN1 expression levels were determined using qRT-PCR normalized to GADPH expression. Each line (180–184 in Figure 10C and 190–194 in Figure 10D) represents a different mouse from the same strain.

[0431] Although the present invention has been described with reference to preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the present invention is limited only by the following claims.

Claims

1. A method for differentiating pluripotent stem cells into thymocytes, a) Encapsulating the pluripotent stem cells in a polymer, b) Differentiating the pluripotent stem cells into endoderm (DE) cells, c) Differentiating the DE cells into anterior foregut endoderm (AFE) cells by culturing the DE cells and contacting or incubating them with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, or a combination thereof, d) Culturing the AFE cells and differentiating the anterior foregut cells into ventral pharyngeal endoderm (VPE) cells, i) Contacting or incubating the AFE cells in a first VPE medium containing ascorbic acid, retinoic acid, FGF, a TGFβ inhibitor, or a combination thereof, ii) Differentiation comprising contacting or incubating the AFE cells in a second VPE medium containing noggin, WNT activator, FGF, retinoic acid, ascorbic acid, or a combination thereof, e) Differentiating the VPE cells into thymocytes by culturing the VPE cells and contacting or incubating the VPE cells with ascorbic acid, FGF, BMP, WNT activator, or a combination thereof, The thymic cells are thymic epithelial precursor cells (TEP) and / or thymic epithelium. A method involving cells (TECs).

2. A method for differentiating pluripotent stem cells into thymocytes, a) A step of differentiating the pluripotent stem cells into endoderm (DE) cells of the embryo, b) A step of culturing the DE cells and differentiating the DE cells into anterior foregut endoderm (AFE) cells by contacting or incubating the DE cells with a BMP inhibitor, a TGFβ inhibitor, FGF, ascorbic acid, or a combination thereof, c) The AFE cells are cultured, i) Contacting or incubating the AFE cells in a first VPE medium containing ascorbic acid, retinoic acid, FGF, a TGFβ inhibitor, or a combination thereof, ii) Differentiating the anterior foregut cells into ventral pharyngeal endoderm (VPE) cells by contacting or incubating the AFE cells in a second VPE medium containing noggin, WNT activator, FGF, retinoic acid, ascorbic acid, or a combination thereof; d) The step of culturing the VPE cells and differentiating the VPE cells into thymocytes by contacting or incubating the VPE cells with ascorbic acid, FGF, BMP, WNT activator, or a combination thereof, A method wherein the thymocytes are thymic epithelial precursor cells (TEP) and / or thymic epithelial cells (TEC).

3. The method according to any one of claims 1 to 2, wherein the TEP cells are further differentiated into thymic epithelial cells (TECs) by contacting or incubating the TEP with interleukin, a WNT activator, RANKL, FGF, BMP, ascorbic acid, or a combination thereof.

4. The aforementioned differentiation of pluripotent stem cells into DE cells a) Contacting or culturing the pluripotent stem cells in a first growth medium, wherein the first growth medium contains activin A, PI-103, CHIR99021, or a combination thereof. b) The method according to any one of claims 1 to 3, comprising culturing the pluripotent stem cells in a second growth medium to generate endoderm cells, wherein the second growth medium comprises activin A, a BMP inhibitor, PI-103, CHIR99021, or a combination thereof.

5. The method according to any one of claims 1 to 4, wherein the first VPE medium further comprises a WNT inhibitor.

6. The method according to any one of claims 1 to 5, wherein the second VPE medium further comprises a BMP inhibitor, an SHH inhibitor, or a combination thereof.

7. The method according to any one of claims 1 and 3 to 6, wherein the polymer comprises a bio-derived polymer, a synthetic polymer, a composite polymer, a hydrogel, or a crosslinked polymer.

8. The method according to any one of claims 1 and 3 to 7, wherein the polymer is an alginate.

9. The method according to any one of claims 1 to 8, wherein the BMP inhibitor is LDN193189.

10. The method according to any one of claims 1 to 9, wherein the TGFβ inhibitor is SB431542.

11. The method according to any one of claims 1 to 10, wherein the FGF is FGF8b, FGF7, FGF10, FGF1, bFGF, or a combination thereof.

