Thymic cell compositions and manufacturing methods

Description

“THYMAL CELL COMPOSITIONS AND MANUFACTURING METHODS” RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 63 / 457,314, filed April 5, 2023. The contents of this application are incorporated herein by reference in their entirety. GOVERNMENT INTERESTS

[0002] This invention was made with the support of the government under Agreement No. AY2AX000004 granted by the Advanced Research Projects Agency for Health (ARPA-H). The Government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The present invention relates generally to the bioengineering of pluripotent stem cells and, more specifically, to bioengineered thymic cells, thymic organoids, humanized animal models that include these thymic cells and thymic organoids, and their methods of use. BACKGROUND INFORMATION

[0004] The thymus is a primary lymphoid organ that plays a central role in the immune system. The thymus microenvironment offers a unique training ground for the development and maturation of effector cells, such as lymphocytes (e.g., T cells). Complex interactions between thymic cells and effector cells can determine the phenotype and functionality of effector cells. Some interactions between thymic cells and effector cells are tuned so that recognition of factors expressed by thymic cells promotes the survival of effector cells. Conversely, other interactions between thymic cells and effector cells can result in the death of effector cells. By controlling these interactions, the thymus plays a key role in establishing a repertoire of effector cells that are capable of mounting an activated immune response to foreign invaders while simultaneously establishing tolerance to the organism itself.

[0005] Interactions between thymic cells and effector cells occur Petition 870250090928, dated 06 / 10 / 2025, page 10 / 189 2 / 134 in the intricate three-dimensional network of the thymus, which creates a complex microenvironment for the development of effector cells. There is still a need for thymic cell-based therapies that recapitulate and mimic the native thymus environment. SUMMARY OF THE INVENTION

[0006] The present invention provides thymic cells, thymic organoids, and animal models that include these thymic cells and thymic organoids. It also offers methods for manufacturing and maintaining thymic cells and thymic organoids in vitro and in vivo.

[0007] In particular, the present invention provides methods for inducing the differentiation of pluripotent stem cells into thymic cells. These methods may include the steps of encapsulating the pluripotent stem cells in a polymer, differentiating the pluripotent stem cells into definitive endoderm (DE) cells. DE cells can be cultured and differentiated into foregut endoderm (AFE) cells by contacting or incubating DE cells with a BMP inhibitor, a TGFβ inhibitor, an FGF, ascorbic acid, and / or a combination thereof. AFE cells can be cultured and differentiated into ventral pharyngeal endoderm (VPE) cells by culturing in a first VPE medium and / or a second VPE medium. The first VPE medium may include ascorbic acid, retinoic acid, an FGF, and / or a TGFβ inhibitor. In some modalities, the first means of VPE may also include a WNT inhibitor.The second VPE medium may include Noggin, a WNT activator, an FGF, retinoic acid, and / or ascorbic acid. In some embodiments, the second VPE medium may also include a BMP inhibitor, an SHH inhibitor, or a combination thereof. VPE cells can be further differentiated into thymic cells, such as thymic epithelial progenitor cells (TEPs), by contacting or incubating VPE cells with ascorbic acid, an FGF, a BMP, and / or a WNT activator. Petition 870250090928, dated 06 / 10 / 2025, page 11 / 189 3 / 134

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

[0009] In one aspect, the methods for inducing differentiation of pluripotent stem cells into thymic cells do not include an encapsulation step, but do include the steps of: differentiation of pluripotent stem cells into definitive endoderm (DE) cells; culture of DE cells and differentiation of DE cells into anterior foregut endoderm (AFE) cells by contacting or incubating DE cells with a BMP inhibitor, a TGFe inhibitor, an FGF, an ascorbic acid, or a combination thereof; culture of AFE cells and differentiation of foregut cells into ventral pharyngeal endoderm (VPE) cells by: (i) contacting or incubating AFE cells in a first VPE medium comprising ascorbic acid, a retinoic acid, an FGF, a TGFe inhibitor, or a combination thereof;and (ii) contact or incubation of AFE cells in a second VPE medium comprising a Noggin, a WNT activator, an FGF, a retinoic acid, an ascorbic acid or a combination thereof; and culture of VPE cells and differentiation of VPE cells into thymic cells by contact or incubation of VPE cells with an ascorbic acid, an FGF, a BMP, a WNT activator or a combination thereof; wherein the thymic cells are thymic epithelial progenitors (TEPs) and / or thymic epithelial cells (TECs).

[0010] In some embodiments, thymic cells can be thymic epithelial progenitors (TEPs) or thymic epithelial cells (TECs). TEPs can be further differentiated into TECs by culturing TEPs with an interleukin, a WNT activator, a RANKL, an FGF, a BMP, and / or ascorbic acid.

[0011] In some embodiments, pluripotent stem cells can be differentiated into DE cells by contact or culture of pluripotent stem cells in a first growth medium containing Activin A, PI-103 and / or CHIR99021. Differentiation into DE cells may also include Petition 870250090928, dated 06 / 10 / 2025, page 12 / 189 4 / 134 cell culture in a second growth medium containing Activin A, a BMP inhibitor, PI-103 and / or CHIR99021.

[0012] In some modalities, the BMP inhibitor may be the LDN193189. In some modalities, the TGFe inhibitor may be SB431542. In some modalities, the FGF may be FGF8b, FGF7, FGF10, FGF1, bFGF, or a combination thereof. In some modalities, the WNT activator may be CHIR99021. In some modalities, the BMP may be BMP2, BMP4, or a combination thereof. In some modalities, the interleukin may be IL22. In some modalities, the WNT inhibitor may be IWR-1. In some modalities, the BMP inhibitor may be LDN193189. In some modalities, the SHH inhibitor may be SANT1.

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

[0014] In some embodiments, pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymic cells can be fixed to a solid substrate. In some embodiments, pluripotent stem cells, DE cells, AFE cells, VPE cells, or thymic cells can be fixed to a solid substrate that includes an extracellular matrix-based medium.

[0015] In some other modalities, pluripotent stem cells and Petition 870250090928, dated 06 / 10 / 2025, page 13 / 189 5 / 134 DE cells can be cultured as aggregates in suspension, and then the resulting DE aggregates can be fixed to a solid substrate that includes an extracellular matrix-based medium and subsequently differentiated into AFE, VPE, and TEP cells via two-dimensional (2D) adherent cultures. In some other 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 other additional embodiments, the human induced pluripotent stem cells are not encapsulated.

[0016] The methods of the invention can be performed for about 15 to 18 days. In some embodiments, the methods can be performed for about 18 to 25 days. In some embodiments, pluripotent stem cells can be differentiated into definitive endodermal cells for about 5 days. In some embodiments, DE cells can be differentiated into AFE cells for about 2 to 3 days. In some embodiments, AFE cells can be differentiated into VPE cells in the first VPE medium for about 2 to 4 days and in the second VPE medium for about 2 to 3 days. In some embodiments, VPE cells can be differentiated into thymic cells for about 3 to 12 days.

[0017] The present invention also provides thymic cells, for example, TEPs and TECs, generated by the methods described here.

[0018] Methods for culturing thymic cells in vitro are also provided herein. These methods may include culturing or incubating thymic cells in a thymic cell medium that includes FGF10, BMP4, FGF8b, CHIR99021, and / or ascorbic acid. In some embodiments, the thymic cell medium may further include FGF7 and RANKL. The methods may also include culturing thymic cells in suspension. As a non-limiting example, thymic cells may be cultured as aggregates in suspension.

[0019] In some embodiments, the present invention provides methods for increasing FOXN1 expression in a thymic cell population. Petition 870250090928, dated 06 / 10 / 2025, page 14 / 189 6 / 134 These methods may include freezing the thymic cell population, thawing the thymic cell population, and measuring and comparing FOXN1 expression in the thymic cell population before freezing and comparing it with FOXN1 expression after thawing the thymic cell population. In some modalities, FOXN1 expression can be increased by approximately 10 to 100 times. In some modalities, FOXN1 expression can be increased by approximately: 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. In some embodiments, FOXN1 expression can be increased by approximately: 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In some embodiments, the thymic cell population can be cultured as aggregates in suspension.

[0020] Pharmaceutical compositions that include a population of thymic cells prepared by the methods described in this document are also provided in this document. The present invention also offers methods of treating or preventing a condition in an individual through the administration of the pharmaceutical compositions of the invention. In some embodiments, the condition is associated with an absence, decline, or aberrant functioning of the thymus in the individual. The condition may be an immunodeficiency, a cancer, an autoimmune disease, an infectious disease, or a graft-versus-host disease (GvHD). In some embodiments, the pharmaceutical compositions are administered parenterally. For example, the compositions may be implanted or injected into one or more lymph nodes of the individual.

[0021] The present invention provides compositions. The compositions may include a population of thymic cells. In some embodiments, the thymic cell population may include one or more cell types. The cell types may be selected from one or more of the following options: iTEC, mTECs, corneocyte-like mTECs, high-cTEC, low-cTEC, Petition 870250090928, dated 06 / 10 / 2025, page 15 / 189 7 / 134 mTEC-low. Compositions may also include a thymic support system (TSS).

[0022] In some embodiments, the thymic cell population can be prepared by differentiating iPS cells into thymic cells.

[0023] In some embodiments, TSS may include a polymer. The polymer may be a biogenic polymer or a synthetic polymer. Biogenic polymers may be polypeptide-based biogenic polymers, polynucleotide-based biogenic polymers, polysaccharide-based polymers, or a combination thereof. Non-limiting examples of polypeptide-based polymers include collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin, and actin. Non-limiting examples of polysaccharides include alginate, 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-co-glycolic acid, poly(ethylene oxide), polyethylene glycol, polyurethane), a poly(siloxane), a poly(ethylene), a poly(vinylpyrrolidone), a poly(2-hydroxyethyl methacrylate), a poly(N-vinylpyrrolidone), a poly(methyl methacrylate), a poly(vinyl alcohol), a poly(acrylic acid), a polyacrylamide, a poly(ethylene-co-vinyl acetate), a poly(ethylene glycol), a poly(methacrylic acid), polyhydroxybutyrate (PHB), polypropylene fumarate (PPF), a polyvinyl alcohol (PVA), a polypropylene carbonate, a polyanhydride, a polyphosphazene, a polygerman, a polyester, a polyester, a polyamide, a polyolefin, a polycarbonate, a polyamide, a polyimide), chitosan, Poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA),hydroxyethoxyethyl methacrylate (HEEMA), hydroxydioxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethoxyethyl methacrylate (MEEMA), methoxydiethoxyethyl methacrylate (MDEEMA), ethylene glycol dimethacrylate (EGDMA), N-vinyl-2-pyrrolidone (NVP), N, Petition 870250090928, dated 06 / 10 / 2025, page 16 / 189 8 / 134 isopropyl AAm (NIPAAm), vinyl acetate (VAc), acrylic acid (AA), N-(2-hydroxypropyl) methacrylamide (HPMA), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), an acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride and / or poly(N-isopropylacrylamide) (PNIPAM).

[0025] The polymers of the invention can form a hydrogel. In some embodiments, the polymers can be crosslinked.

[0026] The TSS of the invention may further include an extracellular matrix component and / or an agent. In some embodiments, the extracellular matrix component may be an extracellular matrix protein or a region or part thereof. Non-limiting examples of extracellular matrix proteins include fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrin, merosin, ankalin, chondronectin, binding protein, bone sialoacid protein, osteocalcin, osteopontin, epinectin, hyaluronectin, unjulin, epiligrin, or carinin. In some embodiments, the extracellular matrix component may be a peptide derived from the extracellular matrix protein. Non-limiting examples of the peptide include an amino acid sequence from any of the SEQ ID NO: 9-18.

[0027] The TSSs of the invention may include a biological agent or a chemical agent.In some modalities, the biological agent may be a ligand, an immune modulator, or a hormone.

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

[0029] Methods of treating or preventing a condition in an individual through the administration of the compositions described in this document are also provided. Petition 870250090928, dated 06 / 10 / 2025, page 17 / 189 9 / 134 BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The aforementioned objectives, features and advantages, as well as others, will be apparent from the following description of the particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale; the emphasis is being placed on illustrating the principles of various embodiments of the invention.

[0031] Figure 1 is a graph showing the expression of the markers of TEP in cells differentiated by iPSC cell encapsulation compared with cells differentiated in two-dimensional cultures, as well as cells in two-dimensional culture and subjected to freezing and thawing.

[0032] Figures 2A-2B are a schematic and a bar graph showing the timing, process, and results of the differentiation of chitosan-coated alginate-encapsulated hiPSC aggregates into thymic epithelial cells. Figure 2A is a schematic illustrating the differentiation process of chitosan-coated alginate-encapsulated hiPSC aggregates into thymic epithelial cells (TEC, also called “THY-100”) using 3D suspension culture. Cells were encapsulated in alginate (by extrusion or emulsification) and cultured in 3D suspension at 45 revolutions per minute (RPM) at 37 °C in 5% CO2. Figure 2A also shows the differentiation stages that occur from Day 0 to Day 21, representing Stage 0 to Stage 4, when thymic epithelial progenitors (TEPs) are frozen (indicated by the dashed line on the right); it also shows a final differentiation stage (Stage 5) that occurs after the TEP cells are thawed.The photographs below the ovals in Figure 2A show the hiPSC cells after encapsulation (far left image) and at each stage, from Stage 0 to Stage 5 (far right image). All images were taken at 10x magnification, and the scale bar in each image represents 100 µm. Figure 2B is a bar graph showing the amount of FOXN1 expressed by Stage 5 TECs after they have been desiccated. Petition 870250090928, dated 06 / 10 / 2025, page 18 / 189 10 / 134 cells were frozen and cultured in 3D suspension in TEC Freeze / Thaw medium for 7 days. These expression data were obtained by qRT-PCR, with FOXN1 expression levels normalized to GAPDH expression and cell concentration at 200,000 cells / mL. The FOXN1 expression level was approximately five 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).

[0033] Figures 3A-3N show flow cytometry results characterizing hiPSC-derived TECs. Figures 3A to 3N present the results for the first of the four stainings.

[0034] Figures 4A-4M show flow cytometry results characterizing hiPSC-derived TECs. Figures 4A to 4M present the results for the second of the four stainings.

[0035] Figures 5A-5O show flow cytometry results characterizing hiPSC-derived TECs. Figures 5A to 5O present the results for the third of the five stainings.

[0036] Figures 6A-6F show flow cytometry results characterizing hiPSC-derived TECs. Figures 6A to 6F present the results for the fourth of the four stainings.

[0037] Figure 7 is a bar graph illustrating the expression levels of various genes for alginate-encapsulated hiPSC-derived TEC cells on Day 23, prior to freezing (see leftmost columns, “AEMF-200K-090123 TEC”). Expression levels were also assessed for a sample of the same cells on Day G (after freezing, thawing, and culture for 7 days in TEC FT medium); these results are shown in the rightmost columns (“AEMF-200K-090123-FT1”). The TEC and FT1 samples had a cell concentration of 200,000 cells / mL. These results demonstrate that differentiated TECs exhibit thymopoietic capacity to support T cell development in vitro. Petition 870250090928, dated 06 / 10 / 2025, page 19 / 189 11 / 134

[0038] Figures 8A-8N are a set of flow cytometry graphs and contour maps showing that human iPSC-derived TEPs can efficiently induce human T cell development in vivo.

[0039] Figures 9A-9G are a set of diagrams and graphs illustrating the differentiation of hiPSCs into TECs using individual non-encapsulated cells cultured in 3D suspension. Figures 9A to 9G illustrate representative experiments of hiPSC differentiation into TECs using individual non-encapsulated cells cultured in 3D suspension (Figures 9A to 9C), as described in Example 5, or individual non-encapsulated cells cultured in 3D in a bioreactor (Figures 9D to 9F) using the same buffers described in Example 5.Successful growth and differentiation into thymic epithelial precursor cells were achieved, as shown qualitatively in the representative micrographs in Figure 9A (micrographs taken at 10x magnification; scale bar = 100 µm) and Figure 9B (micrograph taken at 10x magnification showing thymic cells at stage 5; scale bar = 100 µm). Figure 9C is a bar graph illustrating the in vitro expression of FOXN1 by thymic cells produced as described in Example 5 (after iPSC-derived TEP cells were frozen, thawed, and cultured for 7 days in TEC FT medium). The expression level of FOXN1 (which ranged from 0.0019 to 0.0022) was determined using qRT-PCR in samples at a concentration of approximately 200,000 thymic cells per mL, with FOXN1 expression normalized to GADPH expression.These results demonstrate that thymic cells differentiated as individual cells in 3D suspension produced higher levels of FOXN1 in vitro compared to thymic cells produced using other methods described in this document. The results obtained when hiPSCs were differentiated as individual cells in 3D in a bioreactor according to the process illustrated in Figure 9D were also above the 0.0001 threshold required to obtain sufficient T cell production in vivo when the cells... Petition 870250090928, dated 06 / 10 / 2025, page 20 / 189 12 / 134 thymic cells were transplanted into mice, as in Example 6. Figure 9G is a schematic showing several attempts to induce individual cells at various stages of differentiation (ranging from iPS to TEP) to differentiate in a 3D suspension culture without encapsulation. All experiments in Figure 9G were performed under standard medium conditions. Individual aggregated or dissociated 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 a speckled pattern. Cell death is indicated by diagonal lines.

[0040] Figures 10A-10D. Figure 10A is a set of two diagrams and six graphs illustrating the process, timing, and results when aggregates of unencapsulated hiPSCs are differentiated into TECs using a hybrid (2D / 3D) differentiation protocol and transplanted into humanized NSG MHC-I / II double knockout mice. Figure 10A is a diagram illustrating a hybrid (2D / 3D) process for the differentiation of aggregates of unencapsulated hiPSCs into thymic epithelial cells (TECs, also called “THY-100”). In this hybrid process, unencapsulated hiPSCs were cultured to the DE stage in 3D suspension culture at 45 revolutions per minute (RPM) at 37°C in 5% CO2, and then the DE aggregates were placed in 24-well culture plates coated with Matrigel. The remaining differentiation stages (AFE, VPE, and TEP) were all performed in adherent 2D culture.Figure 10A also shows the differentiation stages that occur from Day 0 to Day 21, representing Stage 0 to Stage 4 when thymic epithelial progenitors (TEPs) are frozen (indicated by the dashed blue line on the right sides); it also shows a final differentiation stage (Stage 5) that occurs after TEP cells are frozen at -80°C and thawed. The images below the ovals in Figure 10A show hiPSC cells at each stage, from pre-stage 0 (far left) to es. Petition 870250090928, dated 06 / 10 / 2025, page 21 / 189 13 / 134 stage 0 (2nd image from the left) to stage 5 (far right image). All images were taken at 10x magnification, and the scale bar within all images represents 100 µm. Figure 10B is a diagram illustrating the same hybrid differentiation process (2D / 3D), with markers for specific differentiation stages indicated in the boxes below the ovals. Figures 10C-10D are graphs representing the development of T cells in humanized NSG MHC-I / II double knockout (Jax) mice transplanted with TEPs derived from hiPS differentiated using the hybrid differentiation process (2D / 3D) described in Figure 10A and Figure 10B.The vertical axes of the three graphs in Figure 10C show TCRae-positive T cells (far left graph), CD4+ T cells (middle graph), and CD8+ T cells (far right graph) expressed as a percentage of the total number of human CD45-positive (hCD45+) cells; the horizontal axis represents the time points for sample collection (weeks post-transplant). The results shown in Figure 10C are representative of the results obtained when TEPs expressed FOXN1 in vitro at a level higher than the 0.0001 threshold that was considered necessary to obtain sufficient T cells in vivo. Specifically, the TEPs in Figure 10C expressed FOXN1 at a level 8 times higher than the threshold (0.0001).The results shown in Figure 10D are representative of the results obtained when the TEPs to be transplanted expressed FOXN1 in vitro at a level below the 0.0001 threshold required to obtain sufficient T cells in vivo (0.00009). In vitro FOXN1 expression levels were determined using qRT-PCR normalized for GADPH expression. Each row (180-184 in Figure 10C and 190-194 in Figure 10D) represents a different mouse. DETAILED DESCRIPTION INTRODUCTION.

[0041] Thymic epithelial cells are important in the differentiation of T cells. Thymic cells prepared as described in this document Petition 870250090928, dated 06 / 10 / 2025, page 22 / 189 14 / 134 may allow exploration of thymus tissue and thymus cell properties, for example, thymus-related immune functions, for therapeutic applications. For example, age-related decline in immune function is known to be caused by changes in the composition and functional capabilities of thymic cells. Furthermore, changes in sex hormones, including androgens and estrogens, cause the thymus itself to atrophy or become senescent. The onset of thymic atrophy can begin as early as the onset of puberty. Thus, regeneration of thymic epithelial cells may provide compositions and methods that mitigate age-related decline in immune function.

[0042] The complexity and compartmentalization of thymic tissue allow the definition of distinct microenvironments with specific signals that direct the development of effector cells. Fetal thymus organ cultures (FTOCs) or reaggregated thymus organ cultures (RTOCs) have been developed as platforms to recreate niche environments that attempt to recapitulate thymic environments. In FTOCs, the entire thymus is placed on a membrane that sits atop a sponge with an incorporated medium (G. Anderson, et al., Cold Spring Harb. Protoc. 2007, 2007; the content of which is incorporated herein by reference in its entirety). In the case of RTOCs, after thymus dissociation, the cells of interest are selected by cell sorting technologies and placed as a concentrated droplet on top of a membrane, at the air-liquid interface. (EJ Jenkinson, et al., J. Exp. Med. 1992, 176, 845; the content of which is incorporated herein in its entirety by reference).Although these methodologies reflect the development of effector cells during fetal ontogeny, compositions that can mimic the native environment and allow the generation of effector cells, especially in adults, are still needed. The present invention provides thymic cell-based compositions that combine thymic cells with thymic support systems to create microenvironments that mimic the thymus. The inventors found that encapsulating iPS cells before differentiation with the protocols of... Petition 870250090928, dated 06 / 10 / 2025, page 23 / 189 The 15 / 134 differentiation described here can result in thymic cells expressing FOXN1, among other markers. Applying the thymic cell differentiation protocol in suspension offers opportunities to scale up thymic cell fabrication for therapeutic applications. COMPOSITIONS CELLS

[0043] The cells of the invention may include, without limitation, thymic cells, effector cells, pluripotent stem cells, their populations and derived cells.

[0044] In some embodiments, the cells of the present invention may be autologous, allogeneic, syngeneic, or xenogeneic with respect to a specific individual or individuals. In some embodiments, the thymic cells may be autologous, allogeneic, syngeneic, or xenogeneic with respect to the individuals who will benefit from their clinical application. In some embodiments, the cells of the invention may be mammalian cells, especially human cells. The cells may be primary cells or immortalized cell lines. In some respects, the cells of the invention may be prepared or derived from syngeneic cell sources. Any of the cells described in this document may be characterized by markers known in the art for that cell type.

[0045] One or more cells of the invention may be engineered to ectopically express a polynucleotide. The polynucleotide may encode a polypeptide of interest. In some embodiments, the polypeptide of interest may be operationally linked to an inducible element and / or an inducible promoter, such that the expression of the polypeptide of interest is controlled by the inducible element and / or the promoter. In some embodiments, the inducible element may include a ligand-binding domain, for example, FKBP ligand-binding domain, cyclophilin receptors, steroid receptors, cyclophilin receptor and / or tetracycline receptor. Petition 870250090928, dated 06 / 10 / 2025, p. 24 / 189 16 / 134 Any of the cell engineering compositions or methods described in International Patent Publication WO2021150837 may be useful in the present invention (the contents of which are incorporated 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. Some examples of cell death-inducing polypeptides include, but are not limited to, the proteins Casp2, Casp3, Casp8, Casp9, Casp10, p53, BAX, DFF40, HSV-TK and / or cytosine deaminase. Any inducible promoter known in the art may also be useful in the present invention.

[0046] In some embodiments, the cells of the invention can be engineered to ectopically express a protein that activates one or more checkpoint pathways to induce exhaustion of the host immune cell and anergy of the host cells to the cells of the invention. For example, the cells can be engineered to express an immune checkpoint protein according to the methods described in European Patent Publication EP3886759A1, the contents of which are incorporated herein by reference in their entirety. The immune checkpoint protein can 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 a combination thereof.

[0047] The cells of the invention can be arranged in structures resembling cylinders, rods, cords, filaments, or networks, as described in U.S. Patent Publication US20210213171, the contents of which are incorporated herein by reference in their entirety. This architecture can lead to improved integration of the invention's compositions into host organisms, as evidenced by improved blood supply and / or vasculature. The cells of the invention can be arranged in clusters and / or islands incorporated into ECM components. Petition 870250090928, dated 06 / 10 / 2025, page 25 / 189 17 / 134

[0048] In one embodiment, the compositions include thymic cells, effector cells, and one or more supporting cells, such as mesenchymal stem cells. The compositions are 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. Thymic cells

[0049] The compositions described in this document may include a population of thymic cells. A thymic cell may be a cell with one or more phenotypic or genotypic markers associated with a thymus-derived cell or a cell destined to become a thymic cell. In some embodiments, the thymic cell population may be derived by differentiation from pluripotent stem cells. In some embodiments, the pluripotent stem cells may be iPSCs.

[0050] In some embodiments, thymic cells can be prepared from the differentiation of pluripotent stem cells that differentiate into thymic stem cells through one or more of the following steps: PSCs can differentiate and / or can be induced to differentiate into cells resembling definitive endoderm (DE). Definitive endodermal cells can differentiate and / or can be induced to differentiate into cells resembling the third endoderm of the pharyngeal sac (PPE), which are also called ventral pharyngeal endoderm (VPE). Definitive endodermal cells can differentiate and / or be induced to differentiate into cells resembling foregut endoderm (AFE). The AFE can differentiate and / or be induced to differentiate into cells similar to the third endoderm of the pharyngeal sac (PPE), which are called the ventral pharyngeal endoderm (VPE).Thymic epithelial progenitor cells (TEPCs) can be generated from PPE cells. TECs can be derived from TEPCs. Each of the cell types described in this document can be characterized by one or more markers. In some embodiments, pluripotent stem cells may be associated with increased expression of markers such as, among others. Petition 870250090928, dated 06 / 10 / 2025, page 26 / 189 18 / 134 others, OCT4, SOX2 and / or NANOG. In some modalities, definitive endodermal cells may be associated with increased expression of markers such as, among others, SOX17, FOXA2, CXCR4 and / or CER1. In some modalities, foregut (FEG) cells may be associated with increased expression of markers such as, among others, FOXA2, SOX2 and / or PAX9. In some modalities, third pharyngeal pouch endodermal cells may be associated with increased expression of markers such as, among others, HOXA3, TBX1, PAX9, EYA1, SIX1, PBX1 and / or PAX1. In some modalities, thymic epithelial progenitor cells may be associated with increased expression of markers such as, among others, FOXN1, K5, K8 and / or HOXA3. In some modalities, thymic cells may be derived from a DE cell, a third PPE cell, an AFE cell, a TEPC cell, and / or a TEC cell.

