Thymus organoids bioengineered form human pluripotent stem cells

Abstract

To provide a method for making a bioengineered thymus organoid.SOLUTION: A method has the steps of: obtaining a cell population having human thymic epithelial progenitor cells or human thymic epithelial cells or both; combining the cell population with human hematopoietic stem cells in a defined ratio to form a combination; seeding the combination into an extracellular matrix of a decellularized thymus scaffold to generate a thymus construct; and culturing the thymus construct under conditions permitting cellular attachment onto the extracellular matrix thereby making the bioengineered thymus organoid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications and Priority Claims This application claims priority to U.S. Provisional Application No. 62 / 939,918, filed November 25, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Government Ownership This invention was made with government support under Grant Nos. R21 AI126335 and R01 AI123392 awarded by the National Institutes of Health, and Grant No. 1804728 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION This document relates to bioengineering and relates to bioengineered thymic organoids, related humanized animal models, and related uses. [Background technology]

[0004] The thymus is a crucial immune organ in the adaptive immune system, responsible for generating a diverse repertoire of T cells capable of effectively responding to invading pathogens while maintaining immune self-tolerance. Various factors, including aging, chemotherapy, radiation exposure, viral infection, and inflammation, can cause thymic involution, resulting in a decrease in thymocytes, increased interstitial fibrosis, and a decrease in naive T cells. Impaired immune surveillance due to thymic dysfunction can lead to a variety of diseases, ranging from autoimmunity to immunodeficiency and malignancies. Restoring or improving immune function impaired by thymic defects is essential.

[0005] One of the major obstacles to translating experimental findings obtained in animal models, such as mice, into clinically applicable therapies is the existence of species-specific differences between mice and humans. Humanized mice, in which human immune cells are transplanted and expanded into immunodeficient mice, provide a powerful tool for studying human immune system development and responses in vivo. One major obstacle to recapitulating the human immune system in mice is the defective development of human T cells due to the limitations of the mouse thymic microenvironment in supporting human T cell development and selection. Co-transplantation of fetal liver and thymus into the human bone marrow-liver-thymus (BLT) mouse model can support potent T cell development, but frequent graft-versus-host disease (GVHD) shortens the host's lifespan, preventing its use for long-term studies. Furthermore, ethical issues and the inability to select human leukocyte antigens (HLA) in fetal tissues further limit the use of BLT mice in modeling human immune responses under various pharmacological, physiological, and pathological conditions. Summary of the Invention

[0006] This book addresses the above needs in a multifaceted way.

[0007] In one aspect, this document provides a method for producing bioengineered thymic organoids. This method includes the steps of obtaining a cell population that contains human thymic epithelial progenitor cells (TEPCs) or human thymic epithelial cells (TECs), or both; combining the cell population with human hematopoietic stem cells (HSCs) at a predetermined ratio to form a combination; seeding the combination on the extracellular matrix of a decellularized thymic scaffold to generate a thymic construct; and culturing the thymic construct under conditions that allow cell adhesion to the extracellular matrix to produce a bioengineered thymic organoid. The TEPCs, TECs, or HSCs can be derived from a human donor. The HSCs can also contain human CD34+ hematopoietic stem cells. The decellularized thymic scaffold can be obtained from a human subject or a non-human donor animal. As described below, various suitable animals can be used as donor animals. Preferred examples include non-human mammals. The ratio of TEPCs or TECs to HSCs can be in the range of 100:1 to 1:100, for example, about 10:1 to 1:10, or about 1:10, 1:1, 2:1, or 5:1.

[0008] In some embodiments, the cell population can be obtained by a process comprising encapsulating human pluripotent stem cells (hPSCs) in a suspension that dissociates them into single cells, culturing the hPSCs in a growth medium to expand the number of the hPSCs or to obtain their progeny without differentiation, differentiating the hPSCs or progeny to produce TEPCs or TECs in the encapsulation medium, and releasing the TEPCs or TECs from the encapsulation medium. Examples of hPSCs can include human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0009] In some examples, thymic constructs can be placed in an in vitro flow cell that is continuously supplied with medium or nutrients and human cells to produce human immune cells. The thymic constructs can contain immune cells, such as B cells or T cells. B cells can be specific for a particular antigen or can produce antigen-specific antibodies. In one embodiment, T cells can be transduced with a viral vector encoding a chimeric antigen receptor (CAR). For example, the viral vector can be added to the flow cell to transduce the T cells. Thymic constructs can be used to evaluate drug candidates. In this case, the thymic constructs can be contacted with a drug candidate to test the effect of the drug candidate on immune cell development.

[0010] In some other examples, the thymus construct can be surgically implanted into a host animal. The host animal can be a pre-treated humanized immunodeficient animal, such as a pre-treated humanized immunodeficient pig, rat, or mouse. The thymus construct can be placed in various suitable locations within the host animal, preferably in an anatomical site with a rich vascular network, such as under the kidney capsule, in the thoracic region of the neck, or in the axillary region. The resulting host animal can be provided with HSCs to produce human immune cells. For example, the host animal can be a non-human animal (e.g., a mouse, such as those developed by ABGENIX or MEDAREX) engineered to express human VDJ antibody sequences. Such animals can produce increased amounts of fully human immunoglobulin G and antibodies. The resulting host animal can also be used to evaluate drug candidates. In this case, the drug candidate can be administered to the host animal to test its effect on immune cell development. For example, TEPCs, TECs, or HSCs can be from a donor individual, and the effect of the drug candidate on the donor individual can be tested. Alternatively, the host animal can be transplanted with cells or tissue from a donor individual, such as cancer cells, and the effect of a drug candidate on the cells or tissue from the donor individual can be evaluated.

[0011] In the above-mentioned method, the donor or host animal can be a vertebrate, including a bird, an amphibian, a reptile, a fish (e.g., a zebrafish), or other non-jawed non-mammal, or a non-human mammal. Examples of non-human mammals include a species selected from the group consisting of cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, and non-human primates.

[0012] In a second aspect, the present disclosure provides a bioengineered thymic organoid comprising (i) human TEPCs (hTEPCs), human TECs (hTECs), or human HSCs (hHSCs), and (ii) a decellularized thymic scaffold with an extracellular matrix, wherein the hTEPCs, hTECs, or hHSCs are attached to the extracellular matrix. Examples of bioengineered thymic organoids include those prepared according to the above-described method. The bioengineered thymic organoids may contain immune cells. The thymic scaffold may be xenogeneic or allogeneic to the hTEPCs, hTECs, or hHSCs. In some examples, the hTEPCs or hTECs may be derived from hPSCs. The bioengineered thymic organoids may be in vitro or in vivo. Therefore, the present disclosure also encompasses non-human animals having the above-described bioengineered thymic organoids. The non-human animal may be a mammal selected from the group consisting of a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, and non-human primate.

[0013] In another aspect, this document provides a method for evaluating drug candidates.This method comprises: (a) contacting drug candidates with above-mentioned bioengineered thymus organoid; and (b) detecting the effect of said drug candidates on the development of the cells that exist in said bioengineered thymus organoid or the cells that migrate from said bioengineered thymus organoid.This method can be carried out in vitro or in vivo in host animal.In one example, bioengineered thymus organoid can be transplanted into host animal, and drug candidates can be administered to host animal.

[0014] For the evaluation of drug candidates described herein, the drug candidate may be one selected from the group consisting of a small molecule, a nucleic acid, a peptide, a polypeptide, an antibody, and an antibody fragment.

[0015] In yet another embodiment, the present specification provides a method for preparing thymic progenitor cells.This method includes: (a) introducing progenitor cells into the above-mentioned bioengineered thymic organoid or the above-mentioned non-human animal; (b) maintaining the bioengineered thymic organoid or non-human animal under the condition that allows the differentiation of the above-mentioned progenitor cells, to generate its progeny cells; (c) discharging the progeny cells from the bioengineered thymic organoid to generate thymic emigrant cells; and (d) isolating the above-mentioned thymic emigrant cells.Therefore, the present specification includes the thymic emigrant cells prepared according to this method.Thymic emigrant cells can have one or more transgenes that encode antigen receptors, such as chimeric antigen receptors (CAR).

[0016] Thymic emigrant cells can be contained in a pharmaceutical composition that has thymic emigrant cells and a pharmaceutically acceptable carrier.Thymic emigrant cells or pharmaceutical compositions can be used in the method for improving the immune function of a subject that needs it.Therefore, the present specification provides a method for improving the immune function of a subject.This method includes: (a) administering an effective amount of thymic emigrant cells to a subject; or (b) transplanting the bioengineered thymic organoid into a subject.In some examples, the subject can have a condition selected from the group consisting of cancer, autoimmune disease, and infectious disease. [Brief explanation of the drawings]

[0017] [Figure 1] Figures 1A and 1B show the differentiation of human iPSCs into TEPCs in 3D alginate hydrogel capsules. a. Size distribution of iPSC aggregates at different stages of TEPC differentiation. The open shapes in the left panel indicate the size of individual aggregates within the capsules. The solid lines indicate the overall size distribution of TEPC aggregates at each stage (left panel), which is also displayed as a boxplot in the right panel. # indicates no significance between means. p<0.05 (one-way ANOVA with post-hoc Tukey's test). b. Representative histogram of flow cytometry analysis of EpCAM+ dissociated TEPCs. [Figure 2]Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H show tissue-engineered functional thymic organoids from iPSC-derived TECs. a) RT-qPCR analysis of the expression of genes essential for antigen presentation. Triplicate results from at least three independent experiments are shown. *p<0.05; **p<0.01; ***p<0.005. b) Representative FCM graphs showing T cell development within thymic organoids (iPSC organoids) or among cells shed from the organoids (iPSC shed). L / D, LIVE / DEAD staining, positively stains dead cells. c-h) Thymic organoids were constructed from iPSC-TECs and transplanted under the kidney capsule of athymic B6 nude mice. Representative FCM graphs showing the presence of CD3+CD45+ cells (c and d), CD3+CD4+ and CD3+CD8+ T cells (e), and γδ T cells (f) in the spleens and lymph nodes of thymic organoid-transplanted mice 18–32 weeks after transplantation. Nu, B6 nude mouse control; Thy, thymic organoid-transplanted B6 nude mouse. g. Activation status of CD4+ (left panel) and CD8+ (right panel) T cells in lymph nodes. h. Mixed lymphocyte reaction (MLR). Representative histograms showing the proliferative response of CD4+ (left panel) and CD8+ (right panel) T cells isolated from thymic organoid-transplanted B6 nude mice upon challenge with mitomycin C-treated syngeneic (B6, upper panel) or allogeneic (Valve / C, lower panel) splenocytes. Representative results from triplicates from three independent experiments are shown. *p<0.05; **p<0.01. [Figure 3] Figures 3A and 3B show the generation of hematopoietic humanized mice transplanted with iPSC-derived TEC thymic organoids. a) Kaplan-Meier survival analysis of humanized mice after transplantation. ****, p<0.001 (log-rank test). b) Percentage of hCD45+ cells in the peripheral blood of four groups of humanized mice (G1-G4) 12 weeks after transplantation. *p<0.05; ****p<0.001 (Mann-Whitney test). [Figure 4]Figures 4A, 4B, 4C, and 4D show the development of multiple hematopoietic lineages in hu.Thor mice. Cells were isolated from the bone marrow (BM) and spleen (SPL) of G1–G4 humanized mice (18–40 weeks post-transplant) and analyzed by FCM for the overall percentage of human cell chimerism (% of hCD45+ cells in total CD45+ cells) (a–c) and the presence and distribution of various hematopoietic lineages (d). d. Representative pie chart showing the distribution of human hematopoietic lineages in control (G3) and hu.Thor (G4) mice. Representative results from three independent experiments are shown. *p<0.05; **p<0.01 (Mann-Whitney test). [Figure 5]Figures 5A, 5B, 5C, 5D, 5E, and 5F show the development of functional human T cell subsets in hu.Thor mice. a. Overall diversity of TCR Vβ gene family expression in hu.Thor mice (n=3). RNA was isolated from hu.Thor mouse splenocytes (red columns) and healthy donor PBMCs (blue columns, n=3). Vβ gene expression was analyzed using the NanoString T cell panel. Percentages of total TCR Vβ gene expression for each Vβ family are shown (mean ± SEM). b. Bone marrow and spleens were harvested from d.hu.Thor mice (n=4) at 31 weeks post-transplant and human T cell subsets were characterized by FCM. All cells were initially gated on the hCD45+ population unless otherwise specified. Representative FCM graphs showing the development of CD45RA+CD45RO- naive and CD45RA-CD45RO+ memory CD4+ and CD8+ T cells (b), as well as CD4+ T helper cell subsets (c). d. Spleen cells harvested from hu.Thor mice were stimulated with PMA + ionomycin (lower panel) or medium + DMSO control (upper panel) and intracellularly stained with antibodies against hIL17A and hIFNγ (right panel). e. MLR experiment showing the proliferative response of hCD45+CD3+ T cells from Y1 hu.Thor mice challenged with HLA-mismatched human umbilical cord blood samples (hCBs6 and 18 in Table 3). Representative FCM graphs of three replicates from two independent experiments are shown. f. Overall diversity of the TCR Vα gene family in hu.Thor mice. RNA was isolated from spleen cells of hu.Thor mice (n=3, red columns) and PBMCs of healthy donors (blue columns, n=3). Vα gene expression was analyzed with the NanoString T cell panel. Percentages of total TCR Vα gene expression for each Vα family are shown (mean ± SD). [Figure 6]Figures 6A and 6B show that pathway analysis of genes related to T cell function demonstrates similar expression profiles between hu.Thor immune cells and PBMCs. Total RNA was isolated from splenocytes from hu.Thor (n=2) and hu.SRC mice (n=4) and from PBMCs from healthy donors (n=2). Expression of a panel of T cell-related genes was examined using nCounter's direct digital detection technology. Pathway score analysis calculated a score as the first principal component of the group of genes associated with each pathway, reflecting its overall characteristics. A summary plot of the pathway score analysis shows that T cell-related pathways show similar trends between hu.Thor and PBMC cells, in stark contrast to hu.SRC cells. Pathway score analysis of specific pathways related to TCR signaling, diversity, T helper subsets (a), and activation (b). Overall expression levels of genes associated with T cell exhaustion. [Figure 7] FIG. 7 is a set of figures showing the effective rejection of allogeneic iPSC-derived teratomas in hu.Thor mice, as measured by tumor weight from the allogeneic CC1 (left panel) and Y1 (right panel) strains. [Figure 8] Figures 8A and 8B show that iPSC-thymic organoid-derived human T cells can regulate humoral immune responses in hu.Thor mice. a. Generation of major human immunoglobulin classes in hu.Thor mice. Serum was collected from hu.Thor mice 16–18 weeks post-transplant. Human immunoglobulin class isotypes were quantified using a LUMINEX isotyping kit. Serum from untreated MSG and hu.SRC mice of similar post-transplant ages was used as controls. b. hu.Thor mice were intramuscularly injected with 50 μl of clinical-grade diphtheria toxoid (DT) vaccine, followed by a booster injection one week later. Serum samples were collected before immunization (pre-bleed, Pre), one week after immunization (Post), and four weeks after the first immunization (Boost). DT-reactive IgG antibodies were measured by ELISA. The fold increase in OD450 values ​​compared to the Pre sample is shown. n = 3–5. *, p < 0.05. [Figure 9] Figures 9A and 9B show the differentiation of human hESCs into TEPCs in 3D alginate hydrogel capsules and the size distribution of iPSC aggregates at different stages of TEPC differentiation. a. Open shapes indicate the size of individual aggregates within the capsules. b. Boxplots of size distribution are shown, with # indicating no significance between means. p<0.05 (one-way ANOVA with post-hoc Tukey's test). [Figure 10] Figure 10 shows tissue-engineered functional thymic organoids from hESC-derived TECs. Human thymic organoids were constructed by co-injecting H1 hESC-TECs and CD34+ umbilical cord blood into decellularized mouse thymic scaffolds. Thymic organoids were cultured for 3 weeks in the top chamber of a transwell culture system. Representative FCM graphs show the development of T cells within the thymic organoids (H1 organoids) or among cells spilling out of the organoids (H1 spill). L / D, LIVE / DEAD staining, positively stains dead cells. DETAILED DESCRIPTION OF THE INVENTION

