Thymic epithelial cells and methods of producing and using the same

Abstract

Provided are methods of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs). Such methods comprise culturing PPEv cells in a TEPC differentiation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF-κB) / Toll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. Also provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs). Such methods comprise culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF-κB) / Toll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. TEPCs and matTEPCs produced according to the methods of the present disclosure are also provided, as are compositions comprising the TEPCs and matTEPCs. Methods of using the TEPCs and matTEPCs, e.g., for therapeutic purposes in athymic or hypothymic subjects, are also provided.

Description

[0001] THYMIC EPITHELIAL CELLS AND METHODS OF PRODUCING AND USING THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 611 ,960, filed December 19, 2023, which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] The thymus is a primary lymphoid organ that plays a critical role in the development of adaptive T cell immunity and central tolerance. Bone marrow-derived common lymphoid progenitor cells (CLPs) migrate into the thymus where they interact with thymic epithelial cells (TECs). Once migrated into the thymus, CLPs are referred to as thymocytes. Thymus-resident TECs stimulate thymocytes to proliferate and mature. In addition, TECs define the T-cell receptor (TCR) repertoire of thymocytes through sequential positive and negative selection of thymocytes based on affinity of the TCR to self-peptides presented on major histocompatibility complex (MHC) class I and II of TECs. Mature T cells that have undergone the thymic selection process express a diverse, MHC-restricted and self-tolerant TCR repertoire that protects against infection and prevents autoimmunity. Accordingly, compromise of thymic function can manifest in immunodeficiency, autoimmunity, malignancy, and death.

[0006] Thymic function can be impaired in various congenital or acquired conditions. Patients born with congenital thymic aplasia, due to 22q1 1 Deletion Syndrome, or mutations in TBX1 , FOXN1 or CHD7, can present with complete absence of T cells and a severe combined immunodeficiency (SCID)-like phenotype. During hematopoietic stem cell transplantation (HSCT), acute graft-versus-host disease (aGVHD) can cause severe thymic injury in the recipient leading to impaired T cell reconstitution and increased risk of morbidity and mortality after HSCT. Thymic function can also be compromised by infection, immunoablative therapies, tumor, irradiation, or iatrogenic surgical removal due to cardiac surgeries. The role of the thymus in building immune identity and function begins long before birth. The thymus peaks in size in infancy and then structurally disappears over time. Aging reduces thymic function drastically and results in immune senescence in the elderly. The absence of effective T cell responses in the elderly (immune senescence) explains their susceptibility to infections with novel antigens.

[0007] The thymus has no known endogenous capacity to regenerate. Therefore, the concept of thymic transplantation serves as an ultimate solution to treat thymic defects of congenital or acquired origin. In the case of 22q11 Deletion Syndrome, allogenic postnatal thymus transplantation has provided proof of principle that HLA-unmatched pediatric donor thymic tissues can lead to successful immune reconstitution with the emergence of a diverse TCR V- beta repertoire. However, post-transplant organ-specific autoimmunity remains a major concern. Allogeneic thymus transplantation is currently only available to a very small number of patients leaving an unmet clinical need for patients born without thymus. Lack of histocompatibility- matching limits allogeneic thymic transplantation to patients with complete absence of the thymus. In patients with residual thymic function, preformed T-cells would reject the transplanted tissue unless the patients were medically immunosuppressed. Patient-specific or histocompatible thymic tissues derived from pluripotent stem cells could address the critically unmet need. Importantly, beyond its use for thymic reconstitution in 22q1 1 Deletion Syndrome and other genetic syndromes presenting with thymic aplasia / hypoplasia, transplantation of histocompatible iPSC-derived functional thymic tissue has a much broader range of clinical applications in conditions in which thymic function is compromised or requires modulation, specifically but not limited to, its use after HSCT to promote T cell immune reconstitution, as well as treat (and mitigate the risk developing) of chronic graft versus host disease (GVHD). In addition, regenerative thymic tissues could treat autoimmune diseases due to thymic defects, e.g., such as APECED syndrome, and contribute to the induction of central tolerance in the setting of donor organ transplantation when matched to the donor organ. Moreover, regenerating thymic function could augment the declining immune function during aging (immune senescence), thereby reducing the age-associated increase in inflammation (i.e. “inflammaging”). In addition, regenerative thymic tissues can improve effective responses to vaccines, both in the context of infection and cancer and are expected to potentiate the effect of all immunotherapies that rely on the body’s own immune system, e.g. checkpoint blockade.

[0008] The thymus is derived from the anterior foregut endoderm. During development, the anterior foregut gives rise to the ventrally located respiratory system and the dorsally located esophagus. The anterior-most segment of the foregut develops into the pharyngeal apparatus. Essential to the development of the mammalian pharyngeal apparatus is the out-pocketing of the pharyngeal pouches. The thymic primordium, also referred to as thymus anlage, forms from the ventro-caudal region of the third pharyngeal pouch, while the dorsal aspect gives rise to the parathyroid. The signals that induce iPSCs into anterior foregut endoderm have long been described, and protocols to differentiate anterior foregut endoderm further into epithelial cells of lung and esophagus in vitro have successfully been established. Recent studies of murine and human thymus using single-cell transcriptomics have greatly expanded our understanding of thymic epithelial cell (TEC) diversity, yet the signals that drive TEC fate beyond the anterior foregut stage remain incompletely understood.