[0051] For the preparation of the thymic cell population of the invention, pluripotent stem cells can be cultured and differentiated into definitive endodermal cells. The definitive endodermal cells can be cultured and differentiated into foregut cells. In some embodiments, foregut cells can be cultured and differentiated into pharyngeal endodermal cells. In some embodiments, pharyngeal endodermal cells can be cultured and differentiated into thymic cells, for example, thymic epithelial cells. In some embodiments, differentiation can be carried out between approximately 14 and 21 days.

[0052] In some embodiments, thymic cells can be prepared from PSCs. In this sense, the methods may include culturing pluripotent stem cells for a period of time and under conditions sufficient to differentiate the pluripotent stem cells into thymic cells. For example, the method may include culturing pluripotent stem cells in the presence of factors and / or inhibitors that promote the differentiation of PSCs into thymic cells. In some embodiments, these factors and / or inhibitors may include Petition 870250090928, dated 06 / 10 / 2025, page 27 / 189 19 / 134 or consist of a specific pluripotent stem cell inhibitor that selectively eliminates human pluripotent stem cells. In some embodiments, the specific pluripotent stem cell inhibitor comprises or consists of an oleate synthesis inhibitor. In some embodiments, the specific pluripotent stem cell inhibitor comprises or consists of a stearoyl-CoA desaturase (SCD1) inhibitor that inhibits SCD1 activity in human pluripotent stem cells. In some embodiments, the specific pluripotent stem cell inhibitor comprises or consists of an N-acylphenylhydrazine derivative comprising a phenylhydrazine moiety (Ph-N[H,C]-NH). In some embodiments, the specific pluripotent stem cell inhibitor comprises or consists of N'-phenylpyridine-4-carbohydrazide (NSC 14613, also known as PluriSIn #1).

[0053] In some embodiments, the methods for differentiating PSCs into thymic cells may be any methods known in the art. The methods for differentiating PSCs into thymic cells may include the use of one or more parameters known in the art for differentiation or combinations thereof. Parameters include, but are not limited to, (i) factors that promote differentiation (ii) inhibitors that promote differentiation (iii) duration of time to promote differentiation (iv) temperature (v) substrate and / or (vi) supporting cells that promote differentiation. Any of the methods or parameters for differentiating PSCs into thymic cells described in the following references may be used herein and includes Parent et al. Cell Stem Cell. August 1, 2013;13(2):219-29; Soh et al. Stem Cell Rep. 2014 Vol. 2 j 925-937; Sun et al. Cell Stem Cell. August 1, 2013;13(2):230-6; Okabe et al. Cell. Reprogram. 2015 Vol. 17 No. 5. Su et al. Sci.Rep. 2015 5, 9882; Otsuka et al.Sci Rep 2020 10:224; International Patent Publications, WO2019060336, WO2020205859, WO2020220040, WO2014134213, WO2010143529. WO2011139628; WO2022076751, WO2014134213, WO2021222297 and Chinese Patent Publication CN201110121243; the content of each of them is incorporated herein by reference in its entirety. Petition 870250090928, dated 06 / 10 / 2025, page 28 / 189 20 / 134

[0054] Thymic cells can be from an embryonic, fetal, or adult thymus.

[0055] In some modalities, the thymic cell population may include one or more cell types. The cell types may be iTEC, mTECs, corneocyte-like mTECs, cTEC-high, cTEC-low, mTEC-low.

[0056] In some modalities, the thymic cell population may include thymic epithelial cells (TECs). In some modalities, TECs may be derived by the differentiation of iPSCs. During embryonic development, TECs may be derived from non-hematopoietic cells that are negative for CD45 expression and positive for the epithelial marker EpCAM. TECs may be cortical thymic epithelial cells (cTECs) and / or medullary thymic epithelial cells (mTECs). mTECs are characterized by the expression of cytokeratin 5 (KRT5 or K5) and cytokeratin 14 (KRT14 or K14), but with a low level of cytokeratin 8 (KRT8 or K8) expression, while cTECs express K8 and K18. In some modalities, thymic cells can be derived from TECs that express both K5 and K8 (K5+K8+), which is typical of TEC cells found at the corticomedullary junction. In some respects, K5+K8+ cells can be progenitors of mTECs and / or cTECs.mTECs can also be positive for the expression of Ulex europaeus agglutinin-1 (UEA-1) on the cell surface, but not Ly51 (e.g., UEA-1+Ly51-), while cTECs can be UEA-1Ly51+.

[0057] In some modalities, the thymic cell population may include medullary thymic epithelial cells (mTECs). In some modalities, mTECs may be derived by differentiation from iPSCs. In some modalities, thymic cells may be or may be derived from mTECs. In some modalities, mTECs may have high expression of markers such as, among others, cytokeratin 5, cytokeratin 14, UEA-1, CD80, cathepsin L and / or cathepsin S.

[0058] In some modalities, thymic cells may be or may Petition 870250090928, dated 06 / 10 / 2025, page 29 / 189 21 / 134 being derived from cTECs that have high expression of markers such as, among others, cytokeratin 8, cytokeratin 18, Ly51, CD205, cathepsin L and / or thymus-specific serine proteases. In some modalities, the thymic cell population may include cortical thymic epithelial cells (cTECs). cTECs are responsible for commitment to the T cell lineage and positive selection of early thymocytes. In some modalities, cTECs may be derived by differentiation from iPSCs. As a non-limiting example, thymic cells may be or may be derived from cTECs that express markers such as: CCL25 and / or K5.

[0059] In some modalities, thymic cells may be, or may be derived from, TECs that express one or more markers, such as FOXN1, PAX9, PAX1, DLIA, ISL1, EYA1, SIX1, IL7, K5, K8, and AIRE.

[0060] Thymic cells may be, or may be derived from, any of the cell types described by Park et al. 2020 Science Vol. 367, Issue 6480 (the contents of which are incorporated herein by reference in their entirety). For example, thymic cells may be derived from myoid cells, for example, myoid cells expressing MYOD1 and MYOG (herein referred to as TEC(myo)) and / or from TECs expressing NEUROD1, SYP, CHGA (herein referred to as TEC(neuro)).

[0061] In some embodiments, the thymic cell population may include any cell type described by Bautista et al. 2021 Nat Commun 12, 1096, the content of which is incorporated herein by reference in its entirety. In some embodiments, the thymic cell population derived from iPSC differentiation may include “cTEClo” cells described by Bautista et al. 2021 and may be characterized by lower levels of functional genes (HLA class II) and by containing more proliferating KI67+ cells. Thymic cells may be derived from “mTEClo” cells described by Bautista et al. 2021 and characterized by CLDN4 expression, lower HLA class II levels, PSMB11, PRSS16, CCL25 expression, and high levels of the chemokine CCL21. Petition 870250090928, dated 06 / 10 / 2025, page 30 / 189 22 / 134

[0062] In some cases, the thymic cell population derived from the differentiation of hiPSCs or iPSCs may include any cell type described on US site 20230159887, the content of which is incorporated here by reference in its entirety.

[0063] In some modalities, the thymic cell population derived from iPSC differentiation may include “mTECi” cells described by Bautista et al. 2021 and characterized by SPIB, AIRE, FEZF2, and higher levels of HLA class II expression. Thymic cells may be or may be derived from corneocyte-like mTECs described by Bautista et al. 2021 and characterized by KRT1 and / or IVL expression.

[0064] In some modalities, the thymic cell population derived from iPSC differentiation may include immature TECs (iTECs) described by Bautista et al. 2021, which express canonical TEC identity genes, for example, FOXN1, PAX9, SIX1.

[0065] In some modalities, the thymic cell population derived from iPSC differentiation may include cTECi (or cTEC high) cells that can be characterized by the expression of cell surface markers such as, among others, CTSV, SLC46A2, HLA-DMA, CXCL12, THY1, ENO1, CALR, ALCAM, ATPIF1 and / or HSPA5.

[0066] In some modalities, the thymic cell population derived from iPSC differentiation may include cells with high AIRE+mTEC content, which can be characterized by the expression of one or more markers, such as, among others, LTF, HLA-DRA, CD74, HLA-DRB1, HLA-DPA, HLA-DPB1, IL2RG and / or FCER2.

[0067] In some modalities, the thymic cell population derived from iPSC differentiation may include TECs that express one or more markers, such as, among others, KRT5, KRT8, AIRE, PSMB11 and / or PRSS16.

[0068] In some modalities, the thymic cell population derived from iPSC differentiation may include TECs expressing one or more markers, such as, among others, AIRE, CK5, CK8, CXCL 12, CCL25, DLL4 and / or Petition 870250090928, dated 06 / 10 / 2025, p. 31 / 189 23 / 134 HLA-DR.

[0069] In some forms, the thymic cell population derived from iPSC differentiation may include corneocyte-like mTECs. The cells are called corneocyte-like because they express genes such as keratin cytoskeleton 1 (KRT1), KRT10, and SPINKS, which are also expressed in corneocytes (terminally differentiated keratinocytes) of the skin. Corneocyte-like cells also express transcripts that overlap with mTEC, such as AIRE. Therefore, they are called corneocyte-like mTECs. They are likely the precursors that give rise to Hassall's corpuscles, a unique cell in the human thymus. These cells are also believed to be derived from mTEC precursor cells (Noam Kadouri et al Nature Review Immunology 2020 v20:239; the content of which is incorporated here by reference in its entirety). PLURIPOTENT STEM CELLS (PSCs)

[0070] In some embodiments, the cells of the present invention may be derived from pluripotent stem cells.

[0071] 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) self-renewal capacity and (ii) pluripotency. Genes associated with pluripotency may include, among others, Oct-3 / 4, Sox2, Nanog, GDF3, REXI, FGF4, ESGI, DPPA2, DPPA4, hTERT, and SSEAI.

[0072] The cells described in this document may be derived from embryonic stem cells. ES cells may include a cell that (a) self-renews, (b) differentiates to produce all cell types in an organism, and / or (c) is derived from a developing organism. The cells Petition 870250090928, dated 06 / 10 / 2025, page 32 / 189 24 / 134 ES cells can be derived from the inner cell mass of the blastula of a developing organism. ES cells can also be derived from the blastomere generated by single blastomere biopsy (SBB), which involves the removal of a single blastomere from the eight-cell stage of a developing organism. ES cells can be characterized by the expression of markers such as, among others, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and / or alkaline phosphatase. Methods for generating and characterizing ES cells are known in the art and can be found, for example, in U.S. Patent No. 7,029,913; U.S. 5,843,780; U.S. 6,200,806 (the contents of each of which are incorporated herein by reference in their entirety).

[0073] Induced pluripotent stem cells (iPSCs) can also be used to generate cells of the present invention. iPSCs may include cells with one or more properties, such as, but not limited to, (a) self-renewal, (b) the ability to differentiate to produce all cell types in an organism, and / or (c) being derived from a somatic cell. iPSCs may express markers such as, but not limited to, SSEA3, SSEA4, SOX2, OCT3 / 4, Nanog, TRA160, TRA1818, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, Zpf42. Methods for generating and characterizing iPS cells can be found, for example, in US patent publications nos. US20090047263, US20090068742, US2009191159, US20090227032, US20090246875 and US20090304646 (the content of each is incorporated by reference in its entirety). In some embodiments, iPSCs may be derived from a T or non-T cell, a B cell or any other peripheral blood mononuclear cell, a hematopoietic progenitor cell or any other type of somatic cell.

[0074] 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 individual, such as the individual. Petition 870250090928, dated 06 / 10 / 2025, page 33 / 189 25 / 134 PSCs can also include embryonic stem cells derived from the placenta or umbilical cord; progenitor cells (e.g., progenitor cells derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood). EFFECTOR CELLS

[0075] An “effector cell” refers to any cell or cell type that, when in contact with or in proximity to a thymic cell, acquires the ability to execute, initiate, or propagate a signal or trigger for cell death. “Contact or proximity” may refer to sufficient spatiotemporal proximity to allow intrinsic or extrinsic cell signaling (e.g., cell-to-cell) or other communication or interaction.

[0076] The effector cells described in this document may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, thymus, or the stem or progenitor cells may have been differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Primary tissue stem or progenitor cells, ESCs or iPSCs may be of human or non-human animal origin (e.g., mice).

[0077] In some modalities, the effector cell may be a hematopoietic cell. In some modalities, the effector cell may be a lymphocyte. In some modalities, the lymphocyte may be a CD45-positive lymphocyte.

[0078] Effector cells can be CD4+CD8- T cells, CD4CD8+ T cells, CD34+ CD7+ CD1a+ cells, CD3+ TCRab+ cells, CD3+ TCRgd+ cells, CD3+ TCRab+ CD4+ CD8- cells, CD3+ TCRab+ CD8+ CD4- cells, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ cells, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ cells, CD3+ TCRab+ CD4+ CD8CD45RO- 30 CD45RA+ CCR7+ cells, CD3+ TCRab+ CD8+ CD4- CD45RO Petition 870250090928, dated 06 / 10 / 2025, page 34 / 189 26 / 134 CD45RA+ cells, CCR7+, CD3+ TCRab+, CD4+ CD8-, CD45RO-, CD45RA+ cells, CD27+, 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 cells that are CD45+, CD1 ... CD3-, CD25+, CD25-, Foxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD3 l+, CD3 l-, CD30+, CD30-, CD38+ and / or CD38- and / or cells that are positive for combinations thereof.

[0079] In some embodiments, the effector cell may be a T cell. T cells can be cultured T cells, for example, primary T cells, or T cells from a cultured T cell line, for example, Jurkat, SupTl, or T cells obtained from a mammal. If obtained from a mammal, effector cells can be obtained from various sources, including but not limited to blood, bone marrow, lymph node, thymus, spleen, or other tissues or fluids. Effector cells can also be enriched or purified. T cells can be any type of T cell and can be at any stage of development, including but not limited to double-positive CD4+ / CD8+ T cells, CD4+ helper T cells, for example, Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells (TILs), memory T cells, naive T cells.

[0080] In some modalities, the effector cells may be CCRXA-, CD3+, CD69-, MHC-1+, CD62L+ and / or CCR7+.

[0081] Effector cells can have a naive T cell (TN) phenotype, a central memory T cell (CMT) phenotype, or an effector memory T cell (EMT) phenotype. The phenotypes of TN, CMT, and EMT cells are known in the art. For example, CCR7 and CD62L are expressed by TN cells and Petition 870250090928, dated 06 / 10 / 2025, page 35 / 189 27 / 134 TcM cells are expressed by TN cells, but not by TEM cells. The transcription factors LEF1, FOXP1, and KLF7 are expressed by TN and TcM cells, but not by TEM cells. CD45RO and KLRG1 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 those of TEM cells.

[0082] In some modalities, the effector cells may be cells TCRa+TCRe+. TCRa+TCRe+ effector cells can be T cells that express a receptor that expresses an alpha (α) chain and / or a beta (β) chain. The alpha and beta chains of the TCR are known in the art.

[0083] The effector cell can be further modified. In other embodiments, stem or progenitor cells can be genetically modified. For example, stem or progenitor cells can express an exogenous T-cell receptor (TCR) or a chimeric antigen receptor (CAR), or both. In other embodiments, stem or progenitor cells can express an exogenous TCR associated with invariant natural killer (iNKT) T cells. In yet another embodiment, stem or progenitor cells express an exogenous antigen-specific TCR or have an exogenous genetic modification of genes that modulate T-cell differentiation, expansion, or function.

[0084] In some modalities, the effector cells may be Tregs FOXP3+. Tregs can be generated through clonal diversion of mTECs, where the expression of Aire on mTECs leads to the expression of tissue-specific antigens that become displayed on the surface (i.e., on antigen-presenting cells (APCs)). 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 Kampe. “Autoimmune polyendocrine syndromes.” New England Journal of Medicine 378.12 (2018): 1132-1141, incorporated herein as a reference). Petition 870250090928, dated 06 / 10 / 2025, page 36 / 189 28 / 134 reference in its entirety). SUPPORT CELLS

[0085] In some embodiments, the cells of the present invention may include or be cultured with supporting cells that assist in the generation of thymic cells and / or the maintenance of thymic cells in culture. Non-limiting examples of supporting cells include non-T cell hematopoietic progenitors such as macrophages and dendritic cells (DCs); non-hematopoietic cells such as epithelial cells and fibroblasts; stromal cells such as skeletal tissue progenitors, components such as bone, cartilage, hematopoiesis-supporting stroma, and adipocytes. In some embodiments, the supporting cells stimulate the proliferation, survival, maturation, or function of thymic cells. In some embodiments, the supporting cells may be of mesenchymal origin.

[0086] Supporting cells can be non-immune cells that may be present in the thymic microenvironment. For example, supporting cells can be fibroblasts, vascular smooth muscle cells (VSMCs), endothelial cells, and / or lymphatic endothelial cells. Supporting cells can also include mesenchymal stem cells (MSCs) of different origins, including bone marrow-derived MSCs, adipose tissue-derived MSCs, thymus-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 content of each is incorporated by reference in its entirety).

[0087] In some modalities, the supporting cells may be neuroendocrine cells (expressing BEX1, NEUROD1), muscle-like myoid cells (expressing MYOD1, DES), and myelin-positive epithelial cells (also called myelin cells) (expressing SOX10, MPZ) described by Bautista et al. 2021 Nat Commun 12, 1096 (2021), the content of which is incorporated by reference in its entirety. In some modalities, mesenchymal cells may be associated with markers such as, Petition 870250090928, dated 06 / 10 / 2025, p. 37 / 189 29 / 134 among others, LAMA2, LAMA4, PDGFRA, PDGFRB, LUM, CSPG4, COL1A2, COL3A1, IGF1, FGF7, FGF10, FST, BMP4, SFRP2, WNT5A. Mesenchymal cells can be positive or negative for one or more of these markers. In some modalities, mesenchymal cells may be positive for some markers described in this document, but may be negative for other markers.

[0088] In some modalities, endothelial cells may be associated with one or more markers, such as, among others, 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, the endothelial cells are adult vein endothelial cells, adult artery endothelial cells, endothelial cells derived from embryonic stem cells, iPS-derived endothelial cells, umbilical vein endothelial cells, umbilical artery endothelial cells, bone marrow-derived endothelial progenitor cells, umbilical cord blood-derived endothelial progenitor cells, peripheral blood-derived endothelial progenitor cells, adipose tissue-derived endothelial progenitor cells, adult skin-derived endothelial cells, or a combination thereof.In some modalities, the endothelial cells of the umbilical vein are human umbilical vein endothelial cells (HUVEC).

[0089] In some embodiments, the supporting cells of the invention may be fibroblasts and / or fibroblast-like cells. In some embodiments, the fibroblasts are human foreskin fibroblasts, human embryonic fibroblasts, mouse embryonic fibroblasts, skin fibroblast cells, vascular fibroblast cells, myofibroblasts, smooth muscle cells, mesenchymal stem cell-derived fibroblast cells (MSCs), or a combination thereof. In some embodiments, the fibroblasts are normal human dermal fibroblasts (NHDFs).

[0090] In some embodiments, the supporting cells may be hair cells that are positive for ATOH1, GFI1, LHX3 and / or FOXJ1. In some Petition 870250090928, dated 06 / 10 / 2025, page 38 / 189 In 30 / 134 modalities, the supporting cells may be myelin cells that closely resemble Schwann cells, which may be positive for SOX10, MPZ, MBP, and / or S100A1. In some modalities, the supporting cells may be tuft cells that may be positive for the markers GNB3, TRPM5, GNAT3, PLCB2, OVOL3, and / or POU2F3.

[0091] In some modalities, the supporting cells may be cells from the ionocyte population that may be positive for FOXI1, ASCL3, CFTR and / or CLCNKB.

[0092] Other non-limiting examples of supporting cells include hepatocytes, pancreatic exocrine cells, myocytes, pancreatic endocrine cells, neurons, enterocytes, adipocytes, splenic cells, renal cells, bile cells, Kupffer cells, stellate cells, cardiac muscle cells, alveolar cells, bronchiolar cells, club cells, urothelial cells, mucous 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. Thymic Organoids

[0093] In some embodiments, the compositions of the invention may include a thymic organoid. An organoid is a three-dimensional, miniature, in vitro recapitulation of an organ. A thymic organoid may be a three-dimensional, miniature, in vitro version of the thymic organ that can mimic the physiology and function of a human thymus. Methods for preparing thymic organoids are described in International Patent Publication WO2019060336, the contents of which are incorporated herein by reference in their entirety.

[0094] In some embodiments, effector cells can be prepared by differentiating pluripotent stem cells or progenitor cells into lymphocytes by culturing PSCs or progenitor cells with cells Petition 870250090928, dated 06 / 10 / 2025, page 39 / 189 31 / 134 thymic cells. In some embodiments, thymic cells may express a Notch ligand. In some embodiments, the Notch ligand may be Delta-like 1 (DLL1). In some embodiments, the Notch ligand is Delta-like 4 (DLL4). In some embodiments, the Notch ligand is one of those described in this document or in the literature, such as in U.S. Patent 7,795,404, which is incorporated by reference in its entirety. The effector cells of the present invention may be prepared using thymic organoid cell culture systems. In some embodiments, the method further includes contacting co-cultured stem or progenitor cells and stromal cells with Flt-3 ligand and / or IL-7 ligand and / or stem cell factor / Kit receptor ligand and / or thrombopoietin.In some embodiments, the differentiation of the stem or progenitor cell into a T cell comprises: culturing a three-dimensional (3D) cell aggregate comprising: a) a selected population of support cells endogenously or exogenously expressing a Notch ligand; b) a selected population of stem or progenitor cells; with a serum-free medium comprising supplement B-27®, xeno-free supplement B-27®, supplement GS2 l™, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem cells or progenitor cells described in International Patent Publication WO2017075389 may be useful in the present invention (the content of which is incorporated herein by reference in its entirety).

[0095] In some embodiments, thymic organoids may be based on the artificial thymic organoids 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, thymic cells may be harvested by trypsinization and resuspended in serum-free culture medium (“RB27”), which may include RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 μM sesquimagnesium 2-phosphate L-ascorbic acid hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin / streptomycin (Gemini Bio Petition 870250090928, dated 06 / 10 / 2025, p. 40 / 189 32 / 134 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 proportions of thymic cells and effector cells can be prepared in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4°C in an oscillating bucket centrifuge. The supernatants were carefully removed, and the cell pellet was resuspended by means of a brief vortex. For each organoid, a 0.4 μm trans Millicell well insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) can be placed in a 6-well plate containing 1 ml of RB27 per well. To place the organoids in plates, the inserts were removed and placed on the edge of the plate to drain excess medium.The cell paste can be adjusted to 5 μl per organoid, aspirated with a 20 μl pipette tip, and plated to form a droplet at the pipette tip end, which is gently deposited onto the cell insert. The cell insert can be placed back into the well containing 1 mL of RB27. The medium can be completely changed every 3 to 4 days by aspirating around the cell insert, followed by replacement with 1 mL of fresh RB27 / cytokines. In some embodiments, organoids can be cultured in this manner for up to 10, 15, 20, 25, or 30 weeks.

[0096] At the indicated times, organoid cells were harvested by adding FACS buffer (PBS / 0.5% bovine serum albumin / 2mM EDTA) to each well and briefly disaggregating the organoids by pipetting with a 1 ml “P1000” pipette, followed by passing through a 50 μm nylon sieve. In some experiments, individual cell suspensions of MS5-hDLL1 cells were γ-irradiated with the indicated doses before use in organoids.

[0097] Thymic organoid effector cell cocultures can be prepared as described 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 Petition 870250090928, dated 06 / 10 / 2025, page 41 / 189 33 / 134 0.1% gelatin one or two days prior to use to achieve 70–80% confluence. The medium can be aspirated from the monolayers, and 1.5*10⁴ FACS-purified effector cells (CD34+CD3 hematopoietic cells) can be placed with thymic organoids in 2 ml of medium composed of MEMa, 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, harvesting the cells, filtering them through a 50 μm nylon sieve, and repeating in fresh medium. When confluent, the cells were divided into multiple wells containing fresh stromal layers.

[0098] In some embodiments, the thymic cells of the invention can be combined with double-negative day 14 T cells to form a group of cells that can be deposited in the trans well as an “organoid” and maintained in an air-liquid interface culture condition. In some embodiments, the cells can be harvested from the medium every few days to assess T cell maturation. Thymic Support System (TSS)

[0099] In some embodiments, the compositions of the invention may include a thymic support system. As used in this document, a thymic support system refers to a system of one or more non-living components that can interact with the cells of the disclosed product to provide structure, support, and / or signals essential for the viability, function, proliferation, and / or differentiation of the cells of the disclosed product. 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 TSSs may mimic the structure of the thymus or thymic tissue in a three-dimensional manner.

[0100] The thymic support system may include one or more components, such as polymers, matrix and matrix components, and optionally, one or more agents.

[0101] In some modalities, TSSs can (i) provide structure Petition 870250090928, dated 06 / 10 / 2025, page 42 / 189 34 / 134 for adhesion, proliferation and differentiation of thymic cells, (ii) create a suitable biomechanical environment and / or (iii) allow the dissemination of nutrients and oxygen.

[0102] In some modalities, TSSs can mimic the tensegrity, viscoelasticity and / or stiffness of the thymus (Engler AJ, et al. Cell. 2006;126(4):677-89; the content of which is incorporated here by reference in its entirety).

[0103] In some embodiments, TSSs may have an architecture that allows the migration of T cells toward and / or away from the TSSs and / or cells of the invention.