[0018] This book focuses on bioengineering, specifically bioengineered thymic organoids and related humanized animal models. Thymic organoids and animal models (e.g., mouse models) have a variety of commercial and clinical applications, including the generation of humanized antibodies, generation of antigen-specific human T cells, induction of transplant tolerance, activation of thymic function, and modeling of human disease.

[0019] The thymus is the primary lymphoid organ responsible for T cell development. It is divided into two morphologically and functionally distinct compartments, the cortex and the medulla, which contain two distinct thymic epithelial cell (TEC) populations: cortical TECs (cTECs) and medullary TECs (mTECs). Other populations of thymic stromal cells (TSCs) include thymic fibroblasts, endothelial cells, and hematopoietic-derived macrophages and dendritic cells. This TSC network provides both homing signals for the migration of bone marrow (BM)-derived common lymphoid progenitors (CLPs) and trophic factors required for thymocyte differentiation and maturation and T lymphopoiesis.

[0020] T lymphopoiesis is a well-coordinated process involving continuous crosstalk between developing thymocytes and TECs. Early stages of T cell development (e.g., lineage commitment, proliferation, TCR gene recombination, and positive selection) occur in the cortical region and are primarily mediated by cTECs. The resulting CD4+CD8+ double-positive (DP) cells express TCRs capable of interacting with self-peptide-presenting MHC (pMHC). DP cells are then negatively selected in the medullary region, and those expressing TCRs with high affinity for self-antigens undergo apoptosis by hematopoietic-derived mTECs and APCs, differentiate into CD4+CD8- or CD4-CD8+ single-positive (SP) cells, and are released into the circulation to contribute to a diverse but self-tolerant T cell repertoire in the periphery.

[0021] The 3D organization of the thymic stroma is crucial for its function. Manipulation of the thymic stromal compartment has proven challenging, both in vitro and ex vivo. This bottleneck is primarily due to the unique structure of the thymic stroma, which is critical for TEC survival and function. Unlike epithelial cells of other visceral organs, which typically form in 2D sheet-like structures, TECs are organized in a sponge-like 3D network. TECs in 2D culture begin to express markers of terminally differentiated, senescent epithelial cells and even transdifferentiate into skin cells. The expression of genes important for TEC differentiation and proliferation (e.g., FoxN1, DLL-4, CLL-22, and Tbata) has also been shown to depend on the 3D organization of the thymic stroma.

[0022] Thymic organoids and how to create them This document provides bioengineered thymic organoids and methods for producing thymic organoids in vitro. Organoids are three-dimensional miniaturized versions of organs in vitro. Thymic organoids produced by this method can mimic the physiology and function of the human thymus. The thymic organoids described herein can contain, among other things, thymocytes (e.g., TEPCs) derived from a human donor source and a scaffold derived from a different donor or non-human animal. Thymic organoids can also contain other cells (e.g., progenitor cells) of human origin, which can be differentiated within the thymic organoid in vitro or in vivo in a host animal to produce cells useful for various purposes.

[0023] TEPC The thymic organoid described herein particularly comprises human-derived thymocytes (such as TEPCs).Various methods can be used to generate TEPCs.In certain embodiments, the method can comprise in vitro differentiation of pluripotent stem cells into TEPCs.In this regard, the method can comprise culturing pluripotent stem cells for a sufficient time and under sufficient conditions to differentiate pluripotent stem cells into TEPCs.For example, the method can comprise culturing pluripotent stem cells at different stages in the presence of a member of TGFβ superfamily (such as activin A), or a combination of Wnt family member 3A (Wnt3A), bone morphogenetic protein 4 (BMP4) and fibroblast growth factor (FGF).

[0024] Various pluripotent stem cells can be used to generate TEPCs. Generally, such pluripotent stem cells have the ability to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent stem cells can include, for example, stem cells, such as embryonic stem cells, nuclear transfer-derived embryonic stem cells, and induced pluripotent stem cells. Pluripotent stem cells, such as iPSCs, can express any one or more of a variety of pluripotency-associated genes or markers. Pluripotency-associated genes or markers include, but are not limited to, Oct-3 / 4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, hTERT, SSEA1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, and Nanog.

[0025] Many protocols are available for differentiating pluripotent stem cells (e.g., ESCs and iPSCs) into TEPCs. Stem cell-derived TEPCs express markers of thymic epithelium, but further in vivo maturation (e.g., engraftment under the kidney capsule of athymic nude mice) can be used to acquire T lymphocyte hematopoietic function. For example, Parent, AV et al. Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development. Cell stem cell 13, 219-229, doi:10.1016 / j.stem.2013.04.004 (2013), Sun, X. et al. Directed differentiation of human embryonic stem cells into thymic epithelial progenitor-like cells reconstitutes the thymic microenvironment in vivo. Cell stem cell 13, 230-236, doi:10.1016 / j.stem.2013.06.014 (2013), and Chhatta, AR et al. De novo generation of a functional human thymus from induced pluripotent stem cells. J Allergy Clin Immunol 144, 1416-1419 e1417, doi:10.1016 / j.jaci.2019.05.042 (2019). However, none of the hPSC-TEPCs generated to date have demonstrated the ability to induce differentiation of human HSCs to the T cell lineage in vitro. Of note, all of these methods used conventional 2D culture conditions.

[0026] The methods described herein can include differentiating pluripotent stem cells in 3D culture, where the cells can further differentiate into TEPCs and / or TECs, including cTECs and mTECs. These cell types can be assessed using one, two, or more corresponding markers, such as EpCAM1, CK5, CK8, CK17, CK18, OCT4, SOX17, SOX2, HOXA3, EYA1, PAX9, FOXN1, PRASS16, ACKR4, CD205, β5T, AIRE, and CSN2. As disclosed herein, 3D culture can be more effective for promoting the development of thymic organoids than 2D culture, such as 2D culture of thymic stromal cells in monolayer culture. Suitable 3D culture systems can include any 3D culture system, such as suspension in alginate capsules, hanging drop plates, and ultra-low attachment multiwell plates.

[0027] The 3D organization of TECs in the thymic microenvironment is crucial for maintaining their thymic epithelial gene signature. For example, expression of Tbata, a key transcriptional regulator for maintaining TEC size and proliferation, is rapidly reduced in 2D TEC cultures. Furthermore, TEC progenitors isolated from mouse embryos display keratinocyte markers in 2D adherent cultures. On the other hand, TECs cultured as aggregates in biocompatible hydrogels maintain their molecular characteristics and can extend their survival in vitro for up to 7 days. These data suggest that the alginate hydrogel capsules used in this study may provide critical 3D matrix support for the survival of iPSC-derived TEPCs.

[0028] For example, compared with 2D TEPCs, the expression of cTEC-specific genes (e.g., PRSS16, ACKR4, CD205, and β5t), which are early transcribed in the late embryonic and / or neonatal thymus, was significantly increased in 3D iPSC-derived TEPCs. A similar increase was detected in the expression of epithelial markers (e.g., CK-5, CK-8, CK-17, and CK-18). These findings suggest that 3D TEPCs represent a later stage of TEC development than those in 2D culture. Exposing 3D iPSC-derived TEPCs to the ECM microenvironment of a decellularized thymic scaffold further promoted TEC maturation in vitro, as suggested by the significantly increased expression of genes related to antigen presentation (e.g., MHC II and CD74). Indeed, human thymic organoids constructed from 3D iPSC-derived TECs successfully supported the differentiation of human CD34+ HSCs into both DP and SP T cells in vitro, highlighting their thymopoietic function. Therefore, in embodiments, the thymic organoid produced by this method can have high expression levels of one, two, more or all of the above markers, particularly CK-5, CK-8, CK-17, CK-18, EYA1, PAX9, FOXN1, PRASS16, ACKR4, CD205, β5T, AIRE and CSN2.

[0029] Thymus scaffold As mentioned above, the unique structure and microenvironment of the thymic stroma are important for the survival and function of TECs.The thymic organoids disclosed herein may comprise a thymic scaffold with extracellular matrix from the thymus, which provides a microenvironment for TEC recolonization and related thymic functions, such as T lymphocyte hematopoiesis.In a preferred embodiment, the thymic scaffold is a decellularized thymic scaffold, such as decellularized thymic tissue or organ or gland from a donor animal.

[0030] Various methods can be used to create scaffolds. For example, detergent perfusion-based methods can be used to clear the cellular compartment of almost any organ at any scale while leaving the ECM components largely intact. Below is an example protocol for removing all cells from the thymus while preserving the 3D ECM structure. In this protocol, decellularization is achieved by several freeze / thaw cycles to induce intracellular ice crystal formation, simultaneously with detergent treatment for cell lysis. Exemplary protocols include:

[0031] (1) Thymuses were removed from 3- to 24-week-old mice, placed in a polystyrene foam box, and frozen at -80°C for 25 minutes.

[0032] (2) The frozen thymus sample is then thawed in a water bath at 30°C for 30 minutes.

[0033] (3) Repeat (1) and (2) once or twice.

[0034] (4) Transfer the thymus sample to a 12-well plate with 2 mL of 0.5% SDS solution and place the plate on a shaker at room temperature. Observe the transparency of the thymus sample every hour and replace the 0.5% SDS every 1.5 to 2 hours. Repeat this process 2 to 3 times. For larger thymus samples, one or two additional rounds of treatment may be required.

[0035] (5) Replace 0.5% SDS with 0.1% SDS and further decellularize the sample overnight on a shaker at 4°C (refrigerated room).

[0036] (6) Place the remaining thymus matrix onto a cell strainer fitted to contain the sample.

[0037] (7) Wash three times for 15 minutes with 2 mL of ddH2O, MgSO4, CaCl2 (5 mM), and TRITON-X (1%). Transfer the remaining thymus matrix to a new well after each wash. The thymus sample will only become opaque after the first wash. If it continues to become opaque, the sample may be unsuitable for use as a scaffold. The sample should remain mostly clear.

[0038] (8) Wash three times with 2 mL of PBS for 15 minutes each. Again, transfer the thymus to a new well for each wash.

[0039] (9) Place the thymus scaffolds in a dry, round 96-well plate and store. Cover with a plate sticker and place at 4°C (refrigerated). Alternatively, store the thymus scaffolds in 2 mL of PBE + Pen-Strep. Place at 4°C (refrigerated).

[0040] To construct thymic organoids, thymocytes, such as TEPCs, can be seeded or injected into acellular thymic scaffolds and cultured in vitro. Cell viability can be assessed using techniques known in the art. As shown in the example below, cells effectively colonize the ECM of the thymic scaffold and remain viable for up to several weeks. As demonstrated by RT-PCR analysis of TEC-specific genes, the molecular characteristics of the thymic stroma are largely maintained in thymic organoids.

[0041] The thymic organoid described herein has one or more of various advantages.For example, the human iPSC-derived thymic organoid produced by this method can produce thymus-emigrating cells, such as T cell lineage cells, which are useful for treating or preventing conditions in mammals, such as cancer.In addition, this method can provide organoids that can mimic the positive selection process that occurs in the thymus.Positive selection refers to the ability of newly formed thymocytes to recognize and interact with MHC (major histocompatibility complex).In the positive selection process in human thymus, only the thymocytes that can bind to MHC survive, move to the medulla, and differentiate into mature T cells.

[0042] In some embodiments, thymic organoid allows thymic epithelial cells to be produced in 3D tissue in a manner similar to that of human thymus.In some embodiments, this method can produce organoids that can differentiate T cells in vitro.In some embodiments, this method and thymic organoid can provide a method for producing autologous T cells.For example, this specification discloses a method for producing immune cells such as T cells and NKT cells of rare blood types for blood banking and for treating conditions or deficiencies, such as anemia and other cytopenias.In some embodiments, this method can produce T cells and NKT cells for immunodeficient patients.

[0043] lymphocyte hematopoiesis The thymic organoid produced by this method is believed to be useful for generating various thymic emigrant cells, including the T cell lineage cells used in adoptive cell therapy.Therefore, one embodiment herein provides the method for preparing thymic emigrant cells in vitro, in vivo or ex vivo.

[0044] This method may include the step of seeding or introducing appropriate progenitor cells into thymic organoids. For example, any progenitor cells that have the potential to develop into T cell lineages can be transferred into thymic organoids. Examples of suitable progenitor cells include, but are not limited to, primitive mesoderm cells, hematopoietic progenitor cells, pluripotent stem cell-derived cells, hematopoietic stem cells, T cell progenitor cells, double-positive T cells, and immature T cell lineage cells. Such progenitor cells can be obtained by various methods. The following is an example of a protocol for obtaining CD34+ HSCs from umbilical cord blood samples:

[0045] (1) Obtain umbilical cord blood samples (~100 mL) from VITALANT. Isolate CD34+ HSCs using MILTENYI CD34+ Human Hematopoietic Stem Cell Magnetic Beads according to the manufacturer's protocol.