[0009] In human embryonic samples, the master thymic transcription factor FOXN1 is detected as early as mid-week 6 when other markers of the third pharyngeal pouch and early thymic primordium are also highly expressed (PAX1 , FOXG1 , PAX9, H0XA3, EYA1 ). Hematopoietic progenitors enter the thymus anlage as early as post-conception week (pew) 7. At pew 8, less than 5% of cells in the developing human thymus are of hematopoietic origin (CD45+), while by pew 10, more than 90% of cells in the thymus are of hematopoietic origin, highlighting the dramatic expansion of thymocytes (i.e., immature T cell precursors) and other immune cells during thymic morphogenesis. Environmental cues, including soluble gradient signaling and direct cell-cell interactions, induce cells of the thymic primordium to mature into mature thymic epithelial cells. Mature thymic epithelial cells not only express markers of the thymic primordium (FOXN1 , PAX1 , PAX9 and H0XA3), but functional markers important for the development and selection of T cells such as AIRE, CD83, DSP, NFAT5, IL-32, RELB, CXCL9, CXCL10, CXCL11 , OAS3, TP63, NFkB and more.

[0010] SUMMARY

[0011] Provided are methods of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs). Such methods comprise culturing PPEv cells in a TEPC differentiation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF-KB) / Toll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. Also provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs). Such methods comprise culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. TEPCs and matTEPCs produced according to the methods of the present disclosure are also provided, as are compositions comprising the TEPCs and matTEPCs. Methods of using the TEPCs and matTEPCs, e.g., to provide or reconstitute T cell development and function in a subject in need thereof, are also provided.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 : Five exemplary stages of thymic epithelial progenitor differentiation in vitro according to some embodiments of the present disclosure. The three boxes indicate that one or any combination of an inflammasome activator, a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator, a TLR signaling activator, andZor a tumor necrosis factor (TNF) signaling activator may be included in the culture medium when differentiating PPEIII cells into PPEv cells (lower left box), differentiating PPEv cells into TEPCs (lower right box), or maturing TEPCs into mature TEPCs (upper box post-D24).

[0014] FIG. 2: Key markers of thymic ontogeny. Gene expression (analyzed by bulk RNA sequencing) of key marker genes defining the five stages of thymic epithelial ontogeny in vitro.

[0015] FIG. 3A-3B: Effects of inflammasome signaling pathway activation on thymic epithelial progenitor differentiation in vitro. 3A: Heatmap of extended list of top differentially expressed genes in primary human fetal thymic epithelial cells relative to other human fetal anterior foregut derived organs (BioRx, doi: 10.1 101Z2022.10.02.510339). Gene expression at TEPC stage differentiated as per U.S. Patent Application Publication No. US20240076618A1 (incorporated herein by reference in its entirety for all purposes) as baseline control (left) relative to gene expression of TEPC cells differentiated with the same protocol plus an inflammasome activator (right). 3B: Cumulative (bulk) RNAseq expression of select differentially expressed genes from (A) at TEPC stage as per US20240076618A1 as baseline control (left) relative to gene expression of TEPC cells differentiated with the same protocol plus an inflammasome activator (right).

[0016] FIG. 4A-4B: Effects of NFkB / TLR signaling pathway activation on thymic epithelial progenitor differentiation in vitro. 4A: Heatmap of extended list of top differentially expressed genes in primary human fetal thymic epithelial cells relative to other human fetal anterior foregut derived organs (BioRx, doi: 10.1 101 / 2022.10.02.510339). Gene expression at TEPC stage differentiated as per US20240076618A1 as baseline control (left) relative to gene expression of TEPC cells differentiated with the same protocol plus an NFkB / TLR activator (right). 4B: Cumulative (bulk) RNAseq expression of select differentially expressed genes from (A) at TEPC stage as per US20240076618A1 as baseline control (left) relative to gene expression of TEPC cells differentiated with the same protocol plus an NFKB / TLR activator (right).

[0017] FIG. 5: T cell development of athymic mice transplanted with TEPCs treated with inflammasome, NFKB, TLR and TNF signaling pathway activators. Athymic NSG-FoxN1 null mice were engrafted with human hematopoietic stem cells and transplanted with iPSC-derived TEPCs differentiated as per US20240076618A1 and the addition of an inflammasome signaling pathway activator (IL-1 b), an NFKB / TLR signaling pathway activator (BO-1 12), and a TNF signaling pathway activator (LTA / B). Note the development of CD3+ human T cells beginning 5- 10 weeks after TEPC transplantation under the kidney capsule.

[0018] DETAILED DESCRIPTION

[0019] Before the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

[0020] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0021] 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 the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described.

[0022] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0023] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0024] It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0025] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0026] METHODS OF PRODUCING THYMIC EPITHELIAL PROGENITOR CELLS (TEPCS)

[0027] Aspects of the present disclosure include methods of producing thymic epithelial progenitor cells (TEPCs). In some aspects, provided are methods of differentiating PPEIII cells to ventral pharyngeal pouch endoderm (PPEv) cells, such methods comprising culturing PPEIII cells in a PPEv differentiation medium comprising an inflammasome activator, a nuclear factorkappa B (NF-KB)ZToll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. In some aspects, provided are methods of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), such methods comprising culturing PPEv cells in a TEPC differentiation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF- KB) / Toll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof. In some aspects, provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs), such methods comprising culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator, a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator, a TLR signaling activator, a tumor necrosis factor (TNF) signaling activator, or any combination thereof.