[0104] In some embodiments, TSSs may be “bioinert”, “biocompatible”, “bioactive” or “resorbable”, depending on their biological response in vivo. The term “biocompatible”, as used in this document, refers to a substance or object that performs the desired function when introduced into an organism without inducing an inflammatory response, immunogenicity or cytotoxicity to native cells, tissues or organs or to cells, tissues or organs introduced with the substance or object.

[0105] In some forms, TSSs may include a porous network through which oxygen, nutrients, and metabolites can be exchanged. With regard to porosity, TSSs may also be biphasic, including a region of the TSS with highly porous morphology to mimic the thymic medulla and another region of the TSS with less porous morphology to mimic the morphology of the thymic cortex.

[0106] Other desirable properties of TSSs include, among others, the ability to deliver a large number of cells to desirable sites, injectable into an individual without disrupting their properties (e.g., pre-formed cell structure), reduced ethical concerns, and / or reduced batch-to-batch variability.

[0107] In some modalities, TSSs may include thymic tissue from Petition 870250090928, dated 06 / 10 / 2025, page 43 / 189 35 / 134 cellularized. This approach offers the opportunity to culture thymic cells in their native environment while maintaining biomechanical and / or biochemical signals. Decellularization can be achieved by any method known in the art, such as whole-organ perfusion followed by detergent washing. Decellularized thymic tissue can also be homogenized and cross-linked to produce TSSs of the invention. In some embodiments, the compositions of the invention may include decellularized extracellular matrix extracted from thymic tissues. Thymic tissues may be fetal, neonatal, juvenile, or adult. Thymic tissues may be autologous, allogeneic, or xenogeneic.

[0108] Cells can be modeled within or on the TSSs of the invention by selective polymerization of the polymers of the invention or by modeling the cells using an electric field or both. Cells can be patterned by locating cells in specific regions of relatively homogeneous polymer preparations (resolution down to about 5 microns) or by creating patterned polymer structures with defined patterns in which living cells are contained (resolution down to about 100 microns). Modeling can be performed without direct mechanical manipulation or physical contact and without relying on active cellular processes such as cell adhesion.

[0109] The methods described in this document can be used for the production of various single or multiple layered patterns, including geometric shapes or a repetitive series of points with features of various sizes. Alternatively, multilayer biopolymer gels can be generated using a single mask rotated in various orientations. The formation of high-resolution 3-dimensional patterned cells can be achieved by methods other than photopolymerization, so that the limitations of the method are overcome.

[0110] The TSSs of the invention can be organized into structures that Petition 870250090928, dated 06 / 10 / 2025, page 44 / 189 36 / 134 resemble cylinders, rods, cords, filaments, or networks. This architecture can lead to better integration of the invention's compositions into host organisms, as evidenced by improved blood supply and / or improved vasculature. TSSs can be patterned to allow the organization of cells into groups or islands. Modeling can be performed according to the process in Figure 1A or Figure 5A of U.S. Patent Publication US20210213171, the contents of which are incorporated herein by reference in their entirety. TSSs can also be manufactured using custom 3D printer technology. POLYMERS

[0111] In some embodiments, TSSs may include a polymer. Any substance or mixture of natural or synthetic origin can be used in thymic support systems. In some modalities, TSSs may be a biogenic polymer, a synthetic polymer, or a compound thereof.

[0112] In some embodiments, the polymer may be a hydrophilic polymer. Hydrophilic polymers contain polar or charged functional groups, which makes them soluble in water. They can interact with or be dissolved by water or other polar substances. In some embodiments, one or more polymers (or their monomers) or at least one of the polymers has one or more hydrophilic groups. In some embodiments, each of the one or more hydrophilic groups may be individually selected from the group of: NH2, -COOH, -OH, -CONH2, -CONH- and -SO3H. Each of the one or more polymers may be individually selected from a biogenic polymer and a synthetic polymer. In some embodiments, all the polymers in the hydrogel are biogenic polymers. In some embodiments, all the polymers in the hydrogel are synthetic polymers.In some embodiments, when two or more polymers are present, at least one polymer is a biogenic polymer and at least one polymer is a synthetic polymer.

[0113] In some embodiments, TSSs may be temperature-responsive polymers (e.g., pNIPAM, PVME) that transition between Petition 870250090928, dated 06 / 10 / 2025, page 45 / 189 37 / 134 hydrophobic and hydrophilic states at certain temperatures, allowing control of cell culture and growth and, subsequently, ECM deposition and the formation of cell sheets that adhere to biological surfaces.

[0114] The molecular weight of the polymers can be any suitable molecular weight. In some embodiments, each polymer present in the hydrogel has an average molecular weight that is independently selected from about 100 to about 1,000 Da, about 100 to about 900 Da, about 100 to about 800 Da, about 100 Da to about 700 Da, about 200 Da to 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, 575 Da to about 600 Da.

[0115] In one embodiment, the cells of the invention, such as, among others, iPS cells, AFE cells, VPE cells, DE cells, TEP cells and / or TECs, are encapsulated in the polymers described in this document. The three-dimensional configuration of TECs in the thymic microenvironment is important for maintaining their thymic epithelial gene signature. Thymic cells prepared as aggregates in biocompatible hydrogel can maintain their molecular properties and prolong their survival for up to 7 days in vitro. Encapsulation of the cells of the invention is also advantageous for scaling up the fabrication of thymic cells for clinical applications. In one embodiment, the polymers of the invention provide essential 3D matrix support for the survival of iPSC-derived thymic cells.

[0116] In some embodiments, the polymer concentration 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. about 0.1% to 0.5%, about Petition 870250090928, dated 06 / 10 / 2025, page 46 / 189 38 / 134 0.5% to 1%, about 1% to 1.5%, about 1.5% to 2%, about 2% to 2.5%, about 2.5% to 3%, about 3% to 3.5%, about 3.5% to 4%, about 4% to 4.5%, about 4.5% to 5%, about 5% to 5.5%, about 5.5% to 6%, about 6% to 6.5%, about 6.5% to 7%, about 7% to 7.5%, about 7.5% to 8%, about 8% to 8.5%, about 8.5% to 9%, about 9% to 9.5%, about 9.5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80% or more. BIOGENIC POLYMERS

[0117] Any polymer derived from a natural source or from an organism can be defined as a biogenic polymer and can be used in thymic support systems.

[0118] Biogenic polymers may include, among others, one or more proteins, a polypeptide, a polysaccharide, a lipid, a nucleic acid, and a glycosaminoglycan. Biogenic polymers may include, among others, one or more of 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, neurofilament, light chain neurofilament (NF-L), medium chain neurofilament (NF-M), heavy chain neurofilament (NF-H), amyloid, alpha-amyloid, beta-amyloid, actin, myosin, titin, gelatin, chitin, hyaluronic acid, D-glucuronic acid, legumin, vicilin and / or DN-acetylglucosamine.

[0119] Based on their monomeric units and structure, biogenic polymers can be categorized as polypeptide-based biogenic polymers, polysaccharide-based biogenic polymers, and polynucleotide-based biogenic polymers. Petition 870250090928, dated 06 / 10 / 2025, page 47 / 189 39 / 134

[0120] In some embodiments, the biogenic polymer may be a polypeptide-based biogenic material. Non-limiting examples of polypeptide-based biogenic polymers include collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin, and / or actin. In some embodiments, the biogenic polymer may be collagen, which is a primary structural element of the ECM and has several functional characteristics that aid in cell binding, proliferation, differentiation, and ECM secretion. In some embodiments, collagen may consist of three identical chains (homotrimers), for example, in collagen types II, III, VII, VIII, and / or X. Collagen may also include two or more different chains (heterotrimers), for example, in collagen types I, IV, V, VI, IX, and XI.The composition of collagen can be modified to improve the biological activity and mechanical properties of the final scaffold by combining it with other molecules, such as hyaluronic acid (HA), chitosan, and chondroitin sulfate (CS). In some embodiments, the biogenic polymer may be gelatin, which is the result of the degradation derived from insoluble collagen by disintegration or denaturation. In some embodiments, the biogenic polymer may be silk, a natural protein-based polymer derived from various Lepidoptera larvae, such as spiders and silkworms.

[0121] In some embodiments, the biogenic polymer may be a polysaccharide-based biogenic polymer. Non-limiting examples of polysaccharide-based biogenic polymers include chitin, chitosan, alginate, hyaluronic acid, cellulose, agarose, dextran, and / or glycosaminoglycans. The polysaccharide-based biogenic polymer may be made from different monosaccharide or disaccharide chain units (e.g., starch, cellulose). The effect is an incredibly large number of structurally diverse polysaccharides, as various distinct saccharide isomers are mixed through the use of a series of chemical bonds. In some embodiments, the biogenic polymer may be based on a structural polysaccharide, for example, cellulose in plants and chitin in shells of Petition 870250090928, dated 06 / 10 / 2025, page 48 / 189 40 / 134 crustaceans. Chitin is commonly found in crustacean shells, and its derivative, chitosan, is obtained by the deacetylation of chitin. These are natural cationic polysaccharides similar to glycosaminoglycans. In one embodiment, the polymer may be sulfated dextran.

[0122] In some respects, the biogenic polymer can be starch and glycogen.

[0123] In some embodiments, the biogenic polymer may be hyaluronic acid (HA), a linear polysaccharide that is ubiquitous and extremely biologically compatible in the mammalian ECM. HA includes functional groups, such as carboxylic acids and alcohols, which can be used for the implementation of functional domains or for the development of a hydrogel connecting them.

[0124] In some embodiments, the biogenic polymer may be a polynucleotide-based biogenic polymer. Non-limiting examples of polynucleotide-based biogenic polymers include DNA, linear plasmid DNA, and / or RNA.

[0125] In some embodiments, the polymers of the invention may be a combination of two biogenic polymers, for example, heparin and dextran, as described in International Patent Publication WO2022015902, the contents of which are incorporated herein by reference in their entirety. SYNTHETIC POLYMERS

[0126] In some embodiments, TSS may include a synthetic polymer. As used in this document, the term "synthetic" means produced, prepared and / or manufactured by human hands. The synthesis of polymers or other molecules of the present invention may include chemical or enzymatic synthetic methods.

[0127] In some embodiments, the synthetic polymer may be poly(α-hydroxyesters), including PCL, PGA, PLA and their copolymer PLGA and poly(ethers), including PEO and PEG, PVA and PU. Petition 870250090928, dated 06 / 10 / 2025, p. 49 / 189 41 / 134

[0128] In some embodiments, the synthetic polymer may be polylactic acid (PLA), a semicrystalline, gradual-crystallizing polymer. PLA may be prepared from lactic acid (LA) monomer by fermentation of natural resources such as wheat and grains, or by various polymerization routes as a petrochemical derivative. 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).

[0129] In some embodiments, the synthetic polymer may be polylactic-co-glycolic acid (PLGA), a random ring-opening copolymer of PLA and PGA. In some embodiments, the synthetic polymer may be polycaprolactone (PCL), a semicrystalline, 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. In some embodiments, the synthetic polymer may be polyurethane (PU), which contains a urethane moiety in its repeating units. The reaction of the diisocyanate with the polyol typically produces these polymers.

[0130] Synthetic polymers may include, among others, one or more of a poly(urethane), a poly(siloxane), a poly(ethylene), a poly(vinylpyrrolidone), a poly(2-hydroxyethyl methacrylate), a poly(N-vinylpyrrolidone), a poly(methyl methacrylate), a poly(vinyl alcohol), a poly(acrylic acid), a polyacrylamide, a poly(ethylene-co-vinyl acetate), a poly(ethylene glycol), a poly(methacrylic acid), a PLA, a PGA, a PLGA, a polyhydroxybutyrate (PHB), a polypropylene fumarate (PPF), a polyvinyl alcohol (PVA), a polypropylene carbonate, a polyanhydride, a polyphosphazene, a polygerman, a polyorthoester, a polyester, a polyamide, a polyolefin, a polycarbonate, a polyaramid, a polyimide, a PCL is a copolymer, derived from, or a combination thereof.

[0131] In some embodiments, one or more polymers are selected Petition 870250090928, dated 06 / 10 / 2025, page 50 / 189 42 / 134 individually of polyethylene glycol (PEG), chitosan, poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), hydroxyethoxyethyl methacrylate (HEEMA), hydroxydioxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethyl 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), ethylene glycol (EG), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), an acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, poly(N-isopropylacrylamide) (PNIPAM), self-assembling peptides, acrylate-modified PEG and acrylate-modified hyaluronic acid, heparin,PEG-star 4-arm functionalized with an amine end or any combination thereof. In some embodiments, at least one of the one or more polymers is PEGDA. In some embodiments, one or more of the polymers is PEGDA.

[0132] In some embodiments, the present invention provides a synthetic polymer comprising a polysaccharide that has been modified by converting one or more groups present in the polysaccharide into negatively charged functional groups, wherein the negatively charged groups provide a sufficient amount of negative charge to the synthetic polymer to promote one or more growth factor binding, growth factor activity, and vascularization. In some embodiments, the functional groups in the polysaccharide that are converted into negatively charged groups may be hydroxyl groups. 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 mimetic described in the International Patent Publication. Petition 870250090928, dated 06 / 10 / 2025, page 51 / 189 43 / 134 WO2022015902, the content of which is incorporated herein in its entirety by reference. COMPOUND POLYMERS

[0133] Biogenic and synthetic polymers can be combined to produce composite polymers that combine the features of both types of polymers to produce composite polymers with enhanced features. For example, thymic cells of the invention can be cultured with the 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). Thymic cells and / or thymic cell organoids can also be prepared using gelatin and PEG composite polymers (see AB Suraiya, ACS Biomater. Sci. Eng. 2020, 6, 2198; the contents of which are incorporated herein by reference in their entirety). hydrogels

[0134] In some embodiments, the polymer may be a hydrogel. In general, hydrogels are prepared by translating hydrophilic polymer solutions into a 3D network structure through physical or chemical crosslinking. During this process, hydrogels can homogeneously encapsulate cells and provide cells with a 3D microenvironment similar to the native extracellular matrix (ECM). The behaviors and functions of cells in vivo are affected by stimuli produced by the surrounding ECM. Similarly, the structures and physicochemical properties of hydrogels provide essential clues to controlling the functions of the embedded cells. The structures and physicochemical properties of hydrogels can be designed and controlled through the selection of different polymers, crosslinking methods, and fabrication strategies.

[0135] Hydrogels can be prepared from biogenic polymers, such as, among others, polypeptide-based polymers (such as gelatin, collagen, fibrin and silk fibroin) and polysaccharide-based biogenic polymers. Petition 870250090928, dated 06 / 10 / 2025, page 52 / 189 44 / 134 hydroxides (such as hyaluronic acid (HA), chondroitin sulfate (CS), alginate, and chitosan). Collagen, as the main component of ECM from various tissues, can be used for the preparation of the hydrogel. In some embodiments, the collagen derivative, gelatin, which has greater solubility, can also be used to prepare hydrogels in the present invention. Hyaluronic acid (HA), a glycosaminoglycan, is commonly predominant in body fluids, and ECM can be used for the preparation of hydrogels. Other biogenic polysaccharide-based polymers, 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 the present invention.

[0136] The hydrogels of the present invention can also be prepared with synthetic polymers, such as, among others, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAM) and polyacrylamide (PAM) (Darnell et al., 2013).

[0137] The hydrogels described in this document may include EAK16-II / EAKIIH6 self-assembling hydrogels, as described by A. Tajima et al. Clin. Immunol. 2015, 160, 82; the content of which is incorporated herein by reference in its entirety.

[0138] In some embodiments, hydrogels can be prepared from hydrophilic monomer polymers. As used in this document, a hydrophilic monomer refers to any monomer that, when polymerized, produces a hydrophilic polymer capable of forming a hydrogel when in contact with an aqueous medium, such as water. Examples of hydrophilic monomers include, but are not limited to, monomers containing hydroxyl groups, such as 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylamide, 2-hydroxyethyl acrylamide, N-2-hydroxyethyl vinyl carbamate, 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, esters containing free carboxyl groups Petition 870250090928, dated 06 / 10 / 2025, page 53 / 189 45 / 134 of unsaturated polycarboxylic acids, such as monomethyl maleate ester, monoethyl maleate ester, monomethyl fumarate ester, monoethyl fumarate ester and salts thereof; amide-containing monomers, such as (meth)acrylamide, crotonic amide, cinnamic amide, maleic diamide and fumaric diamide; thiol-containing monomers, such as methanethiol, ethanethiol, 1-propanethiol, butanethiol, tert-butyl mercaptan and penthanethiols; sulfonic acid-containing monomers, such as pestyrenesulfonic acid, vinylsulfonic acid, p-methylstyrene sulfonic acid, isoprene sulfonete and salts thereof.

[0139] In some embodiments, a hydrogel can be formed by the self-organization of one or more types of hydrophilic polymers in an aqueous medium. The term “self-organization” refers to a process of spontaneous organization of components of a higher-order structure based on the attraction of the components to each other and without the formation of chemical bonds between the components. For example, polymer chains can interact with each other through any of the hydrophobic forces, hydrogen bonding, Van der Waals interaction, electrostatic forces, or polymer chain entanglement, induced in the polymer chains, so that the polymer chains can aggregate or coagulate in an aqueous medium, which can form a three-dimensional network, thus trapping water molecules to form a hydrogel.

[0140] Biogenic polymer compounds and synthetic polymers can also be used. For example, PAM, PVA, PNIPAM, and PEG hydrogels can be mixed with gelatin. Cell propagation and proliferation in these hybrid hydrogels can be enhanced compared to 3D-cultured cells in synthetic polymer-based hydrogels. CROSSLINKED POLYMERS

[0141] Cross-linking, as used in this document, can be defined as the induction of chemical or physical bonds between polymer chains. The polymers of the invention can be cross-linked to modify the mechanical, biological and / or degradation properties of the polymers of the invention. Petition 870250090928, dated 06 / 10 / 2025, page 54 / 189 46 / 134

[0142] In some embodiments, hydrophilic polymers can be crosslinked to form hydrogels.

[0143] Several methods of cross-linking polymer chains are known in the art and can be used here. The methods can be selected depending on the chemistry of the materials and the expected functions. In some embodiments, the cross-links can be formed by covalent or ionic bonds.

[0144] Polymers can be physically crosslinked under very mild conditions, without the use of crosslinking agents that often cause toxicity to cells or can affect the activity of biological molecules. Physical crosslinking methods include, among others, ionic interaction, host-guest interaction, and thermogelation.

[0145] In some embodiments, the polymers described in this document may be chemically crosslinked. In some embodiments, some or all of the crosslinks are formed by means of a Thiol-Michael addition reaction, such as a Thiol-Michael addition click reaction. Chemically crosslinked hydrogels may have stronger binding energy and greater flexibility due to the nature of the crosslinking reactions. Hydrophilic polymers have many functional groups, such as OH, COOH, and NH2. The 3D network can be established by covalent bonding between these functional groups using glutaraldehyde and EDC / NHS.In some embodiments, chemical crosslinking can be achieved through photopolymerization (using photoreactive moieties such as methacrylate or acrylate groups), enzyme-activated crosslinking (using enzymes such as transglutaminase and radish peroxidase), click chemistry, and / or using the Schiff base reaction (through coupling between aldehyde and amine groups in polymer chains).

[0146] Examples of chemical compounds that act as cross-linking agents include, among others, dextran dialdehyde, 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), vinylamine, 2-amino methacrylate Petition 870250090928, dated 06 / 10 / 2025, page 55 / 189 47 / 134 ethyl, 3-aminopropyl methacrylamide, ethylene diamine, ethylene glycol dimethacrylate, methyl methacrylate, N,N'-methylene-bisacylamide, N,N'-methylene-bismethacrylamide, diallyl tartardiamide, allyl di(meth)acrylate, lower alkylene glycol di(meth)acrylate, lower polyalkylene glycol di(meth)acrylate, lower alkylene di(meth)acrylate, divinyl ether, divinyl sulfone, di- or trivinylbenzene, trimethylolpropane tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, bisphenol A di(meth)acrylate, methylenebis(meth)acrylamide, triall phthalate, diallyl phthalate, transglutaminase or mixtures thereof.

[0147] In some embodiments, crosslinks can be degradable or non-degradable.

[0148] In some embodiments, the crosslinkers may be a peptide crosslinker. As a non-limiting example, the crosslinker may be a protease-degradable crosslinker, for example, the peptide VPM GCRDVPMSMRGGDRCG (SEQ ID NO: 1) which is rapidly cleaved by MMP-1 and MMP-2 proteases. In some embodiments, the crosslinker may be a peptide that can be removed by matrix metalloproteinase (MMP). In some embodiments, said peptide comprises an amino acid sequence 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 peptides that can be removed by MMPs can be found in Tu, Y. et al. Smart Pharmaceutical Nanocarriers; 83-116 (2016), the full content of which is incorporated by reference here.

[0149] In some embodiments, the polymers of the invention may be crosslinked PEG polymers. As a non-limiting example, the PEG polymers may be crosslinked with a maleimide functional group. In one embodiment, the crosslinked PEG polymer may be a 4-armed PEG-maleimide (PEG-4MAL). Components of the Extracellular Matrix (ECMC) Petition 870250090928, dated 06 / 10 / 2025, p. 56 / 189 48 / 134

[0150] The compositions of the invention may include one or more extracellular matrix components. In some embodiments, the ECMC may be tied or decorated onto polymers of the invention for presentation to the cells of the invention.

[0151] In some embodiments, ECMCs may be extracellular matrix proteins. In some respects, ECMC may be a biogenic polymer. Non-limiting examples of extracellular matrix proteins include, but are not limited to, fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrin, merosin, ankalin, chondronectin, binding protein, bone sialoacid protein, osteocalcin, osteopontin, epinectin, hyaluronectin, unjulin, epiligrin, and carinin.

[0152] The extracellular matrix proteins useful in the present invention may be biogenic or purified from human or animal tissue. Alternatively, the ECM protein may be a genetically modified recombinant protein or an original synthetic product. The ECM protein may be a whole protein or a peptide fragment. Examples of ECM proteins that may be useful include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin.

[0153] In some embodiments, the extracellular matrix component may be Matrigel (or similar commercially available products such as Geltrex), a laminin-111-rich basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma. In some embodiments, ECMC may be generated from natural materials such as fibrin, collagen, or hyaluronic acid, or from synthetic hydrogel.

[0154] In some embodiments, ECMCs may include peptides that correspond to short essential amino acid sequences of ECM proteins. ECMCs may include peptides that bind to the α5, α6, αν, β1, and β5 integrin receptor subunits. In some embodiments, ECMCs may be three-amino acid Arg-Gly-Asp (RGD) peptides derived from fibronectin, which bind to αvβ3 and αvβ5 integrins. A cyclic form of RGD Petition 870250090928, dated 06 / 10 / 2025, p. 57 / 189 49 / 134 was identified as the most effective peptide for hPSC culture; the cyclic RGD peptide was favorably compared with other peptides derived from laminin, fibronectin, and vitronectin and can be used in the present invention (Lambshead JW et al. Sci. Rep 8, 701 (2018); the contents of which are incorporated herein by reference in their entirety). In some embodiments, the peptide may be CGRGDS (SEQ ID NO: 9).

[0155] Other examples of ECMCs include peptides corresponding to the GFXGER collagen motif (SEQ ID NO: 10), the IKVAV laminin motif (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), AELDVP (SEQ ID NO: 16), VALDEP (SEQ ID NO: 17), NGRAHA (SEQ ID NO: 18), peptides derived from vitronectin and / or bone sialoprotein.

[0156] In some embodiments, ECMC may be a synthetic matrix component.

[0157] In some forms, ECMC may include growth factors such as peptides from the transforming growth factor (TGF) family (e.g., TGF-β), fibroblast growth factors (FGFs), integrins, as well as enzymes such as matrix metalloproteinases (MMPs). AGENTS

[0158] TSSs can be prepared with one or more agents. One or more agents can be released (continuously or in a controlled manner) from the TSSs to become available for interactions with the cells of the invention. The agents can be biological agents or chemical agents. The agents can promote the survival, growth, expansion, differentiation and / or one or more functions of the cells of the invention.

[0159] In some forms, an agent may be a biological agent. As used in this document, "biological agent" refers to any compound, composition, biopolymer, molecule and the like that are produced by a living organism and include, without limitation, polynucleotides (e.g., DNA, RNA), peptides and polypeptides, and chemical compounds. Petition 870250090928, dated 06 / 10 / 2025, p. 58 / 189 50 / 134

[0160] In one embodiment, the biological agent may be a protein tag, for example, a biotin molecule.

[0161] In some embodiments, the biological agent may be an antibody or a fragment thereof. As a non-limiting example, the antibody may be an EpCAM antibody that binds to the polymers of the invention by means of an adapter complex. These antibodies may enable the sequestration of thymic cells of the invention within or on the TSSs of the invention (see A. Tajima, et al. Fan, Clin. Immunol. 2015, 160, 82; the contents of which are incorporated herein by reference in their entirety).

[0162] One or more agents may be selected individually from among 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-inflammatories, antihistamines, anti-infectives, radiation sensitizers, sensitizing agents, imaging agents, chemotherapeutic agents, chemokines, cytokines, antimigration compounds capable of inhibiting chemokine receptors to reduce cell invasion, or any combination thereof.

