Hypoimmune engineering for universal IPSC-derived cells

Abstract

The present invention discloses a method for generating thymic epithelial progenitor (TEP) cells, hematopoietic stem cells (HSCs), adult tissue progenitor / stem cells, or other progenitor cells derived from human-induced pluripotent stem cells (iPSCs) to overexpress PD-L1 and / or HLA-G. This innovative approach aims to reduce allogeneic rejection by donor immune cells in immunocompetent recipients. By enhancing the immune-modulatory properties of iPSC-derived cells through the overexpression of PD-L1 and / or HLA-G, the invention seeks to improve the engraftment and functionality of these cells. This strategy holds significant potential for restoring thymus function and inducing immune tolerance, offering therapeutic benefits for patients with thymus defects, immune dysfunction, or requiring enhanced immune tolerance for graft acceptance.

Description

PATENT ATTORNEY DOCKET NO. THYM1160-2WO HYPOIMMUNE ENGINEERING FOR UNIVERSAL IPSC-DERIVED CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under U.S.C. § 119 (e) of U.S. Provisional Patent Application Serial No.: 63 / 652,390 filed on May 28, 2024 and U.S. Provisional Patent Application Serial No.: 63 / 781,817 filed on April 1, 2025. The contents of the prior applications are considered part of and are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates generally to induced pluripotent stem cells (iPSCs) and more specifically to methods for generating iPSCs-derived cells to overexpress PD-L1 and / or HLA-G to reduce allogeneic rejection by donor immune cells in recipients. BACKGROUND INFORMATION

[0003] Allogeneic cell therapy derived from induced pluripotent stem cells (iPSCs) holds great promise for treating diseases caused by the loss of specialized cell populations. However, ensuring the engraftment, survival, and long-term functionality of the transplanted cells remains a significant challenge, even with the use of immunosuppression. While patient- specific autologous approaches offer a potential solution, they are severely limited by scalability issues and batch-to-batch variability. Encapsulating the cell therapy products has also been explored but has been hindered by changes in functionality or toxicity. Recently, the development of immune-evasive iPSCs for creating “hypoimmune” cell therapies has shown remarkable potential in preclinical animal models, offering a promising avenue for overcoming the limitations of current allogeneic cell therapy approaches.

[0004] Using stem cells to replace lost or damaged tissue is one of the most promising applications of stem cell research. Thymic epithelial cells (TECs) are among the most intriguing and clinically relevant cell types that human pluripotent stem cells have yet to successfully generate. It has been demonstrated that thymic epithelial progenitor cells (TEPs), the precursors to TECs, derived from human-induced pluripotent stem cells (iPSCs), which are referred to herein as iPSC-TEPs, can be engrafted in animals, survive, and produce functional T cells in vivo. The thymus is a critical organ in the immune system that regulates and develops T cells, essential for fighting infection, disease, and mounting effective responses to vaccines. 1 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO As part of the natural aging process, the functional thymus begins to shrink, and its ability to produce naive T cells decreases, leading to immune dysfunction and disease. For children born without a thymus, those with thymus defects, and elderly patients with failing immune function, restoring thymus function could significantly improve their health and quality of life. The thymus supports the development of functional T cells and establishes immune tolerance by eliminating autoreactive T cell subsets. These critical functions are mediated by TECs, the main component of the thymic stroma. Because the thymus undergoes profound degeneration with age and when exposed to stresses such as irradiation and chemotherapy, using stem cells as a potential source of TECs to enhance or restore thymic function is of great therapeutic interest. Given the key role of TECs in establishing self-tolerance, differentiating a functional thymus from stem cells also has the potential to enhance the engraftment of human-stem-cell- derived tissue through the induction of graft-specific immune tolerance.

[0005] Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule with low polymorphism and restricted tissue distribution that displays tolerogenic functions. Four membrane-bound (HLA-G1 through HLA-G4) and three soluble HLA-G isoforms (HLA-G5 through HLA-G7) have been described, all of which derive from alternative splicing of the primary transcript. HLA-G is physiologically expressed in the thymus and extra-embryonic fetal tissues. HLA-G expression by extravillous cytotrophoblasts has been extensively described and proposed to contribute to the tolerance of the fetal semi-allogeneic graft by the maternal immune system. HLA-G possesses widely described immune tolerogenic properties by modulating the functions of several immune effectors: it acts on natural killer cells (NK) by inhibiting their cytotoxicity, rolling adhesion, and transendothelial migration; inhibits antigen-specific CD8+ cytotoxic T cell (CTL) function; and induces apoptosis of phytohemagglutinin-activated CD8+ CTL. The tolerogenic capacity of HLA-G is executed through the interaction of membrane-bound or soluble HLA-G with ILT2 / ILT4.

[0006] Programmed Death Ligand 1 (PD-L1, also termed B7-H1) and its corresponding receptor, PD-1, are both transmembrane proteins that are part of the immunoglobulin superfamily. PD-L1 is constitutively present on the surface of hematopoietic cells such as monocytes and T cells. PD-1 has an extracellular domain that allows for interaction with PD- L1, a localizing transmembrane portion, and an intracellular domain that allows for signal transduction. T cell receptor (TCR) recognition of MHC upregulates the production of the 2 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO receptor, preventing T cell overactivation and limiting immune-mediated damage to native tissue. PD-L1 has been shown to decrease the expansion of CD4+ T cells and NK cells, limit the cytotoxic effects of CD8+ T cells, and simultaneously increase the differentiation of CD4+ T cells into Tregs. The role of PD-L1 in modulating immune responses in autoimmune diseases can be similarly applied to the context of cell transplantation, where PD-L1 expression may help mitigate immune-mediated rejection. In mouse models, the absence of PD-L1 exacerbates various immune-mediated disease processes, including encephalomyelitis, lupus-like nephritis, and autoimmune dilated cardiomyopathy. This shows that overexpressing PD-L1 might be able to lower the function of immune cells and the problems that come with having too many immune cells after an organ transplant or in autoimmune conditions. Through the application of both HLA-G and PD-L1 overexpression in various “hypoimmune” cell therapy and solid organ transplant approaches, generating iPSC-TEPs overexpressing HLA-G and / or PD-L1 could significantly reduce allogeneic rejection by donor immune cells in immunocompetent recipients. SUMMARY OF THE INVENTION

[0007] The present disclosure builds on the pivotal discovery that human-induced pluripotent stem cell (iPSC)-derived thymic epithelial progenitor cells (iPSC-TEPs) can be engrafted in animals, survive, and yield functional T cell production in vivo. Leveraging this discovery, the present disclosure provides methods for generating iPSC-derived cells to overexpress PD-L1 and / or HLA-G to ameliorate or reduce allogeneic rejection by donor immune cells in immunocompetent recipients.

[0008] In one embodiment, the present disclosure provides an isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1 or HLA-G, or a combination thereof.

[0009] In some aspects, the isolated TEP cell expresses an HLA-I molecule.

[0010] In some aspects, the isolated TEP cell expresses HLA-A, HLA-B, and HLA-C.

[0011] In some aspects, the isolated TEP cell expresses an HLA-II molecule.

[0012] In some aspects, the isolated TEP cell expresses HLA-DP, HLA-DQ, and HLA-DR.

[0013] In some aspects, the isolated TEP cell expresses CIITA.

[0014] In some aspects, the isolated TEP cell expresses FOXN1.

[0015] In another embodiment, the present disclosure provides a method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with a nucleic acid sequence encoding PD-L1 and / or 3 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO HLA-G; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium including an activator of retinoic acid receptor, an activator of bone morphogeneticprotein (BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signalingto produce anterior foregut endodermal (AFE) cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein(BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signaling toproduce ventral pharyngeal endodermal (VPE) cells; and d) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1 and / or HLA-G; thereby generating the TEP cells overexpressing PD-L1 and / or HLA-G.

[0016] In some aspects, the DE cells are obtained from pluripotent stem cells by culturing pluripotent stem cells in a medium comprising a growth factor selected from the group consisting of Nodal, Activin A, and Activin B.

[0017] In some aspects, the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling.

[0018] In some aspects, the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

[0019] In some aspects, the TEP cells express FOXN1.