[0046] (2) CD34+ HSCs were stored in liquid nitrogen at 1x10 per tube until use. 5 ~2x10 5 Store frozen individually.

[0047] (3) CD34- cells are used to isolate genomic DNA using QIAGEN's blood / cell DNA isolation kit or other methods. Genomic DNA is then subjected to HLA testing using an HLA typing service. DNA from iPSC and hESC lines, as described elsewhere, is then subjected to HLA typing. In a later step, CD34+ HSCs bearing HLA molecules matching the iPSC or hESC lines are used for thymic reconstitution.

[0048] (4) Five to seven days before thymic organoid construction, CD34+ HSCs are harvested and expanded in a 6-well tissue culture dish using serum-free hematopoietic stem cell culture and expansion medium (available from Miltenyi Biotec or Stemcell Technologies).

[0049] An exemplary protocol for constructing thymic organoids containing CD34 HSCs for T cell generation is provided below:

[0050] (1) Construction of thymic organoids.

[0051] (1.1) Decapsulate the alginate capsules to recover human TEPCs.

[0052] (1.1.1) Spin down the capsules at 250 rcf for 5 minutes and remove the medium.

[0053] (1.2.1) When the liquid level approaches the capsule, tilt the tube slightly to the side and aspirate the medium supernatant, which helps to remove the medium below the capsule surface.

[0054] (1.3.1) Add approximately 7 mL of 100 mM EDTA solution to the alginate capsules and place on a rocker / shaker at room temperature for 5 minutes. After 5 minutes, small white cell aggregates will be visible in the solution.

[0055] (1.4.1) Spin down the solution at 250 rcf for 5 minutes to recover the cells. If there are visible residual alginate capsules, repeat steps 1.2.1 and 1.3.1.

[0056] (1.5.1) Aspirate the supernatant and resuspend the cells in the medium used to seed the scaffolds.

[0057] (1.6.1) A small portion of the TEPCs can be used for quality control. Specifically, approximately 500,000–1,000,000 cells can be stained with anti-EpCAM antibodies (a specific marker for TECs). Antibodies against other epithelial cell markers, such as Krt18, can also be used. In a good batch, there should be at least 60–70% EpCAM+ cells.

[0058] (1.2) Construction of thymic organoids.

[0059] (1.2.1) 1~3x10 5 CD34+ HSCs and 1–10x10 5Mix the TEPCs, centrifuge, aspirate, and resuspend in 20-50 µL of injection solution. The ratio of hematopoietic stem cells to TEPCs used can be varied between 1 / 10 and 10 / 1.

[0060] (1.2.2) Load the cell mix into a 20 µL Hamilton syringe (with a 33-gauge needle) or into a freshly pulled glass needle over a flame from a micropipette (if using a glass needle, connect the tip of the glass needle to rubber tubing and attach a syringe to the other end).

[0061] (1.2.3) Transfer the thymus scaffold to the cover of the Petri dish. Use forceps in one hand to stabilize the thymus scaffold. Pierce the membrane of the thymus scaffold with the tip of the needle and slowly inject approximately 20 μL of cells into one lobe. Repeat the injection with the other lobe.

[0062] (1.2.4) Transfer the implanted thymic scaffold to the top chamber of a 12-well plate. Add 2 mL of T cell culture medium (RPMI-1640 + 10% FBS and other nutritional supplements, plus 1-10 ng / mL IL-7 and 1-10 ng / mL Flt3-L). Culture the scaffold in a 37°C incubator with 5% CO2 for 1-14 days before transplantation (thymic engraftment can also be performed on the same day as thymic organoid reconstruction). The in vitro culture time allows TEPCs to attach to the extracellular matrix. It also facilitates monitoring to ensure the absence of bacterial or other microbial contamination.

[0063] In some embodiments, the step of seeding progenitor cells may include injecting the progenitor cells into thymic organoids. For example, the method may include co-injecting iPSC-TECs and CD34+ HSCs isolated from UCB into decellularized mouse thymic scaffolds. The resulting thymic organoids can be cultured in the top chamber of a transwell culture system for a period of time, for example, up to 4 weeks or more. If necessary, other cells can be injected into the thymic organoids. Examples include mesenchymal stem cells or any other cells that are commonly present in the thymus and are not directly generated from TEPCs, such as endothelial cells and dendritic cells. The following is an example protocol for culturing human thymic organoids in a flow cell to support the in vitro generation of human T cells.

[0064] (1) The microfluidic chip used in these studies was designed based on the general Aline chip design and manufactured by ALINE, Inc. (Rancho Dominguez, California). Briefly, the chip (75 mm L x 25 mm W) consists of a five-layer design with an 8–10 μm porous membrane. The acrylic top layer has four linear barbs designed for medium inflow and outflow. The two-chamber system separated by a porous membrane allows for the simultaneous culture of human thymus organoids in the lower chamber while simultaneously providing a constant medium flow to the upper chamber.

[0065] (2) Thymic organoids in microfluidic chips were grown using a Multi-Syringe Programmable Syringe Pump (available from Braintree Scientific, Braintree, MA) loaded with T cell differentiation medium (STEMSPAN SFEM II base medium supplemented with hIL-7 (1 ng / mL), hFLT3L (100 ng / mL), HKGS (100x, LIFE TECHNOLOGIES), hSCF (100 ng / mL), and hTPO (50 ng / mL). Experiments were performed at a flow rate of 80 μl / h (flow rates of 20–200 μl / h were also possible) using syringes of appropriate volume and diameter depending on the length of the study. The syringes were fitted with 23G 0.5-inch blunt needles (available from SAI Infusion Technologies, Lake Villa, IL). Tygon tubing (available from Warner Instruments, Hamden, CT) was used to connect the syringe to the input port of the chip and to the effluent collection flask. Additional tubing was used to close the remaining two ports on the chip to maintain efficient fluid pressure.

[0066] (3) Cells shed from thymic organoids were collected on collagen I matrix discs housed in the same flow cell or in T-25 tissue culture flasks (available from Corning Incorporated, Kennebunk, ME) containing 3 mL of RPMI medium supplemented with Mouse T-Activator CD3 / CD28 DYNABEADS (available from Life Technologies, Carlsbad, CA) and recombinant mIL-2 (available from StemCell Technologies, Cambridge, MA). All experiments were performed at 37°C and 5% CO2.

[0067] (4) Cells were characterized by staining with fluorochrome-conjugated antibodies that bind to specific surface markers and analyzing them by flow cytometry.

[0068] In some embodiments, preparing cells from thymic organoids (i.e., thymic emigrant cells) may include migrating or separating cells from thymic organoids to obtain thymic emigrant cells. The shedding of cells from thymic organoids can be observed under direct visualization, for example, using a dissecting microscope. Separating thymic emigrant cells from thymic organoids can be performed by any suitable method. For example, the method may include gently removing the shedding cells by removing the medium from the thymic organoid culture. Preferably, isolating thymic emigrant cells from thymic organoids may be performed under direct visualization, for example, using a dissecting microscope. Preferably, thymic emigrant cells are isolated without aspirating or destroying the thymic organoids. The method may also include replacing the removed medium from the thymic organoid culture with fresh medium. The thymic organoids can then be observed shedding additional thymic emigrant cells.

[0069] Thymic emigrant cells may be CD4-, CD8-, CD4+, CD8+, CD4- / CD8-, CD4+ / CD8+, CD4+ / CD8-, CD4- / CD8+, CD45-, or CD45+. Alternatively or additionally, thymic emigrant cells may be any one or more of CD45+, CD3+, CD45+ / CD3+, CD62L+, CD69-, CD62L+ / CD69-, CD62L-, CD69+, CD62L- / CD69+, CD45RA+CD45RO-, CD3+CD4+, and / or CD3+CD8+. In some embodiments, thymic emigrant cells may be or include T-helper cells and subsets thereof, including CXCR3+CCR6- Th1, CXCR3-CCR6+ Th17, and CXCR3-CCR6- Th2 cells, as well as CD4+FoxP3+ T-regulatory cells (Tregs), an important population of CD4+ T cells responsible for maintaining immune tolerance. In other embodiments, thymic emigrant cells may be or include CD4+ T-helper cells, CD8+ cytotoxic TCRαβ+ T cells, or CRγδ+ T cells.

[0070] The method may further comprise differentiating the thymic emigrant cells into any desired type of cell of the T cell lineage. Examples of cell types that can be prepared by differentiating the thymic emigrant cells include, but are not limited to, natural killer T (NKT) cells, T cells (e.g., naive T cells, regulatory T cells, T stem cell memory cells, effector T cells, effector memory RA cells (EMRA), Th1 cells, Th2 cells, or Th17 cells). The thymic emigrant cells prepared by the method of the present invention are believed to be useful for preparing cells for adoptive cell therapy.

[0071] The population of thymic emigrant cells can be a heterogeneous population that includes thymic emigrant cells in addition to cells other than thymic emigrant cells, e.g., PBMCs, B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, etc. Alternatively, the population of cells can be a substantially homogeneous population, the population comprising predominantly thymic emigrant cells (e.g., consisting essentially of thymic emigrant cells).

[0072] humanized animals The thymic organoids described herein can be used to generate humanized animals. Such animals can be used as model systems to study human physiology and disease. These animals can also be used to produce therapeutic cells for human use.

[0073] Although animal models have contributed greatly to our understanding of human physiology and disease, one of the major hurdles in translating these findings into clinical practice is the existence of species-specific differences between humans and animals. null (NSG) or other immunodeficiency IL2rg nullHumanized mice, in which human hematopoietic cells are transplanted into mouse strains, are a powerful model that has been widely used to overcome these challenges. Hematopoietic humanized mice not only enable long-term survival of transplanted human cells but also functionally recapitulate human immune responses, making them suitable for use as preclinical models to study various human pathologies, including infectious diseases, cancer, and autoimmunity. Comparison with other humanized mouse models of human hematopoietic cells highlights the differences and demonstrates the advantages of the methods and models provided herein.

[0074] Based on the type of human hematopoietic cells used and the engraftment route, current humanized mice can generally be categorized into three types. The first model was the hu.PBL model, in which human peripheral blood mononuclear cells (PBMCs) are intravenously (i.v.) injected into NSG mice. While engraftment of both lymphoid and myeloid cells is easily established, hu.PBL mice develop lethal xenograft-versus-host disease (xGVHD) within a few weeks due to the presence of host cell-reactive human T cells. The development of xGVHD limits the experimental period to within 3–4 weeks and can significantly complicate the interpretation of experimental results. To overcome these drawbacks, the hu.SRC (severe combined immunodeficiency-repopulating cell) model was developed, in which recipient mice are injected with CD34+ hematopoietic stem cells. In these mice, human cells of most hematopoietic lineages, except T cells, are efficiently generated. Due to species-related differences between mice and humans (e.g., growth factors and cytokines), the endogenous murine thymus of hu.SRC mice cannot fully support the development of functional human T cells. To achieve robust human T cell development, the hu.BLT (or BLT for bone marrow, liver, and thymus) model was developed. In this model, human fetal thymus and liver fragments were transfected with immunodeficient IL-2rg nullThe recipient is co-transplanted under the kidney capsule and receives an intravenous infusion of CD34+ hematopoietic stem cells from the same fetal donor. However, like the hu.PBL model, hu.BLT mice suffer from a high incidence of xGVHD, making them unsuitable for long-term studies. Ethical issues surrounding the use of human fetal tissue further limit their widespread application in preclinical studies. Therefore, the field desperately needs new mouse models that can support the development of self-tolerant, functional human T cells that mediate long-term adaptive immune responses.

[0075] The thymus does not contain self-renewing HSCs and instead relies on the mobilization of common lymphoid progenitors (CLPs) from the bone marrow to maintain long-term thymopoiesis. CLPs enter the thymus and undergo a series of differentiation processes, including lineage commitment and positive and negative selection, to become mature T cells. Thymic epithelial cells are the major cell population in the thymic stroma and play an important regulatory role throughout thymogenesis. TECs in the cortical region (cTECs) provide important signals for T cell fate determination and positively select T cells capable of functionally interacting with antigen-presenting cells (APCs). On the other hand, TECs in the medullary region (mTECs) have the unique characteristic of expressing and presenting tissue-specific self-antigens (TSAs). These mTECs are important for eliminating self-reactive T cells and maintaining immunological self-tolerance in the thymus.

[0076] Functional thymic organoids can be constructed by repopulating decellularized thymic scaffolds with isolated mouse TECs. See, for example, Tajima, A., Pradhan, I., Geng, X., Trucco, M. & Fan, Y. Construction of Thymus Organoids from Decellularized Thymus Scaffolds. Methods in molecular biology 1576, 33-42, doi:10.1007 / 7651_2016_9 (2019) and Hun, M. et al. Native thymic extracellular matrix improves in vivo thymic organoid T cell output and drives in vitro thymic epithelial cell differentiation. Native thymic extracellular matrix improves in vivo thymic organoid T cell output and drives in vitro thymocyte differentiation. Biomaterials 118, 1-15, doi:10.1016 / j.biomaterials.2016.11.054 (2017). The three-dimensional extracellular matrix network in decellularized thymic scaffolds can maintain the long-term survival and function of TECs both in vitro and in vivo. When transplanted under the kidney capsule of athymic nude mice, tissue-engineered mouse thymic organoids can support the generation of diverse and functional T cell repertoires in recipient mice (Fan, Y. et al. Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts. Mol Ther 23, 1262-1277, doi:10.1038 / mt.2015.77 (2015)). Thus, thymic organoids bioengineered with isolated TECs can provide complete physiological thymic function.However, ethical concerns and a shortage of human thymic donor tissue prohibit the widespread use of isolated human TECs to reconstitute thymic function in humanized mice.

[0077] In one embodiment, this document discloses an approach to circumvent these empirical and ethical challenges through the use of induced pluripotent stem cells (iPSCs). iPSCs are stem cells reprogrammed from somatic cells by transiently overexpressing Yamanaka factors (Oct3 / 4, Sox2, Klf4, c-Myc). See Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, doi:10.1016 / j.cell.2006.07.024 (2006). Like pluripotent embryonic stem cells (ESCs), iPSCs can be induced to differentiate into specific cell types (e.g., pancreatic islet beta cells, hepatocytes, neurons, cardiomyocytes, etc.) and are widely used in regenerative medicine research. Human iPSC-derived TECs can serve as a renewable cell source for generating functional thymic organoids. Significant progress has been made in deriving TECs from human pluripotent stem cells (hPSCs, both embryonic stem cells and induced stem cells).

