Method for generating thymocytes in vitro

Abstract

Provided herein are improved methods for generating thymic epithelial progenitor (TEP) cells from pluripotent stem (PS) cells in vitro. Also provided are isolated in vitro cell populations, compositions, and systems containing in vitro-produced TEP cells. Provided are cell population compositions and systems for thymic epithelial cells and their subpopulations, as well as cells formed during different stages of differentiation of PS cells into thymic epithelial cells and their subpopulations.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 147,126, filed on February 8, 2021, entitled "Methods for Generating Thymic Cells In Vitro" and U.S. Provisional Patent Application No. 63 / 016,527, filed on April 28, 2020, entitled "Methods for Generating Thymic Cells In Vitro", the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Government Licensing Rights This invention was made with government support under grant U01 DK107383 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] This disclosure relates to compositions and methods for generating thymic cells, including, for example, thymic epithelial cells.

Background Art

[0004] The use of stem cells to replace lost or damaged tissue continues to be a promising area of research for the development of therapeutic compositions and methods. Specifically, thymic epithelial progenitor cells derived from stem cells present increasingly important applications for stem - cell - based therapeutic approaches for treating a variety of diseases.

[0005] The thymus is a primary lymphoid organ that plays a central role in the immune system. For example, the thymic microenvironment provides a unique training ground for the development and maturation of effector cells such as lymphocytes (e.g., T cells). The thymus is also a major organ involved in establishing immune tolerance through the elimination of autoreactive T cell subsets and the production of regulatory T cells (as outlined in Anderson et al., Nat Rev Immunol 7, 954-963, 2007). These important functions are mediated by thymic epithelial cells, the main component of the thymic stroma.

[0006] There is a need for improved methods to generate functional thymic epithelial progenitor (TEP) cells, and for cell populations enriched with functional TEP cells capable of differentiating into functional thymic epithelial cells. In particular, there remains a critical need to replicate the complex thymic microenvironment and support the maturation and function of thymocytes in vitro for therapeutic use. [Overview of the Initiative]

[0007] A method for generating thymic epithelial progenitor (TEP) cells in vitro is provided, for example, in International Patent Application Publication No. 2014 / 134213, the disclosure of which is incorporated herein by reference in its entirety. Given the central role of the thymus in the immune system, thymic cells and thymic tissue have great therapeutic potential.

[0008] Accordingly, a method for generating thymic epithelial progenitor (TEP) cells in vitro is provided, comprising culturing a cell population in a first medium containing an activator of bone morphogenetic protein (BMP) signaling, an activator of fibroblast growth factor (FGF) signaling, and an inhibitor of transforming growth factor-β (TGF-β) signaling; and further culturing the cell population to induce further maturation of the TEP cells in vitro, wherein the further culturing comprises culturing the cell population in a second medium containing keratinocyte growth factor (KGF), heparin, and hydrocortisone. Cells produced from the further maturation of TEP cells may be referred to as mature TEP (mTEP) cells.

[0009] Further culturing of the cell population can be performed in a medium further containing triiodo-L-thyronine (T3) supplementation.

[0010] Further culturing of the cell population can be carried out in a medium further containing an insulin-transferrin-selenium (ITS) supplement.

[0011] Further culturing of the cell population can be carried out in a medium containing an additional B27 supplement.

[0012] The cell population may include one or more of the following: endoderm (DE) cells, anterior foregut endoderm (AFE) cells, ventral pharyngeal endoderm (VPE) cells, and TEP cells.

[0013] Further culturing of the cell population to induce further maturation of TEP cells in vitro may include culturing the cell population for an additional 14 days or more.

[0014] Further culturing of the cell population to induce further maturation of TEP cells in vitro may involve transferring the cell population to an extracellular matrix-based medium such as Matrigel.

[0015] This method can be carried out in cell culture medium, where the first and / or second medium is a liquid medium, and the culture conditions include suspension culture.

[0016] The first and / or second culture medium may be minimal essential medium or Dulbecco's minimal essential medium (DMEM).

[0017] This method may further include transplanting TEP cells into a subject. In such embodiments, further culturing of the cell population to induce further maturation of TEP cells in vitro results in thymic epithelial cells (TECs) containing a subpopulation of TECs after in vivo transplantation.

[0018] Subpopulations of TECs may include one or more of the following: corticothymic epithelial cell (cTEC) lineage cells, pluripotent TEP cells, committed medullary thymic epithelial cell (mTEC) progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells.

[0019] This method may further include transferring the cell population to an air-liquid interface culture system before transplantation.

[0020] This method may further include reassembling cells to form a reassembled structure before transplantation.

[0021] This method may further include reducing or removing non-epithelial cells from a culture of differentiated TEP cells. This reduction or removal may be accompanied by enrichment of EPCAM+ TEP cells.

[0022] In some aspects, the method includes adjusting culture conditions or combining cell types in culture to replicate the thymic microenvironment.

[0023] Recreating the thymic microenvironment may involve culturing TEP cells under conditions sufficient to support the survival of lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

[0024] Recreating the thymic microenvironment may involve culturing TEP cells under conditions sufficient to support the differentiation of TECs and their subpopulations. Subpopulations of TECs include cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myoform cells.

[0025] This method may include culturing endoderm (DE) cells in a first medium containing a retinoic acid receptor (RAR) activator, a BMP signaling activator, a FGF signaling activator, and a TGF-β signaling inhibitor. In some embodiments, DE cells are initially differentiated in a first cell culture medium containing a BMP signaling inhibitor before being transferred to a first cell culture medium containing a BMP signaling activator. In some embodiments, a Wnt signaling inhibitor is introduced into the first cell culture medium.