[0020] In some aspects, the TEP cells express an HLA-I molecule. In some aspects, the TEP cells express HLA-II molecules.

[0021] In some aspects, the PSCs are selected from the group consisting of embryonic stem cell, embryonic germ cells, and induced pluripotent stem cell.

[0022] In some aspects, the PSCs are primate pluripotent stem cells (pPSCs).

[0023] In some aspects, the pPSCs are human pluripotent stem cells (hPSCs).

[0024] In some aspects, the hPSCs are human embryonic stem cells (hESCs).

[0025] In some aspects, the hPSCs are induced pluripotent stem cells (iPSCs).

[0026] In some aspects, the nucleic acid sequence is in a vector.

[0027] In some aspects, the vector is a plasmid or a viral vector.

[0028] In some aspects, the nucleic acid sequence in the vector is operably linked to a promoter. In some aspects, the promoter is a constitutive promoter. In some aspects, the promoter is a constitutive promoter or an inducible promoter. In some aspects, the inducible promoter allows for tunable expression of the antigens. 4 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0029] In some aspects, the virial vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

[0030] In another embodiment, the present disclosure provides a method of treating or preventing a condition in a subject, including administering the isolated TEP cell overexpressing PD-L1 or HLA-G, or a combination thereof.

[0031] In some aspects, the condition is associated with the absence, decline or aberrant functioning of the thymus of the subject.

[0032] In some aspects, the condition is selected from Di George syndrome, thymoma (such as type A thymoma or type B thymoma), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymus 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, age- or infection-associated decline in thymic function, thymic-mediated tolerance, transplant tolerance, or autoimmunity. In some aspects, the autoimmunity is general or antigen-mediated.

[0033] In some aspects, the subject has undergone a thymectomy surgery.

[0034] In some aspects, the condition is an immunodeficiency.

[0035] In some aspects, the condition is cancer.

[0036] In some aspects, the condition is an autoimmune disease.

[0037] In some aspects, the condition is an infectious disease.

[0038] In some aspects, the condition is graft versus host disease (GvHD).

[0039] In another embodiment, the present disclosure provides an isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1, HLA-G, and an autoimmune antigen.

[0040] In some aspects, the autoimmune antigen is selected from the group consisting of myelin basic protein (MBP) peptide, proinsulin 2, and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP).

[0041] In another embodiment, the present disclosure provides a method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of 5 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WOtransforming growth factor- (TGF- ) signaling to produce anterior foregut endodermal (AFE)cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforminggrowth factor- (TGF- ) signaling to produce ventral pharyngeal endodermal (VPE) cells; andd) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen; thereby generating the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen. In some aspects, the autoimmune antigen is selected from the group consisting of myelin basic protein (MBP) peptide, proinsulin 2, and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP).

[0042] In another embodiment, the present disclosure provides an isolated hematopoietic stem cells (HSCs) overexpressing PD-L1 or HLA-G, or a combination thereof.

[0043] In another embodiments, the present disclosure provides a method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; thereby generating the HSCs overexpressing PD-L1 and / or HLA-G.

[0044] In another embodiments, the present disclosure provides an isolated adult tissue progenitor / stem cells overexpressing PD-L1 or HLA-G, or a combination thereof.

[0045] In another embodiments, the present disclosure provides a method for generating adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing the cells obtained from a), to further differentiate into progenitor cells of adult tissue, wherein the progenitor cells of adult tissue are selected from the group consisting of cardiomyocytes, neuronal cells, lung cells, pancreatic islet cells, liver cells, kidney cells, and intestinal cells; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G.

[0046] In another embodiment, the present disclosure provides an isolated hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen. 6 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0047] In another embodiment, the present disclosure provides a method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; thereby generating the HSC cells overexpressing PD-L1, HLA-G, and the autoimmune antigen.

[0048] In another embodiment, the present disclosure provides an isolated adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen.

[0049] In another embodiment, the present disclosure provides a method for generating adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing the cells obtained from a), to further differentiate into progenitor cells of adult tissue, wherein the progenitor cells of adult tissue are selected from the group consisting of cardiomyocytes, neuronal cells, lung cells, pancreatic islet cells, liver cells, kidney cells, and intestinal cells; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and the autoimmune antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0051] FIG. 1 illustrates a schematic workflow for engineering iPSC-TEP overexpressing HLA-G and PD-L1.

[0052] FIG.2 illustrates exemplary elements of lentiviral transfection for the overexpression of PD-L1 and HLA-G in iPSC-TEP.

[0053] FIG. 3 illustrates the exemplary design of vectors for the overexpression of PD-L1 and HLA-G in iPSC-TEP.

[0054] FIG.4 illustrates exemplary vector maps for the overexpression of PD-L1 and HLA- G in iPSC-TEP. 7 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0055] FIG.5 illustrates a schematic of an adapted mixed lymphocyte reaction (MLR) assay for in vitro demonstration of hypoimmunity.

[0056] FIG.6 illustrates a schematic of an adapted cytotoxic T lymphocyte (CTL)-mediated lysis (CML) assay for in vitro demonstration of hypoimmunity.

[0057] FIG.7 illustrates a schematic of an in vivo demonstration of hypoimmunity.

[0058] FIG.8 is a schematic diagram illustrating HLA class I and II engineering.

[0059] FIG. 9 is a schematic diagram illustrating positive and negative selection of thymic epithelial cells.

[0060] FIGs. 10A-10C are schematic diagrams illustrating generation of engineered human iPSC cell lines with the constitutive overexpression of human PD-L1 and HLA-G. FIG. 10A is a schematic illustrating design and package plasmids. FIG. 10B is a schematic illustrating a construct for PD-L1 and HLA-G overexpression. FIG.10C is a schematic illustrating TEP cell interaction with T cell.

[0061] FIGs.11A-11C are graphs illustrating MOI selection. FIG.11A is a graph illustrating PD-L1 and endothelial cells (EC) HLA-G expression intensity in Multiplicity of Infection (MOI) 16, MOI 8, MOI 4, and MOI 2. FIG. 11B is a graph illustrating immune checkpoint (IC) and EC HLA-G expression intensity in JEG-3 cells. FIG. 11C is a graph illustrating IC HLA-G expression intensity in MOI 16, MOI 8, MOI 4, and MOI 2.

[0062] FIG. 12 is a schematic diagram illustrating hypoimmune construct subcloning and screening.

[0063] FIGs. 13A-13B are schematic diagrams illustrating poor cytotoxic T cell (CTL) stimulators and targets vs robust CTL stimulators. FIG. 13A is a schematic illustrating poor cytotoxic T lymphocyte (CTL) stimulators and targets. FIG. 13B is a schematic illustrating robust CTL stimulators.

[0064] FIGs. 14A-14B illustrate generation of HLA-A*02:01 specific cytotoxic T lymphocyte precursor (CTLp). FIG. 14A is a schematic diagram illustrating generation of HLA-A*02:01 specific CTLp in contrast to poor CTL stimulators. FIG. 14B is an image illustrating clonal CTL expansion.

[0065] FIGs. 15A-15B are schematic diagrams illustrating quantification of HLA-A 02:01 specific CTLp activity. FIG. 15A is a schematic illustrating phase I: polyclonal expansion of HLA-A*02:01 specific CTLp. FIG.15B is a schematic illustrating phase II: co-culture of CTL 8 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO effectors with various target cells for the qualification and quantification of HLA-specific cell- mediated lysis.

[0066] FIGs.16A-16B illustrate specific cell lysis. FIG.16A is a schematic illustrating cell mediated lysis assay. FIG.16B is a graph illustrating percentage of specific lysis of (+) control, WT endothelial, edited endothelial, and 3rdparty control.

[0067] FIG. 17 is an image that illustrate hypoimmune protection from HLA-A*02:01 specific CTLp. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention stems from the critical discovery that human-induced pluripotent stem cell (iPSC)-derived thymic epithelial progenitor (TEP) cells can engraft, survive, and produce functional T cells in vivo. Building on this discovery, the invention introduces a method for creating iPSC-derived cells to overexpress PD-L1 and / or HLA-G, aiming to minimize allogeneic rejection by donor immune cells in immunocompetent recipients. By enhancing the immune-modulatory capabilities of iPSC-derived cells through PD-L1 and HLA-G overexpression, the invention seeks to improve cell engraftment and functionality. This approach promises to restore thymus function and induce immune tolerance, offering significant therapeutic potential for patients with thymus-related defects, immune dysfunction, or those needing enhanced immune tolerance for graft acceptance.