[0078] hPSC-derived TEPCs, when transplanted into athymic nude mice, further mature and support the development of polyclonal mouse T cells. A similar approach was also used to differentiate human iPSCs into TEPCs. To further improve TEPC differentiation from iPSCs, Chhatta et al. transduced differentiating iPSCs with a lentiviral vector encoding FoxN1, a master transcriptional regulator of TEC development (Chhatta, AR et al. De novo generation of a functional human thymus from induced pluripotent stem cells. J Allergy Clin Immunol 144, 1416-1419 e1417, doi:10.1016 / j.jaci.2019.05.042 (2019)). Similar to hESC-derived TEPCs, iPSC-TEPCs can support the de novo generation of mouse T cells in nude mice. However, whether these iPSC-derived TEPCs can support the differentiation of human T cells from HSCs has not been investigated.

[0079] This paper describes the generation of functional human thymic organoids from human iPSCs, which can support human T cell development from CD34+ HSCs both in vitro and in vivo. For example, as shown in the following examples, human iPSCs embedded in 3D hydrogel capsules were subjected to an optimized multistep differentiation protocol to generate human TEPC aggregates. Human thymic organoids were constructed by repopulating decellularized mouse thymic scaffolds with a combination of iPSC-TEPC aggregates and CD34+ HSCs. When transplanted into hematopoietic humanized mice (hu.Thor), iPSC-derived human thymic organoids support the generation of diverse T cell populations capable of robust alloreactive responses and effective rejection of allogeneic teratomas. Serum from hu.Thor mice contains IgG subsets, suggesting the development of T cell-dependent immunoglobulin class switching in B cells. Furthermore, vaccination with diphtheria toxoid produces antigen-specific IgG. Together, these data suggest that iPSC-derived thymic organoids are capable of supporting the development of a functional human T cell compartment in hu.Thor mice.

[0080] The functional human thymic organoids disclosed herein can be tissue engineered from any suitable human pluripotent stem cells, including induced pluripotent stem cells derived from adult cells and human embryonic stem cells. When hPSC-derived thymic organoids were transplanted into pretreated immunodeficient mice together with CD34+ HSCs isolated from umbilical cord blood, both supported HSC engraftment and established humoral and cellular adaptive immune responses in the mice. This approach provides a platform for generating customized humanized mice to recapitulate the adaptive immune system of individual patients.

[0081] In several examples, this paper describes the creation of a novel humanized mouse model capable of modeling the complete immune compartment simultaneously with diverse and functional T cell responses. At the heart of this model are human iPSC-derived thymic organoids that can support the development of functional human T cells from CD34+ HSCs. Therefore, this humanized mouse model can be used to model conditions and mismatches characterized by severe defects in thymic function and associated life-threatening immunodeficiency states, such as primary DiGeorge syndrome and acquired immunodeficiency disorder (AIDS).

[0082] Personalized Medicine The thymic organoids or animal models described herein can be used for personalized medicine. More specifically, the thymic organoids or animal models can be used as immune models for a particular patient with a particular disorder to test how that patient might respond to a particular therapy or drug for treating that disorder. In this case, cells from the patent can be used to generate thymic organoids using the methods disclosed herein, and the thymic organoids can then be transplanted into an appropriate animal. Both the thymic organoids and the animal can then be used to evaluate a therapy or drug. As an example, if the disorder is a tumor, samples or cells from the patient's tumor can be transplanted into an animal model in vivo or co-cultured with thymic organoids in vitro. Tumor cell growth in the animal or culture can be examined. Increased cell death, lack of growth, or size of the transplanted or cultured tumor sample compared to an appropriate control indicates that the therapy or drug is suitable for treating the patient.

[0083] Recently, a humanized mouse model that has attracted attention in cancer research is the personalized xenograft humanized mouse, in which immunodeficient mice are transplanted with PBMCs and tumor cells from the same patient. This is a powerful tool for drug testing, but like other hu.PBMC models, it develops xenograft-versus-host disease, limiting the experimental timeframe. The humanized mouse model presented here does not have these issues and addresses the need for a more suitable model.

[0084] Drug Evaluation and Discovery Above-mentioned thymic organoid or non-human animal can be used to evaluate or determine whether test compound or candidate compound (for example, drug candidate) can be used to treat symptoms or disorders.Alternatively, thymic organoid or non-human animal can be used to determine the prognosis of target disease or pathology.

[0085] The evaluation methods described herein are useful for identifying drugs that can modulate (either promote or suppress) a disease or disorder. For example, the evaluation methods can be used to identify whether a subject is at risk of developing a disease or disorder associated with impaired immune function (e.g., inflammation, cancer, autoimmune disorders, or infections) in response to a drug. The assays can also be used to determine whether a subject is suitable for receiving a drug (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat such a disorder. Information obtained from performing the above assays is useful in the prognosis, progression identification, and clinical management of diseases and other adverse conditions that affect an individual's health status. In preferred embodiments, the diagnostic assays provide information useful for the prognosis, progression identification, and management of conditions characterized by inflammation, cancer, autoimmune disorders, or infections. This information more specifically assists clinicians in designing treatment regimes for treating or preventing such conditions.

[0086] The evaluation of a test compound can be carried out by culturing thymic organoids in a test solution containing the test compound for a certain period of time, and determining the effect on cells within the thymic organoids or the migration of cells from the thymic organoids. The test compound can also be administered to a non-human animal. After a certain period of time, the effect of the test compound on the animal or its cells can be examined. To this end, the effect of the compound or composition on the animal's thymic organoids, the cells of the thymic organoids, or the cells migrating from the thymic organoids can be examined.

[0087] In one embodiment, this method can include examining the activation of T cells in or from thymic organoid.T cell activation can be determined during and / or after the co-culture of compound and thymic organoid.Suitable assays for T cell activation include DNA replication assay (for example, 3 H-thymidine incorporation), extracellular and / or cytokine production assays (e.g., ELISA, flow cytometry, etc.), and T cell activation marker assays (e.g., flow cytometry).

[0088] In some examples, T cell activation can be measured by extracellular or intracellular cytokine production, such as IFNγ and / or IL-2 production. Extracellular cytokine production can be measured by measuring changes in the level of one or more cytokines in culture medium. Typically, immunoassays (e.g., ELISA assays, sandwich assays, immunoprecipitation assays, or Western blotting) can be used, but other assays may also be suitable. (See, e.g., Harlow and Lane, "Using Antibodies," A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999)). For intracellular cytokine levels, immunoassays and the like can be used. T cells can optionally be separated from organoids or animals (e.g., by collection based on the expression of T cell markers) prior to assaying intracellular cytokine levels. (See, e.g., Harlow and Lane, supra). In a further embodiment, T cell activation can be determined by the regulation of T cell activation markers. Such markers include, for example, CD25, CD69, CD44, CD125, and the like. The modulation of T cell activation markers can be measured, for example, by determining changes in protein levels or mRNA levels. Changes in protein levels can be determined by flow cytometry, immunoassays such as ELISA or Western blotting using labeled antibodies against T cell activation markers, transcription factors, or other proteins associated with T cell activation. Changes in mRNA levels can be determined for messages encoding T cell activation markers, transcription factors, etc. mRNA levels can be determined, for example, by Northern blotting, polymerase chain reaction (e.g., RT-PCR), other hybridization assays (e.g., assays using probe arrays, etc.), or other assays.(See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Publish., Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999); U.S. Patent Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; See Nos. 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.

[0089] In one example, tumor sample / tissue can be co-cultured with thymus organoid or transplanted into non-human animals.After administering compound / composition, if the level of tumor cell death or immune cell infiltration (for example, T cell infiltration) in tumor sample or tissue is higher than control level, test compound or test composition can be identified as the candidate compound or candidate composition suitable for treating tumor.Alternatively, after administering compound / composition, if the level of tumor growth is lower than control level, test compound or test composition can be identified as the candidate compound or candidate composition suitable for treating tumor.

[0090] Candidate compounds / compositions identified by the evaluation methods can be further tested to confirm therapeutic efficacy, modified to optimize efficacy or reduce side effects, and formulated as therapeutic agents. These identified therapeutic agents can be used in treatment protocols to treat diseases such as:

[0091] Examples of such test compounds include organic or inorganic small molecules, proteins, peptides, peptidomimetics, polysaccharides, nucleic acids, nucleic acid analogs and derivatives, or peptoids.Candidate compounds to be screened (e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs) can be isolated from naturally occurring substances or obtained using any of the many approaches in combinatorial library methods known in the art.Such libraries include peptide libraries, peptoid libraries (libraries of molecules that have peptide functionality but have novel non-peptide backbones that are resistant to enzymatic degradation), spatially addressable parallel solid-phase or solution-phase libraries, synthetic libraries obtained by deconvolution or affinity chromatography selection, and "one-bead one-compound" libraries.See, for example, Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. For examples of methods for synthesizing molecular libraries, see, e.g., DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNAS USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994 J. Med. Chem. 37:1233.Libraries of compounds can be grown in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), on beads (Lam, 1991, Nature 354:82-84), on chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, PNAS USA 89:1865-1869), or phages (Scott and Smith 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNAS USA 87:6378-6382; Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,223,409).

[0092] Restoration of thymus function Age-related thymic involution leads to reduced T cell production, narrowed T cell diversity, and impaired adaptive immune responses. Furthermore, the thymus is highly sensitive to external stimuli, such as chemotherapy, radiation, and infection, which can cause irreversible damage. Thus, there is a strong clinical need to restore thymic function.

[0093] Restoring thymic function has broad clinical implications for the treatment of thymic insufficiency. The thymic organoids described herein can be used to restore thymic function in subjects in need. To this end, thymic organoids can be bioengineered from the subject's own PSCs (autologous) or from PSCs from a matched donor (allogeneic). Here, we demonstrate that functional human thymic organoids can be tissue engineered from human iPSCs and can support human T cell development from human HSCs both in vitro and in vivo.

[0094] In some embodiments, the present specification provides the method for restoring thymus function in the subject that needs it.This method comprises transplanting the thymic organoid described herein into the subject.Thymic organoid can be allogeneic, and preferably can be autologous.In some embodiments, immunosuppressive treatment can also be administered as needed.

[0095] Cell-mediated immunotherapy This document also relates to agents, methods, and compositions for conferring and / or augmenting immune responses mediated by cellular immunotherapy, such as by adoptively transferring antigen-specific genetically modified lymphocyte subsets. Such adoptive cell transfer or adoptive cell therapy (ACT) represents a promising therapeutic approach for treating cancer patients. This document provides compositions comprising genetically modified lymphocytes expressing chimeric antigen receptors that have the ability to modulate the immune system and innate and adaptive immune responses. The disclosed agents, methods, and compositions provide genetically modified lymphocytes with enhanced antitumor function, as well as methods for developing such lymphocytes.

[0096] Immune cells, such as T cells and NK cells, genetically engineered to express foreign antigen receptors are effective immunotherapies for cancer and infectious diseases. Isolating self-antigen-specific immune cells, such as T cells, for therapeutic use is laborious and impossible when such cells are absent or rare. Therefore, strategies have been developed to genetically engineer tumor- or virus-specific immune receptors into patients' T cells. These antigen receptors combine the antigen (Ag) recognition domain with the signaling domains of TCRs or Fc receptors. T cells expressing such antigen receptors can reproduce the immune-specific responses mediated by the engineered receptors.

[0097] Chimeric antigen receptors (also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are engineered receptor proteins that confer new capabilities to T cells to target specific proteins. These receptors combine antigen-binding and T cell-activating functions into a single receptor, hence the name chimeric. CARs can have one or more functional domains in addition to the antigen-binding site.

[0098] The thymic organoids and humanized animals described herein can be used to generate genetically modified immune function cells, such as T cells and NK cells, that express CAR.In some embodiments, for example, CD34+ bone marrow progenitor cells can be transformed using lentivirus or retroviral vectors containing one or more transgenes encoding specific T cell receptors (both alpha and beta chains).The transformed cells can then be seeded into the thymic organoids described herein, further differentiated into T cells, and expanded using the thymic organoid system in vitro (e.g., flowable chip) or in vivo (in humanized mice).This method can generate a sufficient amount of CAR-T cells for the treatment of diseases such as infectious diseases, cancer, or tumors.

[0099] Transgenes can be introduced into target cells by a variety of methods, including, but not limited to, transduction of cells with integration-competent gammaretroviruses or lentiviruses, and DNA transfer.

[0100] A wide variety of vectors can be used to express transgenes. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis and integrate into the host cell genome to stably and efficiently express viral genes makes them attractive candidates for introducing foreign nucleic acids into cells. Thus, in certain embodiments, viral vectors are used to introduce nucleotide sequences encoding one or more transgenes or fragments thereof into host cells for expression. Viral vectors may have nucleotide sequences encoding one or more transgenes or fragments thereof operably linked to one or more regulatory sequences, such as promoters. Alternatively, viral vectors may lack regulatory sequences and instead rely on regulatory sequences within the host cell to drive the expression of the transgenes or fragments thereof. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenoviral vectors, adeno-associated viral (AAV) vectors, and retroviral vectors.

[0101] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing exogenous vectors and / or nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0102] Chemical means for introducing polynucleotides into host cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a release vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0103] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome or complex with a liposome, dispersed in a solution containing lipids, mixed with or associated with lipids, contained as a suspension in a complex with lipids, micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to a particular structure in solution. For example, they can exist in bilayer structures, micelles, or "collapsed" structures. They may also simply be dispersed in solution or form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0104] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of the recombinant DNA sequence in the host cell can be confirmed by a series of tests. Such assays include molecular biology assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot), or the assays described herein to identify agents within the scope of this specification.

[0105] Pharmaceutical Composition The therapeutic cells (e.g., thymic emigrant cells, CAT-T cells, or NK-T cells) described above can be formulated into compositions, such as pharmaceutical compositions. Such pharmaceutical compositions may include any of the populations of cells described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can include the population of cells and another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agent. Preferably, the carrier is a pharmaceutically acceptable carrier appropriate for the particular population of cells under consideration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are generally readily available. Suitable formulations include any formulation for parenteral, subcutaneous, intratumoral, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration.