[0163] In some embodiments, the biological agent may be a ligand, which can interact with a specific ligand-equivalent receptor and together transmit a message or signal to assume a specific function or phenotype. For example, the cells described in this document may have an exogenous nucleotide sequence encoding a ligand that may be introduced (or have been previously introduced) into the cells by transfection or transduction. As a non-limiting example, the ligand may be a Notch ligand. The term “Notch ligand,” as used herein, includes intact (full-length), partial (a truncated form), or modified (comprising one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands of any kind or fragments thereof that retain at least one activity or function of a full-length Notch ligand. Petition 870250090928, dated 06 / 10 / 2025, p. 59 / 189 51 / 134 Also included are peptides that mimic notch ligands. Notch ligands can be “canonical Notch ligands” or “non-canonical Notch ligands.” Canonical notch ligands are characterized by extracellular domains typically comprising an N-terminal (NT) domain followed by a Delta / Serrate / LAG-2 (DSL) domain and multiple tandemly arranged Epidermal Growth Factor (EGF)-like repeats. The DSL domain, along with the flanking NT domain and the first two EGF repeats containing the Delta and OSM-11-like (DOS) protein motif, are typically required for canonical ligands to bind to Notch. The intracellular domains of some canonical ligands contain a PSD-95 / Dlg / ZO-1 (PDZL) linker motif at the carboxy-terminal end that plays a role independent of Notch signaling. C. elegans DSL ligands lack a DOS motif, but have been proposed to cooperate with DOS-only ligands to activate Notch signaling.Illustrative canonical notch ligands include, among others, Delta-like ligand 4 (DLL4), Delta-like ligand 1 (DLL1), Jagged 1 (JAG1), Jagged 2 (JAG2), Delta-like ligand 3 (DLL3), and X-delta 2; other similar illustrative canonical ligands are contemplated in other embodiments. Non-canonical notch ligands lack a DSL (Delta / Serrate / LAG-2) domain, are structurally diverse, and include integral and GPI-linked membrane proteins, as well as various secreted proteins. When a “notch ligand fragment” or a “canonical notch ligand fragment” is mentioned in this document, the fragment is considered to be a fragment that binds to Notch. Examples of non-canonical notch ligands include, among others, Contactin-1, NOV / CCN3, Contactin-6, Periostin / OSF2, DLK2 / EGFL9, Pref-1 / DLKl / FAl, DNER, Thrombospondin-2, MAGP-1 / MF AP2, Thrombospondin-3, MAGP-2 / MF AP5, Thrombospondin-4, and Netrin-1.In some forms, the ligand may be JAG1, JAG2, and / or Delta-like-1. In some aspects, the ligand may be VCAM1. Shukla S et al. and Michaels et al. demonstrated that both DLL4 and vascular cell adhesion molecule (VCAM1) play an important role in the differentiation of... Petition 870250090928, dated 06 / 10 / 2025, p. 60 / 189 52 / 134 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 content of each is incorporated here by reference in its entirety). In some modalities, the ligand may be an apoptosis modulator, such as Fas ligand (FasL). The Fas-receptor / Fas ligand pathway has been shown to play a crucial role in autoantigen tolerance, and Fas-L expression at the transplant site has induced allogeneic tissue tolerance (see Ji Lei et al. Sci Adv, 8, 2022; Lau et al. Science 1996 273:109). In some modalities, the agent may be a growth factor. Exemplary growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), bone morphogenetic protein(s) (BMP), a transforming growth factor (TGF), such as transforming growth factor beta, a platelet-derived growth factor (PDGF),an epidermal growth factor (EGF), a nerve growth factor (NGF), an insulin-like growth factor (e.g., insulin-like growth factor I), hepatocyte dispersal / growth factor (HGF), granulocyte / macrophage colony-stimulating factor (GMCSF), a glial growth factor (GGF) and a fibroblast growth factor (FGF), GCSF, erythropoietin, TPO, GDF, neurotrophins, 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, antigen3 associated with lymphocyte function, myeloid-derived growth factor, osteoclast-stimulating factor, progranulin,Colony-stimulating factor and combinations thereof. Growth factors may be one or more of the following: stem cell factor (SCF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor-1, growth factor, VEGF, TGFβ, growth factor, Petition 870250090928, dated 06 / 10 / 2025, page 61 / 189 53 / 134 platelet-derived growth factor (PDGF), angiopoietins (Ang), epidermal growth factor (EGF), bFGF, HNF, NGF, fibroblast growth factor (FGF), hepatocyte growth factor, liver growth factor (LGF), insulin-like growth factor (IGF-1), colony-stimulating factors, thrombopoietin, erythropoietin, fit3 ligand, tumor necrosis factor alpha (TNFα), a growth factor from the bone morphogenetic protein (BMP) family (e.g., BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15), a growth factor that functions 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.

[0164] In some embodiments, the ligand may promote the long-term survival of the thymic cells of the invention. For example, the ligand that promotes the long-term survival of thymic cells may be FGF2, FGF7, RANKL (see Lee HW et al. Expt & Mol. Med. 2008;40(1):59-70; the content of which is incorporated herein by reference in its entirety). In some embodiments, the ligands may promote vascularization, for example, VEGF (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 content of each of which is incorporated herein by reference in its entirety).

[0165] In some embodiments, an agent may be an immunomodulator. Suitable 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-1a, IL-1b, 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 agents is a chemokine. Exemplary chemokines include, without limitation, for example, CCL3, CCL26, CXCL13, CXCL14, CCL6, CCL27, CXCL16, CXCL17, CXCL6, CXCL5, eotaxin, CCL2, CX3CL1, CXCL1, 2, 3, Petition 870250090928, dated 06 / 10 / 2025, p. 62 / 189 54 / 134 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 agents is a cytokine. Exemplary cytokines include, without limitation, interferons (e.g., IFN-α, IFN-γ3, IFN-γ, IFN-γ, IFN-κB, IFN-γ), granulocyte colony-stimulating factor, imiquimod, 4-1BB, adiponectin, AITR, AIF1, B-cell activating factor, beta-defensin, betacellulin, BMP, BST1, B-type 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 regulatory factor), 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), otoraplin, resistin, leukemia inhibitory factor, serum amyloid A, TPO, cloverleaf factor, thymic stromal lymphopoietin, tumor necrosis factor, uteroglobin, visfatin, wingless MMTV nitration site family, AIMP1, CLCF1, CYTL1, EMAP II, TAFA2, Vaspin, and any combination thereof. In some embodiments, the immune modulator may be an agent, such as CXCL12, that promotes the migration of dendritic cells toward or away from the cells and / or TSSs of the invention (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 modalities, the immunomodulator may be a ligand for CCR7 and / or CCR9 that can promote the recruitment of effector cells, such as T cells,for thymic cells or TSSs of the invention (Gameiro J et al. Cell adhesion & migration. 2010;4(3):382-90; the content of which is incorporated herein by reference in its entirety). In some embodiments, the immunomodulator may be thymic stromal lymphopoietin (TSLP) and / or IL10, which may promote the development of effector T cells, for example, regulatory T cells (see Alawam AS, et al. Frontiers in immunology. 2020;11:858; the content of which is, Petition 870250090928, dated 06 / 10 / 2025, p. 63 / 189 55 / 134 incorporated herein by reference in its entirety).

[0166] In some embodiments, the agent may be a hormone. Suitable hormones include, but are not limited to, amino acid-derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, cortisol). Exemplary hormones include, without limitation, endothelin, exendin, follicle-stimulating hormone, growth hormone-releasing hormone, growth hormone-releasing peptides, ipamorelin, glucagon, glucagon-like peptides, insulin, chorionic gonadotropin, inhibin beta C chain, inhibin alpha, inhibin alpha chain, luteinizing hormone,luteinizing hormone-releasing hormone, peptide hormones (e.g., adrenocorticotropic hormone, alarelin, antide, atosiban, buserelin, cetrorelix, desmopressin, deslorelin, elkatonin, ganirelix, ghrelin, goserelin, hexarelin, gistrelin, lanreotide, leuprolide, lipressin, melanotan-I and-II, nafarelin, octreotide, pramlintide, secretin, sincalide, somatostatin, terlipressin, thymopentin, triptorelin, vasopressin, neuropeptide Y, cholecystokinin), procalcitonin, prolactin, oxytocin, parathyroid hormone, estrogen, testosterone, stanozololcin-1 and -2, thymosin, thyrostimulin, thyroid-stimulating hormone, agouti-related protein, calcitonin, corticotropin-releasing hormone-binding protein, prouroguaniline, oxyntomodulin, thyrotropin-releasing hormone, and any combination thereof.

[0167] In some embodiments, the agents of the invention may promote angiogenesis.

[0168] An agent can be a gene-modifying agent. Some examples of gene-modifying agents include, among others, nu systems. Petition 870250090928, dated 06 / 10 / 2025, page 64 / 189 56 / 134 programmable or RNA-guided cleases, such as a CRISPR-Cas system, meganucleases, zinc finger nucleases and / or similar systems. Such systems are generally known in the art.

[0169] As used in this document, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of non-coding RNA, such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), antisense RNA, RNAi (RNA interfering construct), siRNA (short interfering RNA), microRNA (miRNA) or ribozymes, aptamers, guide RNA (gRNA) or coding mRNA (messenger RNA).

[0170] In some embodiments, an agent is a chemical agent. As used in this document, “chemical agent” refers to a substance, molecule, or chemical composition. Exemplary chemical agents are those that are suitable for use as a pharmaceutical agent in an animal, as well as 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. The term “biocompatible,” as used in this document, refers to a substance or object that performs the desired function when introduced into an organism without inducing a significant inflammatory response, immunogenicity, or cytotoxicity to native cells, tissues, or organs or to cells, tissues, or organs introduced with the substance or object.For example, a biocompatible product is a product that performs its intended function when introduced into an organism without inducing a significant inflammatory response, immunogenicity, or cytotoxicity to native cells, tissues, or organs. DEVICES. Petition 870250090928, dated 06 / 10 / 2025, page 65 / 189 57 / 134

[0171] The cells and / or TSSs of the invention can be incorporated into devices. Mechanical parameters, such as flow, shear stresses, pressure, and movements, can affect thymic cell culture, differentiation, and tissue homeostasis. For example, shear stresses induced by blood flow in cells can trigger terminal maturation. Similarly, various physical forces are involved in the normal and pathological function of cells, tissues, organs, and / or organisms. Integration of the compositions of the invention into devices that allow the generation of forces and movements, enabling the compositions to more closely mimic the thymic environment in vivo. Organ-on-a-chip technology has introduced many techniques for generating and maintaining forces and may be useful in the present invention.Organ-on-a-chip devices also allow the growth of epithelia on 2D membranes with microfluidic channels on the apical and basal sides, providing bilateral accessibility and the ability to apply fluid flow. These microfabrication-based devices offer the opportunity to combine biophysical and biochemical stimuli, thus increasing the physiological relevance of in vitro models and the robustness of their generation protocols.

[0172] Microfluidic devices can be used to integrate access channels for waste removal and nutrient delivery in cell populations and to allow independent control of experimental conditions.

[0173] Any of the microfluidic devices described in U.S. Patent Publication US20210115369 and U.S. Patent 10,829,727 may be useful in the present invention. The contents of each of these publications are incorporated herein by reference in their entirety. METHODS

[0174] The present invention offers methods for differentiating pluripotent stem cells into thymic cells. In some embodiments, the present invention offers a method for differentiating pluripotent stem cells. Petition 870250090928, dated 06 / 10 / 2025, page 66 / 189 58 / 134 induced in thymic cells.

[0175] In some modalities, the steps involved in the differentiation of iPSCs into thymic cells may include the encapsulation of individual iPSCs. In some other modalities, the steps involved in the differentiation of iPSCs into thymic cells do not include the encapsulation of individual iPSCs.

[0176] In some modalities, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include the activation of WNT signaling. As a non-limiting example, the activator of WNT signaling may be CHIR99021.

[0177] In some modalities, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include the inhibition of WNT signaling. As a non-limiting example, the WNT signaling inhibitor may be IWR1 (or IWR-1).

[0178] In some modalities, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include the inhibition of BMP signaling. In some modalities, the inhibition of BMP signaling can be achieved using the BMP pathway inhibitor, LDN193189.

[0179] In some modalities, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include the inhibition of SHH signaling. In some modalities, SHH inhibition is achieved using an SHH antagonist, SANT-1.

[0180] In some modalities, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include the inhibition of TGFβ signaling. In some modalities, the inhibition of TGFβ signaling is achieved using the TGFβ inhibitor, SB431542.

[0181] In some embodiments, one or more of the steps involved in the differentiation of iPSCs into thymic cells may include a cell culture medium containing Insulin, Transferrin and Selenium (ITS), knockout replacement serum (KSR), Penicillin and Streptomycin (also referred to here as “Pen Strep”) and non-essential amino acids (NEAA). Petition 870250090928, dated 06 / 10 / 2025, page 67 / 189 59 / 134

[0182] In one embodiment, the cells of the invention are grown in the presence of one or more polymers described in this document. In one embodiment, the cells of the invention are encapsulated in the polymers of the invention. In one embodiment, the polymer is alginate.

[0183] In one embodiment, iPS cells are cultured in suspension and / or encapsulated in alginate. In one embodiment, DE cells are cultured in suspension and / or encapsulated in alginate. In one embodiment, AFE cells are cultured in suspension and / or encapsulated in alginate. In one embodiment, VPE cells are cultured in suspension and / or encapsulated in alginate. In one embodiment, TEP cells are cultured in suspension and / or encapsulated in alginate. In one embodiment, TECs are cultured in suspension and / or encapsulated in alginate.

[0184] In one embodiment, iPS cells are cultured in suspension and are not encapsulated in alginate. In one embodiment, DE cells are cultured in suspension and are not encapsulated in alginate. In one embodiment, AFE cells are cultured in suspension and are not encapsulated in alginate. In one embodiment, VPE cells are cultured in suspension and are not encapsulated in alginate. In one embodiment, TEP cells are cultured in suspension and are not encapsulated in alginate. In one embodiment, TEC cells are cultured in suspension and are not encapsulated in alginate.

[0185] 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, TECs are cultured in suspension and are not encapsulated. Petition 870250090928, dated 06 / 10 / 2025, page 68 / 189 60 / 134

[0186] 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.

[0187] In one embodiment, iPS cells are differentiated in suspension and are not encapsulated in alginate. In one embodiment, DE cells are differentiated in suspension and are not encapsulated in alginate. In one embodiment, AFE cells are differentiated in suspension and are not encapsulated in alginate. In one embodiment, VPE cells are differentiated in suspension and are not encapsulated in alginate. In one embodiment, TEP cells are differentiated in suspension and are not encapsulated in alginate. In one embodiment, TEC cells are differentiated in suspension and are not encapsulated in alginate.

[0188] In one embodiment, iPS cells are differentiated in suspension and are not encapsulated. In one embodiment, DE cells are differentiated in suspension and are not encapsulated. In one embodiment, AFE cells are differentiated in suspension and are not encapsulated. In one embodiment, VPE cells are differentiated in suspension and are not encapsulated. In one embodiment, TEP cells are differentiated in suspension and are not encapsulated. In one embodiment, TECs are differentiated in suspension and are not encapsulated.

[0189] In one embodiment, alginate-encapsulated iPS cells are differentiated in suspension into DE cells. In one embodiment, alginate-encapsulated iPS cells are differentiated in suspension into DE and AFE cells. In one embodiment, alginate-encapsulated iPS cells are Petition 870250090928, dated 06 / 10 / 2025, page 69 / 189 61 / 134 differentiated in suspension into DE or AFE cells. In one embodiment, alginate-encapsulated iPS cells are differentiated in suspension into DE, AFE, VPE, and thymic cells. In another embodiment, alginate-encapsulated iPS cells are differentiated in suspension into DE, AFE, VPE, or thymic cells.

[0190] In one embodiment, DE and AFE cells encapsulated in alginate are differentiated in suspension into VPE cells. In one embodiment, DE and AFE cells encapsulated in alginate are differentiated in suspension into VPE and thymic cells. In one embodiment, DE or AFE cells encapsulated in alginate are differentiated in suspension into VPE or thymic cells. In one embodiment, AFE and VPE cells encapsulated in alginate are differentiated in suspension into thymic cells. In one embodiment, AFE or VPE cells encapsulated in alginate are differentiated in suspension into thymic cells. In one embodiment, VPE cells encapsulated in alginate are differentiated in suspension into thymic cells.

[0191] In one embodiment, non-encapsulated iPS cells are differentiated in suspension into DE cells. In one embodiment, non-encapsulated iPS cells are differentiated in suspension into DE and AFE cells. In one embodiment, non-encapsulated iPS cells are differentiated in suspension into DE or AFE cells. In one embodiment, non-encapsulated iPS cells are differentiated in suspension into DE, AFE, VPE, and thymic cells. In one embodiment, non-encapsulated iPS cells are differentiated in suspension into DE, AFE, VPE, or thymic cells.

[0192] In one embodiment, non-encapsulated DE and AFE cells are differentiated in suspension into VPE cells. In one embodiment, non-encapsulated DE and AFE cells are differentiated in suspension into VPE and thymic cells. In one embodiment, non-encapsulated DE or AFE cells are differentiated in suspension into VPE or thymic cells. In one embodiment, non-encapsulated AFE and VPE cells are differentiated in suspension into thymic cells. In one embodiment, non-encapsulated AFE or VPE cells Petition 870250090928, dated 06 / 10 / 2025, page 70 / 189 62 / 134 are differentiated in suspension into thymic cells. In one embodiment, non-encapsulated VPE cells are differentiated in suspension into thymic cells. Alginate Encapsulation Methods

[0193] In some embodiments, oil / water emulsion methods are used for alginate encapsulation. Emulsion encapsulation allows for uniform spatial distribution of cells within the small microcapsule. Alginate solutions containing cells are agitated with non-toxic paraffin oil and then cross-linked, resulting in cell-laden microcapsules. Using this technique, parameters such as the agitation rate can allow for adjustment of the ideal capsule size for differentiation. In some embodiments, extrusion (droplet) methods can be used for alginate encapsulation.

[0194] In one embodiment, the cells of the invention are encapsulated by extrusion. The autoclaved alginate solution is prepared with alginate, gelatin, HEPES, and / or NaCl. The cells can be resuspended in an alginate solution at varying concentrations, for example, 200,000 cells / mL. The solutions are loaded into a 1 mL syringe fitted with a 27-gauge needle tip. The syringe is then placed in a syringe pump and extruded into a CaCl2 gelling bath at a predetermined rate. The alginate capsules can remain in the bath for a further 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.

[0195] In one embodiment, the cells of the invention are encapsulated by an emulsion method using autoclaved solutions of alginate, gelatin, HEPES, NaCl and / or CaCO3.Mineral oil can be added to a sterile container containing a 35 mm magnetic stir bar. The cells are resuspended in an alginate solution at a variable concentration. The alginate solution containing the cells is added to the oil while stirring and allowing it to emulsify. After emulsification, mineral oil containing the alginate solution can be added. Petition 870250090928, dated 06 / 10 / 2025, page 71 / 189 63 / 134 acetic acid and allow gelation to continue during agitation. After gelation is complete, culture medium can be added to neutralize the pH. Oil separation and capsule collection are performed by centrifugation. The capsules were washed and resuspended in cell culture medium for differentiation.

[0196] In some embodiments, the cells of the invention are encapsulated in alginate. In one embodiment, individual cells are obtained by pretreating a population of confluent cells with Y27632 dihydrochloride (R&D Systems) before dissociation with Accutase (StemPro). In some embodiments, the cells are suspended in low viscosity alginate (Sigma) at 1.1% w / v at a predetermined density (e.g., 5 x 10⁵ per ml of alginate). In some embodiments, the individual cell alginate mixture is dropwise polymerized with a stirred solution of CaCl₂ (e.g., 100 mM CaCl₂) with 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) before being cultured in mTeSR1 (StemCell Technologies) supplemented with 10 μM of Y-27632 dihydrochloride for 4 to 6 days before initiating differentiation. ALGINATE DECAPSULATION METHODS

[0197] At any stage of differentiation, the encapsulated cells of the invention can be decapsulated, i.e., the alginate encapsulation is removed. In one embodiment, iPS cells are encapsulated in alginate and subsequently differentiated into thymic cells using the methods described in this document. The differentiated thymic cells are then decapsulated before in vivo implantation.

[0198] In one embodiment, the cells of the invention are decapsulated in a solution containing sodium citrate, HEPES and / or NaCl. The capsules are suspended in the decapsulation solution during agitation to promote Petition 870250090928, dated 06 / 10 / 2025, page 72 / 189 64 / 134 decapsulation. The aggregates are collected by centrifugation and then treated with accutase to dissociate them into individual cells. The individual cells are collected by centrifugation and used for further testing.

[0199] In one embodiment, the cells of the invention are decapsulated with EDTA. In one embodiment, cells are placed in a Ficoll gradient to remove capsular debris and dead cells. In some embodiments, the layer of live cells is pipetted from the gradient and dissociated for analysis. Preparation and maintenance of thymic cells

[0200] Here are presented methods for differentiating pluripotent stem cells into thymic cells. These methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-target P13K inhibitor). In some embodiments, the first growth medium includes DMEM-F12, Activin A, CHIR99021, insulin transferrin selenium (ITS), and knockout serum replacement (KSR). In some embodiments, the second growth medium includes DMEM-F12, bFGF, Activin A, LDN193189, ITS, and KSR. In some embodiments, cells are cultured in the presence of PI-103. The concentration of PI-103 may be approximately 1 nM to 1,000 nM. In one embodiment, the concentration of PI-103 can be 50nM.

[0201] Definitive endodermal cells can be cultured and differentiated into foregut cells by contacting or incubating the definitive endodermal cells with at least one of SB431542, LDN-193189, and KSR. In some embodiments, foregut cells can be cultured and differentiated into pharyngeal endodermal cells by contacting or incubating the foregut cells with at least one of EGF, retinoic acid, FGF8B, and SHH. In some embodiments, pharyngeal endodermal cells can be cultured and differentiated Petition 870250090928, dated 06 / 10 / 2025, page 73 / 189 65 / 134 in thymic epithelial cells through contact or incubation of pharyngeal endoderm cells with at least one of BMP4, FGF8b, EGF, SANT, CHIR99021, ascorbic acid, or a combination thereof. In some embodiments, differentiation is carried out between approximately 14 and 25 days. For example, differentiation is carried out for about 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days.

[0202] In some embodiments, the present invention provides methods for preparing one or more cells or cell types described in this document. In some embodiments, the cells may be thymic cells.

[0203] A growing body of data in public databases provides individual cell transcriptomes of primary human and murine thymus cells (see Bautista et al. 2021 Nat Commun 12, 1096; Kernfeld, et al. Immunity. June 19, 2018;48(6):1258-1270.e6; Zeng et al. Immunity. November 19, 2019;51(5):930-948.e6; the contents of each of which are incorporated herein by reference in their entirety). This provides a rich source of material for identifying factors that promote the differentiation and / or maturation of the cells of the invention into thymic cells. By analyzing scRNA sequencing data, the present invention identifies potential supporting factors and / or cells that may promote and / or maintain the thymic cell phenotype.

[0204] In some embodiments, the cells of the present invention can be isolated from an organism. In some embodiments, the organism can be a mammal. Mammalian cells can be isolated from human, rodent, swine, and / or bovine sources. The human sources of cells of the invention can be autologous or allogeneic. In some embodiments, tissues containing the cells of the invention can be harvested and used as such for the applications described in this document. The cells of the invention can be obtained from embryonic, fetal, and adult organisms. In some respects, the organism can be living or it can be a cadaveric organism. Petition 870250090928, dated 06 / 10 / 2025, page 74 / 189 66 / 134

[0205] The cells described in this document may be derived from other cell types. As a non-limiting example, the cells of the invention may be derived from pluripotent stem cells (PSCs). In some embodiments, the cells of the invention may be derived from progenitor cells. In some embodiments, the cells of the invention may be derived by differentiation of PSCs and / or progenitor cells.

[0206] In some embodiments, thymic cells can be prepared from PSCs. In this sense, the methods may include culturing pluripotent stem cells for a time and under conditions sufficient to differentiate them into thymic cells. For example, the method may include culturing pluripotent stem cells in the presence of factors and / or inhibitors that promote the differentiation of PSCs into thymic cells. The methods of differentiating PSCs into thymic cells are known in the art. The methods of differentiating PSCs into thymic cells may include the use of one or more parameters known in the art for differentiation or combinations thereof. The parameters include, among others, (i) factors that promote differentiation (ii) inhibitors that promote differentiation (iii) duration of time to promote differentiation (iv) temperature (v) substrate and / or (vi) supporting cells that promote differentiation.Any of the methods or parameters for differentiating PSCs in thymic cells described in the following references may be used here and includes Parent et al. Cell Stem Cell. August 1, 2013;13(2):219-29; Soh et al. Stem Cell Rep. 2014 Vol. 2 j 925-937; Sun et al. Cell Stem Cell. August 1, 2013;13(2):230-6; Okabe et al. Cell. Reprog. 2015 Vol. 17 No. 5. Su et al. Sci. Rep. 2015 5, 9882; Otsuka et al. Sci Rep 2020 10: 224; International Patent Publications, WO2019060336, WO20205859, WO2020220040, WO2014134213, WO2010143529, WO2011139628; and Chinese Patent Publication CN201110121243; the content of each is incorporated herein by reference in its entirety.

[0207] In one embodiment, iPS cells are encapsulated in polymers described in this document, such as alginate, prior to their differentiation into Petition 870250090928, dated 06 / 10 / 2025, p. 75 / 189 67 / 134 thymic cells. Preparation and maintenance of definitive endoderm cells (DE)

[0208] The present invention provides a method for preparing definitive endodermal cells that can be subsequently differentiated into thymic cells. In some embodiments, the definitive cells can be prepared by culturing cells in two-dimensional or three-dimensional cultures. These methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-target P13K inhibitor). In some embodiments, the first growth medium includes Activin A, CHIR99021, insulin, transferrin, selenium (ITS), and / or knockout serum replacement (KSR). In one embodiment, iPS cells are encapsulated in polymers described herein, such as alginate, prior to their differentiation. The encapsulated cells are cultured in three-dimensional cultures.

[0209] In some aspects, the second growth medium includes basic fibroblast growth factor (bFGF), Activin A, LDN193189, ITS, and KSR. In some modalities, the second growth medium includes CHIR99021.

[0210] In some embodiments, the concentration of CHIR99021 is approximately 0.1μM to 100μM. In some embodiments, the concentration of CHIR99021 is approximately 2 μM to 3 μM.

[0211] In some forms, the cells are grown in the presence of PI-103. The concentration of PI-103 can be approximately 1nM to 1,000nM. In one embodiment, the concentration of PI-103 can be 50nM. In some embodiments, the concentration of PI-103 can be 25nM. In some embodiments, pluripotent stem cells can be cultured for about three to five days. PI-103 can be added for one or two days. Pluripotent stem cells can be embryonic stem cells or stem cells. Petition 870250090928, dated 06 / 10 / 2025, p. 76 / 189 68 / 134 induced pluripotent stem cells.