[0069] Before the present TEP cells overexpressing PD-L1 and / or HLA-G and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only to the appended claims.

[0070] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications 9 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0072] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods, and / or steps of the type described herein, which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0073] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0074] As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of a therapeutic compound, a combination of therapeutic compounds, or TEP cells thereof provided herein, which is sufficient to result in the desired outcome.

[0075] As used herein, the terms “subject” and “patient” may be used interchangeably. As used herein, in some aspects, a subject is a mammal. In some aspects, the subject is a human. In some aspects, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In some aspects, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0076] As used herein, the term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0077] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or disorder resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a decrease, alleviation, and / or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite the fact that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease.

[0078] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s). 10 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0079] As used herein, the term “pluripotent stem cell” or “pluripotent cell” refers to a cell that has the ability, under appropriate conditions, to produce progeny of several different cell types that are derivatives of all three germinal layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells (PSCs)are capable of forming teratomas. Examples of pluripotent stem cells are embryonic stem (ES) cells, embryonic germ stem (EG) cells, induced pluripotent stem cells (iPSCs), and adult stem cells. PSCs may be from any organism of interest, including primate, e.g., human; canine; feline; murine; equine; porcine; avian; camel; bovine; ovine, and so on.

[0080] As used here, the term “induced pluripotent stem cell” or “iPSC” refers to a cell that a) can self-renew, b) can differentiate to produce all types of cells in an organism, and c) is derived from a somatic cell. iPSCs have an embryonic stem cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. iPSCs may be generated by providing the cell with “reprogramming factors,” i.e., one or more, e.g., a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to pluripotency.

[0081] As used herein, the term “cell line” refers to a population of largely or substantially identical cells that has typically been derived from a single ancestor cell or from a defined and / or substantially identical population of ancestor cells. The cell line may have been or may be capable of being maintained in culture for an extended period (e.g., months, years, or an unlimited period of time).

[0082] As used herein, the term “endoderm” refers to the germ layer formed during animal embryogenesis that gives rise to the gastrointestinal tract, respiratory tract, endocrine glands and organs, certain structures of the auditory system, and certain structures of the urinary system.

[0083] As used herein, the term “mesoderm” refers to the germ layer formed during animal embryogenesis that gives rise to muscles, cartilage, bones, dermis, the reproductive system, adipose tissue, connective tissues of the gut, peritoneum, certain structures of the urinary system, mesothelium, notochord, and spleen.

[0084] As used herein, the term “ectoderm” refers to the germ layer formed during animal embryogenesis that gives rise to the nervous system, tooth enamel, epidermis, hair, nails, and linings of mucosal tissues. 11 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0085] As used herein the term “TEP cell” or “thymic epithelial progenitor cell” refers to specialized cells that play a crucial role in the development and function of the thymus, an organ essential for T cell maturation. TEP cells originate from non-hematopoietic cells and are characterized by the expression of epithelial cell adhesion molecule (EpCAM) and the absence of CD45. TEP cells differentiate into two main types of thymic epithelial cells (TECs): cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs). TEP cells provide the necessary microenvironment for T cell development. cTECs are involved in the positive selection of thymocytes, ensuring that T cells can recognize self-MHC molecules. mTECs are responsible for negative selection, eliminating self-reactive T cells to prevent autoimmunity.

[0086] As used herein the term “PD-L1” or “programmed death-ligand 1” or “CD274” or “B7-H1” refers to a transmembrane protein that plays a significant role in regulating the immune system. PD-L1 binds to the PD-1 receptor on T cells, which helps modulate T cell activation and proliferation. This interaction is crucial for maintaining immune homeostasis and preventing autoimmunity. Many cancer cells express high levels of PD-L1, which allows them to evade immune detection and destruction. By binding to PD-1 on T cells, PD-L1 sends an inhibitory signal that reduces the T cell's ability to attack the cancer cells.

[0087] As used herein the term “HLA-G” or “human leukocyte antigen-G” refers to a non- classical major histocompatibility complex (MHC) class I molecule with functions in immune regulation. HLA-G help maintain immune tolerance. HLA-G is expressed in various tissues, including the placenta, thymus, and certain immune-privileged sites. HLA-G can be found in both membrane-bound and soluble forms, with different isoforms resulting from alternative splicing. HLA-G is expressed on trophoblast cells, contributing to maternal-fetal tolerance. This expression helps protect the fetus from being recognized and attacked by the maternal immune system. HLA-G has implications in transplantation, where its expression can help reduce the risk of graft rejection.

[0088] As used herein the term “HLA-I” or “human leukocyte antigen class I” refers to a group of proteins encoded by the HLA human leukocyte antigen (HLA) complex, which is part of the major histocompatibility complex (MHC) in humans. HLA-I molecules present endogenous peptides (peptides from within the cell) to CD8+ T cells (cytotoxic T cells). This process is crucial for the immune system to recognize and eliminate infected or malignant cells. Examples of HLA-I molecules include but are not limited to HLA-A, HLA-B, and HLA-C, 12 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO each of which has many alleles. HLA-I molecules are critical in organ transplantation, as mismatches between donor and recipient HLA-I molecules can lead to transplant rejection. During T cell development in the thymus, TEP cells (through their differentiated forms, cTECs and mTECs) present self-antigens via HLA-I molecules to developing thymocytes. This presentation is crucial for the positive and negative selection processes that ensure a functional and self-tolerant T cell repertoire.

[0089] As used herein the term “HLA-II” or “human leukocyte antigen class II” refers a group of proteins encoded by the HLA complex, which is part of the major histocompatibility complex (MHC) in humans. HLA-II molecules present exogenous peptides (peptides from outside the cell) to CD4+ T cells (helper T cells). This process is crucial for initiating and regulating immune responses, particularly in recognizing and responding to extracellular pathogens. HLA-II molecules include but are not limited to HLA-DP, HLA-DQ, and HLA- DR, each of which has multiple alleles, contributing to the diversity of the immune response. HLA-II molecules are critical in organ transplantation, as mismatches between donor and recipient HLA-II molecules can lead to transplant rejection. During T cell development in the thymus, TEP cells (through their differentiated forms, cTECs and mTECs) present self- antigens via HLA-II molecules to developing thymocytes. This presentation is crucial for the positive and negative selection processes that ensure a functional and self-tolerant T cell repertoire.

[0090] Maintaining the expression of classical HLA molecules is important for proper antigen presentation. Their presence helps prevent NK cell-mediated lysis, which can occur when cells lack self-markers eliminating the requirement for CD47 overexpression.

[0091] As used herein, the term “bone morphogenic proteins” or “BMPs” refers to the family of growth factors that is a subfamily of the transforming growth factor (TGF ) superfamily. BMPs (e.g. BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9 / GDF, BMP10, BMP11 / GDF11, BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15 / GDF9B) were first discovered by their ability to induce the formation of bone and cartilage. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in the mobilization of members of the SMAD family of proteins, which in turn modulate the transcription of target genes. Of particular interest in the present invention are activators of BMP signaling, which can readily be identified by one of ordinary skill in the art by any of a 13 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO number of methods, for example, competitive binding assays for binding to BMP or BMP receptors, functional assays, e.g., measuring enhancement of activity of downstream signaling proteins such as relocalization of SMADs, such as, BR-Smad to the nucleus, and transcriptional activation of downstream gene targets as known in the art.