[0106] In one example, the cell composition can be administered by injection, e.g., intravenous administration. When a population of therapeutic cells is administered, a pharmaceutically acceptable carrier for the therapeutic cells for injection can include any isotonic carrier, such as, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution, PLASMA-LYTE A, about 5% dextrose in water, or lactated Ringer's. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0107] The amount or dose of the therapeutic cell population or pharmaceutical composition administered (e.g., the number of cells when the cell population is administered) should be sufficient to produce a sufficiently effective, e.g., therapeutic or prophylactic, response in the patient over a reasonable time frame. For example, the dose of the cell population or pharmaceutical composition should be sufficient to treat or prevent the condition for a period of about 2 hours or more, e.g., 12 to 24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose may be determined by the efficacy of the particular cell population or pharmaceutical composition administered, the condition of the patient, and the weight of the patient being treated. Assays for determining doses are known in the art. For example, an assay comprising comparing the extent to which target cells are lysed upon administration of a given dose of therapeutic cells to a set of mammals each receiving different doses of cells can be used to determine the starting dose to be administered to the patient. The extent to which target cells are lysed upon administration of a particular dose can be assayed by methods known in the art.

[0108] The dosage of a population of cells or pharmaceutical composition will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular population of cells or pharmaceutical composition. Typically, the attending physician will determine the dosage of a population of cells or pharmaceutical composition to treat an individual patient, taking into account various factors such as age, weight, general health, diet, sex, the population of cells or pharmaceutical composition being administered, the route of administration, and the severity of the condition being treated.

[0109] This document also provides a method for treating or preventing a condition in a mammal, comprising administering to the mammal an amount of the therapeutic cells described above effective to treat or prevent the condition in the mammal. In one embodiment herein, the condition is cancer, an immunodeficiency, an autoimmune condition, an infectious disease, or a hematological condition.

[0110] Examples of cancer include acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain tumor, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid, Hodgkin's lymphoma, hypopharyngeal cancer, and kidney cancer. The cancer may include, but is not limited to, any of the following: cancer of the bladder, bladder cancer, rectal cancer, rectal cancer, rectal cancer, rectal cancer, uterine cancer, uterine carcinoma, uterine cancer, thyroid cancer, ureteral cancer, urinary tract ...

[0111] An example of an immunodeficiency may be any condition in which the body's ability to defend itself against external pathogens is impaired. An immunodeficiency may also be any condition in which a patient's immune system is impaired and requires reconstruction after immune loss due to radiation or chemotherapy. An immunodeficiency may include, for example, the exhaustion of the adaptive immune system in elderly populations. Therefore, the thymic organoids described herein can produce therapeutic cells that are useful for treating both primary and secondary immunodeficiencies. Examples of immunodeficiencies that can be treated or prevented include, but are not limited to, X-linked agama globulinemia (XLA), variable immunodeficiency syndrome (CVID), severe combined immunodeficiency syndrome (SCID), AIDS, and hepatitis.

[0112] Autoimmune disease can be any condition in which the body's immune system attacks healthy cells.The thymus organoid described herein can produce therapeutic cells useful for treating autoimmune disease.Examples of autoimmune conditions that can be treated or prevented include, but are not limited to, rheumatoid arthritis, lupus, type 1 diabetes, multiple sclerosis, celiac disease, temporal arteritis, vasculitis, alopecia areata, ankylosing spondylitis, Sjogren's syndrome and polymyalgia rheumatica.

[0113] The infectious disease may be, for example, a viral infection, a bacterial infection, a fungal infection, or a protozoal infection. As used herein, "viral infection" refers to a condition that can be transmitted from person to person or from organism to organism and is caused by a virus. In embodiments herein, the viral disease may be caused by a virus selected from the group consisting of herpesvirus, poxvirus, hepadnavirus, papillomavirus, adenovirus, coronavirus, orthomyxovirus, paramyxovirus, flavivirus, and calicivirus. For example, the viral disease may be caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, herpes simplex virus, Epstein-Barr virus, varicella virus, cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), human T-lymphotropic virus, calicivirus, adenovirus, and arenavirus. The viral infection can be, for example, influenza, pneumonia, herpes, hepatitis, hepatitis A, hepatitis B, hepatitis C, chronic fatigue syndrome, sudden acute respiratory syndrome (SARS), gastroenteritis, enteritis, carditis, encephalitis, bronchitis, papillary airway disease, meningitis, HIV / AIDS, mononucleosis, and the like.

[0114] The hematological condition can be any non-cancerous condition affecting the blood, such as cytopenias (e.g., anemia, leukopenia, neutropenia), bleeding disorders such as hemophilia, and thrombosis.

[0115] definition As used herein, the term "antigen receptor" or "antigen-recognizing receptor" refers to a receptor that can activate immune cells (e.g., T cells) in response to antigen binding. In particular, the term "antigen receptor" also includes artificial receptors that confer specificity to immune effector cells, such as T cells. An antigen receptor according to the present specification can be present on a T cell, for example, instead of or in addition to the T cell's own T cell receptor. Such T cells do not necessarily require antigen processing and presentation to recognize the target cell, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, the antigen receptor is expressed on the cell surface. Specifically, the term refers to an artificial or recombinant receptor having a single molecule or a complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell (e.g., by binding of an antigen-binding site or antigen-binding domain to an antigen expressed on the target cell surface) and confer specificity to immune effector cells, such as T cells, that express the antigen receptor on their cell surface. Preferably, recognition of the target structure by the antigen receptor results in activation of the immune effector cell that expresses the antigen receptor. An antigen receptor may have one or more protein units with one or more domains as described herein. The term "antigen receptor" preferably does not include naturally occurring T cell receptors. According to the present specification, the term "antigen receptor" is preferably synonymous with the terms "chimeric antigen receptor," "chimeric T cell receptor," and "artificial T cell receptor." Exemplary antigen-recognizing receptors may be native or genetically engineered TCRs, or genetically engineered TCR-like mAbs (Hoydahl et al. Antibodies 2019 8:32), or CARs in which a tumor antigen-binding domain is fused to an intracellular signaling domain that can activate immune cells (e.g., T cells).

[0116] The term "chimeric antigen receptor" or "CAR" refers to a set of polypeptides, usually two in the simplest embodiment, that, when present in an immune effector cell, provides the cell with target cell specificity and intracellular signal generation. In some embodiments, the CAR has at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") with a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule, as defined below. In some embodiments, the set of polypeptides are on the same polypeptide chain, e.g., a chimeric fusion protein. In some embodiments, the set of polypeptides are not contiguous with each other, e.g., on different polypeptide chains. In some embodiments, the set of polypeptides includes a dimerization switch that allows the polypeptides to bind to each other in the presence of a dimerization molecule, e.g., the antigen-binding domain to be linked to the intracellular signaling domain. In one aspect, the stimulatory molecule of the CAR is a zeta chain (e.g., CD3-zeta) associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain has a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0117] The term "immune cells" refers to cells derived from the hematopoietic system that are involved in the specific recognition of antigens. Immune cells include antigen-presenting cells (APCs) such as dendritic cells and macrophages, B cells, T cells, and NK cells such as NK-92 cells. T cells include Teff cells and Treg cells.

[0118] As used herein, the term "lymphocyte" may include natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte and represent a major component of the innate immune system. NK cells reject tumor and virus-infected cells through the process of apoptosis, or programmed cell death. T cells play a major role in cell-mediated immunity (antibody-independent). T cells are distinguished from other lymphocytes by their T cell receptors (TCRs). The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are several types of T cells: helper T cells (e.g., CD4+ cells), effector T cells, and T cells. EFF cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, killer T cells), memory T cells ((i) stem memory T cells, similar to naive cells scm The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but express high amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit many functional properties characteristic of memory cells; (ii) central memory T CM The cells express L-selectin, are CCR7+ and CD45RO+, and secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T EM These include T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. T cells present within tumors are called "tumor-infiltrating lymphocytes" or "TILs." Meanwhile, B cells play a key role in humoral immunity (antibody-mediated immunity). They produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0119] The term "stem cell" refers to a cell that has the ability to self-renew and pluripotency or multipotency. Typically, stem cells can regenerate damaged tissues. The stem cells referred to herein may be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells, or tissue stem cells (also called tissue-specific stem cells or somatic stem cells).

[0120] "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, usually adult somatic cells, or terminally differentiated cells such as fibroblasts, hematopoietic cells, muscle cells, nerve cells, and epidermal cells, by introducing specific factors known as reprogramming factors.

[0121] "Pluripotent" refers to stem cells that have the potential to differentiate into all cells that make up one or more tissues or organs, particularly endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urinary tract), or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cells" refer to cells that can differentiate into cells derived from any of the three germ layers, e.g., the direct descendants of totipotent cells or induced pluripotent cells.

[0122] "Peripheral blood cells" refers to the cellular components of blood, including red blood cells, white blood cells, and platelets, found within the circulating pool of blood.

[0123] "Hematopoietic stem and progenitor cells" or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic system but can further differentiate into hematopoietic lineages, including hematopoietic stem cells, pluripotent hematopoietic stem cells (hematopoietic blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. "Hematopoietic stem cells (HSCs)" are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lineages.

[0124] As used herein, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any treatment. As used herein, the terms "subject" and "subjects" can refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, non-human primates (e.g., monkeys such as cynomolgus monkeys and chimpanzees), and humans). The subject may be human or non-human. In more exemplary aspects, the mammal is a human. In some embodiments, the subject is human. In some embodiments, the subject has cancer. In some embodiments, the subject is immunocompromised.

[0125] As used herein, "treating" or "treatment" refers to the administration of a compound or agent to a subject having a disorder for the purpose of curing, alleviating, ameliorating, delaying the onset of, preventing, or ameliorating the disorder, the symptoms of the disorder, a disease state secondary to the disorder, or the predisposition to the disorder.

[0126] The terms "treat" and "prevent" do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one skilled in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of the present invention can provide any level of treatment or prevention of a condition in a patient, and any amount of treatment or prevention. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the condition being treated or prevented. For example, treatment or prevention can include promoting tumor regression. As used herein, "prevention" can also encompass preventing the recurrence of a condition, delaying the onset of a condition, or its symptoms or conditions.

[0127] An "effective amount" or "therapeutically effective amount" refers to an amount of a compound or agent (e.g., T cells or DC cells) capable of producing a medically desirable result in a treated subject. Treatment methods can be performed in vivo or ex vivo, alone or in combination with other agents or treatments. A therapeutically effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. The ability of T cells or DC cells to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as assaying the activity of an agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0128] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or properties of the composition and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting therapeutic agents or cells in or to a subject and performing their intended function.

[0129] The term "autologous" refers to any material derived from the same subject or individual that is later reintroduced. For example, the autologous cell therapy methods described herein involve collecting lymphocytes, immune cells, or progenitor cells thereof from a donor, e.g., a patient, and then engineering them to express, e.g., a CAR construct, and then re-administering them to the same donor, e.g., patient.

[0130] The term "xenogeneic" refers to any material (e.g., a cell or tissue scaffold) derived from a different subject or individual. As used herein, "xenogeneic" or "non-endogenous" or "foreign" also refers to any material (e.g., a gene, protein, compound, molecule, cell, or tissue or tissue component) or activity that is not native to the host cell or host subject, or any gene, protein, compound, molecule, cell, tissue or tissue component, or activity that is native to the host or host cell but has been altered or mutated such that the structure, activity, or both differ between the native and mutated version.

[0131] The term "allogeneic" refers to the introduction of material (e.g., cells or tissue scaffolds) obtained from one individual into another individual of the same species, e.g., allogeneic cell transplantation. For example, cells may be obtained from a first subject, modified ex vivo according to the methods described herein, and then administered to a second subject to treat a disease. In such embodiments, the cells administered to the subject are allogeneic and xenogeneic cells.

[0132] A "vector" is a nucleic acid molecule or particle capable of transporting another nucleic acid. A vector may be, for example, a plasmid, cosmid, virus, or phage. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells. An "expression vector" is a vector that, when present in the appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector. In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, gammaretroviral vectors, and lentiviral vectors. A "retrovirus" is a virus having an RNA genome. "Gammaretrovirus" refers to a genus in the Retroviridae family. Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentivirus" refers to a genus of retroviruses capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types 1 and 2), equine infectious anemia virus, feline immunodeficiency virus (Hy), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed-end linear double-stranded (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). When a non-viral delivery system is used, the delivery vehicle can be a liposome. Lipid formulations can be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo.The nucleic acid may be encapsulated within the interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the nucleic acid, comprised in or complexed with a micelle, or otherwise associated with a lipid.

[0133] Example Example 1 Materials and Methods This example describes the materials and methods used in Examples 2-8 below.

[0134] mouse All animal procedures were approved by the Allegheny General Hospital Animal Care and Use Committee. scid Il2rgtm1Wjl / SzJ (NSG) mice (strain #005557) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were 8–12 weeks old on the day of transplantation. NSG mice were housed in a pathogen-free facility. 4–8 week-old FVB mice were purchased from Jackson Laboratory and were housed and cared for in accordance with the assessment and certification of the National Institutes of Health and the International Association for Laboratory Animal Care.

[0135] Generation of iPS-derived thymic epithelial cells (TECs) The stepwise induction protocol for thymic epithelial progenitor (TEP) differentiation of hESCs was adapted from a previous study (Parent, AV et al. Cell Stem Cell 13, 219-229, doi:10.1016 / j.stem.2013.04.004 (2013)). Stage 1 definitive endoderm (DE) differentiation was performed using a Melton beta cell differentiation protocol. Stage 2 foregut endoderm (AFE) differentiation was performed in RPMI medium supplemented with 0.5% B27 (GIBCO, Gaithersburg, MD). For stage 2 (AFE), the following factors were used: 100 ng / ml activin A on day 5, 0.25 μM retinoic acid on days 5–7, 50 ng / ml BMP4 (MILTENYI BIOTEC, Auburn, CA) on days 6–7, and 5 μM LY364947 (MILLIPORE SIGMA, Burlington, MA) on days 6–7. Stages 3 and 4 of the ventral pharyngeal endoderm (VPE) and TEP were cultured in DMEM / F12 medium (GIBCO) supplemented with 0.5% B27. For stage 3 (VPE) and stage 4 (TEP), the following factors were used: 50 ng / ml Wnt3A on days 8–11, 0.1 μM retinoic acid on days 8–11, 50 ng / ml BMP4 on days 8–11, 5 μM LY364947 on days 8–9, 50 ng / ml FGF8b (MILTENYI BIOTEC) on days 8–11, and 0.5 μM KAAD-cyclopamine (MILLIPORE) on days 8–11.