[0028] PPEv cells (sometimes referred to herein as “PPE-ventral” cells) are characterized by TBXI dim, PAX1 +, PAX9+ or FOXG1 +, FOXNI dim gene expression (see FIG. 2). TEPCs are characterized by FOXN1 +, TP63+, and ASCL1 + gene expression (see FIG. 2). Mature TEPCs (matTEPCs) are characterized by FOXN1 +, TP63+, and ASCL1 + with gain in expression of but not limited to CXCL10, CXCL1 1 and RELB genes. Lists of genes that gain expression through the addition of maturation factors (i.e., an inflammasome activator, an NFkB / TLR signaling activator or a TNFsignaling activator) are provided (see FIG. 3)

[0029] As used herein, a “TEPC differentiation medium” is a basal medium containing factors sufficient to differentiate PPEv cells to TEPCs. A suitable TEPC differentiation medium is one that includes, e.g., an IFN type I or II signaling pathway activator (e.g., IFN gamma), an FGF signaling pathway activator (e.g., FGF7 and / or FGF10), an IGF signaling pathway activator (e.g., IGF2), BMP4, a WNT signaling pathway activator, or any combination thereof.

[0030] Basal medium can be, without limitation, chemically defined medium (CDM2), RPMI1640 or DMEM / F12. Basal medium can be supplemented with, but not limited to, Knockout serum replacement (KOSR), B27 or N2 supplements. This medium composition applies to all the stages of the TEPC differentiation and TEPC maturation methods. Other basal media with different bases as defined above can be used. In certain embodiments, provided are methods of differentiating PPEv cells to TEPCs, the methods comprising culturing the PPEv cells in a TEPC differentiation medium comprising an inflammasome activator. According to some embodiments, provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator. Inflammasomes are cytoplasmic multiprotein complexes comprising a sensor protein, inflammatory caspases, and in some but not all cases an adapter protein connecting the two. They can be activated by a repertoire of endogenous and exogenous stimuli, leading to enzymatic activation of canonical caspase-1 , noncanonical caspase-11 (or the equivalent caspase-4 and caspase-5 in humans) or caspase-8, resulting in secretion of IL-1 p and IL-18, as well as apoptotic and pyroptotic cell death. Details regarding the inflammasome and activation thereof are described in Zheng et al. (2020) Cell Discovery volume 6, Article number: 36.

[0031] According to any aspects and embodiments of the present disclosure, when an inflammasome activator is employed, the medium may comprise as the inflammasome activator one or any combination of IL-1 alpha, IL-1 beta, IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL- 36 gamma, IL-36ra, IL-37, IL-37b, and / or IL38. In some instances, the medium comprises IL-1 beta. When the medium comprises IL-1 beta, the IL-1 beta may be present at a concentration of 0.2-20 ng / mL. In one non-limiting example, the IL-1 beta may be present at 2 ng / mL.

[0032] For methods in which the medium (differentiation or maturation medium) comprises an inflammasome activator, the medium may further comprise one, two or each of a nuclear factorkappa B (NF-KB) / Toll-like receptor (TLR) signaling activator, a TLR signaling activator, and / or a tumor necrosis factor (TNF) signaling activator. Such activators are described in further detail below.

[0033] In certain embodiments, provided are methods of differentiating PPEv cells to TEPCs, the methods comprising culturing the PPEv cells in a TEPC differentiation medium comprising an inflammasome activator. According to some embodiments, provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs), the methods comprising culturing TEPCs in a TEPC maturation medium comprising a nuclear factor-kappa B (NF-KB) / Toll-like receptor (TLR) signaling activator. As used herein, an “NF-KB / TLR signaling activator” is an agent that activates signaling of both the NF-KB and TLR signaling pathways.

[0034] According to any aspects and embodiments of the present disclosure, when an NF- KB / TLR signaling activator is employed, the medium may comprise as an NF-KB / TLR signaling activator one or any combination of BO-1 12 (a nanoplexed form of poly i nosi n ic: polycytidy lie acid (poly l:C)), and / or polyinosinic:polycytidylic acid (poly l :C). In some instances, the medium comprises poly l:C as an NF-KB / TLR signaling activator. In some instances, the medium comprises BO-112 as an NF-KB / TLR signaling activator. When the medium comprises BO-112 as an NF-KB / TLR signaling activator, the BO-1 12 may be present at a concentration of 0.01 - 6ug / mL. In one non-limiting example, the BO-1 12 is present at 0.2 ug / mL. For methods in which the medium (differentiation or maturation medium) comprises an an NF-KB / TLR signaling activator, the medium may further comprise one, two or each of an inflammasome activator, a TLR signaling activator, and / or a tumor necrosis factor (TNF) signaling activator. TLR and TNF signaling activators are described in further detail below.

[0035] In certain embodiments, provided are methods of differentiating PPEv cells to TEPCs, the methods comprising culturing the PPEv cells in a TEPC differentiation medium comprising a TLR signaling activator. According to some embodiments, provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs), the methods comprising culturing TEPCs in a TEPC maturation medium comprising a TLR signaling activator. As used herein, a “TLR signaling activator” is an agent that activates a TLR signaling pathway without also activating an NF-KB pathway.

[0036] According to any aspects and embodiments of the present disclosure, when a TLR signaling activator is employed, the medium may comprise as a TLR signaling activator one or any combination of CU-CPT17e, CU-T12-9, and / or Diprovocim. In some instances, the medium comprises CU-CPT17e (e.g., at a concentration of from 0.1 -10 uM, such as 1 uM) as a TLR signaling activator. In some instances, the medium comprises CU-T12-9 (e.g., at a concentration of from 10nM-10uM) as a TLR signaling activator. In some instances, the medium comprises Diprovocim (e.g., at a concentration of from 0.1 -10uM) as a TLR signaling activator.