[0092] As used here, the terms “transforming growth factor betas,” “TGF- s,” and “TGFBs”refer to the TGFB secreted proteins belonging to the subfamily of the transforming growthfactor (TGF ) superfamily. TGFBs (TGFB1, TGFB2, TGFB3) are multifunctional peptidesthat regulate proliferation, differentiation, adhesion, and migration and in many cell types. The mature peptides may be found as homodimers or as heterodimers with other TGFB familymembers. TGFBs interact with transforming growth factor beta receptors (TGF- Rs, orTGFBRs) on the cell surface, which binding activates MAP kinase-, Akt-, Rho- and Rac / cdc42- directed signal transduction pathways, the reorganization of the cellular architecture and nuclear localization of SMAD proteins, and the modulation of target gene transcription. Of particular interest in the present invention are inhibitors of TGFB signaling, which can be readily be identified by one of ordinary skill in the art by any of a number of methods, for example competitive binding assays for binding to TGFB or TGFB receptors, or functional assays, e.g. measuring suppression of activity of downstream signaling proteins such as MAPK, Akt, Rho, Rac, and SMADs, e.g., AR-Smad, etc., as well known in the art.

[0093] As used herein, the term “Wnts” refers to the family of highly conserved secreted signaling molecules which play key roles in both embryogenesis and mature tissues. The human Wnt gene family has at least 19 members (Wnt-1, Wnt-2, Wnt-2B / Wnt-13, Wnt-3, Wnt3a, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-9A / Wnt- 14, Wnt-9B / Wnt-15, Wnt-10A, Wnt-10B, Wnt-11, Wnt-16). Wnt proteins modulate cell activity by binding to Wnt receptor complexes that include a polypeptide from the Frizzled (Fz) family of proteins and a polypeptide of the low-density lipoprotein receptor (LDLR)- related protein (LRP) family of proteins. Once activated by Wnt binding, the Wnt receptor complex will activate one or more intracellular signaling cascades. These include the canonical Wnt signaling pathway; the Wnt / planar cell polarity (Wnt / PCP) pathway; and the Wnt-calcium (Wnt / Ca2+) pathway.

[0094] As used herein, the term “medium” in context of cell culture or the phrase “cell culture medium” or “cell medium” refers to a cellular growth medium suitable for culturing of PS cells, DE cells, AFE cells, VPE cells, TEP cells. Examples of cell culture medium include 14 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO Minimum Essential Medium (MEM), Eagle’s Medium, Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12), F10 Nutrient Mixture, Ham’s F10 Nutrient Mix, Ham’s F12 Nutrient Mixture, Medium 199, RPMI, RPMI 1640, reduced serum medium, basal medium (BME), DMEM / F12 (1:1), and the like, and combinations thereof. The medium or cell culture medium may be modified by adding one or more additives. Additives may include serum, such as, fetal bovine serum and / or serum replacement agents, such as, B27, N2, KSR, and combinations thereof, and differentiation factors, such as, activators of RA receptor, nodal, Act-A, Act-B, Wnt family members,activators of BMP signaling, inhibitors of TGF- signaling, FGF, inhibitors of hedgehogsignaling, and the like, and combinations thereof.

[0095] As used herein, the term “isolated” in context of cells or cell population refers to cells that are in an environment other than their native environment, such as, apart from tissue of an organism.

[0096] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.

[0097] As used herein, the term “immunodeficiency” may refer to any condition in which a subject’s immune system is compromised and / or in need of reconstitution, e.g., after irradiation or chemotherapy. Immunodeficiency may be a primary immunodeficiency, caused by an inherited or genetic factor, or a secondary immunodeficiency, caused by an environmental factor. In some aspects, the TEP cell of the present disclosure may be used to treat primary immunodeficiencies such as, but not limited to, 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 aspects, the TEP cells of the present disclosure may be used to treat secondary immunodeficiencies caused by diseases such as AIDS and / or hepatitis. 15 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0098] As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue, metastasize to new body sites, and refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas / leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.

[0099] As used herein, the term “plasmid” refers generally to a construction comprised of extra-chromosomal genetic material, usually of a circular duplex of DNA that can replicate independently of chromosomal DNA. Plasmids, or fragments thereof, may be used as vectors. Plasmids are double-stranded DNA molecule that occur or are derived from bacteria and (rarely) other microorganisms. However, mitochondrial and chloroplast DNA, yeast killer and other cases are commonly excluded.

[0100] As used herein, the term “vector” refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell wherein, in some aspects, it can be replicated. A nucleic acid sequence can be native to the animal, or it can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), linear DNA fragments, and artificial chromosomes (e.g., YACs), although in some aspects the vector contains substantially no viral sequences. One of skill in the art would be well equipped to construct a vector through standard recombinant techniques.

[0101] As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host cell. In addition to control sequences that govern 16 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. Expression vectors can include promoters which are operably linked to a nucleic acid sequence which encodes any of the proteins described herein. In some aspects, the promoter is a constitutive promoter. In some aspects, the promoter is a constitutive promoter or an inducible promoter. In some aspects, the inducible promoter allows for tunable expression of the antigens. In one embodiment, the present disclosure provides an isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1 or HLA-G, or a combination thereof.

[0103] In some aspects, the isolated TEP cell expresses an HLA-I molecule.

[0104] In some aspects, the isolated TEP cell constitutively overexpress PD-L1 and / or HLA- G.

[0105] In some aspects, the isolated TEP cell expresses an HLA-II molecule.

[0106] In some aspects, the isolated TEP cell overexpress PD-L1 and / or HLA-G in an inducible manner.

[0107] In some aspects, the isolated TEP cell expresses HLA-A, HLA-B, and HLA-C.

[0108] In some aspects, the isolated TEP cell expresses HLA-DP, HLA-DQ, and HLA-DR.

[0109] In some aspects, the isolated TEP cell expresses CIITA.

[0110] In some aspects, the isolated TEP cell expresses FOXN1.

[0111] In another embodiment, the present disclosure provides a method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with a nucleic acid sequence encoding PD-L1 and / or HLA-G; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogeneticprotein (BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signalingto produce anterior foregut endodermal (AFE) cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein(BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signaling toproduce ventral pharyngeal endodermal (VPE) cells; and d) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1 and / or HLA-G; thereby generating the TEP cells overexpressing PD-L1 and / or HLA-G. 17 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO Transfection of pluripotent stem cells (PSCs) with a Nucleic Acid Sequence Encoding PD-L1 and / or HLA-G

[0112] In some aspects, the PSCs can be transfected with a vector or a plasmid encoding nucleic acid sequences of PD-L1 and / or HLA-G. In some aspects, the vector can be a viral vector. In some aspects, the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. In some aspects, the viral vector is a lentiviral vector. In some aspects, the lentiviral vector is a polycistronic third generation lentiviral vector.

[0113] In some aspects, the PSCs are primate pluripotent stem cells (pPSCs).

[0114] In some aspects, pPSCs are human pluripotent stem cells (hPSCs).

[0115] In some aspects, the hPSCs are human embryonic stem cells (hESCs).

[0116] In some aspects, the hPSCs are induced pluripotent stem cells (iPSCs). Preparation of Definitive Endoderm (DE) cells

[0117] In some aspects, DE cells can be prepared by culturing cells in two-dimensional or three-dimensional cultures. Such methods can include culturing pluripotent stem cells in a first growth medium, a second growth medium, or a combination thereof. In some aspects, the first or second growth medium can include PI-103 (a multitargeted P13K inhibitor). In some aspects, the first growth medium includes Activin A, CHIR99021, insulin transferrin selenium (ITS), and / or knockout serum replacement (KSR). In some aspects, the pluripotent stem cells (PSCs) are encapsulated in polymers, such as alginate, prior to their differentiation. Encapsulated cells are grown in three dimensional cultures. In some aspects, the second growth medium includes basic fibroblast growth factor (bFGF), Activin A, LDN193189, ITS, and KSR. In some aspects, the second growth medium includes CHIR99021. In some aspects, theconcentration of CHIR99021 ranges from about 0.1 M to 100 M. In some aspects, theconcentration of CHIR99021 is about 2 M–3 M. In some aspects, the cells are cultured in the presence of PI-103. The concentration of PI-103 can range from about 1 nM to 1000 nM. In some aspects, the concentration of PI-103 can be 50 nM. In some aspects, the concentration of PI-103 can be 25 nM. In some aspects, the PSCs can be cultured for about three to five days. The PI-103 can be added for 1-2 days. The PSCs can be embryonic stem cells or induced pluripotent stem cells.