[0136] Humanized mouse conditioning and cell preparation Hu.SRC mice were generated by transplanting human umbilical cord-derived CD34+ cells into conditioned NSG mice. Briefly, recipient NSG mice were intraperitoneally injected with 250 μL of 2 mg / mL anti-mouse CD117 (c-kit) antibody (BIOLEGEND, San Jose, CA) 1 week prior to transplantation. All NSG mice involved in this study were intraperitoneally injected with 30 μg / g busulfan (SAGENT PHARMACEUTICALS, Schaumburg, IL) 48 and 24 hours prior to the injection of human umbilical cord blood cells.

[0137] Human umbilical cord blood was purchased from VITALANT (Pittsburgh, PA). Lymphocyte populations were isolated from the blood using Ficoll separation, and CD34+ cell populations were isolated using the Human CD34 Microbead Kit ULTRAPURE (MILTENYI BIOTEC). Cells were then identified using flow cytometer staining for human CD45-APC (BD BIOSCIENCES) and human CD34-VIOBLUE (MILTENYI BIOTEC). Cells were then frozen or cultured until transplantation. On the day of transplantation, 1x10 cells were collected. 5 ~1x10 6 CD34+ cells were injected retroorbitally into recipient mice.

[0138] Generation and transplantation of human thymic organoids Decellularization of mouse thymus was performed using chemical detergent washes as previously described (Tajima, A., Pradhan, I., Geng, X., Trucco, M. & Fan, Y. Construction of Thymus Organoids from Decellularized Thymus Scaffolds. Methods in Molecular Biology 1576, 33-42, 2019). Briefly, thymuses from 3-4 week-old C57BL / 6J.CD45.1 mice were harvested in 0.1% sodium dodecyl sulfate (INVITROGEN, Grand Island, NY) in deionized water with continuous rotation (LAB LINE, THERMO SCIENTIFIC, Waltham, MA) until the tissue became translucent and white (~24 hours). The organs were then washed three times with phosphate-buffered saline (PBS) followed by incubation in 1% TRITON X-100 (Sigma-Aldrich, St. Louis, MO). After three more PBS washes, the organs were given a final wash in PBS + penicillin / streptomycin (100 U / ml) and rotated for an additional 48 hours. Decellularized thymus scaffolds were stored in PBS at 4°C for up to one month and then switched to Roswell Park Memorial Institute (RPMI)-10 culture medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mmol / L l-glutamine, and 10 mmol / L HEPES 24 hours before use.

[0139] Decellularized scaffolds were reconstituted with isolated CD34+ umbilical cord blood cells and deencapsulated thymic epithelial cells. To deencapsulate TECs, alginate capsules were gently pelleted and incubated in 100 mM EDTA solution for 5 minutes at room temperature. After washing with PBE, cells were pelleted and counted. After combining with CD34+ cells at a ratio of ~1:10 (TEC:CD34+), they were pelleted and resuspended in 40 μL of STEMSPAN SFEM II basal medium supplemented with human SCF (100 ng / mL), human FLT3L (100 ng / mL), human TPO (50 ng / mL) (MILTENYI), and human keratinocyte growth factor supplement (THERMOFISHER SCIENTIFIC, Waltham, MA). Cells were then injected into both lobes of the thymic scaffold using a pulled glass needle and cultured in a transwell system for 5–7 days before implantation. On the day of implantation, the scaffold was gently washed three times with saline before surgery. Recipient hu.Thor mice were anesthetized with isofluorane, and the scaffold was implanted under the left kidney capsule.

[0140] Flow cytometry analysis Decapsulated thymic epithelial cells were stained with the following antibodies to measure surface protein expression: anti-human CD45-APC, MHC class II-PerCP-Cy5.5 (BD BIOSCIENCES), and EpCAM-PE (INVITROGEN). All samples were compared with a fluorochrome-matched IgG control.

[0141] To determine both humanization and T cell generation, blood was collected from hu.Thor mice at various time points. Blood was stained with the following monoclonal antibodies: anti-human CD45-APC, CD3-PE, CD4-V450, CD8-FITC, and anti-mouse CD45-APC / Cy7 (BD BIOSCIENCES, San Jose, CA). Red blood cells were lysed. The remaining cell population was fixed with 2% paraformaldehyde and analyzed using a FACS Influx system (BD BIOSCIENCES). On the day of animal sacrifice and tissue collection, mouse bone marrow and spleen cells were stained with the following additional antibodies: anti-human CD20-V450, CD14-PE, CD33-V450, CD45RA-PE, CD45RO-FITC, CD25-V450, CD11c-FITC, CD56-FITC, CD117-PE, TCRαβ-PE, TCRγδ-FITC, CCR6-BV421, and CXCR3-BV510 (BD BIOSCIENCES). Anti-human FOXP3-PE was also used for intracellular staining using the FOXP3 / Transcription Factor Staining Buffer Set (THERMOFISHER) according to the manufacturer's protocol. Additional information about the stains used is listed in Table 1. Dead cells were excluded from analysis using the LIVE / DEAD Violet Fixable dead cell stain kit (THERMOFISHER). All corresponding flow cytometry analyses were performed with FLOWJO 10 (Version 10.5.3) software (Ashland, OR). [Table 1]

[0142] RNA isolation and gene expression analysis Total RNA was extracted using TRIZOL (INVITROGEN, Waltham, MA), and cDNA was synthesized using the SUPERSCRIPT III First-Strand Synthesis System for RT-PCR (INVITROGEN). Quantitative real-time PCR was performed on a ROCHE LIGHTCYCLER 480 system (ROCHE APPLIED SCIENCE, Indianapolis, IN) using All-in-One qPCR Mix (GENECOPOEIA, Rockville, MD). The quantitative real-time PCR primer sequences used in this study are listed in Table 2. PCR reactions were performed in triplicate in at least three separate experiments. Relative gene expression was normalized to GAPDH. [Table 2]

[0143] For gene expression profiling analysis, RNA was extracted from splenocytes of hu.Thor and hu.SRC humanized mice using the TRIzol™ method (INVITROGEN) according to the manufacturer's protocol. The concentration, quality, and integrity of each RNA sample were first tested using a Nanodrop Spectrophotometer and then using a BIOLANALYZER 2100 (RNA Pico kit, AGILENT). Only samples with DV200 values ​​of 70% or higher were used in the study. 150–500 ng of total RNA was loaded onto a CAR-T cell gene profiling hybridization cartridge capable of capturing reporter probes for 771 genes (with unique barcodes) specific to various pathways of T cell function and characterized using the nCounter Max Gen 2 System (NANOSTRING TECHNOLOGIES, Seattle, WA). Splenocytes isolated from NSG mice served as a negative control, with less than 15% of genes above background and were excluded from the analysis. All reads were analyzed using NSOLVER 4.0 Advanced analysis software. Specifically, pathway score analysis was performed, which condenses the gene expression profile of each sample into a small set of pathway scores. Pathway scores were fitted using the first principal component of the data for each gene set. Increasing scores are oriented to correspond to increased expression (specifically, each pathway score has a positive weight for at least half of the genes). Summary plots explore pathway binding behavior, and covariate plots compare pathway scores and covariates.

[0144] To characterize TCR Vβ gene family usage, we calculated the total number of Vβ gene reads from the T cell gene panel and calculated the percentage of individual Vβ family expression. We also calculated Vα gene family usage in the same way.

[0145] T cell subset phenotype Spleen cells were obtained from hu.Thor mice. 1x10 6Cells / mL were plated in RPMI-10 in non-tissue culture treated 6-well plates. H 1 / T H Cells were stimulated with both ionomycin (1 μg / mL) and PMA (50 ng / mL) and treated with Golgi block as described in the 17 Phenotyping Kit (BD BIOSCIENCES). Control wells were left unstimulated and unblocked. Cells were incubated at 37°C for 5 hours, then collected into polypropylene FACS tubes and pelleted. Cells were then resuspended in cold BD Cytofix buffer and incubated at room temperature. Cells were pelleted and washed with PBE, then resuspended in 1x BD Perm / Wash buffer and incubated at room temperature. After a final centrifugation, cells were stained with the following antibody cocktail: anti-human CD4-PerCP / Cy5.5, IL-17A-PE, IFNγ-FITC, CD45-APC, and anti-mouse APC / Cy7 (BD BIOSCIENCES). Cells were fixed with 2% paraformaldehyde before FACS analysis.

[0146] mixed lymphocyte reaction Hu.Thor mice were sacrificed, and spleens and bone marrow were harvested. Spleen and bone marrow cells from the same animal were combined, counted, and labeled with carboxyfluorescein succinimidyl ester (CFSE) (INVITROGEN), except for a small portion that was set aside unlabeled as a negative control. Briefly, 1 x 10 7 ~1x10 8Hu.Thor killer cells were resuspended in a 10 μM working solution of CFSE in PBS. Cells were incubated at 37°C protected from light, and staining was quenched by the addition of RPMI-10 containing 10% FBS. Cells were then pelleted, resuspended in RPMI-10, and allowed to incubate for 10 minutes. A small portion of these labeled cells was then Fc-blocked (BD BIOSCIENCES) and stained with the following antibodies, which served as positive controls: anti-human CD45-APC, CD3-PE, CD4-PerCP / Cy5.5, CD8-BUV395, and Violet LIVE / DEAD (BD BIOSCIENCES). The remaining cells were counted, and 1-2 x 10 of these labeled responder cells were collected. 5 were plated in triplicate in 96-well round-bottom plates.

[0147] Allogeneic stimulator cells were prepared from human umbilical cord blood samples by mitomycin C treatment. Briefly, CD34+ cells isolated from umbilical cord blood were cultured and counted. Cell suspensions were prepared at 5x10 in PBS. 7 The cells were incubated at 37°C with 50 μg / mL mitomycin C (SIGMA-ALDRICH) and protected from light. The reaction was quenched by adding excess complete RPMI-10 medium. The cells were then thoroughly washed three times by centrifugation at 300 x g for 10 minutes to remove all traces of mitomycin C from the cells and reduce the possibility of a proliferative response due to the addition of stimulator cells to the reaction. The cells were then resuspended in complete RPMI-10 and counted. Cells were plated at 2–5 x 10 densities depending on the plating density of the responder cells. 5Stimulator cells were plated at a ratio of 1:3 (responder:stimulator cells) per well. Plates were protected from light and cultured at 37°C for 7 days. On day 7, wells were stained with the following antibody panel: anti-human CD45-APC, CD3-PE, CD4-PerCP / Cy5.5, CD8-BUV395, and Violet LIVE / DEAD (BD BIOSCIENCES). Samples were then run on a BD Influx FACS system, and data were analyzed using FLOWJO 10 software.

[0148] Teratoma analysis The CC1 iPS cell line (CW70296CC1) was purchased from CELLULAR DYNAMICS. The Y1 iPS cell line was established from skin fibroblasts of a healthy donor and successfully induced differentiation into (pro)insulin-producing pancreatic β-cell-like cells, thymic epithelial progenitor cells, and fibroblast-like cells. Both lines were maintained in mTeSR plus medium (STEMCELL TECHNOLOGY, 05825). Upon reaching confluence, cells were dissociated as aggregates using RELESR (STEMCELL TECHNOLOGY, 05872). The collected cells were collected at a concentration of 1 x 10 per 25 μL. 6 The cells were resuspended in culture medium at a concentration of 1 x 10, mixed with an equal volume of growth factor reduced (GFR) Matrigel (CORNING, 356231, thawed on ice), and slowly drawn into an insulin syringe for intramuscular injection. 6 iPSC aggregates were slowly injected into the gastrocnemius muscles of supine hu.Thor and hu.SRC recipients, with CC1 cells on the left side and Y1 cells on the right side. Mice were sacrificed 26 days after injection, and teratomas were removed. Resected teratomas were measured and weighed before proceeding to image analysis. Immunodeficient FVB mice served as a negative control for teratoma formation, and immunodeficient NSG mice served as a positive control.

[0149] Histological analysis of teratomas Teratoma tissues were harvested for immunofluorescence (IF) and H&E histological analysis. Tissues used for IF staining were refrigerated in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA) for 3 hours, washed in PBS, and then placed in 30% sucrose solution at 4°C for at least 3 days. When ready to section, the tissues were briefly washed in PBS, embedded in Tissue Plus Optimal Cutting Temperature Clear embedding medium (Fisher Healthcare, Houston, TX) on dry ice, and cryopreserved into 8 μm sections using a LEICA CM1950 cryostat (LEICA, Wetzlar, Germany). When ready to stain, the slides were removed from -20°C and rehydrated in PBS. The tissues were then fixed to slides with 4% PFA, washed with PBS, and dialyzed against 0.5% TRITON X-100 (Sigma-Aldrich, St. Louis, MO). The slides were washed again and blocked for 1 hour with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in PBS. After blocking, the tissues were washed and incubated overnight at 4°C with the following primary antibodies (ABCAM, Cambridge, UK) at a 1:100 dilution: human anti-CD3 (ab11089) and anti-HLA-A (ab52922). After aspirating the primary antibody and washing the tissue with PBS, the following secondary antibodies (THERMO FISHER, Waltham, MA) were added at a 1:1000 dilution and incubated for 1 hour at room temperature: ALEXAFLUOR 488 goat anti-rabbit IgG (A11034) and ALEXAFLUOR 555 goat anti-rat IgG (A21434). The secondary antibodies were aspirated, and the tissue was washed with PBS. Nuclei were stained using ProLong Glass Antifade Mountant with NUCBLUE (LIFE TECHNOLOGIES CORPORATION, Eugene, OR). Slides were imaged using an OLYMPUS FLUOVIEW FV1000 confocal microscope (OLYMPUS, Shinjuku, Tokyo, Japan).

[0150] Teratoma tissue for H&E staining was collected and placed in 10% neutral buffered formalin (RICHARD-ALLEN SCIENTIFIC, Kalamazoo, MI). Tissue was then processed and stained within the Allegheny General Hospital Pathology Department. Stained slides were reviewed by multiple board-certified pathologists for appropriate interpretation and analysis.

[0151] Serum assays to detect immunoglobulin class switching Serum was collected from hu.Thor mice via facial vein or cardiac puncture. Normal class switching function was detected by examining IgM, IgG, IgA, and IgE levels in hu.Thor serum using the Antibody Isotyping 7-Plex Human PROCARTAPLEX Panel (INVITROGEN). Assays were performed according to the manufacturer's protocol and analyzed using a LUMINEX FLEXMAP 3D system (LUMINEX Corporation, Austin, TX). Hu.Thor samples were compared with control, untreated NSG mice to reduce false-positive results.