[0037] For methods in which the medium (differentiation or maturation medium) comprises a TLR signaling activator, the medium may further comprise one, two or each of an inflammasome activator, an NF-KB / TLR signaling activator, and / or a tumor necrosis factor (TNF) signaling activator. TNF signaling activators are described in further detail below.

[0038] In certain embodiments, provided are methods of differentiating PPEv cells to TEPCs, the methods comprising culturing the PPEv cells in a TEPC differentiation medium comprising a tumor necrosis factor (TNF) signaling activator. According to some embodiments, provided are methods of producing mature thymic epithelial progenitor cells (matTEPCs), the methods comprising culturing TEPCs in a TEPC maturation medium comprising a tumor necrosis factor (TNF) signaling activator. As used herein, a “TNF signaling activator” is an agent that activates a tumor necrosis factor (TNF) signaling pathway.

[0039] According to any aspects and embodiments of the present disclosure, when a TNF signaling activator is employed, the medium may comprise as a TNF signaling activator one or any combination of lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, and / or GITRL. In some instances, the medium comprises LTA / B. The LTA / B may be present at a concentration of 1-1000 ng / mL, a non-limiting example of which is 10 ng / mL. In some instances, the medium comprises LTB. The LTB may be present at a concentration of 1-1000 ng / mL, a non-limiting example of which is 10 ng / mL. In some instances, the medium comprises LTA. The LTA may be present at a concentration of 1 -1000 ng / mL, a non-limiting example of which is 10 ng / mL.

[0040] For methods in which the medium (differentiation or maturation medium) comprises a TNF signaling activator, the medium may further comprise one, two or each of an inflammasome activator, an NF-KB / TLR signaling activator, and / or a TLR signaling activator.

[0041] Any of the aforementioned culturing steps are carried out for a suitable period of time. In some embodiments, the culturing (e.g., to differentiate a PPEv to a TEPC) is for a duration of from 4 to 20 days, from 5 to 15 days, or from 6 to 12 days, e.g., from 6 to 10 days.

[0042] Any of the differentiation and / or maturation methods of the present disclosure may further include administering the produced TEPCs or matTEPCs to a subject in need thereof. Subjects of interest include those described in Methods of Use section below.

[0043] The term “medium” in the context of cell culture or the phrase “cell culture medium” or “cell medium” refers to a cellular growth medium suitable for culturing of pluripotent stem cells (PSCs), DE cells, AFG cells, AFG-plus cells, bipotent PPEIII cells, ventral PPEIII cells, and / or TEPCs. Examples of cell culture medium include, without limitation to, Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12), Ham's F10 Nutrient Mixture, Ham's F12 Nutrient Mixture, Medium 199, RPMI, RPMI 1640, reduced serum medium, Basal Medium Eagle's (BME), 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, KOSR, and combinations thereof, and differentiation and / or maturation factors as described herein.

[0044] Numerous alternative approaches are available to produce PPEv cells (e.g, from induced pluripotent stems cells (iPSCs) or embryonic stem cells (ESCs)) which may then be differentiated into TEPCs according to the methods of the present disclosure. Stages and differentiation factors are described in detail in Example 1 herein and outlined in FIG. 1. In addition, non-limiting example approaches for producing PPEv cells are described U.S. Patent Application Publication No. US20240076618A1 , the disclosure of which is incorporated herein by reference in its entirety.

[0045] In some embodiments, methods of differentiating a Definitive Endoderm (DE) cell or an Anterior Foregut (AFG) cell or an Anterior Foregut (AFG)-plus cell, to a HOXA3+, TBX1 + / high, PPEIII cell that has the potency to further differentiate into thymus are provided. In some embodiments, the method comprises culturing the DE cell or AFG cell or AFG-plus cell in a medium comprising at least: a Transforming Growth Factor (TGF-[3) signaling pathway inhibitor; a Bone Morphogenic Protein (BMP) signaling pathway inhibitor; a Retinoic Acid (RA) signaling pathway activator; a PI3K / AKT signaling pathway inhibitor, wherein the culturing of the DE or AFG or AFG-plus cell promotes differentiation to a PPEIII cell specified for PPEIII primed for thymus fate. In some embodiments, the medium further comprises a Fibroblast Growth Factor (FGF) signaling pathway activator. In some embodiments, the medium further comprises a canonical WNT signaling pathway inhibitor.

[0046] In some embodiments, the method further comprises differentiating the HOXA3+, TBX1 + / high PPEIII cell into a human TBX1 dim, PAX1 +, PAX9+, FOXG1 + PPE-ventral cell by culturing the PPEIII cell in a second medium comprising: a WNT signaling pathway activator; a BMP signaling pathway activator; wherein the culturing promotes differentiation of the PPEIII cell to a PPE-ventral cell.

[0047] In some embodiments, the second medium further comprises one, some or all of the following: an FGF signaling pathway activator, an IGF signaling pathway activator, and a Sonic Hedgehog (SHH) signaling pathway inhibitor. In some embodiments, the second medium comprises an FGF signaling pathway activator. In some embodiments, the second medium comprises an IGF signaling pathway activator. In some embodiments, the second medium comprises a Sonic Hedgehog (SHH) signaling pathway inhibitor.

[0048] In some embodiments, the method further comprises differentiating the PPE-ventral cell to a FOXN1 +, TP63+, ASCL1 + TEPC cell, wherein the differentiation protocol comprises culturing the ventral PPEIII cell in a third medium comprising: an IFN type I or II signaling pathway activator; a FGF signaling pathway activator; and an IGF signaling pathway activator.

[0049] In some embodiments, the third medium further comprises one, some or all three of the following: a Sonic Hedgehog (SHH) signaling pathway inhibitor, a canonical WNT signaling pathway inhibitor and a RANK ligand.