[0212] In some embodiments, pluripotent stem cells can be cultured for about three to five days. Stem cells can be cultured in the first growth medium for about one to two days and in the second growth medium for about two to three days. Pluripotent stem cells can be cultured in the first growth medium for two days and in the second growth medium for three days. In some embodiments, the concentration of Activin A can be approximately 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 one day.

[0213] Here are presented methods for differentiating pluripotent stem cells into thymic cells. These methods may include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some embodiments, the first or second growth medium may include PI-103 (a multi-target P13K inhibitor). In some embodiments, the first growth medium includes DMEM-F12, Activin A, CHIR99021, insulin transferrin selenium (ITS), and knockout serum replacement (KSR). In some embodiments, 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 approximately 0.00M to 100μM. 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 range from approximately 1nM to 1,000nM. In one embodiment, the concentration of PI-103 can be 50nM. Preparation of endodermal cells from the foregut Petition 870250090928, dated 06 / 10 / 2025, page 77 / 189 69 / 134 (AFE)

[0214] Definitive endodermal cells can be cultured and differentiated into foregut cells. In some embodiments, AFE cells can be prepared by means of two-dimensional or three-dimensional cell culture. Definitive endodermal cells can be differentiated into AFE cells by contacting DE cells with a BMP inhibitor, a TGFe inhibitor, at least one FGF and / or ascorbic acid.

[0215] In one embodiment, cells are encapsulated in polymers such as those described herein, like alginate, before their differentiation. The encapsulated cells are grown in three-dimensional cultures.

[0216] In some cases, the cell culture medium used for the differentiation of DE cells into AFE cells may include N2 supplement (GIBCO, Waltham, Massachusetts), Eagle Basal Medium (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement, non-essential amino acids, KSR and / or ITS.

[0217] In some cases, the cell culture medium used for the differentiation of DE cells into AFE cells does not include the serum-free B27™ supplement.

[0218] In some modalities, the BMP inhibitor may be the LDN193189. In some embodiments, the concentration of LDN193189 is from about 0.1nM to about 1,000nM. In some aspects, the concentration of LDN193189 is approximately 100 to 200nM.

[0219] In some modalities, the TGFβ inhibitor may be SB431542. In some embodiments, the concentration of SB431542 is from about 1 pM to about 100 pM. As a non-limiting example, the concentration of SB431542 is 10 pM.

[0220] In some modalities, FGF may be FGF8. In some modalities, the concentration of FGF8 is from about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF8b is from about 25 to 50 ng / ml. Petition 870250090928, dated 06 / 10 / 2025, p. 78 / 189 70 / 134

[0221] DE cells can be differentiated into AFE cells for about 1 day, 2 days, 3 days, 4 days or 5 days. Preparation of ventral pharyngeal endodermal cells (VPE)

[0222] The differentiation of AFE cells into VPE cells is performed in a single-step process or in a multi-step process. The multi-step process can be a two-step process. In a first step, AFEs are cultured in a VPE1 medium, and in the second step, the cells are cultured in a VPE2 medium. In one embodiment, the cells are encapsulated in polymers such as those described herein, such as alginate, before their differentiation. The encapsulated cells are cultured in three-dimensional cultures.

[0223] In some embodiments, VPE cells can be prepared by means of cell culture in two-dimensional or three-dimensional culture. The VPE1 step may include cell culture for approximately 1 day, 2 days, 3 days, 4 days, or 5 days. The VPE2 step may include cell culture for approximately 2 days, 3 days, 4 days, 5 days, or 6 days.

[0224] In some embodiments, the VPE1 medium may include retinoic acid, at least one FGF, a WNT inhibitor, a TGFe inhibitor and / or ascorbic acid.

[0225] In some embodiments, the VPE2 medium may include Noggin, a BMP inhibitor, a WNT activator (e.g., CHIR99021), at least one FGF, retinoic acid, an SHH antagonist, and / or ascorbic acid.

[0226] In some modalities, the FGF may be FGF8, FGF7 and / or FGF10. In some modalities, the concentration of FGF8 is approximately 1 ng / ml to approximately 100 ng / ml. As a non-limiting example, the concentration of FGF8b is approximately 25 to 50 ng / ml.

[0227] In some embodiments, the WNT inhibitor is IWR1. The concentration of IWR1 can be approximately 0.01 to 10 μM. As a non-limiting example, the concentration of IWR1 is 2.5 μM.

[0228] In some modalities, the TGFβ inhibitor may be SB431542. Petition 870250090928, dated 06 / 10 / 2025, p. 79 / 189 71 / 134 In some embodiments, the concentration of SB431542 is from about 1 μM to about 100 μM. As a non-limiting example, the concentration of SB431542 is 10 μM.

[0229] In some embodiments, the concentration of ascorbic acid is approximately 0.1 to 30 μM. As a non-limiting example, the concentration of ascorbic acid may be 10 μM.

[0230] In some modalities, the BMP inhibitor may be the LDN193189. In some embodiments, the concentration of LDN193189 is from about 0.1nM to about 1,000nM. In some aspects, the concentration of LDN193189 is approximately 100 to 200nM.

[0231] In some forms, the SHH inhibitor may be SANT-1. In some embodiments, the concentration of SANT-1 is from about 0.01 μM to about 10 μM. As a non-limiting example, the concentration of SANT1 is 0.25 μM.

[0232] In some embodiments, foregut cells can be cultured and differentiated into pharyngeal endoderm cells by contacting or incubating foregut cells with at least one of the following: EGF, retinoic acid, FGF8B and / or SHH.

[0233] In some modalities, the VPE1 and / or VPE2 medium may include N2- supplement (GIBCO, Waltham, Massachusetts), Eagle Basal Medium (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ non-whey supplement (with or without vitamin A), non-essential amino acids, KSR and / or ITS. PREPARATION OF THYMIC EPITHELIAL PROGENITOR CELLS (TEP)

[0234] Differentiation of VPE cells into TEP cells can be performed by culturing the cells in a TEP medium. In some embodiments, TEP cells can be prepared by culturing cells in two-dimensional or three-dimensional culture. The TEP step may include culturing cells for approximately 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0235] In one embodiment, the cells are encapsulated in polymers such as those described herein, such as alginate, before their differentiation. The cells Petition 870250090928, dated 06 / 10 / 2025, page 80 / 189 72 / 134 encapsulated particles are grown in three-dimensional cultures.

[0236] In some modalities, VPE cells can be differentiated into TEP cells using BMP (e.g., BMP4, BMP2), a WNT activator (e.g., CHIR99021), at least one FGF and / or ascorbic acid.

[0237] In some modalities, the TEP medium may include the supplement N2- (GIBCO, Waltham, Massachusetts), Eagle Basal Medium (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ whey-free supplement (with or without vitamin A), non-essential amino acids, KSR and / or ITS.

[0238] In some forms, BMP can be BMP2 or BMP4. The concentration of BMP can be from 1 ng / ml to about 100 ng / ml. In some forms, the concentration of BMP can be 50 ng / ml.

[0239] In some modalities, the FGF may be FGF8, FGF7, FGF1 and / or FGF10. In some modalities, the concentration of FGF is approximately 1 ng / ml to approximately 100 ng / ml. As a non-limiting example, the concentration of FGF is approximately 25 to 50 ng / ml.

[0240] In some embodiments, pharyngeal endoderm cells can be cultured and differentiated into thymic epithelial cells by contact or incubation of pharyngeal endoderm cells with at least one of BMP4, FGF8b, EGF, SANT-1 (SHH antagonist), CHIR99021, ascorbic acid, or a combination thereof. Preparation of thymic epithelial cells (TEC)

[0241] TEP cells can be differentiated in vitro into TECs. Differentiation into TECs can be performed in 2D or 3D culture. In some modalities, TEP differentiation can be performed for approximately 2, 3, 4, 5, 6, or 7 days.

[0242] In one embodiment, cells are encapsulated in polymers such as those described herein, like alginate, before their differentiation. The encapsulated cells are grown in three-dimensional cultures.

[0243] In some modalities, the differentiation of TEPs into TECs is carried out in a TEC medium. Petition 870250090928, dated 06 / 10 / 2025, p. 81 / 189 73 / 134

[0244] The TEC medium may include RANKL, Interleukin, for example, (IL22), at least one FGF, at least one BMP (e.g., BMP4), a WNT activator and / or ascorbic acid.

[0245] In some forms, the RANKL concentration can be from about 1 ng / ml to about 100 ng / ml. In some aspects, the RANKL concentration can be from approximately 20 ng / ml to approximately 50 ng / ml.

[0246] In some modalities, the FGF may be FGF8, FGF7, FGF1 and / or FGF10. In some modalities, the concentration of FGF is approximately 1 ng / ml to approximately 100 ng / ml. As a non-limiting example, the concentration of FGF is approximately 25 to 50 ng / ml.

[0247] In some modalities, the concentration of interleukins is from about 1 ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of IL22 is approximately 20 ng / ml.

[0248] TEC medium may include N2- supplement (GIBCO, Waltham, Massachusetts), Eagle Basal Medium (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ non-whey supplement (with or without vitamin A), non-essential amino acids, KSR and / or ITS.

[0249] In some embodiments, differentiation is carried out between approximately 14 and 17 days. In some embodiments, the cells of the invention can be cultured as aggregates. In some aspects, the invention of the cells can be cultured in a medium based on an extracellular matrix, for example, Geltrex. PREPARATION OF EFFECTOR CELLS

[0250] Methods for preparing effector cells are also provided here. In some embodiments, the effector cells may be lymphocytes. Effector cells may be obtained from primary mammalian cells or from an established cell line. If obtained from a mammal, effector cells may be obtained from various sources, including, but not limited to, blood, bone marrow, lymph node, thymus, spleen, or other tissues or fluids. Effector cells may also be enriched or purified. In some embodiments, the Petition 870250090928, dated 06 / 10 / 2025, page 82 / 189 74 / 134 effector cells can be T cells. T cells can be of any type and can be at any stage of development, including, but not limited to, double-positive CD4+ / CD8+ T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells (TILs), memory T cells, naive T cells. Methods for isolating and / or enriching lymphocytes are known in the art. Methods for enriching a population of lymphocytes obtained from a mammal or a donor can be performed by any suitable separation method, including, but not limited to, the use of a separation medium (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA total lymphocyte enrichment cocktail, lymphocyte separation medium (LSA) (MP Biomedical Cat.No. 0850494X) or similar), separation of cell size, shape or density by filtration or elutriation, immunomagnetic separation (e.g., magnetism-activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence-activated cell sorting system, FACS) and / or bead-based column separation.

[0251] In some embodiments, the effector cells described in this document may be derived from pluripotent stem cells. In some embodiments, the effector cells may be derived from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, umbilical cord blood, peripheral blood, thymus, or the stem or progenitor cells may have been differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Primary tissue stem or progenitor cells or ESCs or iPSCs may be of human or non-human animal origin (e.g., mice).

[0252] In some embodiments, effector cells can be prepared by differentiating pluripotent stem cells or progenitor cells into lymphocytes by culturing PSCs or progenitor cells with cells of Petition 870250090928, dated 06 / 10 / 2025, page 83 / 189 75 / 134 support cells that ectopically express a Notch ligand. In some embodiments, the support 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 of those described in this document or in the literature, such as in U.S. Patent 7,795,404, which is incorporated by reference in its entirety. The effector cells of the present invention may be prepared with the ATO (Artificial Thymic Organoid) cell culture system, which utilizes support cells that ectopically express OP9-DLL1. In some modalities, the method also includes contacting co-cultured stem or progenitor cells with the ligand Flt-3 and / or IL-7 and / or stem cell factor / receptor ligand Kit and / or thrombopoietin.In some embodiments, the differentiation of the stem or progenitor cell into a T cell comprises: the culture of a three-dimensional (3D) cell aggregate comprising: a) a selected population of support cells expressing an exogenous Notch ligand; b) a selected population of stem or progenitor cells; with a serum-free medium comprising supplement B-27®, xeno-free supplement B-27®, supplement GS2 l™, ascorbic acid, Flt-3 ligand, IL-7, or a combination thereof. Any of the methods for generating lymphocytes from stem cells or progenitor cells described in International Patent Publication WO2017075389 may be useful in the present invention (the content of which is incorporated herein by reference in its entirety).

[0253] In some embodiments, the effector cell may be or may be derived from a hematopoietic cell. Methods of preparing hematopoietic cells from pluripotent stem cells are known in the art, for example, as described in U.S. Patent 9,834,754, and may include one or more of the following steps, such as (i) inducing hematopoietic differentiation in a population of human pluripotent stem cells, wherein activin / nodal signaling is inhibited between day 1 and day 4 of differentiation; (ii) classifying the induced population based on CD34 expression and Petition 870250090928, dated 06 / 10 / 2025, page 84 / 189 76 / 134 CD43; and / or (iii) selecting a fraction of the cell population that is positive for CD34 and / or negative for CD43, wherein the sorting and selection of the cell fraction are performed on a day selected between approximately day 6 and day 11 of differentiation (the contents of U.S. Patent 9,834,754 are incorporated herein by reference in their entirety).

[0254] In some embodiments, thymic cells and / or effector cells can be cultured in the presence of extracellular vesicles (e.g., exosomes) derived from thymic cells and / or effector cells. Methods for preparing thymic cell exosomes are described in U.S. Patent Publication US2020299641 and (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the exosomes can be derived from thymic cells engineered to ectopically express DLL1.

[0255] In some modalities, effector cells, such as T cells, can be derived by the differentiation of other cell types. T cell differentiation can include four stages: 1) mesoderm induction (around days 1 to 4), 2) hematopoietic specification (around days 4 to 8), 3) hematopoietic commitment and expansion (around days 8-10), and / or 4) T lymphoid differentiation. PSCs (iPSCs or ESCs) can be used as the initial cell population for mesoderm differentiation. These cells can differentiate into mesodermal cells. Mesodermal cells can further differentiate into hematopoietic cells, which can be expanded in cell number.Cell culture systems for use in the present invention include, among others, 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 lymphoid differentiation. The first cell culture medium may include BMP4 (e.g., human BMP4) and bFGF (e.g., human bFGF). PSCs or ESCs may be used as the initial cell population. Undifferentiated PSCs or ESCs may be transferred to... Petition 870250090928, dated 06 / 10 / 2025, page 85 / 189 77 / 134 low-fixation plates to allow the formation of embryonic bodies (EBs). EB formation during the first stage can be facilitated 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. The second cell culture medium may include VEGF (e.g., hVEGF) and a cocktail of hematopoietic cytokines. The hematopoietic cytokine cocktail may include SCF (e.g., hSCF), Flt3L (e.g., hFlt3L), at least one cytokine, and bFGF for hematopoietic specification. The cytokine may be a Th1 cytokine, which includes, among others, IL3, IL15, IL7, IL12, and IL21. EBs can be cultured in the second cell culture medium for hematopoietic specification until approximately Day 10.EBs can be immunophenotypically analyzed by FACS for the expression of CD34, CD31, CD43, CD45, CD41a, ckit, Notch1, and IL7Ra. In some modalities, CD34+ EB cells approximately one day old express the highest levels of key transcription factors for lymphoid differentiation, for example, CD127 (IL7Ra) and Notch1. The third cell culture medium may include a feeder cell and SCF, Flt3L, and at least one cytokine. The cytokine may be a Th1 cytokine, which includes, among others, IL3, IL15, IL7, IL12, and IL21. In some modalities, around Day 10, EBs can be dissociated and hematopoietic precursors can be transferred to a feeder cell to induce T lymphoid differentiation in an established co-culture system in the presence of SCF, Flt3L, and Th1 cytokine(s) (e.g., IL-7).In some embodiments, the co-culture system may include thymic cells and / or feeder cultures, for example, OP9-DL11 feeder cells.

[0256] In some embodiments, co-culture can be performed using a co-culture medium. In some cases, the co-culture medium may include StemSpan SFEM II and StemSpanTMT Cell Progenitor Maturation Supplement. Petition 870250090928, dated 06 / 10 / 2025, page 86 / 189 78 / 134 In some modalities, the co-culture medium may include aMEM, 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 1X Pen Strep. In some embodiments, the co-culture medium may include DMEM / F12, 1% B27 supplement without vitamin A, 50 μM ascorbic acid, 50 ng / ml FGF8b, 50 ng / ml BMP, 50 ng / ml FGF10, 2 μM CHIR99021, 0.1% ITS, 0.0025% KSR, 0.5X Pen Strep, 1x NEAA, 1% N2, 1% Glutamax, 1% β-ME, 50 ng / ml IL7, 50 ng / ml FLT3L, 50 ng / ml TPO and / or 50 ng / ml SCF.

[0257] In some embodiments, effector cell progenitors are co-cultured with thymic cells of the invention to promote their differentiation. The co-culture may also include TSS and one or more supporting cells. AGGREGATE SIZE

[0258] In some embodiments, the cells of the invention can be grown in three-dimensional culture. In some embodiments, the cells of the invention may be in the form of aggregates or spheroids. The term “spheroid” refers to groups of cells and / or colonies of cells. Spheroids can be formed from various cell types, for example, thymic cells, pluripotent cells, effector cells, stem cells and / or supporting cells. Spheroids may have spherical or irregular shapes. Spheroids may contain heterogeneous populations of cells, cell types, cells of different states, such as proliferating cells, quiescent cells and necrotic cells.

[0259] In some modalities, the size of the spheroid / aggregate can be adjusted. For example, the aggregate size in pluripotent stem cells can be critical during the expansion period, since the aggregate size can determine the oxygen distribution within the cellular spheroid, resulting in discrete zones composed of outer, intermediate, and inner regions of the spheroid along the oxygen supply, from top to bottom, exhibiting proliferative, quiescent, viable, and apoptotic nuclei properties, respectively (Langan et al. Plos One. 2016;11(2); content of which is Petition 870250090928, dated 06 / 10 / 2025, page 87 / 189 79 / 134 incorporated herein by reference in its entirety). In some embodiments, the aggregates may be from about 50μm to 500μm, from about 100μm to 1,000μm, from about 200μm to 2,000μm, from about 250μm to 2,500μm, from about 300μm to 3,000μm, from about 400μm to 4,000μm. In one embodiment, the size of the spheroid / aggregate may be 250μm.

[0260] The present invention provides methods for treating or preventing a condition in an individual. The methods may include administering to the mammal any of the cell populations described in this document or a pharmaceutical composition that includes any of the cell populations described in this document, in an amount effective to treat or prevent the condition in the individual. The condition may be cancer, an immunodeficiency, an autoimmune condition, an infection, or a blood condition. Thymic Indications

[0261] The thymic cells, effector cells and / or compositions described in this document may be used in the treatment or prevention of one or more diseases or conditions associated with the absence, decline or aberrant functioning of the individual's thymus. In some embodiments, the thymic cells, effector cells and / or compositions described herein may be used in the treatment of athymia, a condition in which an individual may be born with a severely deficient thymus or in which the thymus is completely absent in the individual.Conditions associated with athymia include, but are not limited to, complete or partial DiGeorge syndrome, complete or partial CHARGE syndrome, thymomas, thymic cancer, type A thymoma, type B thymoma, thymic atrophy, age-related thymic atrophy, thymic cyst, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, thymic irradiation, myasthenia gravis, thymic carcinoma, thymic hyperplasia, thymic irradiation, age-related decline in thymic function, or infection. Thymic cells, effector cells, and / or composites may be used for the treatment of athymia associated with mutations, defects, or deletions in genes involved in the development of... Petition 870250090928, dated 06 / 10 / 2025, page 88 / 189 80 / 134 thymic tissue. Thymic cells, effector cells and / or compositions can be used for the treatment of athymia associated with mutations, defects or deletions in the PAX1 gene (herein referred to as PAX1 deficiency). Thymic cells, effector cells and / or compositions can be used for the treatment of athymia associated with mutations, defects or deletions in the TBX1 gene (herein referred to as TBX1 deficiency). Thymic cells, effector cells and / or compositions can be used for the treatment of athymia associated with mutations, defects or deletions in the FOXN1 gene (herein referred to as FOXN1 deficiency).

[0262] In some embodiments, thymic cells, effector cells and / or compositions described in this document may be used in the treatment of age-related thymic insufficiency, chemotherapy, radiotherapy, treatment with immunosuppressive drugs, graft-versus-host disease, hematopoietic stem cell transplantation with T-cell depletion and / or HIV infection. SURGERY

[0263] In certain modalities, the thymic cells, effector cells and / or compositions described herein may be used to treat an individual who may undergo thymectomy surgery. In certain modalities, the individual may have a congenital heart defect and may have received or be receiving open-heart surgery. An individual may undergo thymectomy for the treatment of one or more indications associated with the thymus, for example, myasthenia gravis or thymoma. IMMUNODEFICIENCY

[0264] In some embodiments, the compositions of the present invention can be used to treat immunodeficiency. The term immunodeficiency can refer to any condition in which an individual's immune system is compromised and / or requires reconstitution, for example, after irradiation or chemotherapy. Immunodeficiency can be primary immunodeficiency, caused by a genetic or hereditary factor, or secondary immunodeficiency, caused by an environmental factor. In some embodiments, the Petition 870250090928, dated 06 / 10 / 2025, page 89 / 189 81 / 134 compositions of the present invention can be used to treat primary immunodeficiencies, such as, among others, Wiscott-Aldrich syndrome, severe combined immunodeficiency disease (SCID), DiGeorge syndrome, ataxia-telangiectasia, chronic granulomatous disease, transient hypogammaglobulinemia of infancy, agammaglobulinemia, complement deficiencies, T-cell lymphopenia and / or selective IgA deficiency. In some embodiments, the compositions of the present invention can be used to treat secondary immunodeficiencies caused by diseases such as AIDS and / or hepatitis.

[0265] Lymphopenia, as used in this document, 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 reduction in the number of T cells, it may be called T-cell lymphopenia. The compositions of the invention can be used to treat inherent or acquired lymphopenia that may be caused by hematopoietic stem cell therapy, bone marrow transplant therapy, radiation, chemotherapy, surgery, immunosenescence and / or aging. CANCER

[0266] Various types of cancer can be treated with the pharmaceutical compositions of the present invention. As used in this document, the term “cancer” refers to any of several malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new locations in the body, and refers to the pathological condition characterized by these malignant neoplastic growths. Cancers can be hematological tumors or malignancies and include, among others, all types of lymphomas / leukemias, carcinomas, and sarcomas, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, liver, kidney, colon / rectal, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis. Petition 870250090928, dated 06 / 10 / 2025, page 90 / 189 82 / 134 prostate, skin, small intestine, stomach, spinal cord, coccyx, testicles, thyroid and uterus.

[0267] The types of carcinomas that can be treated with the compositions of the present invention include, among others, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma, and sinonasal undifferentiated carcinoma.

[0268] The types of carcinomas that can be treated with the compositions of the present invention include, but are not limited to, soft tissue sarcoma, such as alveolar soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and chondrosarcoma.

[0269] As a non-limiting example, the carcinoma that can be treated may be: Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myeloid leukemia, Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-cell lymphoma, Bile duct cancer, Bladder cancer, Bone cancer, Intestinal cancer, Brain cancer, Brainstem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myeloid leukemia, Colon cancer, Petition 870250090928, of 06 / 10 / 2025, page 91 / 189 83 / 134 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, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Bowel cancer, Intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell carcinoma, Jaw cancer, Kaposi's sarcoma, Kidney cancer, Laryngeal cancer, LeiomyosarcomaLeptomeningeal metastases, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma, Mesenchymal, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma, Non-small cell lung cancer, Oat cell carcinoma, Eye cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer oropharyngeal, osteogenic sarcoma, osteosarcoma, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor,primary ovarian peritoneal carcinoma, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, cancer Petition 870250090928, dated 06 / 10 / 2025, page 92 / 189 84 / 134 penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary gland cancer, primary lymphoma of the central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft tissue sarcoma, uterine sarcoma, maxillary sinus cancer, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, spinal 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, cell cancer Transitional cells, Transitional cell carcinoma, Triple-negative breast cancer, Tubal cancerTubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer. AUTOIMMUNE DISEASES.

[0270] In some embodiments, thymic cells, effector cells, or any of the compositions described herein may be useful in the treatment of autoimmune diseases. Autoimmune diseases can arise in an individual when self-antigen(s) is / are recognized by effector cells in extra-thymic tissue and / or when such recognition triggers an activated immune response in an individual. The present invention provides methods for preparing an effector cell capable of effecting an immune tolerance response in an individual. Effector cells and pharmaceutical compositions including them may be useful in training the individual's immune system. In some embodiments, the engineered thymic cells of the present invention may also be administered to an individual for the treatment of autoimmune diseases.

[0271] In some embodiments, the compositions of the present invention can be used to treat autoimmune polyglandular syndrome type 1 (APS). Petition 870250090928, dated 06 / 10 / 2025, p. 93 / 189 85 / 134 1) or autoimmune polyendocrinopathy-candidiasis-ectodermal dysplasia (APECED) syndrome. More than 60 mutations of the autoimmune regulator gene (AIRE) are associated with the development of autoimmune polyglandular syndrome type 1 (APS-1). AIRE plays an important role in the formation of the T cell repertoire through its function in eliminating T cells that are reactive to self-antigens in the thymus. Clinical manifestations associated with APS-1 classically involve 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 invention can be administered with current standard therapies for the treatment of APS1, such as, but not limited to, lifelong antifungals, calcium modulators, endocrine hormone replacement, corticosteroids with or without 5 azacitidine, 5 azacitidine with mycophenolate (autoimmune hepatitis) and / or 5 azacitidine with or without rituximab (autoimmune pneumonitis).

[0272] 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 pulmonary inflammation, 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.

[0273] In some forms, the autoimmune disease may be X-linked polyendocrinopathy enteropathy syndrome (IPEX).

[0274] In some embodiments, the compositions of the present invention may be used in combination with a second therapeutic agent for Petition 870250090928, dated 06 / 10 / 2025, page 94 / 189 86 / 134 treatment of autoimmune diseases. The second therapeutic agents may be immunosuppressants or anti-inflammatory agents, which may include, among others, an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a folic acid analogue, a purine analogue, an antibody, a TNF-binding protein, an interferon, an opioid, a mycophenolate, and a calcineurin inhibitor. The compositions of the present invention may also be used in combination with peripheral lymphodepletion.

[0275] In some respects, thymic cells can be engineered to express MHC molecules, the expression of which may be associated with a disease or disorder. In some modalities, the expression of MHC molecules may be protective against a particular disease or disorder in an individual. In some respects, the disease or disorder may be an autoimmune disease.