[0152] statistical analysis All values ​​are expressed as mean ± standard deviation unless otherwise specified. Statistical analysis and comparisons were performed using an unpaired Student's t-test (GRAPHPAD Software) with GRAPHPAD PRISM Version 8.0 unless otherwise specified. The significance of p-values ​​for replicates is as follows: *<0.05, **<0.01, ***<0.001. All experiments were performed with a minimum of n=3 to obtain statistical significance.

[0153] Example 2: 3D alginate microenvironment promotes differentiation of human pluripotent stem cells into thymic epithelial progenitor cells Encapsulation of hESCs in 3D alginate capsules has been shown to promote cell proliferation in a pluripotent state and to result in efficient differentiation into pancreatic islet-like cells through stepwise lineage-specific induction (Richardson, T., Kumta, PN & Banerjee, I. Alginate encapsulation of human embryonic stem cells to enhance directed differentiation to pancreatic islet-like cells. Tissue Eng Part A 20, 3198-3211, 2014). Synergistic chemical and biophysical induction in 3D aggregate cultures significantly promoted islet specification of hESCs compared to 2D cultures. A similar approach was employed to promote the differentiation of iPSCs into TEPCs.

[0154] Briefly, iPSCs are embedded in alginate capsules and then cultured in a thymic microenvironment. (Parent, AV et al. Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development. Cell stem cell 13, 219-229, doi:10.1016 / j.stem.2013.04.004 (2013)), Sun, X. et al. Directed differentiation of human embryonic stem cells into thymic epithelial progenitor-like cells reconstitutes the thymic microenvironment in vivo. Cell stem cell 13, 230-236, doi:10.1016 / j.stem.2013.06.014 (2013) and Richardson, T., Kumta, PN & Banerjee, I. Alginate encapsulation of human embryonic stem cells to enhance directed differentiation to pancreatic islet-like cells. Tissue Eng Part A We followed a four-step differentiation protocol, modified from that reported in [20, 3198-3211, 2014]. The 3D environment of the alginate capsules maintained the viability of encapsulated iPSCs as single cells, which proliferated into small aggregates approximately 50 μm in diameter after 4–6 days of culture (Figure 1a). Further chemical induction of encapsulated aggregates toward definitive endoderm (D1–D4) resulted in an increase in average aggregate size (boxplot, Figure 1a) and a shift in the distribution toward higher aggregate sizes (Figure 1a, solid line in the left panel). Further differentiation toward the TEPC lineage did not induce further proliferation, as judged by the slight change in aggregate size, but the cell phenotype significantly changed (Figures 1a and 1b).The overall efficiency of iPSC-to-TEPC differentiation was assessed by flow cytometry (FCM) for surface expression of EpCAM, an important marker for TECs (Figure 1b). Approximately 84.6 ± 13% (n = 5) of iPSC-derived TEPCs were EpCAM+. A similar pattern of TEPC aggregate formation and differentiation efficiency was observed in the differentiation of hESCs to TEPCs (Figure 1b and Figure 9). These results highlight the effectiveness of 3D encapsulation for promoting the derivation of TEPCs from hPSCs.

[0155] To further characterize iPSC-derived TEPCs, we analyzed the expression of TEC-specific markers by RT-qPCR analysis. We confirmed that the expression of thymic epithelial-specific cytokeratin markers was significantly increased in TEPCs generated in 3D alginate capsules compared to those obtained from 2D culture. Specifically, CK8 expression was increased two-fold, and CK17 and CK18 expression was increased approximately six-fold and five-fold, respectively. Conversely, both 2D and 3D TEPCs exhibited a significant loss of stem cell (OCT4 and SOX2) and definitive endoderm (SOX17) markers. Furthermore, the expression of ventral pharyngeal endoderm (VPE) and TEC progenitor markers was significantly increased, further suggesting successful iPSC differentiation toward the TEPC lineage. Notably, the expression of FOXN1, a key regulator of TEC lineage development, was increased more than five-fold compared to 2D TEPCs, strongly confirming the high efficiency of TEPC differentiation under 3D alginate encapsulation conditions.

[0156] Differentiation of epithelial progenitor cells into mature TEC subsets (cTECs and mTECs) is crucial for the formation and function of the thymic organ. To further explore the progression of TEC differentiation, we examined the expression of TEC subset-specific genes. Genes specific to the cTEC subset, important for autoantigen processing and positive selection function (e.g., PRSS16, ACKR4, and β5t), were expressed at significantly higher levels in 3D TECs than in 2D TECs. Specifically, PRSS16 and ACKR4 showed a two-fold and ten-fold increase in expression, respectively. Similarly, the expression of AIRE, a key transcriptional regulator of TSA expression in mTECs, which is important for the establishment of immunological self-tolerance, was enhanced only in 3D TECs. A similar expression pattern was observed for CSN2, a TSA whose expression in mTECs is regulated by AIRE. Collectively, these data strongly suggest that 3D culture is more efficient at promoting the differentiation and maturation of TEC lineages from iPSCs than conventional adherent 2D culture. Furthermore, a similar gene expression pattern was observed when hESCs were differentiated into TEPCs.

[0157] Example 3 The microenvironment of decellularized thymic scaffolds supports further differentiation and maturation of iPSC-derived TEPCs It has been demonstrated that functional thymic organoids can be tissue engineered by repopulating decellularized thymic scaffolds with isolated adult mouse TECs. The extracellular matrix (ECM) of the thymic scaffold can effectively support the survival and proliferation of adult TECs. To investigate whether the thymic ECM can further support the maturation and function of iPSC-derived TECs, TECs were injected into decellularized mouse thymic scaffolds along with CD34+ HSCs isolated from human umbilical cord blood (UCB). The reconstituted human thymic organoids were cultured in vitro in the top chamber of a transwell culture system. Thymocytes within the thymic organoids were able to survive long-term culture. RT-qPCR analysis revealed significantly increased expression of both MHC II and CD74. These genes are essential for TECs to present self-antigens and mediate positive and negative selection of developing T cells (Figure 2a).

[0158] To demonstrate that iPSC-derived TECs can support the de novo generation of human T cells from HSCs, we isolated cells from human thymic organoids after 21 days of in vitro culture and examined the surface expression of T cell developmental markers by FCM. Thymocytes at various developmental stages were detected, including CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8-, or CD4-CD8+ single positive (SP) cells, suggesting that human thymic organoids tissue-engineered from iPSC-TECs recapitulate T lymphohematopoietic functions in vitro (Figure 2b and Figure 10).

[0159] To further demonstrate their T lymphopoiesis function, iPSC-derived TECs (alone, without human HSCs) were injected into decellularized thymic scaffolds and transplanted under the kidney capsule of athymic nude mice. iPSC-derived TEC-engrafted mice were sacrificed at 18–32 weeks, and CD45+CD3+ T cells in the spleen and lymph nodes were further characterized by FCM (Figure 2c and Figure 2d). Both CD4+ T-helper cells and CD8+ cytotoxic TCRαβ+ T cells were detected, as well as TCRγδ+ T cells (Figure 2e and Figure 2f). Similar to previous findings in thymic-engrafted nude mice, the majority of CD8+ T cells exhibited a naive phenotype (CD62L+CD69-), whereas CD4+ T-helper cells displayed a CD62L-CD69+ memory T cell phenotype (Figure 2g), likely due to the expansion of CD4+ helper cells in the lymphopenic environment. Furthermore, T cells isolated from nude mice transplanted with thymic organoids mounted robust proliferative responses when challenged with alloantigens in a mixed lymphocyte reaction (MLR) assay, suggesting that iPSC-derived TEC thymic organoids support the differentiation of endogenous mouse bone marrow progenitors into functional mature T cells in vivo (Figure 2h).

[0160] Example 4 Promotion of human hematopoietic cell engraftment in humanized NSG mice transplanted with iPSC-derived thymic organoids One of the major hurdles to recapitulating human adaptive immune responses in NSG mice transplanted with human CD34+ HSCs (commonly known as hu.SRC) is the absence of a human thymus capable of supporting human T cell development. To overcome this challenge, we transplanted iPSC-derived thymic organoids under the kidney capsule of hu.SRC mice to generate humanized thymic organoid-transplanted mice (hu.Thor). These mice were divided into four groups and received the following treatments: TIFF2025160223000003.tif69164

[0161] Prior to HSC and thymic organoid transplantation, NSG recipients were chemically pretreated with the myeloablative alkylating agent busulfan (Group 2, Group 1 as a control). Because treatment of mice with anti-c-kit antibodies has been shown to deplete endogenous mouse HSCs in the bone marrow and promote donor stem cell engraftment, we also evaluated a c-kit depletion regimen for the generation of hu.Thor mice (Group 4, Group 3 as a control). To ensure that human T cells generated from iPSC-derived thymic organoids are functionally compatible with HSC-derived APCs (e.g., dendritic cells, macrophages, and B cells), only CD34+ HSCs isolated from umbilical cord blood (UCB) samples with HLA alleles partially matching those of the iPSCs were used in the study, unless otherwise noted (Table 3). [Table 3]

[0162] Among the four groups of mice (G1–G4), the control groups (G1, G3) all had significantly worse overall survival rates than the hu.Thor groups (G2, G4) transplanted with thymic organoids (Figure 3a). 56.1% of G1 (n=16 / 31) and 57.9% of G3 (n=11 / 19) mice died within 30 days of CD34+ HSC injection. Conversely, only three hu.Thor mice (G2, n=0 / 8; G4, n=3 / 39) died within 30 days of transplantation. These findings suggest that transplanted human thymic organoids protect recipients from the deleterious effects associated with the conditioning regimen.

[0163] To improve survival of G1 and G3 mice and ensure sufficient controls for the study, the CD34+ HSC transplant was increased five-fold to approximately 1x10 6Cells / mouse. Notably, signs of GVHD, such as weight loss, hair loss, and lymphocytic infiltration into organs and tissues, were not observed in hu.Thor mice 350 days after the start of the study, suggesting cross-tolerance between the engrafted hCD34+ human hematopoietic cells and the cells of the NSG recipient. Despite the low number of transplanted CD34+ HSCs, higher levels of hCD45+ cells were observed in the peripheral blood of hu.Thor mice 12 weeks post-transplant compared with controls (Figure 3b). For example, G4 hu.Thor mice showed an average of 55% hCD45+ cells in their circulating blood, significantly higher than the 4% seen in control G3 mice (Figure 3b).

[0164] Similar to the results of peripheral blood analysis, higher levels of human cell chimerism were observed in the primary (bone marrow) and secondary (spleen) lymphoid organs of hu.Thor mice (Figure 4a-b). Notably, G4 mice expressed 80% and 76% hCD45+ cells in the bone marrow and spleen, respectively, a significant increase compared to control G3 mice, which only showed 20% hCD45+ expression in both tissues (Figure 4a and Figure 4b). hu.Thor mice (G2+G4) showed a more than two-fold increase in hCD45+ cells in the bone marrow (2.4-fold) and spleen (2.3-fold) compared to hu.SRC mice (G1+G3) (Figure 4c). Further characterization of hu.Thor mice using FCM revealed the development of both lymphoid and myeloid lineages in the human hematopoietic compartment (Figure 4d). Because more robust human cell engraftment and T cell development were observed in G4 mice, we made efforts to focus on G4 hu.Thor mice to further clarify the impact of iPSC-derived thymic organoid transplantation on human T cell development.

[0165] Example 5 iPSC-derived thymic organoids can support the development of functional human T helper cell subsets in hu.Thor mice A diverse TCR repertoire is essential for an effective adaptive immune response. To assess the overall diversity of the T cell population in hu.Thor mice, we examined the expression of Vβ and Vα gene families in a NanoString TCR multiplex assay panel (Figure 5a and Figure 5f). For each Vβ and Vα family, similar levels of reads were detected between hu.Thor spleen cells and PBMCs from healthy human donors, demonstrating the complexity of the T cell repertoire in hu.Thor mice. Notably, a significant population of splenic CD4+ T-helper cells and CD8+ cytotoxic T lymphocytes (CTLs) displayed a native CD45RA+CD45RO- phenotype (Figure 5b). Further characterization of T-helper cells showed the development of multiple subsets, including CXCR3+CCR6-Th1, CXCR3-CCR6+Th17, and CXCR3-CCR6-Th2 cells, as well as the presence of CD4+FoxP3+T-regulatory cells (Tregs), an important population of CD4+T cells responsible for maintaining immune tolerance (Figure 5C).

[0166] To further demonstrate the functionality of major T cell subsets in hu.Thor mice, hu.Thor T cells were stimulated with phorbol 13-myristate acetate (PMA) / ionomycin, intracellularly stained with anti-IFN and anti-IL-17a antibodies, and analyzed by FCM. Both IFN-producing Th1 cells and IL-17a-producing Th17 cells were readily detected, demonstrating the successful development of multiple functional T helper lineages in hu.Thor mice (Figure 5d). Furthermore, hu.Thor T cells expanded when stimulated with transplanted CD34+ HSCs or allogeneic human umbilical cord blood cells bearing HLA genes distinct from those of the iPSC-derived thymic organoids (Table 3) (Figure 5e). These results demonstrate that iPSC-derived thymic organoids can support the development of a diverse and functional repertoire of human T cells in hu.Thor mice.

[0167] Example 6 Hu.Thor immune cells exhibit similar gene expression profiling to human PBMCs To further characterize hu.Thor immune cells, we performed gene expression profiling, focusing on pathways essential for T cell biology, including T cell diversity, activation, TCR signaling, metabolism, and exhaustion. Hu.Thor immune cells displayed an overall T cell gene expression profile similar to that of hPBMCs, but significantly different from that of hu.SRC cells. Notably, both hu.Thor and PBMCs exhibited higher levels of TCR diversity than hu.SRC cells, suggesting a more diverse and complex TCR repertoire (Figure 6a). Furthermore, both hu.Thor and PBMCs exhibited a more potent TCR signaling phenotype. Consistently, pathway scores for T helper subsets, including Th2, Th9, Th17, and Treg cells, were comparable between hu.Thor and PBMC cells and significantly higher than those of hu.SRC cells (Figure 6a). Interestingly, expression of activation markers was also elevated in hu.Thor and PBMC cells, whereas expression of T cell exhaustion markers was elevated in hu.SRC cells (Figure 6b). Indeed, higher transcript read counts for T cell exhaustion-related genes (e.g., PDCD1, TNFRSF9 / CD137, CD244, HAVCR2 / TIM3, and LAG3) and lower read counts for the terminally differentiated memory effector marker KLRG1 were detected in hu.SRC cells. Gene expression profiling and pathway analysis further demonstrated the superiority of hu.Thor mice over hu.SRC mice in recapitulating the molecular characteristics of human T cell-mediated immune pathways.