[0050] In some embodiments, the DE or AFG cell is a primary DE or AFG cell, respectively.

[0051] Also provided are methods of differentiating a HOXA3+, TBX1 + / high, PPEIII cell to a TBX1 dim, PAX1 +, PAX9+ FOXG1 + PPE-ventral cell that can further differentiate into thymic epithelial progenitor cells. In some embodiments, the method comprises culturing the PPEIII cell in a medium comprising at least: a WNT signaling pathway activator; a BMP signaling pathway activator, wherein the culturing promotes differentiation of the PPEIII cell to a PPE- ventral cell.

[0052] In some embodiments, the medium further comprises one, some or all of the following: an FGF signaling pathway activator, an IGF signaling pathway activator, and a Sonic Hedgehog (SHH) signaling pathway inhibitor. In some embodiments, the medium comprises an FGF signaling pathway activator. In some embodiments, the medium comprises an IGF signaling pathway activator. In some embodiments, the medium comprises a Sonic Hedgehog (SHH) signaling pathway inhibitor.

[0053] CELLS AND COMPOSITIONS

[0054] Aspects of the present disclosure further include cells and compositions. In some aspects, provided is a population of TEPCs and / or matTEPCs produced according to the differentiation and / or maturation methods of the present disclosure, e.g., those described in the preceding section relating to Methods of Producing Thymic Epithelial Progenitor Cells (TEPCs). Also provided are compositions comprising such cell populations.

[0055] Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like.

[0056] In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In some instances, a cell therapy dose includes, e.g., at least 100,000, 250,000, 500,000, 1 million, 5 million, 10 million, at least 25 million, at least 50 million, at least 75 million, at least 100 million, at least 250 million, at least 500 million, at least 750 million, at least 1 billion, at least 2 billion, at least 3 billion, at least 4 billion, at least 5 billion, at least 6 billion, at least 7 billion, at least 8 billion, at least 9 billion, at least 10 billion, at least 15 billion, at least 20 billion, at least 30 billion, at least 40 billion, at least 50 billion, at least 60 billion, at least 70 billion, at least 80 billion, at least 90 billion, or at least 100 billion therapeutic cells.

[0057] In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI2, KOI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0058] The compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder, e.g., athymia, hypothymia, immune senescence, or the like. An effective amount can be administered in one or more administrations.

[0059] A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a human subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain aspects, a pharmaceutical composition of the present disclosure includes from 1 x106to 5x1010of the cells produced according to the methods of the present disclosure.

[0060] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like.

[0061] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0062] The cells may be formulated for parenteral (e.g., subcutaneous, intramuscular, etc.) administration, or any other suitable route of administration.

[0063] An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0064] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM. In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, tricresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).

[0065] METHODS OF USE

[0066] Aspects of the present disclosure further include methods of using a population of cells of the present disclosure. Accordingly, provided are methods comprising administering an effective amount of a population of TEPCs and / or matTEPCs produced according to the differentiation and / or maturation methods of the present disclosure to a subject (e.g., a human subject) in need thereof.

[0067] The therapeutic cells may be autologous / autogeneic (“self”) or non-autologous (“nonself,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human.

[0068] Aspects of the present disclosure include methods of providing or reconstituting T cell development and function in a subject in need thereof, the method comprising administering an effective amount of the TEPCs or matTEPCs to the subject.

[0069] The cells described herein can be used to ameliorate, for example, any diseases or disorders involving defective thymic epithelial cells. Exemplary subjects receiving the cells can include, for example, subjects born with congenital thymic aplasia, e.g., due to 22q11 Deletion Syndrome, or mutations in TBX1 , FOXN1 or CHD7, for example who can present with T-cell immunodeficiency. In other embodiments, the cells described herein can be administered to human patients before or during or after hematopoietic stem cell transplantation (HSCT), for example to mitigate the injury caused to thymic cells by acute graft-versus-host disease (aGVHD). In other embodiments, the cells described herein can be administered to subjects having compromised thymic function, for example but not limited to, caused by infection, cancer, irradiation, medication or iatrogenic surgical removal due to cardiac surgeries. In other embodiments, the cells described herein can be administered to elderly individuals, e.g., over 50, 55, 60, 65, 70, 75, or 80 years old, for example individuals experiencing reduced thymic function due to age (immune senescence).

[0070] In some embodiments, the cells as described herein can be used to induce tolerance to transplanted solid organs (for example but not limited to kidney, heart, lung, islet cells or liver). In some embodiments, autologous or HLA-matched allogeneic cells as described herein are used to induce stable donor-specific immune tolerance during allogeneic solid organ transplant. This can reduce the dependence of patients on immunosuppressive drugs and address the current shortage of HLA-matched donor organs.

[0071] In some embodiments, the cells as described herein can be administered to a recipient of pancreatic islet (p-cells) to treat type I diabetes mellitus (DM). Thymic dysfunction contributes to the development of type I DM through impaired presentation of tissue-restricted antigens and / or negative selection of autoreactive T cells, thus is permissive of the production of self- reactive T cells that target and destroy islet cells. The thymic transplant can eliminate these self- reactive T cells through negative selection (clonal deletion) of autoreactive T cells on AIRE- positive thymic epithelial cells that express tissue restricted antigens (including pro-insulin) and through the generation of regulatory T cells to further protect islet function through induction of central tolerance.

[0072] For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

[0073] 1 . A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising an inflammasome activator.

[0074] 2. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator.

[0075] 3. The method of clause 1 or 2, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

[0076] 4. The method of clause 3, wherein the inflammasome activator is IL-1 beta.