[0276] In some respects, the MHC haplotypes expressed by the thymic cells of the invention may be associated with a disease or disorder in an individual. In some embodiments, the expression of the MHC haplotype may be protective against a particular disease or disorder. As a non-limiting example, thymic cells may be engineered to express the DR1501-DQ6 haplotype as a therapeutic strategy for the treatment and / or prevention of type I diabetes in an individual (see Wen et al., Science Immunology 5.44 (2020), the content of which is incorporated by reference in its entirety). CELL, TISSUE AND ORGAN TRANSPLANTATION

[0277] In some embodiments, the cells, compositions and pharmaceutical compositions of the present invention can be used to improve the acceptance of cell, tissue or organ transplantation and / or prevent rejection of a cell, tissue or organ transplant. In some embodiments, the cells, compositions and / or pharmaceutical compositions of the present invention can be used to treat or prevent graft-versus-host disease (GvHD). In some embodiments, the thymic cells of the present invention Petition 870250090928, dated 06 / 10 / 2025, page 95 / 189 87 / 134 compositions can be administered to the individual prior to cell, tissue, or organ transplantation. Pre-grafting of thymic cells may result in an immune tolerance response in the individual. In some respects, the compositions can 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.

[0278] In some embodiments, the cells of the invention, for example, thymic cells, can be administered concomitantly with or after cell, tissue or organ transplantation has occurred.

[0279] In some modalities, organ transplantation may be a solid organ transplant (SOT), which represents a treatment modality for end-stage organ failure of the kidney, liver, pancreas, heart, and lung. SOT may also include small intestine and vascularized composite allografts.

[0280] In some modalities, organ transplantation may be a hematopoietic stem cell transplant (HSCT). Non-limiting examples of indications requiring HSCT include non-malignant indications such as aplastic anemia, Fanconi anemia, Diamond-Blackfan syndrome, sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, Chediak-Higashi syndrome, chronic granulomatous disease, Glanzmann thrombasthenia, osteopetrosis, lysosomal storage disorders, Gaucher disease, Niemann-Pick disease, mucopolysaccharidosis, glycoproteinoses, immunodeficiency, ataxia telangiectasia, DiGeorge syndrome, severe combined immunodeficiency (SCID), Wiscott-Aldrich syndrome, Kostmann syndrome, Shwachman-Diamond syndrome. Non-limiting examples of indications requiring HSCT include malignant indications, such as, but not limited to, leukemias, such as Petition 870250090928, dated 06 / 10 / 2025, page 96 / 189 88 / 134 acute myelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, myelodysplasia; lymphomas, such as Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myeloproliferative neoplasms, myelofibrosis, chronic myeloid leukemia; solid tumors, such as neuroblastoma, desmoplastic small round cell tumor, Ewing's sarcoma and / or choriocarcinoma.

[0281] In some modalities, tissue transplantation may be a bone marrow transplant (BMT).

[0282] The compositions and cells, for example, thymic cells of the invention, can be used to treat T-cell lymphopenia observed after hematopoietic stem cell transplantation or bone marrow transplantation. INFECTIOUS DISEASES

[0283] The cells, compositions and pharmaceutical compositions of the invention can be used to treat infectious diseases. Infectious disease-causing organisms include, among others, any one or more bacterial species (spp.), including, for example, Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia 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., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp, Corynebacterium diptheriae), Enterococcus spp. (e.g. Enterococcus faecalis, Enterococcus faecum), Escherichia spp.(e.g. Escherichia coli), Francisella spp. (e.g. Francisella tularensis), Haemophilus spp, Haemophilus influenza), Helicobacter spp. (e.g. Helicobacter pylori), Legionella spp. (e.g. Legionella pneumophila), Leptospira spp. (e.g. Leptospira interrogans), Listeria spp. (e.g. Listeria monocyto. Petition 870250090928, dated 06 / 10 / 2025, p. 97 / 189 89 / 134 genes), Mycobacterium spp, Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseriaspp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Porphyromonas spp. (e.g., P. Gingavalis), Porphyromonas spp, P. Gingavalis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp, Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus coagulase negative (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 infectious diseases may include, for example, one or more parasitic organisms (spp.) (e.g., target(s) of the parasite) including, for example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., 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., sandfly), Onchocercidae spp., Opisthorchis spp., O. viverrini, O. felineus, O. guayaquilensis and O. noverca), Plasmodium spp.(e.g. P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp., 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. Petition 870250090928, dated 06 / 10 / 2025, p. 98 / 189 90 / 134 example, T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans and / or Wuchereria bancrofti. PHARMACEUTICAL COMPOSITIONS

[0284] The pharmaceutical compositions of the present invention may include compositions with one or more cells described in this document and one or more pharmacologically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate-buffered saline and the like; carbohydrates, such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are, in one aspect, prepared for intravenous administration.

[0285] In some embodiments, pharmaceutical formulations may include any isotonic vehicle, such as, for example, normal saline solution (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® electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water or Ringer's lactate. In one embodiment, the pharmaceutically acceptable vehicle may be supplemented with human serum albumin.

[0286] In some embodiments, the pharmaceutical composition may be substantially free of, for example, there are no detectable levels of a contaminant, for example, selected from endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual coated granules of anti-CD3 / anti-CD28, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In some embodiments, the bacterium is Petition 870250090928, dated 06 / 10 / 2025, p. 99 / 189 91 / 134 at least one selected from Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia and Streptococcus pyogenes group A. SHOCK ABSORBERS

[0287] In some embodiments, the pharmaceutical compositions of the present invention are prepared with one or more buffering agents.

[0288] Some examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, hydroxide magnesium, aluminum hydroxide, alginic acid, pyrogen-free water,Isotonic saline solution, Ringer's solution, ethyl alcohol and / or combinations thereof.

[0289] Non-limiting examples include aqueous formulations, such as the phosphate-buffered formulation pH 7.4 or the citrate-buffered formulation pH 6.2; formulations for lyophilization, such as the citrate-buffered formulation pH 6.2 with 3% mannitol, the citrate-buffered formulation pH 6.2 with 4% mannitol / 1% sucrose; or a formulation prepared by the process disclosed in U.S. Patent No. 8883737 to Reddy et al., the contents of which are incorporated herein by reference in their entirety.

[0290] In some embodiments, the pharmaceutical compositions of the pre Petition 870250090928, dated 06 / 10 / 2025, page 100 / 189 92 / 134 The present invention is formulated in parenteral dosage forms. Parenteral formulations may be aqueous solutions containing carriers or excipients, such as salts, carbohydrates, and buffering agents (e.g., at a pH of 3 to 9), or sterile non-aqueous solutions, or dry forms that may be used in conjunction with a suitable vehicle, such as sterile, pyrogen-free water. For example, an aqueous solution of the therapeutic agents of the present invention comprises an isotonic saline solution, 5% glucose, or other pharmaceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters, and amides, for example, as disclosed in U.S. Patent No. 7,910,594 to Vlahov et al. (Endocyte), the contents of which are incorporated herein by reference in their entirety.In another example, an aqueous solution of the therapeutic agents of the present invention comprises a phosphate-buffered formulation (pH 7.4) for intravenous administration, as disclosed in Example 23 of WO2011014821 to Leamon et al., the contents of which are incorporated herein by reference in their entirety. The parenteral dosage form may be in the form of a reconstitutable lyophilized powder comprising the dose of the therapeutic agents of the present invention. Any sustained-release dosage forms known in the art may be used, such as, for example, the biodegradable carbohydrate matrices described in U.S. Patent Nos. 4,713,249; 5,266,333; and 5,417,982, disclosures of which are incorporated herein by reference, or alternatively, a slow pump (e.g., an osmotic pump) may be used. NUTRIENTS.

[0291] In some embodiments, the pharmaceutical compositions of the present invention include one or more nutrients that promote the health, survival and / or proliferation of the cells described herein.

[0292] In some embodiments, the pharmaceutical formulations include vitamins. In some embodiments, the pharmaceutical compositions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following items (and any range) Petition 870250090928, dated 06 / 10 / 2025, p. 101 / 189 93 / 134 derivable from them): biotin, DL alpha-tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the pharmaceutical compositions include combinations of these or salts thereof. In some embodiments, the pharmaceutical compositions include or consist essentially 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 consist essentially of biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations or salts thereof.

[0293] In some embodiments, the pharmaceutical compositions also include proteins. In some embodiments, the proteins include albumin or bovine serum albumin, a BSA fraction, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the pharmaceutical compositions include one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodothyronine, or combinations thereof.

[0294] In some embodiments, the pharmaceutical compositions include amino acids, inorganic ions and / or monosaccharides. In some embodiments, the amino acids comprise 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 compositions also include one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or combinations thereof. In some embodiments, the pharmaceutical compositions also include one or more of the following Petition 870250090928, dated 06 / 10 / 2025, page 102 / 189 94 / 134 tes: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodoI-thyronine, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper or manganese. PRESERVATIVES

[0295] Exemplary preservatives may include, among others, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives and / or other preservatives. Some examples of antioxidants include, among others, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulfite. Some examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, eutectic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate.Some 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, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Some 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. Some examples of alcohol-based preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Exemplary acidic preservatives include... Petition 870250090928, dated 06 / 10 / 2025, page 103 / 189 95 / 134 among others, 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, among others, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (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®. Dosage and Administration

[0296] The cells and compositions of the present invention described above can be administered by any delivery route, systemic delivery or local delivery, resulting in a therapeutically effective outcome.

[0297] In some modalities, thymic cells and effector cells can be co-delivered to the same anatomical location in an individual. In some modalities, thymic cells and effector cells can be delivered to different anatomical locations in an individual.

[0298] In some modalities, thymic cells and effector cells can be delivered to the individual at the same time via the same delivery route or via a different delivery route.

[0299] In some modalities, thymic cells may be administered to the individual prior to the administration of effector cells.

[0300] In some modalities, thymic cells can be administered to the individual after the administration of effector cells.

[0301] Non-limiting examples of routes of administration include enteral (into the intestine), gastroenteral, epidural (into the dura mater), oral (by mouth), transdermal, intracerebral (into the brain), intracerebroventricular (into the cerebral ventricles), epicutaneous (application to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a Petition 870250090928, dated 06 / 10 / 2025, p. 104 / 189 96 / 134 vein), intravenous bolus, intravenous infusion, intra-arterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous injection (into a pathological cavity), intracavitary (at the base of the penis), intravaginal administration, intrauterine administration, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (nasal inhalation / aspiration), sublingual, sublabial, enema, eye drops (in the conjunctiva), ear drops, auricular (in or through the ear), buccal (directed towards the cheek), conjunctival, cutaneous, dental (for a tooth or teeth), electro-osmosis, endocervical, endosinusal, endotracheal, extracorporeal, hemodialysis, infiltration,interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equina), intracisternal (within the cerebellomedullary cisterna magna), intracorneal (within the cornea), intracorneal dental, intracoronary (within the coronary arteries), intracorporeal cavernous (within the dilatable spaces of the corpora cavernosa of the penis), intradiscal (within an intervertebral disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or below the dura mater), intraepidermal (in the epidermis), intraesophageal (in the esophagus), intragastric (within the stomach), intragingival (within the gum), intraileal (within the distal portion of the small intestine), intralesional (within or administered directly into a localized lesion), intraluminal (within the lumen of a tube), intralymphatic (within the lymphatic system),intramedullary (within the medullary cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or bronchi), intrasinusal (within the nasal or periorbital sinuses). Petition 870250090928, dated 06 / 10 / 2025, page 105 / 189 97 / 134 intraspinal (within the spinal column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumoral (within a tumor), intratympanic (within the middle ear), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current, where ions of soluble salts migrate to the body tissues), irrigation (washing or rinsing open wounds or body cavities), laryngeal (directly onto the larynx), nasogastric (through the nose to the stomach), occlusive dressing technique (topical administration followed by covering with dressing that occludes the area), ophthalmic (for the external part of the eye), oropharyngeal (directly into the mouth and pharynx), parenteral,percutaneous, periarticular, epidural, perineural, periodontal, rectal, respiratory (within the respiratory tract by oral or nasal inhalation, with local or systemic effect), retrobulbar (behind the pons or eyeball), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or by means of the placenta), transtracheal (through the tracheal wall), transtympanic (through or by means of the tympanic cavity), ureteral (into the ureter), urethral (into the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, spinal.

[0302] In some embodiments, the compositions containing cells of the invention can be administered intrathymically (into the thymus).

[0303] In some embodiments, the compositions containing cells of the invention can be surgically placed in the individual. As non-limiting examples, the cells can be surgically placed in the kidney capsule or in the quadriceps muscles of the thigh.

[0304] In some embodiments, thymic cells and / or cell-containing compositions of the invention may be administered intrahepatically, by intrasplenic injection or by intraportal injection. Petition 870250090928, dated 06 / 10 / 2025, page 106 / 189 98 / 134

[0305] The thymic cells and / or compositions described in this document may be provided to the individual by direct injection into the bone marrow (herein referred to as intraosseous infusion). The bone may be a long bone, such as the tibia, fibula, femur, metatarsals and phalanges of the lower limbs, the humerus, radius, ulna, metacarpals and / or phalanges of the upper limb. PARENTERAL AND INJECTABLE ADMINISTRATION

[0306] In some modalities, the cells and compositions described in this document can be administered parenterally.

[0307] Injectable preparations, for example, sterile aqueous or oily injectable suspensions, may be formulated according to known techniques, using suitable dispersing agents, wetting agents and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions and / or emulsions in non-toxic and parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as a solvent or suspension medium. For this purpose, any mild fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid may be used in the preparation of injectables.

[0308] Injectable formulations can be sterilized, for example, by filtration through a bacteria-retention filter and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0309] To prolong the effect of the active ingredients, it is generally desirable to delay the absorption of the active ingredients from subcutaneous or intramuscular injections. This can be done using liquid suspensions of crystalline or amorphous material with low water solubility. The absorption rate of the active ingredients depends on the dissolution rate, which in turn can Petition 870250090928, dated 06 / 10 / 2025, page 107 / 189 99 / 134 depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot formulations are made by forming microencapsulated matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide. Depending on the drug-to-polymer ratio and the nature of the specific polymer employed, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. LYMPH NODE ADMINISTRATION

[0310] In certain embodiments, thymic cells and / or compositions of the present invention can be administered to the individual for grafting into the lymph node. In some embodiments, thymic cells and / or compositions of the present invention can be administered to the individual in an amount effective to form ectopic thymic tissue in the lymph node. In certain embodiments, the methods and compositions described herein are used to deliver thymic cells and / or compositions of the invention into a lymph node of the individual, allowing the thymic cells to develop and produce an ectopic thymus in the lymph node. In certain embodiments, the ectopic thymus can restore thymic function to the individual, for example, supplementing or augmenting one or more functions that a normal, healthy thymus organ can perform.For example, but not exclusively, ectopic thymus may participate in the body's immunomodulation, promoting the growth, development, maturation, and selection of T cells.

[0311] In certain modalities, thymic cells can be administered to the patient as a liquid suspension.

[0312] Non-limiting examples of lymph nodes in which thymic cells Petition 870250090928, dated 06 / 10 / 2025, pp. 108 / 189 100 / 134 can be delivered including abdominal lymph nodes, celiac lymph nodes, para-aortic lymph nodes, splenic hilar lymph nodes, porta hepatis lymph nodes, left gastric lymph nodes, right gastric lymph nodes, left gastroepiploic lymph nodes, right gastroepiploic lymph nodes, retroperitoneal lymph nodes, pyloric lymph nodes (e.g., suprapyloric lymph nodes, subpyloric lymph nodes, retropyloric lymph nodes), pancreatic lymph nodes (e.g., superior pancreatic lymph nodes, inferior pancreatic lymph nodes, splenic linear lymph nodes), splenic lymph nodes, hepatic lymph nodes (e.g., cystic lymph nodes, foraminal lymph nodes, foramen of Winslow), pancreaticoduodenal lymph nodes (e.g., superior pancreaticoduodenal lymph nodes, inferior pancreaticoduodenal lymph nodes), superior mesenteric lymph nodes, ileocolic lymph nodes, prececal lymph nodes, retrocecal lymph nodes,appendicular lymph nodes, mesocolic lymph nodes (e.g., paracolic lymph nodes, left colic lymph nodes, middle colic lymph nodes, right colic lymph nodes, inferior mesenteric lymph nodes, sigmoid 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, promontory common iliac lymph nodes), and external iliac lymph nodes (e.g., medial external iliac lymph nodes, intermediate external iliac lymph nodes, lateral external iliac lymph nodes, medial lacunar-femoral lymph nodes, intermediate lacunar-femoral lymph nodes, lymph nodes lateral femoral lacunar lymph nodes, interiliac external iliac lymph nodes, obturator lymph nodes (obturator external iliac lymph nodes).

[0313] By way of non-limiting example, any of the thymic tissue transplantation methods to the lymph node described in International Patent Publication WO2021026195 may be useful in the present invention (the content of which is incorporated by reference in its entirety). WAREHOUSE MANAGEMENT

[0314] As described herein, in some embodiments, the cells and Petition 870250090928, dated 06 / 10 / 2025, pp. 109 / 189 101 / 134 compositions, including the pharmaceutical compositions of the present invention, are formulated in depots for sustained release. In general, specific organs or tissues (“target tissues”) are targeted for administration. In some embodiments, localized release is affected by the use of a biocompatible device. For example, the biocompatible device may restrict cell diffusion within the individual.

[0315] In some aspects of the present invention, the cells, compositions, and pharmaceutical compositions are spatially retained within or near the target tissues. Methods are provided for delivering pharmaceutical compositions to mammalian target tissues by contacting target tissues (including one or more target cells) with pharmaceutical compositions under conditions such that they are substantially retained in the target tissues, 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 in the target tissues. 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 compositions administered to individuals are present at a time after administration. DOSAGE AND REGIMEN

[0316] The present invention provides methods of administering cells, compositions, and pharmaceutical compositions according to the present invention to an individual in need thereof. The pharmaceutical compositions comprising the described cells may be administered to an individual using any quantity and any route of administration effective to prevent, treat, manage, or diagnose diseases, disorders, and / or conditions. The exact quantity required will vary from individual to individual depending on the individual's species, age, and general condition, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The individual may be a human being, a mammal, or an animal. The specific dose level may be therapeutically effective, prophylactically effective, or diagnostic. Petition 870250090928, dated 06 / 10 / 2025, page 110 / 189 The appropriate dosage of 102 / 134 for any specific individual will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the patient's age, body weight, general health, sex, and diet; the time of administration and the route of administration.

[0317] In certain embodiments, the cells, compositions and pharmaceutical compositions described herein according to the present invention may be administered at dosage levels sufficient to administer from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg of the individual's body weight per day, once or more times a day, to obtain the desired therapeutic, diagnostic or prophylactic effect.

[0318] In some embodiments, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be approximately 1 x 106, 1.1 x 106, 2 x 106, 3.6 x 106, 5 x 106, 1 x 107, 1.8 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, 3 x 108 or 5 x 108 cells / kg. In some embodiments, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be at least about 1 x 10⁶, 1.1 x 10⁶, 2 x 10⁶, 3.6 x 10⁶, 5 x 10⁶, 1 x 10⁷, 1.8 x 10⁷, 2 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸ or 5 x 10⁸ cells / kg. In some embodiments, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be up to approximately 1 x 10⁶, 1.1 x 10⁶, 2 x 10⁶, 3.6 x 10⁶, 5 x 10⁶, 1 x 10⁷, 1.8 x 10⁷, 2 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸ or 5 x 10⁸ cells / kg.In some cases, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be approximately 1.1 x 106-1.8 x 107 cells / kg. Petition 870250090928, dated 06 / 10 / 2025, p. 111 / 189 103 / 134 In some embodiments, a dose of cells, compositions and / or pharmaceutical compositions described herein may be approximately 1 x 10⁷, 2 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸, 5 x 10⁸, 1 x 10⁹, 2 x 10⁹ or 5 x 10⁹ cells / kg. In some embodiments, a dose of cells, compositions and / or pharmaceutical compositions described herein may be approximately 1 x 10⁷, 2 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸, 5 x 10⁸, 1 x 10⁹, 2 x 10⁹ or 5 x 10⁹ cells / kg. In some cases, a dose of cells, compositions and / or pharmaceutical compositions described herein may be approximately 1 x 10⁷, 2 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸, 5 x 10⁸, 1 x 10⁹, 2 x 10⁹ or 5 x 10⁹ cells / kg.In some cases, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be approximately 1 x 10⁷, 1.5 x 10⁷, 2 x 10⁷, 2.5 x 10⁷, 3 x 10⁷, 3.5 x 10⁷, 4 x 10⁷, 5 x 10⁷, 1 x 10⁸, 1.5 x 10⁸, 2 x 10⁸, 2.5 x 10⁸, 3 x 10⁸, 3.5 x 10⁸, 4 x 10⁸, 5 x 10⁸, 1 x 10⁹, 2 x 10⁹ or 5 x 10⁹ cells / kg. In some cases, a dose of cells, compositions and / or pharmaceutical compositions described in this document may be approximately 1-3 x 107 to 1-3 x 108 cells / kg.

[0319] In certain embodiments, the cell described in this document or the pharmaceutical compositions according to the present invention can be administered at about 10 to about 600 plc / site, 50 to about 500 plc / site, 100 to about 400 plc / site, 120 to about 300 plc / site, 140 to about 200 plc / site, about 160 plc / site.

[0320] The desired dose may be delivered at least once, three times a day, twice a day, once a day, on alternate days, every three days, every week, every two weeks, every three weeks or every four weeks. In certain embodiments, the desired dose may be administered using multiple administrations (e.g. two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations).

[0321] The desired dosage of the cells of the present invention may be administered once or several times. Cells, compositions and pharmaceutical formulations may be administered regularly at a defined frequency. Petition 870250090928, dated 06 / 10 / 2025, p. 112 / 189 104 / 134 over a period of time or continuously as a "continuous flow." A total daily dose, an amount given or prescribed over a 24-hour period, may be administered by any of these methods or as a combination thereof.

[0322] In some modalities, administering the cell(s) to an individual provides a therapeutic effect for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.

[0323] The cells of the present invention can be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, sequentially or concomitantly. In general, each agent will be administered at a dose and / or on a schedule determined for that agent. In some embodiments, the present invention encompasses the provision 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.

[0324] For example, the cells of the present invention are administered as a biocompatible device that restricts diffusion in the individual to increase bioavailability in the target treatment area. The cell(s) of the present invention may also be administered locally.

[0325] The term “conditioning regimen” refers to a course of therapy to which the patient is subjected before stem cell transplantation. For example, before hematopoietic stem cell transplantation, a patient may undergo myeloablative, non-myeloablative therapy or reduced-intensity conditioning to prevent stem cell transplant rejection, even if the stem cells originate from the same source. Petition 870250090928, dated 06 / 10 / 2025, page 113 / 189 105 / 134 patient. The conditioning regimen may involve the administration of cytotoxic agents. The conditioning regimen may also include immunosuppression, antibodies, and irradiation. Other possible conditioning regimens include antibody-mediated conditioning (see, for example, 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 CART-mediated conditioning (see, for example, Arai et al., 26(5) Molecular Therapy 1181-1197 (2018); each of which is incorporated by reference in its entirety). Conditioning needs to be used to create space in the brain so that engineered HSC-derived microglia can migrate to deliver the protein of interest (recent gene therapy trials for ALD and MLD).The conditioning regimen is also designed to create a “space” niche to allow the transplanted cells to have a place in the body to engraft and proliferate. In hematopoietic stem cell transplantation, for example, the conditioning regimen creates a space niche in the bone marrow for the transplanted hematopoietic stem cells to develop. Without a conditioning regimen, the transplanted hematopoietic stem cells cannot develop. In some embodiments, an individual may receive a dose of cells, compositions, and / or pharmaceutical formulation of the present invention after treatment with a conditioning regimen.

[0326] The use of the cells described in the present invention for the treatment of a disease, disorder or condition is also included in the invention.

[0327] Certain modalities provide the disease, disorder or condition selected from cancer, Parkinson's disease, graft-versus-host disease (GvHD), autoimmune conditions, hyperproliferative disorder or condition, malignant transformation, liver conditions, genetic conditions including hereditary genetic defects, juvenile-onset diabetes mellitus and ocular compartment conditions.

[0328] In certain modalities, the disease, disorder or condition Petition 870250090928, dated 06 / 10 / 2025, page 114 / 189 106 / 134 affects at least one selected body system from among the muscular, skeletal, circulatory, nervous, lymphatic, respiratory, endocrine, digestive, excretory, and reproductive systems.

[0329] Effector cell: As used in this document, an “effector cell” refers to any cell or cell type that, when in contact with or in proximity to a thymic cell, acquires the ability to execute, initiate, or propagate a signal or trigger for cell death. “Contact or proximity” refers to sufficient spatiotemporal proximity to allow intrinsic or extrinsic cell signaling (e.g., cell-to-cell) or other communication or interaction.

[0330] Immune tolerance response: As used in this document, “immune tolerance response” refers to a class of immune response characterized by the reduced or eliminated tendency of the immune system to mount an activated immune response to at least one antigen.

[0331] Infectious agent antigen: As used in this document, the term “infectious agent antigen” refers to a class of antigens derived from agents or microorganisms that cause infectious diseases. Non-limiting examples of infectious agent antigens include viral antigens, bacterial antigens, protozoan antigens, prion antigens, and / or fungal antigens.

[0332] Lymphocyte: As used in this document, a “lymphocyte” encompasses the meanings and uses that one skilled in the art would understand the term to encompass and, furthermore, refers to a type of immune cell originating in the bone marrow that resides in lymphoid tissues or in the blood. In some modalities, lymphocytes undergo maturation in the thymus.

[0333] Negative: As used in this document, the term “negative” (which may be abbreviated as “-”), as used in this document with reference to the expression of the indicated cell marker, means that the cell does not express the indicated cell marker at a detectable level. Petition 870250090928, dated 06 / 10 / 2025, page 115 / 189 107 / 134

[0334] Neoantigen: As used in this document, the term “neoantigen” encompasses the meanings and uses that one skilled in the art would understand the term to encompass and, furthermore, refers to a class of tumor antigens that arise from tumor-specific mutations in expressed proteins.