12. The method according to any one of claims 1 to 11, wherein the WNT activator is CHIR99021.

13. The method according to any one of claims 1 to 12, wherein the BMP is BMP2, BMP4, or a combination thereof.

14. The method according to any one of claims 1 to 13, wherein the interleukin is IL22.

15. The method according to any one of claims 1 to 14, wherein the WNT inhibitor is IWR-1.

16. The method according to any one of claims 10 to 15, wherein the BMP inhibitor is LDN193189.

17. The method according to claim 5, wherein the SHH inhibitor is SANT-1.

18. The method according to any one of claims 1 to 17, wherein the pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes are cultured in 3D culture.

19. The method according to any one of claims 1 to 18, wherein the pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymocytes are cultured as aggregates in a suspension.

20. The method according to any one of claims 1 to 19, wherein the method is carried out for approximately 15 to 30 days.

21. The method according to any one of claims 1 to 20, wherein the method is carried out for approximately 18 to 25 days.

22. The method according to any one of claims 1 to 21, wherein the pluripotent stem cells are differentiated into endoderm cells of the embryo for about 5 days.

23. The method according to any one of claims 1 to 22, wherein the DE cells are differentiated into AFE cells for about 2 to 3 days.

24. The method according to any one of claims 1 to 23, wherein the AFE cells are cultured in the first VPE medium for about 2 to 4 days.

25. The method according to any one of claims 1 to 24, wherein the AFE cells are cultured in the second VPE medium for about 2 to 3 days.

26. The method according to any one of claims 1 to 25, wherein the VPE cells are differentiated into thymocytes for about 3 to 6 days.

27. The method according to any one of claims 1 to 26, wherein the TEP is differentiated into TEC over a period of about four days.

28. The method according to any one of claims 1 or 3 to 27, wherein the polymer is removed from the TEP.

29. The method according to any one of claims 1 or 3 to 28, wherein the polymer is removed from the TEC.

30. A population of thymocytes prepared according to the method described in any one of claims 1 to 29.

31. A pharmaceutical composition comprising a population of thymocytes according to claim 30 and at least one excipient.

32. A method for treating or preventing a condition in a subject, comprising administering the subject the pharmaceutical composition described in claim 31.

33. The method according to claim 32, wherein the condition is a condition related to a defect, reduction, or abnormal function of the thymus of the subject, immunodeficiency, cancer, autoimmune disease, infection, or graft-versus-host disease (GvHD).

34. A composition, a) A population of thymocytes, wherein the population of thymocytes includes one or more of iTEC cells, mTEC cells, keratinocyte-like mTEC cells, cTEC-high cells, cTEC-low cells, and mTEC-low cells, b) A composition comprising the thymic support system (TSS).

35. The composition according to claim 34, wherein the population of thymocytes is prepared by the differentiation of iPS cells into thymocytes.

36. The composition according to claim 35, wherein the TSS comprises a polymer.

37. The composition according to claim 36, wherein the polymer is a bio-derived polymer or a synthetic polymer.

38. The composition according to any one of claims 31 to 37, wherein the polymer is a bio-derived polymer, and the bio-derived polymer is a polypeptide-based bio-derived polymer, a polynucleotide-based bio-derived polymer, or a polysaccharide-based polymer.

39. The composition according to claim 38, wherein the polymer is a polypeptide-based polymer, and the polypeptide-based polymer is selected from the group consisting of collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin, and actin.

40. The composition according to claim 38, wherein the polymer is a polysaccharide-based polymer, and the polysaccharide-based polymer is selected from the group consisting of chitin, chitosan, alginate, hyaluronic acid, cellulose, agarose, starch, cellulose, dextran, hyaluronic acid, glycogen, and glycosaminoglycans.