[0077] 5. The method of any one of clauses 1-4, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator.

[0078] 6. The method of clause 5, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

[0079] 7. The method of any one of clauses 1-6, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

[0080] 8. The method of clause 7, wherein the TLR signaling activator is CU-CPT 17e, CU-T 12-9, Diprovocim, or any combination thereof. 9. The method of any one of clauses 1-8, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.

[0081] 10. The method of clause 9, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

[0082] 11 . The method of clause 10, wherein the TNF signaling activator is LTB.

[0083] 12. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator.

[0084] 13. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a nuclear factorkappa B (NF-KB) / Toll-like receptor (TLR) signaling activator.

[0085] 14. The method of clause 12 or 13, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

[0086] 15. The method of any one of clauses 12-14, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator.

[0087] 16. The method of clause 15, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

[0088] 17. The method of clause 16, wherein the inflammasome activator is IL-1 beta.

[0089] 18. The method of any one of clauses 12-17, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

[0090] 19. The method of clause 18, wherein the TLR signaling activator is CU-CPT 17e, CU-T 12- 9, Diprovocim, or any combination thereof.

[0091] 20. The method of any one of clauses 12-19, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.

[0092] 21 . The method of clause 20, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

[0093] 22. The method of clause 21 , wherein the TNF signaling activator is LTB.

[0094] 23. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a Toll-like receptor (TLR) signaling activator. 24. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a Toll-like receptor (TLR) signaling activator.

[0095] 25. The method of clause 23 or 24, wherein the TLR signaling activator is CU-CPT17e, CU- T12-9, Diprovocim, or any combination thereof.

[0096] 26. The method of any one of clauses 23-25, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator.

[0097] 27. The method of clause 26, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

[0098] 28. The method of clause 27, wherein the inflammasome activator is IL-1 beta.

[0099] 29. The method of any one of clauses 23-28, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB)ZTLR signaling activator.

[0100] 30. The method of clause 29, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly l:C), or a combination thereof.

[0101] 31 . The method of any one of clauses 23-30, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.

[0102] 32. The method of clause 31 , wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

[0103] 33. The method of clause 32, wherein the TNF signaling activator is LTB.

[0104] 34. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a tumor necrosis factor (TNF) signaling activator.

[0105] 35. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a tumor necrosis factor (TNF) signaling activator.

[0106] 36. The method of clause 34 or 35, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

[0107] 37. The method of clause 36, wherein the TNF signaling activator is LTB.

[0108] 38. The method of any one of clauses 34-36, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator. 39. The method of clause 38, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

[0109] 40. The method of clause 39, wherein the inflammasome activator is IL-1 beta.

[0110] 41 . The method of any one of clauses 34-40, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator.

[0111] 42. The method of clause 41 , wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

[0112] 43. The method of any one of clauses 34-42, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

[0113] 44. The method of clause 43, wherein the TLR signaling activator is CU-CPT 17e, CU-T 12- 9, Diprovocim, or any combination thereof.

[0114] 45. The method of any one of clauses 1-44, wherein the culturing is for a duration of from 5 to 15 days.

[0115] 46. The method of any one of clauses 1-45, further comprising administering the TEPCs or matTEPCs to a subject in need thereof.

[0116] 47. A method comprising administering an effective amount of TEPCs or matTEPCs produced according to the method of any one of clauses 1 -45 to a subject in need thereof.

[0117] 48. A method of providing or reconstituting T cell development and function in a subject in need thereof, the method comprising administering an effective amount of TEPCs or matTEPCs produced according to the method of any one of clauses 1 -45 to the subject.

[0118] 49. The method of any one of clauses 46-48, wherein the subject has athymia or hypothymia.

[0119] 50. The method of clause 49, wherein the subject has 22q11 Deletion Syndrome characterized by TBX11 deficiency, or FOXN1 or PAX1 or CHD7 deficiency.

[0120] 51 . The method of any one of clauses 46-48, wherein the subject is an elderly subject having immune senescence.

[0121] 52. The method of any one of clauses 46-48, wherein the subject has an acquired thymic defect.

[0122] 53. The method of clause 52, wherein the subject has undergone hematopoietic stem cell transplantation.

[0123] 54. A population of TEPCs or matTEPCs produced according to the method of any one of clauses 1 -45.

[0124] 55. A composition comprising a population of TEPCs or matTEPCs produced according to the method of any one of clauses 1-45. 56. The composition of clause 55, wherein the composition is formulated for administration to a subject in need thereof.

[0125] The following examples are offered by way of illustration and not by way of limitation.

[0126] EXPERIMENTAL

[0127] Example 1 - Production of Thymic Epithelial Progenitor Cells

[0128] Outlined in the present example is a stepwise differentiation protocol to thymic epithelial progenitor cells using human pluripotent stem cells is illustrated in FIG. 1. The entire course of in vitro differentiation can be divided into 5 stages based on the phenotype of the differentiated cells achieved at each stage under corresponding inductive signals:

[0129] Stage 1 : generation of DE cells from human pluripotent stem cells

[0130] Stage 2: generation of AFG or AFG-plus cells from DE cells

[0131] Stage 3: generation of bipotent PPEIII cells from AFG cells

[0132] Stage 4: generation of PPE ventral cells from bipotent PPEIII cells

[0133] Stage 5: generation of thymic epithelial progenitor cells (TEPCs) from ventral PPE cells, The described signaling pathways in this disclosure improve the fidelity of TEPCs to primary human TECs.

[0134] In vitro differentiation stage 1 generation of DE cells from human pluripotent stem cells.