[0335] Positive: As used in this document, the term “positive” (which may be abbreviated as “+”), with reference to the expression of the indicated cell marker, means that the cell expresses the indicated cell marker at any detectable level, which may include, for example, expression at a low (but detectable) level as well as expression at a high (hi) level.

[0336] Pre-T cell: As used in this document, a “pre-T cell” refers to a lymphocyte capable of maturing or differentiating into a T cell.

[0337] Autoantigen: As used in this document, the term “autoantigen” encompasses the meanings and uses that one skilled in the art would understand the term to encompass and, furthermore, refers to a class of antigens derived from one or more cells or cell types of a parent organism that induces an immune response in another organism but does not induce an immune response in a healthy parent organism from which it was derived.

[0338] Thymic origin or lineage: As used in this document, “thymic origin or lineage” refers to a cell with one or more phenotypic or genotypic markers associated with a thymus-derived cell or a cell destined to become a thymus cell. As used herein, the thymus may be an embryonic, fetal, or adult thymus.

[0339] Tumor antigen: As used in this document, the term “tumor antigen” encompasses the meanings and uses that one skilled in the art would understand the term to encompass and, furthermore, refers to a class of antigens derived from cancer cells.

[0340] Tumor-associated antigen (TAA): As used in this document, “tumor-associated antigen” refers to a class of tumor antigens derived from tumor cells that may also be derived from a Petition 870250090928, dated 06 / 10 / 2025, p. 116 / 189 108 / 134 or more non-tumor cells or cell types.

[0341] Tumor-specific antigen (TSA): As used in this document, “tumor-specific antigen” refers to a class of tumor antigen characterized by being specific to a particular tumor or cancer cell.

[0342] Variant: The term “variant,” as used in reference to a biomolecule, refers to a biomolecule related to or derived from a parent molecule. The variant may be, for example, a modified form, a truncated form, a mutant form, a homologous form, or another altered form of the parent molecule. The term variant may be used to describe polynucleotides or polypeptides.

[0343] Details of one or more embodiments of the invention are presented in the attached description below. Although any materials and methods similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred materials and methods are described herein. Other features, objects and advantages of the invention will become apparent in the description. In the description, singular forms also include the plural, unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which this invention pertains. In case of conflict, the present description shall prevail. Equivalents and Scope

[0344] Those skilled in the art will recognize or be able to verify, using only routine experimentation, many equivalents to the specific embodiments according to the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather as set forth in the appended claims.

[0345] In claims, articles such as “a / an”, “some” and “the” may mean one or more than one, unless otherwise indicated or Petition 870250090928, dated 06 / 10 / 2025, p. 117 / 189 109 / 134 unless the context makes otherwise evident. Claims or descriptions that include “or” among one or more members of a group are considered satisfied if one, more than one, or all members of the group are present, employed, or otherwise relevant to a particular product or process, unless otherwise indicated or otherwise evident from the context. The description includes embodiments in which exactly one member of the group is present, employed, or otherwise relevant to a particular product or process. The invention includes embodiments in which more than one or all members of the group are present, employed, or otherwise relevant to a particular product or process.

[0346] It is also noted that the term “comprising” is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term “comprising” is used in this document, the term “consisting of” is also encompassed and disclosed.

[0347] When intervals are provided, endpoints are included. Furthermore, it should be understood that, unless otherwise indicated or otherwise evident from the context and understanding of a person skilled in the art, the values ​​that are expressed as intervals may assume any specific value or subinterval within the intervals stated in different embodiments of the invention, down to the tenth of the unit of the lower limit of the interval, unless the context clearly determines otherwise.

[0348] In addition, it should be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. As such embodiments are considered known to those skilled in the art, they may be excluded even if the exclusion is not explicitly stated herein.Any particular embodiment of the compositions of the invention may be excluded from one or more claims, for any reason whatsoever, whether or not it relates to the existence of prior art.

[0349] It should be understood that the words that were used are words Petition 870250090928, dated 06 / 10 / 2025, pp. 118 / 189 110 / 134 of description and not of limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and essence of the invention in its broadest aspects.

[0350] Although the present invention has been described extensively and with some particularities with respect to the various embodiments described, it is not intended that it be limited to such particularities or embodiments or to any specific embodiment, but should be interpreted with reference to the appended claims, so as to provide the broadest possible interpretation of such claims in view of the state of the art and, therefore, to effectively encompass the intended scope of the invention. EXAMPLES EXAMPLE 1. PREPARATION OF THYMIC CELLS WITH ALGINATE ENCAPSULATION Expansion of the iPSC line

[0351] On day 0, iPSC lines (3 to 4 days in suspension; 300 to 400 microns in diameter) were treated with Accutase to prepare individual cells from aggregates. The cells were then resuspended in Stemscale medium (Thermo Fisher) containing 1% pen strep and 10 pM Y-27632 and placed on ice for 30 minutes before encapsulation. iPSC encapsulation by extrusion

[0352] The autoclaved alginate solution was prepared with 1.1% alginate, 0.2% gelatin, 10 mM HEPES, and 0.9% NaCl. Cells were resuspended in an alginate solution at a concentration of 200,000 cells / mL. The solutions were loaded into a 1 mL syringe fitted with a 27-gauge needle tip. The syringe was then placed in a syringe pump and extruded into a 100 mM CaCl2 gelling bath at a rate of 0.25 mL / minute. The alginate capsules were allowed to remain in the bath for an additional 5 minutes. The capsules were then washed with PBS and suspended in Stem Scale medium containing 1% pen strep and 10 pM of Petition 870250090928, dated 06 / 10 / 2025, pp. 119 / 189 111 / 134 Y-27632. The cells were cultured in this medium for 5 days before differentiation. Differentiation was performed using 3 mL of each differentiation medium per mL of capsules. Complete medium changes were made daily. Differentiation of iPSCs into definitive endoderm (DE)

[0353] On day 1, the cells were washed with PBS and resuspended in Medium A. Medium A was prepared to include the Baseline Medium: DMEM-F12, Activin A (100 ng / mL), CHIR99021 2μM, Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:100), PI-103 (25nM), and NEAA (1:400). On day 2, 1 ml of supernatant from each well was replaced with 1 ml of freshly prepared Medium A.

[0354] On day 3, the cells were washed with PBS and resuspended in Medium B. Medium B was prepared to include the Baseline Medium: DMEM-F12, Activin A (100 ng / mL), LDN193189 (200nM), PI-103 (25nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200) and NEAA (1:400).

[0355] 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 include the Baseline Medium: DMEM-F12, Activin A (100 ng / mL), LDN193189 (200nM), PI-103 (25nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200) and NEAA (1:400). Differentiation of the definitive endoderm (DE) in the endoderm of the foregut (AFE)

[0356] The 24-well plates were coated with Geltrex (1:100) and left at room temperature for one hour. On the sixth day, the supernatant and alginate capsules were transferred to 15 ml conical tubes. The alginate capsules were centrifuged at 250 G for 5 minutes at room temperature and the supernatant was aspirated. Alginate capsules were washed with PBS and resuspended in AFE medium and cultured between days 6 and 8. The AFE medium was prepared to include the following: Basal Medium: DMEM-F12, LDN193189 (200nM), SB431542 (10pM), FGF8b (50ng / ml), ascorbic acid (10pM), Pen Strep (1:200), B27 (without RA) (1:200), N2 (1:100), Glutamax (1:100), BME Petition 870250090928, dated 06 / 10 / 2025, pp. 120 / 189 112 / 134 (1:100) and NEAA (1:400).

[0357] On the seventh day, an equal amount of freshly prepared AFE media (500 μL) was added per well. On the eighth day, 50% of the supernatant was removed and freshly prepared AFE media (500 μL) was added to each well. Differentiation of the endoderm of the foregut (AFE) to the endoderm of the ventral pharynx (VPE)

[0358] On day 9, an equal amount of freshly prepared VPE1 medium (500 µL) was gently replaced without disturbing the alginate capsules. The VPE1 was prepared to include the following basal medium: DMEM-F12, SB431542 (10 µM), FGF8b (50 ng / ml), Retinoic acid (0.0 M), Ascorbic acid (10 µM), Insulin-Transferrin Selenium (ITS-G) (1:1000), KSR (0.05%), Pen strep (1:200), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), and NEAA (1:400). On day 10, an equal amount of freshly prepared VPE1 medium (500 µL) was added per well.

[0359] On day 12, an equal amount of newly created VPE2 medium (500µl) was gently replaced without disturbing the alginate capsules. The VPE2 medium was prepared to include the following basal medium: DMEM-F12, Noggin (50 ng / ml), CHIR99021 (2 pM), FGF8b (50 ng / ml), Retinoic acid (0.1 pM), Ascorbic acid (10 pM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), NEAA (1:400), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100)

[0360] On day 13, an equal amount of freshly prepared VPE2 medium (500 μθ) was added per well. Differentiation of the ventral pharyngeal endoderm (VPE) into thymic epithelial progenitors (TEP)

[0361] On day 14, an equal amount of freshly prepared TEP medium (500 μg) was gently replaced without disturbing the alginate capsules. Cells were cultured in TEP medium from days 14 to 18. The TEP medium was prepared to include the Basal Medium: DMEM-F12, FGF10 (50 ng / ml), BMP4 (50 ng / ml), Petition 870250090928, dated 06 / 10 / 2025, pp. 121 / 189 113 / 134 FGF8b (50 ng / ml), CHIR99021 (2μM), ascorbic acid (10μM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), Pen strep (1:200), B27 (without VIT A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400). On day 15, an equal amount of freshly prepared TEP medium (500µl) was added per well. On days 16 to 18, 50% of the supernatant was removed and freshly prepared TEP medium (500µl) was added per well.

[0362] From the 19th to the 22nd, the cells were cultured in TEC medium. On the day 19, an equal amount of newly created TEC medium (500 μm) was gently substituted without disturbing the alginate capsules. The TEC medium was prepared to include the following basal media: 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 (1:M), 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 (100X) and BME (100X). On day 20, the same amount of freshly prepared TEC medium (500µl) was added along with Plurisin#1 (20μM) (Groups - Untreated and Treated). On day 21, an equal amount of freshly prepared TEC medium (500µl) was gently replaced without disturbing the alginate capsules. REMOVAL OF ALGINATE TO COLLECT DIFFERENTIATED CELLS

[0363] 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 agitation at 100 rpm. The aggregates were collected by centrifugation (1,100 rpm for 4 minutes) and then treated with Accutase to dissociate into individual cells. Individual cells were collected by centrifugation (1,100 rpm for 4 minutes) and used for further testing. EXAMPLE 2. CHARACTERIZATION OF TEPS DERIVED FROM THE DIFFERENTIATION OF iPS CELLS ENCAPSULATED IN 3D ALGINATE SUSPENSION Petition 870250090928, dated 06 / 10 / 2025, pp. 122 / 189 114 / 134

[0364] Single-cell iPS cells were encapsulated in low-density alginate microspheres and cultured in 3D suspension. On day 4, aggregates of different sizes were observed, indicating that iPS cells can proliferate within the alginate and form colonies. Differentiation was induced as described in Example 1 by the addition of culture media for DE, AFE, VPE, TPE, and TEC. At the TEC stage (days 15–16), the aggregates were larger (approximately 600 microns) and fewer in number.

[0365] The expression of TEP markers was compared between iPSC aggregates that were differentiated into alginate capsules using 3D suspension culture and iPS differentiated under 2D conditions, and also after 2D culture and subsequent freezing and thawing. FOXN1 expression was observed in alginate TEC at a level comparable to that of two-dimensional culture conditions before and after being frozen and refrozen in an aggregated suspension culture in TEC medium for 5 days. FOXN1 was expressed at the highest level after freezing and thawing. HOXA3 and DLL4 expression was higher in alginate TEC, and only alginate TEC showed PAX1 expression. The data showed that iPS cells can be differentiated into 3D aggregates from iPS to TEC (Figure 1). EXAMPLE 3. DIFFERENTIATION OF CHITOSAN-COATED, ALGINATE-ENCAPSULATED iPSC AGGREGATES IN THYMIC CELLS USING 3D SUSPENSION CULTURE iPSC Line Expansion (Day -4 or Day -3)

[0366] An iPSC strain was thawed and cultured in medium Stemscale (Thermo Fisher) in six-well plates containing Pen Strep (1:100) and 10 pM Y-27632 in an incubator at 37 °C, 5% CO2, on a platform with agitation at 70 revolutions per minute (rpm). When the aggregates reached an average diameter of 300 to 400 µm (3 to 4 days), the expanded iPSCs were placed on ice for 30 minutes before encapsulation. Petition 870250090928, dated 06 / 10 / 2025, pp. 123 / 189 115 / 134 Preparation of solutions for encapsulation in chitosan-alginate.

[0367] A buffered saline solution containing 10 mM HEPES and 0.9% NaCl was prepared. An encapsulation solution containing 10 mM HEPES, 0.9% NaCl, 0.2% type A gelatin, 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.

[0368] Sterile glacial acetic acid was filtered through a 22 µm filter and added to sterile mineral oil to create a 0.4% glacial acetic acid and mineral oil mixture, which was then stirred at maximum speed for 1 to 2 minutes and autoclaved.

[0369] A 1.66% low molecular weight chitosan coating solution was prepared using 0.5 M HCl. It was allowed to dissolve for two hours while stirring at 1,000 rpm and then autoclaved.

[0370] A decapsulation solution was prepared containing 10 mM HEPES, 95 mM NaCl and 55 mM sodium citrate. Encapsulation of hiPSC aggregates in chitosan-coated alginate microspheres by emulsification.

[0371] Approximately 200,000 hiPS cells were resuspended in mL of encapsulation solution and vortexed for 20–30 seconds to create a homogeneous mixture. All 10 mL of this homogenized mixture were slowly added to 20 mL of mineral oil and stirred at 500 rpm for 12 minutes to create an emulsion. Under continuous stirring, 10 mL of a 0.4% glacial acetic acid and mineral oil mixture were added to the emulsion, and stirring was maintained for 8 minutes. After gelation was complete, 40 mL of DMEM / F12 medium were added and stirred for 1 minute to neutralize the pH. The emulsion was separated into two 50 mL conical tubes and centrifuged at 700 x g for 4 minutes to separate the oil and collect the cells. Petition 870250090928, dated 06 / 10 / 2025, pp. 124 / 189 116 / 134 capsules. The oil layer was removed and a vacuum trap was used to remove as much media as possible. The alginate beads were aspirated from each conical tube and then resuspended in 40 mL of buffered saline solution.

[0372] Separately, 9 mL of chitosan coating solution were combined with 21 mL of buffered saline to create 30 mL of chitosan saline solution. 10 mL of this chitosan saline solution were added to each conical tube to resuspend the hiPS-containing alginate microspheres. The resuspended alginate microspheres were slowly added to the remaining chitosan + buffered saline solution and stirred at 500 rpm for 1 minute. The solution was then filtered using 40 µm cell filters. The chitosan-coated alginate microspheres, which contained hiPSC aggregates, were washed with 10 mL of buffered saline and placed in shaker flasks with 24 mL of StemScale medium supplemented with 1% PenStrep and 2 mM Y-27632. The shaker-equipped flasks were placed on a shaking platform at 70 rpm and incubated for 3 to 4 days at 37 °C and 5% CO2 before the start of differentiation. PREPARATION OF CELL CULTURE MEDIA

[0373] Medium A was prepared with DMEM-F12 as basal medium and the following reagents: Activin A (100 ng / mL), CHIR99021 (2 uM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400).

[0374] Medium B was prepared with DMEM-F12 as basal medium and the following reagents: Activin A (100 ng / mL), LDN 193189 (200 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400).

[0375] AFE medium was prepared with DMEM-F12 as basal medium and the following reagents: LDN 193189 (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), Glutamax Petition 870250090928, dated 06 / 10 / 2025, pp. 125 / 189 117 / 134 (1:100).

[0376] VPE1 medium was prepared with DMEM-F12 as basal medium and the following reagents: SB431542 (10 uM), FGF8b (50 ng / mL), Retinoic acid (0.1 nM), Ascorbic acid (10 uM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400).

[0377] VPE2 medium was prepared with DMEM-F12 as basal medium and the following reagents: Noggin (50 ng / mL), CHIR99021 (2 uM), FGF8b (50 ng / mL), Retinoic acid (0.1 nM), Ascorbic acid (10 uM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), NEAA (1:400), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100) and N2 (1:100).

[0378] TEP medium was prepared with DMEM-F12 as basal medium and the following reagents: FGF10 (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (2 uM), ascorbic acid (10 uM), insulin-transferrin-selenium (ITSG) (1:1000), KSR (0.05%), PenStrep (1:200) and NEAA (1:400).

[0379] TEC medium was prepared with DMEM-F12 as 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 uM), ascorbic acid (10 uM) and insulin-transferrin-selenium (ITS-G) (1:1000).

[0380] TEC Freeze / Thaw (FT) medium was prepared with DMEM-F12 as 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 uM), ascorbic acid (10 uM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 without vitamin A (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400). Differentiation of chitosan-coated, alginate-encapsulated HIPSCS in definitive endoderm (DE)

[0381] On day 0, the hiPSCs were disaggregated using Accutase. 3 to 5 Petition 870250090928, dated 06 / 10 / 2025, pp. 126 / 189 118 / 134 million individual iPS cells were suspended in 6-well ultra-low adhesion plates along with Medium A and ROCK inhibitor Y-27632, and incubated at 37 °C and 5% CO2 at 70 rpm. Incubator conditions remained constant during the iPS differentiation steps described in this document, and cells placed on plates were returned to the incubator after each differentiation step unless expressly indicated otherwise.

[0382] On Day 1, 1 mL of supernatant was removed from each well of each 6-well plate and centrifuged. The pellet cells were washed with PBS and resuspended in 1 mL of pre-warmed Medium A and Y-27632 before being added back to the sixth well of each plate (well #6).

[0383] On Day 2, approximately 1.8 mL of the supernatant from each well was removed and 2 mL of pre-warmed Medium A (without Y-27632) was added to each well. The supernatant was centrifuged, and the pellet cells were washed with PBS and resuspended in 2 mL of Medium A (without Y-27632) before being added to the sixth well of each plate.

[0384] On day 3, cells and supernatant were collected in a 50 mL conical tube and left to stand. 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 plates were placed back in 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, and the tubes were gently vortexed and spun at 1,100 rpm for 4 minutes. The cells were resuspended in the required amount of Medium B (based on the number of plates) and evenly distributed into the wells of each plate.

[0385] 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 centrifuged and the residual cells were added to the sixth well of each plate. Petition 870250090928, dated 06 / 10 / 2025, pp. 127 / 189 119 / 134 (well #6). Differentiation of cells into endodermal cells of the foregut (AFE)

[0386] On day 6, the aggregates were collected along with the supernatant and left to stand. 1-2 mL of PBS was added to each well and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared AFE medium was added to each well and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of AFE medium and distributed evenly into the six-well plates.

[0387] On days 7 and 8, 1 mL of supernatant from each well was replaced with 1 mL of freshly prepared AFE medium. The 1 mL of supernatant from each well was rotated and removed, and the residual cells were resuspended in AFE medium and added back to the last well. DIFFERENTIATION OF FORE GUT ENDODERM CELLS (FGE) INTO VENTRAL PHARYNGEAL ENDODERM CELLS (VPE)

[0388] At the beginning of the ninth day, aggregates were collected with the supernatant in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE1 medium was added to each well and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE1 medium and distributed equally into six-well plates.

[0389] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of VPE1 medium and added to the sixth well of each plate. Petition 870250090928, dated 06 / 10 / 2025, pp. 128 / 189 120 / 134

[0390] On day 11, the aggregates were collected with the supernatant in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well, and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE2 medium was added to each well, and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells, and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE2 medium and distributed evenly into the six-well plates.

[0391] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of VPE2 medium and added back to the sixth well of each plate. Differentiation of the ventral pharyngeal endoderm (VPE) into thymic epithelial progenitors (TEP)

[0392] On day 13, the aggregates and supernatant were collected in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well, and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared TEP medium was added to each well, and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells, and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of TEP medium and distributed evenly into the six-well plates.

[0393] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of TEP medium and added to the sixth well of each plate. Petition 870250090928, dated 06 / 10 / 2025, pp. 129 / 189 121 / 134

[0394] On day 17, approximately 1.8 mL of the supernatant from each well was collected and 2 mL of pre-warmed TEP medium were added in its place. The collected supernatant was centrifuged and removed, and the residual cells were resuspended in TEP medium and added back to the last well (well #6). In cases of a large number of aggregates, the residual cells were added back to two or more wells.

[0395] On day 18, 1 mL of supernatant from each well was collected and 1 mL of pre-warmed TEP medium was added in its place. The collected supernatant was shaken and removed, and the residual cells were resuspended in pre-warmed TEP medium and added back to the last well (well #6). This step was repeated on day 19, day 20, day 21, and day 22. Chitosan-alginate removal for collecting and freezing differentiated cells.

[0396] On day 23, chitosan-coated alginate capsules were uncapped by suspending them for 10 minutes and shaking them at 100 rpm in the uncapping solution (55 mM sodium citrate, 10 mM HEPES, and 0.9% NaCl). TEP aggregates were collected, and the plate was 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 individual cells, and a 0.5–1.0 mL sample was removed for cell counting and qRT-PCR.

[0397] 1 to 2 mL of fresh TEP medium and 10 mL of medium were added. Pre-warmed DMEM-F12 was added to the cells, and they were agitated at 1,110 rpm for 4 minutes. 10 mL of fresh TEP medium was added, and the TEP cells were counted with a cell counter. The remaining cells were centrifuged, and 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). 6 to 10 million cells were included in each cryopreservation vial, and the vials were frozen in liquid nitrogen and stored at -80 °C. Petition 870250090928, dated 06 / 10 / 2025, pp. 130 / 189 122 / 134 Differentiation of thymic epithelial progenitors into thymic epithelial cells (TEP to TEC)

[0398] On day A, the TEPs were thawed and 1 mL of TEC Medium was added to Freeze / Thaw (FT) was added to each vial. The thawed cells were transferred to a conical tube and spun at 1,110 rpm for 4 minutes. The supernatant was removed and 6 mL of TEC FT medium (with Y27632 at 1:1000) was added to each tube to resuspend the TEPs. TEC FT medium with Y27632 at 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 the incubator (shake at 70 rpm, 37 °C, 5% CO2).

[0399] 24 hours later, 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 centrifuged and aspirated, and 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 on each of the following three days: (days C, D and E).

[0400] On Day F, 1 mL of supernatant was removed from the well of each plate and 1 mL of TEC FT medium pre-warmed with PluriSIn (20 uM) was added to each well. The supernatant was centrifuged and aspirated, and the residual cells were resuspended in TEC FT medium with PluriSIn (20 uM) and added back to the last well (well #6).

[0401] 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 to 1 mL) were collected and used for cell counting, flow cytometry, qRT-PCR, and CFU analyses. For qPCR analyses, DPBS medium was removed from the corresponding sample in 0.5 to 1.0 mL portions, and 200 to 400 µL of RLT buffer was added. TEC cells in the qPCR sample were lysed and stored at -80°C for RNA extraction. For flow cytometry analyses, the Petition 870250090928, dated 06 / 10 / 2025, pp. 131 / 189 123 / 134 TEC aggregates in the 0.5 to 1.0 mL sample were washed once with DPBS and spun at 1,100 rpm for 4 minutes. The supernatant was aspirated and 1 to 2 mL of Accutase was added to each tube. After a 4-minute incubation, the TEC aggregates were dissociated, washed with 1 mL of DPBS, and then centrifuged. The Accutase / supernatant was aspirated and DPBS was added to the cells before they were placed on ice for staining.

[0402] Figure 2A presents a schematic illustrating the differentiation process of chitosan-coated alginate-encapsulated hiPSC aggregates using 3D suspension culture (“3D Differentiation of Alginate Aggregates”). The typical FOXN1 expression level by these TECs on Day G (after being frozen, thawed, and cultured for 7 days in TEC FT medium), as measured by GADPH-normalized qRT-PCR at a cell concentration of 200,000 TEC cells / mL, is given in the right-hand column shown in Figure 2B (0.0002). This level is approximately five 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.

[0403] The flow cytometry results for the TEC aggregates are presented in Figure 3, Figure 4, Figure 5 and Figure 6. Three replicates were performed (Staining 1, Figure 3A to 3N; Staining 2, Figure 4A to 4M; Staining 3, Figure 5A to 5O; Staining 4, Figure 6A to 6F).

[0404] The expression levels of the following genes were evaluated for TEC cells on Day 23, before freezing (the leftmost columns represent 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 the TEC cells that were frozen on Day 23 were thawed and cultured for 7 days in TEC FT medium); these results are shown in the rightmost columns in Figure 7: Oct4, NANOG, HOXA3, EPCAM, PAX9, and FOXN1 (“AEMF-200K Petition 870250090928, dated 06 / 10 / 2025, pp. 132 / 189 124 / 134 090123-FT1”). These results demonstrate that differentiated TECs possess thymopoietic capacity to support T cell development in vitro. EXAMPLE 4. INTRAMUSCULAR TRANSPLANTATION OF DIFFERENTIATED TEPs SUPPORTS HUMAN THYMOPOIESIS IN A HUMANIZED ANIMAL MODEL

[0405] Human iPSC cells encapsulated in alginate and coated with chitosan were cultured in 3D suspension culture and differentiated into TEP cells, as described in Example 4 (before the freeze / thaw step). Approximately 0.83 million TEP cells plus 300K mesenchymal stem cells were injected intramuscularly into humanized NOD / SCID / IL2RgNull (NSG) MHC-I / II double knockout mice (Jackson Laboratories) (n=3, identified by numbers 235, 236 and 237). Humanization was performed as follows: mice were sublethally irradiated, followed by injection of CD34+ hematopoietic stem cell progenitors derived from human umbilical cord blood; Two weeks later, 0.83 million TEP cells were injected intramuscularly into the quadriceps of each mouse (along with 300,000 mesenchymal stem cells).Humanized NSG mice exhibit inefficient human T cell development due to residual thymic tissue. To create an athymic background in NSG mice, the mice can be thymectomized, but this step was avoided by using an NSG variant strain in which both MHC Class I and MHC Class II genes were knocked out. Since MHC is required for thymic function, the double knockout strain creates a functionally athymic background, and the NSG mouse does not produce FOXN1.