[0118] In some aspects, the PSCs can be cultured for about three to five days. The PSCs can be cultured in the first growth medium for about one to two days and in the second growth 18 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO medium for about two to three days. The PSCs can be cultured in the first growth medium for two days and in the second growth medium for three days. In some aspects, the concentration of Activin A can be about 100ng / ml. In some aspects, the concentration of CHIR99021 can be2 M. In some aspects, the concentration of bFGF can be 10ng / ml. In some aspects, theconcentration of LDN193189 can be 200nM. In some aspects, CHIR99021 can be added to the second growth medium for about one day.

[0119] In some aspects, provided herein are methods of differentiating pluripotent stem cells (PSCs) into thymic cells. Such methods can include culturing PSCs in a first growth medium, a second growth medium, or a combination thereof. In some aspects, the first or second growth medium can include PI-103 (a multitargeted P13K inhibitor). In some aspects, the first growth medium includes DMEM-F12, Activin A, CHIR99021, insulin transferrin selenium (ITS), and knockout serum replacement (KSR). In some aspects, the second growth medium includes DMEM-F12, bFGF, Activin A, LDN193189, ITS and KSR. In some aspects, the second growth medium includes CHIR99021. In some aspects, the concentration of CHIR99021 ranges fromabout 0.1 M to 100 M. In some aspects, the concentration of CHIR99021 is 2 M. In someaspects, the cells are cultured in the presence of PI-103. The concentration of PI-103 can range from about 1nM to 1000nM. In some aspects, the concentration of PI-103 can be 50nM.

[0120] In some aspects, DE cells are obtained from pluripotent stem cells by culturing pluripotent stem cells in a medium comprising a growth factor selected from the group consisting of Nodal, Activin A, and Activin B. Preparation of Anterior Foregut Endodermal (AFE) cells

[0121] The definitive endoderm (DE) cells can be further cultured and differentiated into anterior foregut endodermal (AFE) cells. In some aspects, the AFE cells can be prepared by culturing cells in two-dimensional or three-dimensional culture. The DE cells can bedifferentiated into AFE cells by contacting DE cells with a BMP inhibitor, a TGF inhibitor,at least one FGF, and / or Ascorbic acid.

[0122] In some aspects, the cells are encapsulated in polymers as described herein, such as alginate, prior to their differentiation. Encapsulated cells are grown in three-dimensional cultures.

[0123] In some aspects, the cell culture medium utilized for the differentiation of DE cells to AFE cells can include N2- supplement (GIBCO, Waltham, Massachusetts), Basal Medium 19 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement, non-essential amino acids, KSR and / or ITS.

[0124] In some aspects, the BMP inhibitor can be LDN193189. In some aspects, the concentration of LDN193189 ranges from about 0.1nM to about 1000nM. In some aspects, the concentration of LDN193189 ranges from about 100 to 200nM.

[0125] In some aspects, the TGF inhibitor can be SB431542. In some aspects, theconcentration of SB431542 ranges from about 1 μM to about 100 μM. As a non-limiting example, the concentration of SB431542 is 10μM.

[0126] In some aspects, the FGF can be FGF8. In some aspects, the concentration of FGF8 is from about 1ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF8b is about 25-50ng / ml. In some aspects, the DE cells can be differentiated into AFE cells for about 1 day, 2 days, 3 days, 4 days, or 5 days.

[0127] In some aspects, the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling. Preparation of Ventral Pharyngeal Endoderm (VPE) cells

[0128] In some aspects, differentiation of AFE to VPE cells is performed as a single step process or as a multi-step process. The multi-step process can be a two-step process. In the first step, the AFEs are cultured in VPE1 media, and in the second step, the cells are cultured in VPE2 media. In some aspects, the cells are encapsulated in polymers, such as alginate, prior to their differentiation. Encapsulated cells are grown in three-dimensional cultures.

[0129] In some aspects, the VPE cells can be prepared by culturing cells in two-dimensional or three-dimensional culture. The VPE1 step can include culturing cells for about 1 day, 2 days, 3 days, 4 days, or 5 days. The VPE2 step can include culturing cells for about 2 days, 3 days, 4 days, 5 days, or 6 days.

[0130] In some aspects, the VPE1 media can include Retinoic Acid, at least one FGF, a WNTinhibitor, TGF inhibitor, and / or Ascorbic acid.

[0131] In some aspects, the VPE2 media can include Noggin, BMP inhibitor, WNT activator (e.g., CHIR99021), at least one FGF, Retinoic Acid, an SHH antagonist, and / or Ascorbic acid.

[0132] In some aspects, the FGF can be FGF8, FGF7, and / or FGF10. In some aspects, the concentration of FGF8 ranges from about 1ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF8b is about 25-50ng / ml. 20 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0133] In some aspects, the WNT inhibitor is IWR1. The concentration of IWR1 can range from about 0.01 to 10μM. As a non-limiting example, the concentration of IWR1 is 2.5μM.

[0134] In some aspects, the TGF inhibitor can be SB431542. In some aspects, theconcentration of SB431542 ranges from about 1 μM to about 100 μM. As a non-limiting example, the concentration of SB431542 is 10μM.

[0135] In some aspects, the concentration of Ascorbic Acid is from about 0.1 to 30μM. As a non-limiting example, the concentration of Ascorbic Acid can be 10μM.

[0136] In some aspects, the BMP inhibitor can be LDN193189. In some aspects, the concentration of LDN193189 ranges from about 0.1nM to about 1000nM. In some aspects, the concentration of LDN193189 ranges from about 100 to 200nM.

[0137] In some aspects, the SHH inhibitor can be SANT-1. In some aspects, the concentration of SANT-1 ranges from about 0.01 μM to about 10 μM. As a non-limiting example, the concentration of SANT-1 is 0.25μM.

[0138] In some aspects, the AFE cells can be cultured and differentiated into pharyngeal endoderm cells by contacting or incubating the anterior foregut cells with at least one of EGF, retinoic acid, FGF8B, and / or SHH.

[0139] In some aspects, the VPE1 and / or VPE2 media can include N2- supplement (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement (with or without Vitamin A), non-essential amino acids, KSR and / or ITS.

[0140] In some aspects, the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling. Preparation of Thymic Epithelial Progenitor (TEP) cells

[0141] In some aspects, differentiation of VPE cells into TEP cells can be performed by culturing the cells in TEP media. In some aspects, the TEP cells can be prepared by culturing cells in two-dimensional or three-dimensional culture. The TEP step can include culturing cells for about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0142] In some aspects, the cells are encapsulated in polymers, such as alginate, prior to their differentiation. Encapsulated cells are grown in three-dimensional cultures.

[0143] In some aspects, the 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. 21 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0144] In some aspects, the TEP media can include N2- supplement (GIBCO, Waltham, Massachusetts), Basal Medium Eagle (BME), GLUTAMAX (GIBCO, Waltham, Massachusetts), B27™ serum-free supplement (with or without Vitamin A), non-essential amino acids, KSR and / or ITS.

[0145] In some aspects, the BMP can be BMP2 or BMP4. The concentration of BMP can range from 1ng / ml to about 100ng / ml. In some aspects, the concentration of BMP can be 50ng / ml.

[0146] In some aspects, the FGF can be FGF8, FGF7, FGF1, and / or FGF10. In some aspects, the concentration of FGF ranges from about 1ng / ml to about 100 ng / ml. As a non-limiting example, the concentration of FGF is about 25-50ng / ml.

[0147] In some aspects, the pharyngeal endoderm cells can be cultured and differentiated into, thymic epithelial cells by contacting or incubating the pharyngeal endoderm cells with at least one of BMP4, FGF8b, EGF, SANT-1 (SHH antagonist), CHIR99021, Ascorbic Acid, or a combination thereof.

[0148] In some aspects, the TEP cells express FOXN1.

[0149] In another embodiment, the present disclosure provides a method of treating or preventing a condition in a subject, including administering the isolated TEP cell overexpressing PD-L1 and / or HLA-G.