[0135] The method to generate DE cells is adapted from the work of Loh (Loh, Ang et al, 2014, Cell Stem Cell). As shown in FIG. 1 , such DE cells express endodermal markers such as SOX17, CXCR4.

[0136] In vitro differentiation stage 2 generation of AFG or AFG-plus cells from DE cells.

[0137] The present method of AFG generation is based on inhibition of both TGF[3 and BMP signaling pathways (a.k.a, dual SMAD inhibition). Dual SMAD inhibition was first reported by Green et al (Green, Chen et al. 2011 Nature Biotechnology) to induce AFG cells.

[0138] In the disclosed method, DE cells were cultured in basal medium containing dual SMAD inhibition reagents to induce AFG cells with or without low concentrations of ATRA for 2 days. For example, TGF[3 signal inhibitor A 83-01 (1 uM) combined with BMP signal inhibitor LDN193189 (250nM), can specify DE cells into AFG cells that express SOX17, and CXCR4 (FIG.2). Optional addition of ATRA (5nM-5uM) can induce AFG-plus cells.

[0139] In vitro differentiation stage 3 generation of bipotent PPEIII cells from AFG or AFG-plus cells using continued dual SMAD inhibition together with PI3K / AKT pathway inhibition (e.g., PI 828 at 5uM) plus canonical WNT signal inhibition (e.g., endo-IWR 1 at 100nM) plus FGF / ERK / MAPK pathway activation (e.g., FGF 7 at 20ng / ml and FGF10 at 20ng / ml) and optimal retinoic acid signaling activation (e.g., ATRA at 5-100nM) for 3-10 days drives AFG or AFG-plus cells into bipotent PPEIII cell stage. Compared to AFG or AFG-plus cells, the expression of TBX1 , HOXA3 and PAX9 in bipotent PPEIII cells is significantly increased. The highly controlled modulation of ATRA in combination with the aforementioned signals is necessary to achieve the synchronized expression of the bipotent PPEIII markers (HOXA3, PAX9, FOXG1 and TBX1 ), which are detected in >95% of cells in our bipotent PPEIII protocol. This method is described in U.S. Patent Application Publication No. US20240076618A1 which is incorporated herein by reference in its entirety for all purposes.

[0140] In vitro differentiation stage 4: generation of PPE-ventral cells from bipotent PPEIII cells.

[0141] TBXIdim, PAX1 +, PAX9+, FOXG1 + PPE- ventral cells were generated through combined activation of WNT signal (e.g., CHIR99021 at 2uM), BMP signal (e.g., BMP4 at 50ng / ml) and IGF signal (e.g., IGF2 at 20ng / ml) together with FGF signaling activation (e.g. FGF7 at 20ng / mL and FGF10 at 20ng / mL), and SHH signal inhibition (e.g., Vismodegib at 200nM) for 8 days as described in US20240076618A1 .

[0142] In vitro differentiation stage 5: generation of TEPCs from PPE ventral cells.

[0143] PPE-ventral cells can be differentiated into TEPCs using an IFN type II activator (e.g., IFN gamma), an FGF signaling pathway activator (e.g., FGF7 and FGF10 (e.g., 20 ng / ml each)), and an IGF signaling pathway activator (e.g., IGF2 (e.g., 20 ng / ml)) for 8 days.

[0144] The present inventors discovered that inflammasome signaling activators, NFkB / TLR signaling activators and TNF signaling activators provide signals that further drive the maturation of TEPCs herein defined as any FOXN1 + iPSC or ESC-derived cells. It was determined that these signals markedly enhance the function of TEPCs upon transplantation in vivo. These signaling molecules, one or more of which may be used alone or in combination with a TEPC differentiation or maturation medium (e.g., comprising IFN gamma, FGF7, FGF10 and / or IGF2), include:

[0145] 1 ) an inflammasome signaling activator, e.g., IL-1 b (2 ng / mL),

[0146] 2) an NFkB / TLR signaling activator, e.g., a poly l:C nanoplex (BO-112) (0.2 pg / mL),

[0147] 3) a TLR signaling activator, e.g. CU-CPT17e (e.g., 1 pM), or a TNF signaling activator, e.g., LTA / LTB (e.g., 10 ng / mL).

[0148] Example 2 - Effects of inflammasome signaling pathway activation and NFKB / TLR signaling pathway activation on thymic epithelial progenitor differentiation in vitro

[0149] Conducted in this example was expression analysis by RNAseq of TEPCs differentiated according to the protocol described in US20240076618A1 as compared to TEPCs differentiated in the presence of inflammasome activation (FIG. 3A-3B) or NFkB / TLR signaling pathway activation (FIG. 4A-4B). As shown in FIGs. 3 and 4, the TEPCs differentiated in the presence of inflammasome activation or NFkB / TLR signaling pathway activation more closely resemble human fetal thymic epithelial cells.

[0150] Example 3 - The TEPCs give rise to functional T cells in vivo

[0151] In this example, athymic NSG-FoxN1 null mice were engrafted with human hematopoietic stem cells and transplanted with iPSC-derived TEPCs differentiated as per US20240076618A1 with the addition of an inflammasome signaling pathway activator (IL-1 b), an NFKB / TLR signaling pathway activator (BO-112), and a TNF signaling pathway activator (LTA / B) as described in Example 1. As shown in FIG. 5, development of CD3+ human T cells was observed beginning 5- 10 weeks after TEPC transplantation under the kidney capsule.

[0152] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising an inflammasome activator.

2. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising an inflammasome activator.