[0406] The efficiency of thymopoiesis and T cell generation were assessed every three weeks by flow cytometry in order to track the emergence of T cells and their immunophenotypes. Human hematopoietic stem cell (hCD45) and human T cell markers, such as CD3 and Petition 870250090928, dated 06 / 10 / 2025, pp. 133 / 189 125 / 134 TCRae, as shown in Figures 8A to 8N. At week three, none of the mice expressed any CD3+ (Figures 8G, 8I, 8K). At week 16, the cells expressing CD3. Thirteen weeks after intramuscular injection of TEP, human CD3+ TCRαβ+ T cells ranged from 2% to 18% of the total human blood cells (CD45+), and at 16 weeks, the range was from 15% to 43%. Importantly, no sham transplanted mice (negative control - not shown) developed detectable T cells in the blood, verifying that the mice were effectively athymic and therefore an excellent model for testing the thymopoietic capabilities of iPS-TEPs. These results demonstrated that human iPS-TEPs can efficiently induce the development of human T cells in vivo. EXAMPLE 5. DIFFERENTIATION OF INDIVIDUAL NON-ENCAPSULATED hiPS CELLS INTO THYMIC CELLS USING 3D SUSPENSION CULTURE PREPARATION OF CELL CULTURE MEDIA

[0407] Medium A was prepared with DMEM-F12 as basal medium and the following reagents: Activin A (100 ng / mL), CHIR99021 (2 uM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400) and B27 without vitamin A (1:400).

[0408] Medium B was prepared with DMEM-F12 as basal medium and the following reagents: Activin A (100 ng / mL), LDN 193189 (200 nM), Insulin-Transferrin-Selenium (ITS-G) (1:1000), KSR (0.05%), PenStrep (1:200), PI-103 (25 nM), NEAA (1:400) and B27 without vitamin A (1:400).

[0409] AFE medium was prepared with DMEM-F12 as basal medium and the following reagents: LDN 193189 (200 nM), SB431542 (10 uM), FGF8b (50 ng / mL), ascorbic acid (10 uM), PenStrep (1:200), B27 (without retinoic acid) (1:200), N2 (1:100), BME (1:100), NEAA (1:400), Glutamax (1:100).

[0410] VPE1 medium was prepared with DMEM-F12 as basal medium and the following reagents: SB431542 (10 uM), FGF8b (50 ng / mL), Retinoic acid (0.1 uM), Ascorbic acid (10 uM), Insulin-Transferrin-Selenium (ITS-G) Petition 870250090928, dated 06 / 10 / 2025, pp. 134 / 189 126 / 134 (1:1000), KSR (0.05%), PenStrep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100), NEAA (1:400).

[0411] VPE2 medium was prepared with DMEM-F12 as basal medium and the following reagents: Noggin (50 ng / mL), CHIR99021 (2 uM), FGF8b (50 ng / mL), retinoic acid (0.1 uM), ascorbic acid (10 uM), insulin-transferrinselenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), NEAA (1:400), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100) and N2 (1:100).

[0412] TEP medium was prepared with DMEM-F12 as basal medium and the following reagents: FGF10 (50 ng / mL), BMP4 (50 ng / mL), FGF8b (50 ng / mL), CHIR99021 (2 uM), ascorbic acid (10 uM), insulin-transferrin-selenium (ITSG) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400).

[0413] TEC medium was prepared with DMEM-F12 as 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 uM), ascorbic acid (10 uM) and insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 (without vitamin A) (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400).

[0414] The Freeze / Thaw (FT) TEC medium was prepared with 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 uM), ascorbic acid (10 uM), insulin-transferrin-selenium (ITS-G) (1:1000), KSR (0.0025%), PenStrep (1:200), B27 without vitamin A (1:200), Glutamax (1:100), BME (1:100), N2 (1:100) and NEAA (1:400). Differentiation of individual non-encapsulated hiPSCs in definitive endoderm (DE)

[0415] On day 0, the iPSCs were thawed and disaggregated with Accutase. 3 to 5 million individual iPS cells were suspended in 6-well ultra-low adhesion plates along with Medium A. Petition 870250090928, dated 06 / 10 / 2025, pp. 135 / 189 127 / 134 and the ROCK inhibitor Y-27632, and incubated at 37 °C and 5% CO2 at 70 rpm. Incubator conditions remained constant during the iPS differentiation steps described in this document, and cells placed on plates were returned to the incubator after each differentiation step, unless expressly indicated otherwise.

[0416] On Day 1, 1 mL of supernatant was removed from each well of each 6-well plate and centrifuged. The pellet cells were washed with PBS and resuspended in 1 mL of pre-warmed Medium A and Y-27632 before being added back to the sixth well of each plate (well #6).

[0417] On Day 2, approximately 1.8 mL of the supernatant from each well was removed and 2 mL of pre-warmed Medium A (without Y-27632) was added to each well. The supernatant was centrifuged, and the pellet cells were washed with PBS and resuspended in 2 mL of Medium A (without Y-27632) before being added to the sixth well of each plate.

[0418] On day 3, cells and supernatant were collected in a 50 mL conical tube and left to stand. 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 plates were placed back in 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, and the tubes were gently vortexed and spun at 1100 rpm for 4 minutes. Cells were resuspended in the required amount of Medium B (based on the number of plates) and evenly distributed into the wells of each plate.

[0419] 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 centrifuged and the residual cells were added to the sixth well of each plate (well #6). Differentiation of cells in the endoderm of the foregut (AFE) Petition 870250090928, dated 06 / 10 / 2025, pp. 136 / 189 128 / 134

[0420] On day 6, the aggregates were collected along with the supernatant and left to stand. 1-2 mL of PBS was added to each well, and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared AFE medium was added to each well, and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells, and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of AFE medium and distributed evenly into the six-well plates.

[0421] On days 7 and 8, 1 mL of supernatant from each well was replaced with 1 mL of freshly prepared AFE medium. The 1 mL of supernatant from each well was rotated and removed, and the residual cells were resuspended in AFE medium and added back to the last well. Differentiation of the endoderm of the foregut (AFE) to the endoderm of the ventral pharynx (VPE)

[0422] At the beginning of the ninth day, the aggregates were collected with the supernatant in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well, and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared VPE1 medium was added to each well, and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells, and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE1 medium and distributed evenly into the six-well plates.

[0423] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of VPE1 medium and added to the sixth well of each plate.

[0424] On day 11, the aggregates were collected with the supernatant in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well and the remaining aggregates were collected in a tube. Petition 870250090928, dated 06 / 10 / 2025, pp. 137 / 189 129 / 134 new. 1 mL of freshly prepared VPE2 medium was added to each well and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of VPE2 medium and distributed equally into the six-well plates.

[0425] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of VPE2 medium and added back to the sixth well of each plate. Differentiation of the ventral pharyngeal endoderm (VPE) into thymic epithelial progenitors (TEP)

[0426] On day 13, VPE aggregates, along with the supernatant, were collected in 50 mL conical tubes and left to stand. 1 to 2 mL of PBS was added to each well, and the remaining aggregates were collected in a new tube. 1 mL of freshly prepared TEP medium was added to each well, and the plates were placed back in the incubator. The supernatant was aspirated from the settled cells, and 20 mL of PBS was added before spinning the cells at 1,100 rpm for 4 minutes. The pelleted cells were resuspended in the required amount of TEP medium and distributed evenly into six-well plates.

[0427] 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 centrifuged and removed, and the residual cells were resuspended in the required amount of TEP medium and added to the sixth well of each plate.

[0428] On day 17, approximately 1.8 mL of supernatant from each well was collected and 2 mL of pre-warmed TEC medium were added in its place. The collected supernatant was centrifuged and removed, and the residual cells were Petition 870250090928, dated 06 / 10 / 2025, pp. 138 / 189 130 / 134 resuspended in the TEC medium and added back to the last well (well #6). In cases of a large number of aggregates, the residual cells were added back to two or more wells.

[0429] On day 18, 1 mL of supernatant from each well was collected and 1 mL of pre-warmed TEC medium was added in its place. The collected supernatant was shaken and removed, and the residual cells were resuspended in pre-warmed TEC medium and added back to the last well (well #6). This step was repeated on day 19, day 20, day 21, and day 22. Collection, sampling, and freezing of differentiated cells.

[0430] On day 23, TEP aggregates were collected and the plate was 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 individual cells, and a 0.5–1.0 mL sample was removed for cell counting and qRTPCR.

[0431] 1-2 mL of fresh TEC medium and 10 mL of medium were added. Pre-warmed DMEM-F12 was added to the cells, and they were agitated at 1,110 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 centrifuged, and 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). 6 to 10 million cells were included in each cryopreservation vial, and the vials were frozen in liquid nitrogen and stored at -80 °C. Differentiation of thymic epithelial progenitors into thymic epithelial cells (TEP to TEC)

[0432] On day A, the TECs were thawed and 1 mL of TEC Medium was added to Freeze / Thaw (FT) was added to each vial. The thawed cells were transferred to a conical tube and spun at 1,110 rpm. Petition 870250090928, dated 06 / 10 / 2025, pp. 139 / 189 131 / 134 for 4 minutes. The supernatant was removed and 6 mL of TEC FT medium (with Y27632 at 1:1000) was added to each tube to resuspend the TECs. TEC FT medium with Y27632 at 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 the incubator (shake at 70 rpm, 37 °C, 5% CO2).

[0433] 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 centrifuged and aspirated, and the residual cells were resuspended in TEC FT medium and added back to the last well (well #6) of each plate.

[0434] On Day F, 1 mL of supernatant was removed from the well of each plate and 1 mL of TEC FT medium pre-warmed with PluriSIn (20 uM) was added to each well. The supernatant was centrifuged and aspirated, and the residual cells were resuspended in TEC FT medium with PluriSIn (20 uM) and added back to the last well (well #6).

[0435] 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 to 1 mL) were collected and used for cell counting, flow cytometry, qRT-PCR, and CFU analyses. For qPCR analyses, DPBS medium was removed from the corresponding 0.5 to 1.0 mL sample and 200 to 400 µL of RLT buffer was added. TEC cells in the qPCR sample were lysed and stored at -80°C for RNA extraction. For flow cytometry analyses, TEC aggregates in the 0.5 to 1.0 mL sample were washed once with DPBS and spun at 1,100 rpm for 4 minutes. The supernatant was aspirated and 1 to 2 mL of Accutase was added to each tube. After a 4-minute incubation, the TEC aggregates were dissociated, washed with 1 mL of DPBS, and then centrifuged.Accutase / supernatant was aspirated and DPBS was added to the cells before they were placed on ice for staining. Petition 870250090928, dated 06 / 10 / 2025, pp. 140 / 189 132 / 134

[0436] Figure 9A illustrates the hiPSC to TEC differentiation process described in this example, in which individual non-encapsulated cells were cultured in 3D suspension. Successful growth and differentiation into thymic epithelial precursor cells were achieved, as illustrated in the representative micrographs at the bottom of Figure 9A and in Figure 9B (showing an enlarged version of the far right micrograph in Figure 9A, when the thymic cells were at Stage 5 of the differentiation process; scale bar = 100 µm).Thymic cells produced from hiPSCs differentiated as individual cells in 3D suspension culture expressed FOXN1 levels in vitro (ranging from 0.0019 to 0.0022) that were approximately 19 to 22 times higher than the threshold level (0.0001) required to achieve sufficient T cell production in vivo, once the thymic cells were transplanted into mice as in Example 6 (as illustrated by the data in Figure 10A). The FOXN1 expression level (shown in Figure 9C) was determined using qRT-PCR in thymic cell samples at a concentration of approximately 200,000 thymic cells per mL, with FOXN1 expression normalized to GADPH expression. As shown in Figure 9G, differentiation of non-encapsulated individual cells using 3D suspension culture was successful when B27 was added at the DE stage.

[0437] The expression levels of the following genes were also evaluated for TEP cells on Day 23 (before freezing) and again on Day G (after being frozen, thawed, and cultured for 7 days 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 indicated that differentiated TECs exhibit thymopoietic capacity to support T cell development in vitro.

[0438] In vitro expression levels of FOXN1 when hiPSCs were differentiated as individual 3D cells in a bioreactor according to Petition 870250090928, dated 06 / 10 / 2025, pp. 141 / 189 133 / 134 the process illustrated in Figure 9D was also above the limit of 0.0001, as shown in Figure 9F. EXAMPLE 6. INTRAMUSCULAR TRANSPLANTATION OF DIFFERENTIATED TEPs DEMONSTRATES THAT IN VITRO FOXN1 EXPRESSION LEVELS ARE RELATED TO IN VIVO T CELL PRODUCTION

[0439] Non-encapsulated human iPSC cells were differentiated into TECs using a hybrid (2D / 3D) protocol, as illustrated in FIGS. 10A and 10B, using the same culture media as in Example 5.

[0440] Approximately 1 million TEP cells plus 300,000 mesenchymal stem cells were injected intramuscularly into NOD / SCID / IL2RgNull (NSG) humanized mice with MHC-I / II double knockout (Jackson Laboratories).

[0441] The efficiency of thymopoiesis and T cell generation were assessed every three weeks by flow cytometry in order to monitor the emergence of T cells and their immunophenotypes. Markers for human hematopoietic stem cells (hCD45) and human T cells (CD4, CD8, and TCRae) were observed, as shown in Figures 10C and 10D. The results shown in Figure 10C are representative of the results obtained in similar experiments when TECs expressed FOXN1 in vitro at a level higher than the 0.0001 threshold that was considered necessary to obtain high levels of T cells in vivo. Specifically, the TECs in Figure 10C expressed FOXN1 at a level 8 times higher 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 that received these TECs (see, for example, 181, 182, and 183 in Figure 10C). In comparison, when TECs that expressed FOXN1 in vitro at a level below the 0.0001 threshold were transplanted, the levels of total T cells, CD4+ T cells, and CD8+ T cells were significantly lower. Petition 870250090928, dated 06 / 10 / 2025, pp. 142 / 189 134 / 134 FOXN1 expression in vitro was determined using qRT-PCR normalized for GADPH expression. Each row (180 to 184 in Figure 10C and 190 to 194 in Figure 10D) represents a different mouse from the same strain.

[0442] Although the invention has been described with reference to currently preferred embodiments, it should be understood that various modifications can be made without departing from the essence of the invention. Consequently, the invention is limited only by the following claims.

Claims

CLAIMS 1. A method for differentiating pluripotent stem cells into thymic cells, characterized in that it comprises: a) encapsulating pluripotent stem cells in a polymer; b) differentiating pluripotent stem cells into definitive endoderm (DE) cells; c) culturing DE cells and differentiating DE cells into foregut endoderm (AFE) cells by contacting or incubating DE cells with a BMP inhibitor, a TGFβ inhibitor, an FGF, ascorbic acid, or a combination thereof; d) culturing AFE cells and differentiating foregut cells into ventral pharyngeal endoderm (VPE) cells, which comprises: i) contacting or incubating AFE cells in a first VPE medium containing ascorbic acid, retinoic acid, FGF, a TGFβ inhibitor, or a combination thereof;and ii) contact or incubate AFE cells in a second VPE medium that includes a Noggin, a WNT activator, an FGF, a retinoic acid, an ascorbic acid, or a combination thereof; and ee) culture VPE cells and differentiate VPE cells into thymic cells by contacting or incubating VPE cells with an ascorbic acid, an FGF, a BMP, a WNT activator, or a combination thereof; wherein thymic cells are thymic epithelial progenitor cells (TEPs) and / or thymic epithelial cells (TECs).

2. Method for differentiating pluripotent stem cells into thymic cells, characterized in that it comprises the steps of: a) differentiating pluripotent stem cells into definitive endoderm (DE) cells; Petition 870250090928, dated 06 / 10 / 2025, page 144 / 189 2 / 9 b) culturing DE cells and differentiating DE cells into foregut endoderm (FGE) cells by contacting or incubating DE cells with a BMP inhibitor, a TGFe inhibitor, an FGF, an ascorbic acid, or a combination thereof; c) Cultivate AFE cells and differentiate foregut cells into ventral pharyngeal endoderm (VPE) cells by: i) contacting or incubating AFE cells in a VPE first medium containing ascorbic acid, retinoic acid, an FGF, a TGFβ inhibitor, or a combination thereof;and ii) contact or incubate AFE cells in a second VPE medium that includes a Noggin, a WNT activator, an FGF, a retinoic acid, an ascorbic acid, or a combination thereof; and ed) culture VPE cells and differentiate VPE cells into thymic cells by contacting or incubating VPE cells with an ascorbic acid, an FGF, a BMP, a WNT activator, or a combination thereof; wherein thymic cells are thymic epithelial progenitor cells (TEPs) and / or thymic epithelial cells (TECs).

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

4. Method, according to any one of claims 1 to 3, characterized in that the differentiation of pluripotent stem cells into DE cells comprises: a) contacting or culturing the pluripotent stem cells in a first growth medium, wherein this first growth medium comprises Activin A, PI-103, CHIR99021 or a combination thereof; and Petition 870250090928, dated 06 / 10 / 2025, page 145 / 189 3 / 9 b) culturing the pluripotent stem cells in a second growth medium to generate definitive endodermal cells, wherein the second growth medium comprises: Activin A, a BMP inhibitor, PI103, CHIR99021 or a combination thereof.

5. Method, according to any one of claims 1 to 4, characterized in that the first VPE medium further comprises a WNT inhibitor.

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

7. A method according to any one of claims 1 and 3 to 6, characterized in that the polymer comprises a biogenic polymer, a synthetic polymer, a composite polymer, a hydrogel, or a crosslinked polymer.

8. Method, according to any one of claims 1 and 3 to 7, characterized in that the polymer is alginate.

9. Method, according to any one of claims 1 to 8, characterized in that the BMP inhibitor is LDN193189.

10. Method, according to any one of claims 1 to 9, characterized in that the TGFe inhibitor is SB431542.

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

12. Method, according to any one of claims 1 to 11, characterized in that the WNT activator is CHIR99021.

13. Method, according to any one of claims 1 to 12, characterized in that the BMP is BMP2, BMP4 or a combination thereof.

14. Method, according to any one of claims 1 to 13, characterized in that the interleukin is IL22. Petition 870250090928, dated 06 / 10 / 2025, pp. 146 / 189 4 / 9 15. Method, according to any one of claims 1 to 14, characterized in that the WNT inhibitor is IWR-1.

16. Method, according to any one of claims 10 to 15, characterized in that the BMP inhibitor is LDN193189.

17. Method according to claim 5, characterized in that the SHH inhibitor is SANT-1.

18. Method, according to any one of claims 1 to 17, characterized in that pluripotent stem cells, DE cells, AFE cells, VPE cells or thymic cells are cultured in 3D culture.

19. Method, according to any one of claims 1 to 18, characterized in that pluripotent stem cells, DE cells, AFE cells, VPE cells or thymic cells are cultured as aggregates in suspension.

20. Method, according to any one of claims 1 to 19, characterized in that the method is carried out for about 15 to 30 days.

21. Method, according to any one of claims 1 to 20, characterized in that the method is carried out for about 18 to 25 days.

22. A method, according to any one of claims 1 to 21, characterized in that pluripotent stem cells are differentiated into definitive endodermal cells in about 5 days.

23. Method, according to any one of claims 1 to 22, characterized in that DE cells are differentiated into AFE cells for about 2 to 3 days.

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

25. Method, according to any one of claims 1 to 24, Petition 870250090928, dated 06 / 10 / 2025, pp. 147 / 189 5 / 9 characterized in that AFE cells are cultured in the second VPE medium for about 2 to 3 days.

26. Method, according to any one of claims 1 to 25, characterized in that VPE cells are differentiated into thymic cells for about 3 to 6 days.

27. Method, according to any one of claims 1 to 26, characterized in that TEPs are differentiated into TECs by about 4 days.

28. Method, according to any one of claims 1 or 3 to 27, characterized in that the polymer is removed from the TEPs.

29. Method, according to any one of claims 1 or 3 to 28, characterized in that the polymer is removed from the TECs.

30. Thymic cell population, characterized in that it is prepared according to the method as defined in any one of claims 1 to 29.

31. Pharmaceutical composition, characterized in that it comprises a population of thymic cells, as defined in claim 30, and at least one excipient.

32. A method of treating or preventing a condition in an individual, characterized in that it comprises administering to the individual the pharmaceutical composition as defined in claim 31.

33. Method, according to claim 32, characterized in that the condition is a condition associated with an absence, decline, or aberrant functioning of the individual's thymus, an immunodeficiency, a cancer, an autoimmune disease, an infectious disease, or graft-versus-host disease (GvHD).

34. Composition, characterized in that it comprises: a) a thymic cell population, wherein the thymic cell population comprises one or more of the following: iTEC, mTECs, corneocyte-like mTEC, cTEC-high, cTEC-low, mTEC-low; and Petition 870250090928, dated 06 / 10 / 2025, page 148 / 189 6 / 9 b) a thymic support system (TSS).

35. Composition according to claim 34, characterized in that the thymic cell population is prepared by differentiating iPS cells into thymic cells.

36. Composition according to claim 35, characterized in that the TSS comprises a polymer.

37. Composition according to claim 36, characterized in that the polymer is either a biogenic polymer or a synthetic polymer.

38. Composition, according to any one of claims 31 and 34 to 37, characterized in that the polymer is a biogenic polymer, and in that the biogenic polymer is a polypeptide-based biogenic polymer, a polynucleotide-based biogenic polymer or a polysaccharide-based polymer.

39. Composition according to claim 38, characterized in that the polymer is a polypeptide-based polymer, said polypeptide-based polymer being selected from the group consisting of collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin and actin.

40. Composition according to claim 38, characterized in that the polymer is a polysaccharide-based polymer, said polysaccharide-based polymer being selected from the group consisting of chitin, chitosan, alginate, hyaluronic acid, cellulose, agarose, starch, cellulose, dextran, hyaluronic acid, glycogen and glycosaminoglycans.

41. Composition according to claim 37, characterized in that the polymer is a synthetic polymer, said synthetic polymer being selected from the group consisting of polycaprolactone, polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, poly(ethylene oxide), polyethylene glycol, polyurethane, a poly(siloxane), a poly(ethylene), a poly(vinylpyrrolidone), a poly(2-hydroxyethyl methacrylate), a poly(N-vinylpyrrolidone), a poly(methyl methacrylate), a poly(vinyl alcohol), a poly(acrylic acid), a Petition 870250090928, dated 06 / 10 / 2025, page. 149 / 189 7 / 9 polyacrylamide, a poly(ethylene-co-vinyl acetate), a poly(ethylene glycol), a poly(methacrylic acid), polyhydroxybutyrate (PHB), polypropylene fumarate (PPF), a polyvinyl alcohol (PVA), a polypropylene carbonate, a polyanhydride, a polyphosphazene, a polygermane, a polyorthoester, a polyester, a polyamide, a polyolefin, a polycarbonate, a polyaramid,a polyimide, 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-(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), an acrylamide / acrylic acid copolymer, linear cationic polyallyl ammonium chloride and poly(N-isopropylacrylamide) (PNIPAM).

42. Composition, according to any one of claims 34 to 41, characterized in that the polymer forms a hydrogel.

43. Composition according to any one of claims 34 to 42, characterized in that the polymer is cross-linked.

44. Composition, according to any one of claims 34 to 43, characterized in that the TSS further comprises one or more extracellular matrix components and an agent.

45. Composition according to claim 44, characterized in that the extracellular matrix component is an extracellular matrix protein, or a region or part thereof.

46. ​​Composition according to claim 45, characterized in that the extracellular matrix protein is fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrobrin, merosin, ankalin, chondronectin, binding protein, bone sialoacid protein, osteocalcin, osteopontin, epinectin, hyaluronectin, unjulin, epiligrin or carinin.

47. Composition, according to any one of claims 44 to 46, characterized in that the extracellular matrix component is a peptide derived from extracellular matrix protein.

48. Composition according to claim 47, characterized in that the peptide is SEQ ID NO: 9-18.

49. Composition according to claim 48, characterized in that the agent is either a biological agent or a chemical agent.

50. Composition according to claim 49, characterized in that the biological agent is a ligand, an immune modulator or a hormone.

51. Composition according to claim 34, characterized in that it further comprises a population of supporting cells, said supporting cells comprising one or more myelin cells, a myoid cell, a neuroendocrine cell, a Tuft cell, an ionocyte, an endothelial cell, a mesenchymal stem cell or a fibroblast.

52. Composition according to claim 34, characterized in that it further comprises a population of effector cells.

53. Composition according to claim 34, characterized in that it further comprises a population of stem cells.

54. Composition, according to any one of claims 31 and 34 to 53, characterized in that the level of FOXN1 production in vitro by a sample of the thymic cell population with a concentration of about 200,000 thymic cells per milliliter is greater than 0.0001 when measured by qRT-PCR and normalized with respect to GADPH.

55. A method of treating or preventing a condition in an individual, characterized by the fact that it comprises administering to the individual a composition, as defined in any of the claims Petition 870250090928, dated 06 / 10 / 2025, pp. 151 / 189 9 / 9 31 and 34 to 54.

56. Method, according to claim 55, characterized in that the condition is associated with the absence, decline, or aberrant functioning of the individual's thymus, such as Di George syndrome, thymoma (such as type A thymoma or type B thymoma), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymic cancer, thymic atrophy (such as age-related thymic atrophy), thymic cyst, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, thymic irradiation, myasthenia gravis, thymic carcinoma, thymic hyperplasia, thymic irradiation, or decline in thymic function associated with age or infection.

57. Method according to claim 55, characterized in that the individual underwent a thymectomy surgery.

58. Method according to claim 55, characterized in that the condition is an immunodeficiency.

59. Method according to claim 55, characterized in that the condition is cancer.

60. Method, according to claim 55, characterized in that the condition is an autoimmune disease.

61. Method according to claim 55, characterized in that the condition is an infectious disease.

62. Method according to claim 55, characterized in that the condition is graft-versus-host disease (GvHD).

63. A method for improving the acceptance of an organ transplant and / or preventing the rejection of an organ transplant in an individual receiving an organ transplant, characterized in that it comprises administering to the individual a composition as defined in any one of claims 31 and 34 to 54.

Images