[0150] In some aspects, the condition is associated with the absence, decline or aberrant functioning of the thymus of the subject. In some aspects, the condition is selected from Di George syndrome, thymoma (such as type A thymoma or type B thymoma), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymus 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, age- or infection-associated decline in thymic function, thymic-mediated tolerance, transplant tolerance, or autoimmunity. In some aspects, the autoimmunity is general or antigen-mediated.

[0151] In some aspects, the condition is associated with the absence, decline or aberrant functioning of the thymus of the subject. In some aspects, the condition is associated with immunosenescence associated with natural involution and ageing of the thymus.

[0152] In some aspects, the subject has undergone a thymectomy surgery. 22 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0153] In some aspects, the condition is an immunodeficiency. The term immunodeficiency may refer to any condition in which a subject’s immune system is compromised and / or in need of reconstitution, e.g., after irradiation or chemotherapy. Immunodeficiency may be a primary immunodeficiency, caused by an inherited or a genetic factor or a secondary immunodeficiency, caused by an environmental factor. In some aspects, the TEP cell of the present disclosure may be used to treat primary immunodeficiencies such as, but not limited to, 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 aspects, the TEP cell of the present disclosure may be used to treat secondary immunodeficiencies caused by diseases such as AIDS and / or hepatitis.

[0154] Lymphopenia as used herein, 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 the lymphopenia is associated with a reduction in the number of T cells, it may be referred to as T cell lymphopenia. The TEP cell of the disclosure may be used to treat inherent or acquired lymphopenia which may be caused by hematopoietic stem cell therapy, bone marrow transplantation therapy, radiation, chemotherapy, surgery, immunosenescence and / or aging.

[0155] In some aspects, the condition is cancer. Various cancers may be treated with the TEP cell of the present disclosure. As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue, metastasize to new body sites, and refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas / leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.

[0156] Types of carcinomas which may be treated with the TEP cell of the present disclosure include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma / leukemia, 23 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.

[0157] Types of carcinomas which may be treated with the TEP cell of the present disclosure include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extra skeletal chondrosarcoma, extra skeletal 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.

[0158] As a non-limiting example, the carcinoma which may be treated may be Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous 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, Bowel cancer, Brain cancer, Brain stem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing 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 lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal 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 24 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO chondrosarcoma, Mesenchymal cancer, 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- Hodgkin's lymphoma, Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer, Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumor, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma, Testicular cancer, Throat cancer, Thymoma / thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer.

[0159] In some aspects, the condition is an autoimmune disease. Autoimmune diseases may arise in a subject when self-antigen(s) is / are recognized by the effector cells in extra-thymic tissue and / or when such recognition triggers an activated immune response in a subject. The present disclosure provides methods of preparing an effector cell capable of effecting an immune tolerance response in a subject. Effector cells and the TEP cell including the same may be useful in training the immune system of the subject. In some aspects, engineered thymic cells of the present disclosure may also be administered to a subject for the treatment of autoimmune diseases. In some aspects, the engineered thymic cells lead to generation of T-Reg (T-regulatory ) cells specific for the autoantigen expressed.

[0160] In some aspects, the condition is type 1 autoimmune polyglandular syndrome (APS- 1) or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) 25 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO syndrome. More than 60 mutations of the autoimmune regulator (AIRE) gene are associated with the development of type 1 autoimmune polyglandular syndrome (APS-1). AIRE plays an important part in shaping the T-cell repertoire through its role in the elimination of T cells that are reactive to self-antigens in the thymus. The 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 aspects, the TEP cell of the present disclosure may be administered with the current standard of care therapies of APS-1, such as, but not limited to, lifelong anti-fungals, calcium modulators, endocrine hormone replacement, corticosteroids with or without 5 azacytidine, 5 azacytidine with mycophenolate (autoimmune hepatitis), and / or 5 azacytidine with or without rituximab (autoimmune pneumonitis).

[0161] Autoimmune diseases may be 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-Barre 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. In some aspects, the autoimmune disease may be immuno dysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX).

[0162] In some aspects, the condition is an infectious disease. Infectious disease causing organisms include, but are not limited to, 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., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, Enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus 26 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO influenza), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Porphyromonas spp. (e.g., P. gingivalis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhimurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, coagulase negative staphylococcus (e.g., U.S. Pat. No. 7,473,762)), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogens), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). Additional microorganisms causing infectious diseases may include, for example, one or more parasitic organisms (spp.) (e.g., parasite target (s)) 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., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium), Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and / or Wuchereria bancrofti.

[0163] In some aspects, the condition is graft versus host disease (GvHD).

[0164] In another embodiment, the present disclosure provides an isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1, HLA-G, and an autoimmune antigen. In some aspects, the autoimmune antigen is selected from the group consisting of myelin basic protein 27 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO (MBP) peptide, proinsulin 2, and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP).

[0165] In another embodiment, the present disclosure provided a method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor oftransforming growth factor- (TGF- ) signaling to produce anterior foregut endodermal (AFE)cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforminggrowth factor- (TGF- ) signaling to produce ventral pharyngeal endodermal (VPE) cells; andd) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen; thereby generating the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen.

[0166] In another embodiment, the present disclosure provided an isolated hematopoietic stem cells (HSCs) overexpressing PD-L1 or HLA-G, or a combination thereof.

[0167] In another embodiment, the present disclosure provided an isolated hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen.

[0168] In another embodiment, the present disclosure provided a method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; thereby generating the HSCs overexpressing PD-L1 and / or HLA-G.

[0169] In another embodiment, the present disclosure provided a method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; thereby generating the HSC cells overexpressing PD-L1, HLA-G, and the autoimmune antigen. 28 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO

[0170] In some aspects, HSCs overexpressing PD-L1, HLA-G, and optionally an autoimmune antigen may be used for treating or preventing a condition in a subject.

[0171] In some aspects, the condition of the subject is 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, Mucopolysaccharidosis, Glycoproteinoses, Immune deficiencies, Ataxia telangiectasia, DiGeorge syndrome, Severe combined immunodeficiency (SCID), Wiscott–Aldrich, Kostmann syndrome, Shwachman–Diamond syndrome.

[0172] In some aspects, the condition of the subject is malignant indications such as, but not limited to, leukemias such as acute myelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, myelodysplasia; lymphomas such as Hodgkin disease, Non-Hodgkin lymphoma, multiple myeloma, myeloproliferative neoplasms, myelofibrosis, myelofibrosis, chronic myelogenous leukemia; solid tumors such as neuroblastoma, desmoplastic small round cell tumor, Ewing sarcoma, and / or choriocarcinoma.

[0173] In another embodiment, the present disclosure provided an isolated adult tissue progenitor / stem cells overexpressing PD-L1 or HLA-G, or a combination thereof.

[0174] In another embodiment, the present disclosure provided an isolated adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen.

[0175] In another embodiment, the present disclosure provided a method for generating adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing the cells obtained from a), to further differentiate into progenitor cells of adult tissue, wherein the adult tissues are selected from the group consisting of heart muscle tissue, brain tissue, spinal cord tissue, peripheral nervous tissue, lung tissue, pancreatic islets, liver tissue, kidney tissue, and intestinal lining; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G.

[0176] In another embodiment, the present disclosure provided a method for generating adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, including: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing the cells obtained from a), to further 29 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO differentiate into progenitor cells of adult tissues, wherein the adult tissues are selected from the group consisting of heart muscle tissue, brain tissue, spinal cord tissue, peripheral nervous tissue, lung tissue, pancreatic islets, liver tissue, kidney tissue, and intestinal lining; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and the autoimmune antigen.

[0177] In some aspects, iPSC-derived progenitor cells include, but are not limited to, neural progenitor cells, hematopoietic progenitor cells, mesenchymal progenitor cells, cardiovascular progenitor cells, epidermal progenitor cells, hepatic progenitor cells, pancreatic progenitor cells, intestinal progenitor cells, oligodendrocyte progenitor cells, satellite cells, thymic epithelial progenitor cells, and endothelial progenitor cells.