41. The polymer is a synthetic polymer, and the synthetic polymer is polycaprolactone, polyglycolic acid, polylactic acid, polylactic acid-coglycolic acid, poly(ethylene oxide) polyethylene glycol, polyurethane, poly(siloxane), poly(ethylene), poly(vinylpyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polyhydroxybutyrate (PHB), polypropylene fumarate (PPF), polyvinyl alcohol (PVA), polypropylene carbonate, polyanhydride, polyphosphazene, polygermane, polyorthoester, polyester, polyamide, polyolefin, polycarbonate, polyaramid, polyimide, chitosan, poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), hydroxyethoxyethyl methacrylate Acrylate (HEEMA), Hydroxydiethoxyethyl methacrylate (HDEEMA), Methoxyethyl methacrylate (MEMA), Methoxyethoxyethyl methacrylate (MEEMA), Methoxy-diethoxyethyl methacrylate (MDEEMA), Ethylene glycol dimethacrylate (EGDMA), N-vinyl-2-pyrrolidone (NVP), N-isopropyl Aam (NIPAAm), Vinyl acetate (Vac), Acrylic acid (AA), N-(2-hydroxypropyl) methacrylamide (HPMA), Ethyl The composition according to claim 37, selected from the group consisting of lenglycol (EG), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, and poly(N-isopropylacrylamide) (PNIPAM).

42. The composition according to any one of claims 34 to 41, wherein the polymer forms a hydrogel.

43. The composition according to any one of claims 34 to 42, wherein the polymer is crosslinked.

44. The composition according to any one of claims 34 to 43, wherein the TSS further comprises one or more of the extracellular matrix components and a drug.

45. The composition according to claim 44, wherein the extracellular matrix component is an extracellular matrix protein, or a region or portion thereof.

46. The composition according to claim 45, wherein the extracellular matrix protein is fibronectin, laminin, vitronectin, tenacin, entactin, thrombospondin, elastin, gelatin, collagen, fibrin, merosin, ancarin, chondronectin, link protein, bone sialocate protein, osteocalcin, osteopontin, epinectin, hyaluronectin, undurin, epiligrin, or kalinin.

47. The composition according to any one of claims 44 to 46, wherein the extracellular matrix component is a peptide derived from the extracellular matrix protein.

48. The composition according to claim 47, wherein the peptide is sequence number 9 to 18.

49. The composition according to claim 48, wherein the agent is a biological agent or a chemical agent.

50. The composition according to claim 49, wherein the biological agent is a ligand, an immunomodulator, or a hormone.

51. The composition according to claim 34, further comprising a population of supporting cells, wherein the supporting cells comprise one or more of myelin cells, myosoidal cells, neuroendocrine cells, tuft cells, ionocytes, endothelial cells, mesenchymal stem cells, or fibroblasts.

52. The composition according to claim 34, further comprising a population of effector cells.

53. The composition according to claim 34, further comprising a population of stem cells.

54. The composition according to any one of claims 31 and 34-53, wherein the level of in vitro FOXN1 production from a sample of the thymocyte population having a concentration of approximately 200,000 thymocytes per milliliter is greater than 0.0001 when measured by qRT-PCR and normalized to GADPH.

55. A method for treating or preventing a condition in a subject, comprising administering to the subject a composition according to any one of claims 30 to 31 and 34 to 54.

56. The method according to claim 55, wherein the condition is related to a defect, reduction, or abnormal function of the thymus in the subject, such as DiGeorge syndrome, thymoma (such as type A or type B thymoma), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymic carcinoma, thymic atrophy (such as age-related thymic atrophy), thymic cyst, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, thymic radiation, myasthenia gravis, thymic carcinoma, thymic hyperplasia, thymic radiation, or age- or infection-related decline in thymic function.

57. The method according to claim 55, wherein the subject has undergone thymectomy.

58. The method according to claim 55, wherein the aforementioned condition is immunodeficiency.

59. The method according to claim 55, wherein the aforementioned condition is cancer.

60. The method according to claim 55, wherein the condition is an autoimmune disease.

61. The method according to claim 55, wherein the condition is an infectious disease.

62. The method according to claim 55, wherein the condition is graft-versus-host disease (GvHD).

63. A method for improving organ transplant acceptance and / or preventing organ transplant rejection in a subject receiving organ transplants, comprising administering to the subject a composition according to any one of claims 30-31 and 34-54.

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