3. The method of claim 1 or 2, wherein the inflammasome activator is IL-1 alpha, IL-1 beta,IL-1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

4. The method of claim 3, wherein the inflammasome activator is IL-1 beta.

5. The method of any one of claims 1 -4, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB) / Toll-like receptor (TLR) signaling activator.

6. The method of claim 5, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

7. The method of any one of claims 1 -6, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

8. The method of claim 7, wherein the TLR signaling activator is CU-CPT17e, CU-T12-9, Diprovocim, or any combination thereof.

9. The method of any one of claims 1 -8, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.

10. The method of claim 9, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1A, GITRL, or any combination thereof.11 . The method of claim 10, wherein the TNF signaling activator is LTB.

12. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a nuclear factor-kappa B (NF-KB) / Toll-like receptor (TLR) signaling activator.

13. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a nuclear factorkappa B (NF-KB) / Toll-like receptor (TLR) signaling activator.

14. The method of claim 12 or 13, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

15. The method of any one of claims 12-14, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator.

16. The method of claim 15, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL- 1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

17. The method of claim 16, wherein the inflammasome activator is IL-1 beta.

18. The method of any one of claims 12-17, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

19. The method of claim 18, wherein the TLR signaling activator is CU-CPT 17e, CU-T 12-9, Diprovocim, or any combination thereof.

20. The method of any one of claims 12-19, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.21 . The method of claim 20, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

22. The method of claim 21 , wherein the TNF signaling activator is LTB.

23. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a Toll-like receptor (TLR) signaling activator.

24. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a Toll-like receptor (TLR) signaling activator.

25. The method of claim 23 or 24, wherein the TLR signaling activator is CU-CPT 17e, CU- T12-9, Diprovocim, or any combination thereof.

26. The method of any one of claims 23-25, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator.

27. The method of claim 26, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL- 1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

28. The method of claim 27, wherein the inflammasome activator is IL-1 beta.

29. The method of any one of claims 23-28, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB)ZTLR signaling activator.

30. The method of claim 29, wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.31 . The method of any one of claims 23-30, wherein the TEPC differentiation or maturation medium further comprises a tumor necrosis factor (TNF) signaling activator.

32. The method of claim 31 , wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

33. The method of claim 32, wherein the TNF signaling activator is LTB.

34. A method of differentiating ventral pharyngeal pouch endoderm (PPEv) cells to thymic epithelial progenitor cells (TEPCs), the method comprising culturing the PPEv cells in a TEPC differentiation medium comprising a tumor necrosis factor (TNF) signaling activator.

35. A method of producing mature thymic epithelial progenitor cells (matTEPCs), the method comprising culturing TEPCs in a TEPC maturation medium comprising a tumor necrosis factor (TNF) signaling activator.

36. The method of claim 34 or 35, wherein the TNF signaling activator is lymphotoxin alpha2 / beta1 (LTA / B), lymphotoxin alpha (LTA), lymphotoxin beta (LTB), TNF-alpha, CD40L, OX40L, CD95L, CD27L, CD30L, 4-1 BBL, TRAIL, RANK ligand (RANKL), TWEAK, APRIL, BAFF, LIGHT, TL1 A, GITRL, or any combination thereof.

37. The method of claim 36, wherein the TNF signaling activator is LTB.

38. The method of any one of claims 34-36, wherein the TEPC differentiation or maturation medium further comprises an inflammasome activator.

39. The method of claim 38, wherein the inflammasome activator is IL-1 alpha, IL-1 beta, IL- 1 ra, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-36ra, IL-37, IL-37b, IL38, or any combination thereof.

40. The method of claim 39, wherein the inflammasome activator is IL-1 beta.41 . The method of any one of claims 34-40, wherein the TEPC differentiation or maturation medium further comprises a nuclear factor-kappa B (NF-KB)ZToll-like receptor (TLR) signaling activator.

42. The method of claim 41 , wherein the NF-KB / TLR signaling activator is BO-112, polyinosinic:polycytidylic acid (poly I :C), or a combination thereof.

43. The method of any one of claims 34-42, wherein the TEPC differentiation or maturation medium further comprises a TLR signaling activator.

44. The method of claim 43, wherein the TLR signaling activator is CU-CPT 17e, CU-T 12-9, Diprovocim, or any combination thereof.

45. The method of any one of claims 1 -44, wherein the culturing is for a duration of from 5 to 15 days.

46. The method of any one of claims 1 -45, further comprising administering the TEPCs or matTEPCs to a subject in need thereof.

47. A method comprising administering an effective amount of TEPCs or matTEPCs produced according to the method of any one of claims 1-45 to a subject in need thereof.

48. A method of providing or reconstituting T cell development and function in a subject in need thereof, the method comprising administering an effective amount of TEPCs or matTEPCs produced according to the method of any one of claims 1-45 to the subject.

49. The method of any one of claims 46-48, wherein the subject has athymia or hypothymia.

50. The method of claim 49, wherein the subject has 22q11 Deletion Syndrome characterized by TBX11 deficiency, or FOXN1 or PAX1 or CHD7 deficiency.51 . The method of any one of claims 46-48, wherein the subject is an elderly subject having immune senescence.

52. The method of any one of claims 46-48, wherein the subject has an acquired thymic defect.

53. The method of claim 52, wherein the subject has undergone hematopoietic stem cell transplantation.

54. A population of TEPCs or matTEPCs produced according to the method of any one of claims 1 -45.

55. A composition comprising a population of TEPCs or matTEPCs produced according to the method of any one of claims 1-45.

56. The composition of claim 55, wherein the composition is formulated for administration to a subject in need thereof.

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