[0178] The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

[0179] The invention is further illustrated by the following non-limiting examples. EXAMPLE 1 Hypoimmune Engineering for Universal iPSC-TEP Workflow

[0180] This example describes the workflow for generating thymic epithelial progenitor cells (TEPs) derived from human-induced pluripotent stem cells (iPSCs) that overexpress human PD-L1 and human HLA-G. As shown in FIG.1, iPSCs are transduced with a nucleic sequence encoding human PD-L1 and human HLA-G using viral vectors or plasmids. Approximately 72 hours post-transduction, the iPSCs undergo antibiotic selection for 7 to 8 days, followed by an initial expansion step before validation via flow cytometry. After this initial validation, the transduced iPSCs are expanded further, either for storage or for differentiation into TEPs. The differentiated TEPs (engineered iPSC-TEPs) are validated in vitro through functional assays, meso scale discovery (MSD), ELISA, flow cytometry, and microscopic analysis. Subsequent to in vitro validation, the engineered iPSC-TEPs undergo further validation in vivo using animal models. 30 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO EXAMPLE 2 Cell Engineering for iPSC-TEPs Overexpressing PD-L1 and HLA-G

[0181] This example describes the generation of iPSC-TEPs constitutively overexpressing PD-L1 and HLA-G.

[0182] To generate iPSC-TEPs constitutively overexpressing PD-L1 and HLA-G, proof of concept (PoC) experiments will be conducted using 3rd generation lentiviral packaging for the transfection of the engineered vector, as illustrated in FIG.2. Polycistronic gene encoding will be employed to ensure protein co-expression and minimize promoter silencing through two successive rounds of monocistronic lentiviral transfection. If balancing the expression of PD- L1 and HLA-G proves problematic, monocistronic lentiviral transfections will be used to fine- tune protein expression. The polycistronic 3rd generation lentiviral vector will be used to overexpress PD-L1 and HLA-G in iPSC-TEP, aiming to reduce allogeneic rejection by donor immune cells in immunocompetent recipients.

[0183] Additionally, to achieve constitutive overexpression of human PD-L1 and human HLA-G, several lentiviral vectors will be designed to maximize the chances of generating a stable line, as depicted in FIG. 3. The pSFFV or EF1A promoters will be used, as shown in FIG.4. The polycistronic PD-L1 / HLA-G transgene cassette will have the stop codon removed in the upstream gene and will utilize a P2A linker, with each gene tested in both positions (pre- and post-linker). A polycistronic selection cassette for EGFP and Puromycin Resistance (T2A linker) will also be included, along with control vectors under both promoters being tested. EXAMPLE 3 Demonstration of Hypoimmunity of iPSC-TEP overexpressing PD-L1 and HLA-G In vitro demonstration

[0184] Due to the mechanism of action of PD-L1 and HLA-G at the interface of cytotoxic T lymphocytes (CTL) and target cells, a traditional mixed lymphocyte reaction (MLR) assay is likely unsuitable for measuring tolerance, as CTL proliferation occurs upstream of target cell lysis. Instead, an adapted MLR assay as illustrated in FIG. 5 and an adapted CTL mediated lysis (CML) assay as illustrated in FIG. 6 will be used to determine hypoimmunity of iPSC- TEPs overexpressing PD-L1 and HLA-G in vitro. In vivo demonstration

[0185] The hypoimmunity of iPSC-TEP overexpressing PD-L1 and HLA-G will be determined in vivo, as illustrated in FIG. 7. The triple transgenic NSG-SGM3 mice will be 31 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO injected with human peripheral blood mononuclear cells (hu-PBMCs) for PBMC humanization 10 days prior to the transplantation of iPSC-TEP. After the transplantation, the terminal tissues will be harvested to assess hypoimmunity at around 10-, 20-, and 30-days post transplantation. Immunotyping of the lymphoid and myeloid cells and post-transplant graft histology will be performed to determine hypoimmunity. EXAMPLE 4 Enhancement of Dendritic Cell Recruitment and Trafficking to iPSC-TEP

[0186] This example describes the enhancement of dendritic cell recruitment and trafficking to iPSC-TEP. In the native thymus, a variety of stromal and lymphoid cell lineages are involved in T cell education. In a transplanted thymus, recipient dendritic cells and fibroblasts that reside in the donor thymus are critical for educating thymocytes about “true self” antigens (recipient HLA and minor antigens), thus preventing autoimmune sequelae. Enhancing the trafficking of recipient dendritic cells (particularly the DC-10 subset expressing HLA-G) to the transplanted iPSC-TEP grafts is a promising approach to improving positive and negative selection in the transplanted thymus. Various methods to augment the trafficking of recipient dendritic cell subsets to the transplanted iPSC-TEP grafts will be explored. EXAMPLE 5 Immunomodulatory Strategies for Thymic Epithelial Cells

[0187] Traditional approaches to generate hypoimmune cell therapy products involve knocking out HLA-I and HLA-II molecules to diminish antigen presentation and thereby “shielding” their allogeneic identify. While HLA-I / II strategies have shown immense preclinical promise, they do not provide a platform for the hypoimmune engineering of all cell types. This is particularly true in the context of Thymic Epithelial Cells (TECs), where intact HLA molecules are critical to the form and function of the cells to perform thymopoiesis (FIG. 8 and as disclosed in Malik et al., Regenerative Medicine, 14:11, 983-989; the contents of which are herein incorporated by reference in their entirety as well as FIG.9).

[0188] Engineered human iPSC cell lines with the constitutive overexpression of human PD- L1 and HLA-G were generated (FIGs. 10A-10C).

[0189] MOI Selection uses PD-L1Hi & HLA-G+ [%] and MFI measurement. For reference, Multiplicity of infection (MOI) refers to the ratio of infectious agents (such as viruses or bacteria) to target cells or host. For example, if 100 viruses are added to a cell culture containing 32 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO 10,000 cells, the MOI is 0.01 (100 viruses / 10,000 cells). Therefore, an MOI 2 is 20,000viruse / 10,000 cells. In Figure 11C, the Intracellular(IC) staining of HLA-G increased from MOI2 to MOI4, but no further increase with MOI8 or MOI16.

[0190] EC HLA-G is absent due to inadequate MHC-I stabilization or presentation (FIGs. 11A-11C). Bimodal expression of PD-L1 in the polyclonal cultures necessitated single-cell sorting (FIGs.11A-11C and FIG.12). EXAMPLE 6 Development of a Hypoimmune Immunogenicity Assay: Adapting a Cell-Mediated Lysis Protocol

[0191] To properly assess the hypoimmune capacity of the herein described clonal engineered iPSC lines, we first need to develop an assay capable of demonstrating robust in vitro cell-mediated immune responses specifically targeting our cell product(s). iPSCs and current generation VPE / TEP cells are not adequate for the application of T cell proliferation or targeted CTL lysis assays due to the absence of Class I / II HLA molecule expression (FIGs. 13A-13B). Generation of HLA-A*02:01 Specific CTLp

[0192] Fuji iPSCs are HLA- / -and poor CTL stimulators (FIG. 14A). HLA-A02:01 specific CTLp was generated by stimulating MALME-3M cells (IRR) which leads to activation of responder APC and interaction with responder T cell to obtain anti-HLA-A02-01 with clonal expansion (FIGs.14A-14B).

[0193] Quantification of HLA-A 02:01 Specific CTLp Activity

[0194] In phase I polyclonal expansion of HLA-A*02:01 specific cytotoxic T lymphocyte precursors (CTLp) were generated (FIG. 15A). In phase II co-culture of CTL effectors were generated with various target cells for the qualification and quantification of HLA-specific cell- mediated lysis (FIG.15B). Cell-Mediated Lysis Assay

[0195] Effector and eFluor 670 labeled target cells were co-cultured at 5% CO2, 37°C for 5 hours with target cells in triplicate at ratios of 100:1, 50:1, 25:1, and 12.5:1 in 96-well round- bottom tissue culture plates (Corning). Following incubation, cell suspensions were transferred to 5 mL polystyrene round bottom tubes (Corning). The cells were washed once in HBSS containing 0.1% Bovine Serum Albumin (BSA Fraction V, Fisher Scientific). Following the 33 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO wash, cells were re-suspended in 300uL of HBSS containing 0.1% BSA and 0.05% NaN3. Prior to acquisition of the samples, 25ul of CountBright absolute counting beads (25,000 beads; Invitrogen) were added to each tube. Tubes were mixed well just prior to collection on a Becton Dickinson FACSCalibur™ running CellQuest™ software Version 3.3 (San Jose, CA); all events within 3200 beads were collected per tube to ensure that the same volume was being analyzed in each sample. Data analysis was performed using FlowJo V_10 (Treestar) software. The results were expressed as a percentage of overall cytotoxic lysis and calculated as follows:

[0196] % = 100 [(#experimental targets / #spontaneous ctrl targets) × 100].