[0168] Example 7 De novo generated human T cells in hu.Thor mice can effectively reject allogeneic tumor grafts Neither hu.BLT nor hu.SRC mice have been shown to effectively reject stem cell-derived allografts or teratomas, primarily due to the progressive differentiation of human T cells into an "exhausted" state characterized by increased expression of inhibitory receptors and reduced effector function (Koorman, NG et al. Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics. Cell Reports 20, 1978-1990, 2017). In contrast, hu.Thor T cells mounted effective responses when activated with either antigen-specific (Figure 5e) or non-specific (Figure 5d) stimuli, displaying an increased T cell activation pathway profile (Figure 6b).

[0169] To further develop this model and validate the function of hu.Thor T cells in vivo, teratomas derived from the allogeneic CC1 iPS cell line were generated in hu.Thor mice. Teratomas derived from Y1 iPS cells, which were then transplanted into thymic organoids, served as syngeneic controls. Dissociated small clusters of syngeneic Y1 stem cells and allogeneic CC1 stem cells were injected intramuscularly into the left and right hind limbs of hu.Thor mice, respectively. Teratomas were harvested 3 weeks after inoculation and measured and weighed (Figure 7). While robust growth of allogeneic CC1 teratomas was observed in NSG and hu.SRC mice, significantly smaller tumors were observed in hu.Thor mice (Figure 7, left panel). Histological examination revealed lymphocytic infiltration in CC1 tumors in both hu.SRC and hu.Thor mice, consistent with previous reports. Immunofluorescence analysis of teratoma sections confirmed increased infiltration of human (HLA-A+) CD3+ T cells in CC1 tumors harvested from hu.Thor mice. These results suggest that hu.Thor T cells can effectively mount an alloreactive immune response to reject allogeneic iPSC-derived tumors, a feasibility of hu.SRC T cells. Furthermore, no significant differences in syngeneic Y1 tumor growth were observed between NSG, hu.SRC, and hu.Thor mice (Figure 7, right panel), suggesting that Y1 thymic organoids may induce immune tolerance of allogeneic grafts in hu.Thor mice.

[0170] Example 8 De novo generated human T cells from iPSC-thymic organoids can mediate humoral responses in hu.Thor mice T cell-dependent activation of B cells plays a key role in both primary and secondary humoral adaptive immune responses. After initial antigen exposure, cytokines secreted by T2 cells induce immunoglobulin class switching in plasma cells from IgM to IgG, IgA, or IgE. Upon re-exposure to antigen, memory B cells further mature, undergo V(D)J somatic mutations at the immunoglobulin locus, and generate IgG with higher affinity for the target antigen.

[0171] To examine the levels of immunoglobulin classes and subclasses in the serum of hu.Thor mice, we performed a human antibody isotyping multiplex assay and compared them with hu.SRC controls. Major human immunoglobulin classes, including IgG, IgM, IgA, and IgE, were detected in hu.Thor serum (Figure 8a). In particular, we observed significantly higher levels of IgM and IgG subclasses (IgG1 and IgG3) compared with hu.SRC samples. These results suggest that human T cells generated from transplanted iPSC-derived thymic organoids can promote B cell maturation and isotype switching function.

[0172] Furthermore, to evaluate their ability to mount effective humoral responses to specific antigens, hu.Thor mice were vaccinated against diphtheria toxoid (DT). DT-specific IgG was generated after the first immunization and significantly increased after the booster dose (Figure 8b). These results further demonstrate that T helper cells generated from iPSC-derived thymic organoids of hu.Thor mice can promote human B cell maturation and be used as a model for the humoral response of the human adaptive immune system.

[0173] Hematopoietic-humanized mice are powerful small animal models for studying the human immune system. While considerable progress has been made to improve the engraftment and differentiation of multilineage immune cells, the development of a functional human T cell compartment remains a major challenge, significantly hindering successful modeling of the human adaptive immune response. Over the years, considerable efforts have been made to improve the generation of human T cells in these mice. Genetic transfer of NSG-SGM3 mice with human SCF, GM-CSF, and IL-3 promotes the stable engraftment of diverse hematopoietic cells, including CD3+ T cells, CD19+ B cells, and CD33+ myeloid cells. Recently, the RG SKI hIL-6 mouse (Rag2 - / - Il2rg - / - SIRPa h / m IL-6 h / h) express human IL-6, which better supports lymphoid cell survival. These mice have larger thymuses and higher T cell numbers, suggesting that hIL-6 promotes thymopoiesis. Furthermore, successful IgG class switching in antibody-producing B cells has been observed, suggesting effective T helper function. Transgenic expression of human cytokines and factors promotes human T cell proliferation and survival, but T cell education remains dependent on the mouse thymic microenvironment and mouse MHC. Human T cells positively selected by mouse TECs are largely restricted to interacting with mouse MHC-expressing APCs, compromising their ability to model human immune responses. Transgenic expression of human HLA molecules in mouse cells has yielded interesting results, but the human HLA gene structure is more complex than that of mice (e.g., two MHC I and one MHC II genes in mice, compared with three MHC I and three MHC II genes in humans). Furthermore, the formation of a functional immunological synapse depends on the interaction of human costimulatory molecules (e.g., CD28 and CD40) on human T cells with their ligands (e.g., CD80 / 86 and CD40L) on mouse APCs, further compromising the ability of humanized mice to recapitulate human immune responses. In contrast, the iPSC-derived TECs in the thymic organoids of hu.Thor mice described here support the selection of human T cells within the human thymic microenvironment. Our data demonstrate the development and functionality of T helper subsets, including Th1 and Th17 cells, capable of producing IFNγ and IL17A, respectively. Furthermore, the serum of hu.Thor mice contains low but detectable levels of human interleukin-6 (IL-6), further suggesting that hu.Thor mice are adept at recapitulating human immune responses.

[0174] The presence of endogenous mouse thymus, albeit degenerated, may compete with transplanted thymic organoids for HSC homing. To mitigate this effect, 8- to 12-week-old NSG mice can be used as recipients, as the hypoplastic thymus of NSG mice has been shown to undergo irreversible age-related fibrosis. Indeed, no signs of endogenous mouse thymus recovery were observed in any of the hu.Thor mice examined in the study described herein. To model the condition of patients undergoing chemotherapy, a chemically induced myeloablative regimen was used instead of lethal total body irradiation as preconditioning for NSG mice. The recipient age and chemical regimen used in this study may negatively affect the level of human cell chimerism achieved in hu.Thor mice. Interestingly, a higher survival rate was observed in hu.Thor mice compared to hu.SRC controls, suggesting that human cytokines or factors produced by human thymic organoids may mitigate busulfan chemotoxicity.

[0175] As shown in the examples, partially HLA-matched HSCs can be used as iPSC-thymus cells, but fully matching the HLA between thymic organoids and transplanted HSCs can better recapitulate human adaptive immunity in hu.Thor mice. Therefore, humanized mice using both TEPCs and HSCs from a single patient can further improve modeling of the patient's adaptive immune system. Furthermore, some hu.Thor mice have lived for more than 12 months after transplantation, and no clinical symptoms of GVHD have been observed.

[0176] Here, we describe the development of hu.Thor mice, which are capable of generating vast populations of multiple T cell subsets capable of maintaining self-tolerance and mounting robust immune responses upon challenge with foreign antigens. Human thymic organoids constructed from iPSC lines are capable of differentiating CD34+ human HSCs into functional and diverse CD4+ and CD8+ T cells both in vitro and in vivo. This work highlights the feasibility of recapitulating T cell-mediated human adaptive immune responses from individual patients in small animal models for personalized medicine.

[0177] The foregoing examples and descriptions of preferred embodiments should be taken as illustrative rather than limiting of this document as defined by the claims. As will be readily understood, numerous variations and combinations of the features defined above can be utilized without departing from this document as defined in the claims. Such variations are not considered to be a departure from the scope of this document, and all such variations are intended to be included within the scope of the following claims. All documents cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for producing bioengineered thymic organoids, said method comprising: Obtaining a cell population comprising human thymic epithelial progenitor cells (TEPCs) or human thymic epithelial cells (TECs), or both; combining the cell population with human hematopoietic stem cells (HSCs) in a defined ratio to form a combination; seeding the combination onto the extracellular matrix of a decellularized thymic scaffold to generate a thymic construct; and culturing the thymic construct under conditions that allow cell adhesion to the extracellular matrix to produce a bioengineered thymic organoid; A method comprising:

2. The method of claim 1, wherein the TEPC, the TEC, or the HSC is derived from a donor individual.

3. The method of claim 1, wherein the HSCs comprise human CD34+ hematopoietic stem cells.

4. 10. The method of claim 1, wherein the decellularized thymus scaffold is from a donor animal.

5. 10. The method of claim 1, wherein the cell population comprises: encapsulating human pluripotent stem cells (hPSCs) in a suspension and dissociating the hPSCs into single cells; culturing the hPSCs in an expansion medium to expand their number without differentiating them; differentiating the hPSCs to generate TEPCs or TECs in an encapsulation medium; and liberating the TEPC or TEC from the encapsulation medium; The method is obtained by a process having the steps:

6. 6. The method of claim 5, wherein the hPSCs comprise human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

7. 7. The method of any one of claims 1 to 6, wherein the thymic construct is placed in a flow cell that provides a continuous supply of nutrients and human cells to produce human immune cells.

8. The method of claim 7, wherein the thymic construct comprises immune cells.

9. 9. The method of claim 8, wherein the immune cells comprise B cells and T cells.

10. 9. The method of claim 8, wherein the T cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR).

11. 11. The method of claim 10, wherein the viral vector is added to a flow cell to transduce the T cells.

12. The method of any one of claims 7 to 11, wherein the thymic construct is contacted with a drug candidate to test the effect of the drug candidate on immune cell development.

13. The method of any one of claims 1 to 6, wherein the thymus construct is surgically implanted into a host animal.

14. 14. The method of claim 13, wherein the host animal is a pretreated humanized immunodeficient animal.

15. 15. The method of claim 14, wherein the host animal is a pretreated humanized immunodeficient mouse.

16. 16. The method of claim 15, wherein the thymus construct is placed under the kidney capsule.

17. The method according to any one of claims 13 to 16, wherein the obtained host animal is provided with hematopoietic stem cells to produce human immune cells.

18. 18. The method of claim 17, wherein the resulting host animal produces increased amounts of fully human immunoglobulin G.

19. 19. The method of any one of claims 13 to 18, wherein the host animal is administered a drug candidate to test the effect of the drug candidate on immune cell development.

20. 20. The method of claim 19, wherein the TEPCs, TECs, or HSCs are from a donor individual to test the effect of the drug candidate on the donor individual.

21. 21. The method of claim 20, wherein the host animal is to receive cells or tissue from the donor individual.

22. 22. The method of claim 21, wherein the cells or tissue comprise cancer cells.

23. 14. The method of claim 4 or 13, wherein the donor animal or the host animal is a non-human mammal.

24. 24. The method of claim 23, wherein the non-human mammal is selected from the group consisting of cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, and non-human primates.

25. 1. A bioengineered thymic organoid, comprising: (i) human TEPC (hTEPC), human TEC (hTEC), or human HSC (hHSC), and (ii) a decellularized thymic scaffold having an extracellular matrix, wherein the hTEPCs, hTECs, or hHSCs adhere to the extracellular matrix; Bioengineered thymic organoids comprising:

26. A bioengineered thymic organoid prepared by the method of any one of claims 1 to 24.

27. 27. The bioengineered thymic organoid of claim 25 or 26, wherein the bioengineered thymic organoid comprises immune cells.

28. 27. The bioengineered thymic organoid of claim 25 or 26, wherein the thymic scaffold is xenogeneic or allogeneic to the hTEPC, hTEC, or hHSC.

29. 27. The bioengineered thymic organoid of claim 25 or 26, wherein the hTEPC or hTEC is derived from an hPSC.

30. A non-human animal comprising a bioengineered thymic organoid according to any one of claims 25 to 29.

31. 31. The non-human animal of claim 30, wherein the non-human animal is a mammal selected from the group consisting of cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, and non-human primates.

32. 32. The non-human animal of claim 31, wherein the non-human animal is a mouse.

33. 1. A method for evaluating a drug candidate, the method comprising: (a) contacting a drug candidate with the bioengineered thymic organoid of any one of claims 25 to 29; and (b) detecting the effect of the drug candidate on the development of cells present in or migrating from the bioengineered thymic organoids; A method comprising:

34. 34. The method of claim 33, wherein the bioengineered thymic organoid is transplanted into a host animal and the drug candidate is administered to the host animal.

35. 35. The method of any one of claims 12, 19 and 33-34, wherein the drug candidate is selected from the group consisting of a small molecule, a nucleic acid, a peptide, a polypeptide, an antibody, and an antibody fragment.

36. 1. A method for preparing thymic emigrant cells, said method comprising: (a) introducing progenitor cells into the bioengineered thymic organoid of any one of claims 25 to 29 or the non-human animal of any one of claims 30 to 32; (b) maintaining the bioengineered thymic organoids or the non-human animal under conditions that allow differentiation of the progenitor cells to generate their progeny; (c) expelling the progeny cells from the bioengineered thymic organoids to generate thymic emigrant cells; and (d) isolating the thymic emigrant cells; A method having the following.

37. 37. Thymic emigrant cells prepared by the method of claim 36.

38. 38. The thymic emigrant cell of claim 37, comprising one or more transgenes encoding antigen receptors.

39. 39. The thymic emigrant cell of claim 38, wherein the antigen receptor is a chimeric antigen receptor (CAR).

40. A pharmaceutical composition comprising the thymic emigrant cells of any one of claims 36 to 39 and a pharmaceutically acceptable carrier.

41. 1. A method for improving immune function in a subject in need thereof, the method comprising: (a) administering to said subject an effective amount of thymic emigrant cells according to any one of claims 37 to 39; or (b) transplanting the bioengineered thymic organoid of any one of claims 25 to 29 into the subject. A method comprising:

42. 42. The method of claim 41, wherein the subject has a condition selected from the group consisting of cancer, an autoimmune disease, and an infectious disease.

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