[0197] The described herein hypoimmune engineering approach provide protection against antigen-specific CTLs achieved with edited cells in suspension (CML Assay) (FIGs. 16A- 16B). Protection against antigen-specific CTLs achieved with edited cells in adherent culture demonstrated robust, near-total protection [19-hour Co-culture]. Given the edited cell line phenotypic data, PD-L1 is likely driving the majority of observed CTL suppression. Hypoimmune Protection from HLA-A*02:01 Specific CTLp

[0198] CTL negative controls, A431 cells, showed not CTL cell death. CTL positive controls, MALME-3M cells, showed complete CTL cell death. WT controls showed visible cytotoxicity, while cells with the hypoimmune constructs showed significant reduction in CTL cell death compared to WT (FIG.17).

[0199] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 34 1620353579.1

Claims

PATENT ATTORNEY DOCKET NO. THYM1160-2WO WHAT IS CLAIMED IS:

1. An isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1 or HLA- G, or a combination thereof.

2. The isolated TEP cell of claim 1, wherein the isolated TEP cell expresses an HLA- I molecule.

3. The isolated TEP cell of claim 2, wherein the isolated TEP cell expresses HLA-A, HLA-B, and HLA-C.

4. The isolated TEP cell of claim 1, wherein the isolated TEP cell expresses an HLA- II molecule.

5. The isolated TEP cell of claim 4, wherein the isolated TEP cell expresses HLA-DP, HLA-DQ, and HLA-DR.

6. The isolated TEP cell of claim 1, wherein the isolated TEP cell expresses CIITA.

7. The isolated TEP cell of claim 1, wherein the isolated TEP cell expresses FOXN1.

8. A method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1 and / or HLA-G, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD- L1 and / or HLA-G; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein(BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signaling toproduce anterior foregut endodermal (AFE) cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforminggrowth factor- (TGF- ) signaling to produce ventral pharyngeal endodermal (VPE) cells; andd) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1 and / or HLA-G; thereby generating the TEP cells overexpressing PD-L1 and / or HLA-G. 35 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO 9. The method of claim 8, wherein the DE cells are obtained from pluripotent stem cells by culturing pluripotent stem cells in a medium comprising a growth factor selected from the group consisting of Nodal, Activin A, and Activin B.

10. The method of claim 9, wherein the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling.

11. The method of claim 10, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

12. The method of claim 10, wherein the TEP cells express FOXN1.

13. The method of claim 8, wherein the TEP cells express an HLA-I molecule.

14. The method of claim 8, wherein the TEP cells express an HLA-II molecule.

15. The method of claim 9, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

16. The method of claim 15, wherein the TEP cells express FOXN1.

17. The method of claim 9, wherein the TEP cells express FOXN1.

18. The method of claim 8, wherein the PSCs are selected from the group consisting of embryonic stem cell, embryonic germ cells, and induced pluripotent stem cell.

19. The method of claim 18, wherein the PSCs are primate pluripotent stem cells (pPSCs).

20. The method of claim 19, wherein the pPSCs are human pluripotent stem cells (hPSCs).

21. The method of claim 20, wherein the hPSCs are human embryonic stem cells (hESCs).

22. The method of claim 20, wherein the hPSCs cells are induced pluripotent stem cells (iPSCs).

23. The method of claim 8, wherein the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling. 36 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO 24. The method of claim 23, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

25. The method of claim 23, wherein the TEP cells express FOXN1.

26. The method of claim 8, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

27. The method of claim 26, wherein the TEP cells express FOXN1.

28. The method of claim 8, wherein the TEP cells express FOXN1.

29. The method of claim 8, wherein the nucleic acid sequence is in a vector.

30. The method of claim 29, wherein the vector is a plasmid or a viral vector.

31. The method of claim 30, wherein the virial vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

32. A method of treating or preventing a condition in a subject, comprising administering the isolated TEP cell of claim 1 to the subject.

33. The method of claim 32, wherein the condition is associated with the absence, decline or aberrant functioning of the thymus of the subject.

34. The method of claim 33, wherein the condition is selected from Di George syndrome, thymoma (such as type A thymoma or type B thymoma), CHARGE syndrome, FOXN1 deficiency, PAX1 deficiency, TBX1 deficiency, thymus 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, age- or infection-associated decline in thymic function, thymic- mediated tolerance, transplant tolerance, or autoimmunity.

35. The method of claim 34, wherein the autoimmunity is general or antigen-mediated.

36. The method of claim 33, wherein the subject has undergone a thymectomy surgery.

37. The method of claim 33, wherein the condition is an immunodeficiency.

38. The method of claim 33, wherein the condition is cancer.

39. The method of claim 33, wherein the condition is an autoimmune disease.

40. The method of claim 33, wherein the condition is an infectious disease. 37 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO 41. The method of claim 33, wherein the condition is graft versus host disease (GvHD).

42. An isolated thymic epithelial progenitor (TEP) cell overexpressing PD-L1, HLA- G, and an autoimmune antigen.

43. The isolated TEP cell of claim of 42, wherein the autoimmune antigen is selected from the group consisting of myelin basic protein (MBP) peptide, proinsulin 2, and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP).

44. A method for generating thymic epithelial progenitor (TEP) cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; b) culturing definitive endodermal (DE) cells, obtained from the cells of a), in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signaling to produce anterior foregut endodermal(AFE) cells; c) culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforming growth factor- (TGF- ) signaling to produce ventralpharyngeal endodermal (VPE) cells; and d) culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen; thereby generating the TEP cells overexpressing PD-L1, HLA-G, and the autoimmune antigen.

45. The method of claim 44, wherein the autoimmune antigen is selected from the group consisting of myelin basic protein (MBP) peptide, proinsulin 2, and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP).

46. An isolated hematopoietic stem cells (HSCs) overexpressing PD-L1 or HLA-G, or a combination thereof. 38 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO 47. A method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1 and / or HLA-G, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; thereby generating the HSCs overexpressing PD-L1 and / or HLA-G.

48. An isolated adult tissue progenitor / stem cells overexpressing PD-L1 or HLA-G, or a combination thereof.

49. A method for generating adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1 and / or HLA-G; and b) culturing the cells obtained from a), to further differentiate into progenitor cells of adult tissue, wherein the adult tissues are selected from the group consisting of heart muscle tissue, brain tissue, spinal cord tissue, peripheral nervous tissue, lung tissue, pancreatic islets, liver tissue, kidney tissue, and intestinal lining; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1 and / or HLA-G.

50. An isolated hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen.

51. A method for generating hematopoietic stem cells (HSCs) overexpressing PD-L1, HLA-G, and an autoimmune antigen, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; and b) culturing hemogenic endothelium (HE) cells with the cells obtained from a), to further differentiate into HSCs; 39 1620353579.1PATENT ATTORNEY DOCKET NO. THYM1160-2WO thereby generating the HSC cells overexpressing PD-L1, HLA-G, and the autoimmune antigen.

52. An isolated adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen.

53. A method for generating adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and an autoimmune antigen, comprising: a) transfecting pluripotent stem cells (PSCs) with nucleic acid sequences encoding PD-L1, HLA-G, and the autoimmune antigen; and b) culturing the cells obtained from a), to further differentiate into progenitor cells of adult tissue, wherein the adult tissues are selected from the group consisting of heart muscle tissue, brain tissue, spinal cord tissue, peripheral nervous tissue, lung tissue, pancreatic islets, liver tissue, kidney tissue, and intestinal lining; thereby generating the adult tissue progenitor / stem cells overexpressing PD-L1, HLA-G, and the autoimmune antigen. 40 1620353579.1

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