[0026] This method may include culturing anterior foregut endoderm (AFE) cells produced by the culture of DE cells, the AFE cells being cultured in a first medium containing a BMP signaling activator, a FGF signaling activator, and a TGF-β signaling inhibitor.

[0027] Ventral pharyngeal endoderm (VPE) cells produced by the culture of AFE cells can be cultured in a first medium containing a BMP signaling activator, a FGF signaling activator, and a TGF-β signaling inhibitor to produce TEP cells. In some embodiments, the AFE cell culture medium is substantially the same as the first cell culture medium, except that it substantially does not contain a RAR signaling activator.

[0028] This method can be carried out starting with cells obtained from pluripotent stem (PS) cells.

[0029] PS cells can be embryonic stem cells, embryonic germ cells, or induced pluripotent stem cells.

[0030] PS cells can be primate pluripotent stem (pPS) cells.

[0031] PS cells can be human pluripotent stem (hPS) cells.

[0032] Also provided herein is a method for generating TEP cells in vitro, which includes culturing a cell population containing anterior foregut endoderm (AFE) cells in a first cell culture medium containing an activator of BMP signaling, an activator of FGF signaling, and an inhibitor of TGF-β signaling. In some embodiments, the AFE cell culture medium is substantially the same as the first cell culture medium, except that it does not substantially contain at least one of an activator of retinoic acid receptor signaling, an activator of Wnt signaling, and an inhibitor of hedgehog signaling.

[0033] Also provided herein is a method for generating TEP cells in vitro, which includes culturing a cell population containing definitive endoderm (DE) cells in a first cell culture medium containing an activator of BMP signaling, an activator of retinoic acid receptor signaling, an activator of FGF signaling, an inhibitor of TGF-β signaling, and an inhibitor of Wnt signaling to generate a cell population containing AFE cells, and culturing the AFE cell population in a cell culture medium containing an activator of BMP signaling, an activator of FGF signaling, and an inhibitor of TGF-β, and substantially not containing an inhibitor of Wnt signaling to produce TEP cells.

[0034] Some embodiments of the methods provided herein further include directing the development of one or more subpopulations of thymic epithelial cells in a culture by introducing one or more factors related to WNT signaling, BMP signaling, TGF-beta signaling, IGF signaling, FGF signaling, NOTCH signaling, TNF receptors or their ligands, p53 signaling, and / or Toll-like receptors into the culture medium. Factors may be introduced into the culture medium, for example, by introducing soluble forms of one or more factors and / or by introducing cells expressing one or more factors. As used herein, “factor related to” means a factor that initiates, promotes, inhibits, upregulates, downregulates, or otherwise modulates the activity of a pathway or components of a pathway. For example, factors related to WNT signaling include WNT downstream or upstream factors whose expression / activity modulates one or more components of the WNT signaling pathway. In some cases, these factors include proteins whose expression increases upon activation of signaling pathways, while their expression may be low or undetectable in the absence of signaling pathway activation.

[0035] In some embodiments, the thymic microenvironment can be evaluated or characterized by determining the presence of cells expressing one or more factors related to WNT signaling, BMP signaling, TGF-beta signaling, IGF signaling, FGF signaling, NOTCH signaling, TNF receptors or their ligands, p53 signaling, and / or Toll-like receptors.

[0036] In some embodiments of the methods described herein, factors related to WNT signaling include WNT5A, WNT6, ROR1, ROR2, RYK, FRZB, RSPO1, RSPO3, SFRP2, and / or SFRP5. Factors related to BMP signaling include BMP4, BMP5, and / or FST. Factors related to TGF beta signaling include TGFB1, TGFBR2, CXCL12, and / or CCL21. Factors related to IGF signaling include IGF1R. Factors related to FGF signaling include FGFR2 and / or FGF7 / KGF. Factors related to NOTCH signaling include NOTCH1, NOTCH2, NOTCH3, HES1, HES6, DLL4, JAG2, JAG1, HES2, HES4, HEY1, NRARP, DLK1, and / or DLK2. TNF receptors include RANK / TNFRSF11A, CD40, LTBR, ​​TNFRSF4, TNFRSF9, LTB, and / or CD70. Factors involved in p53 signaling include PERP, SFN, CTSD, CDKN2A, and / or CDKN2B. Toll-like receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, and / or TLR10.

[0037] In some embodiments, the Disclosure provides compositions comprising differentiated cells as described herein. For example, the Disclosure provides compositions comprising a differentiated population of TEP cells produced according to the method described herein.

[0038] The disclosure also provides a composition comprising reassembled thymic epithelial progenitor cells (TEPs) differentiated from PS cells, the composition further comprising one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

[0039] The disclosure also provides compositions comprising TECs differentiated from PS cells, the compositions further comprising one or more of the following: cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells.

[0040] The disclosure also provides compositions comprising reassembled thymic epithelial cells (TECs) differentiated from PS cells, the compositions further comprising one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof. In some embodiments, the reassembled compositions comprise a subpopulation comprising one or more of cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells. [Brief explanation of the drawing]

[0041] [Figure 1] A schematic diagram of the processes that characterize the transcriptome profile of thymocytes is shown. In Figure 1, "MACS" represents magnetically activated cell sorting, and FACS represents fluorescence-activated cell sorting.

[0042] [Figure 2] A heatmap is shown illustrating the mean expression levels of soluble factors, extracellular matrix / adhesion molecules, and chemokines in each of the 12 identified stromal clusters.

[0043] [Figure 3] A heatmap showing the expression of marker genes in each immature TEC ("imm.TEC") cluster is shown.

[0044] [Figure 4] A heatmap is shown illustrating the expression of newly identified marker genes in each of the nine identified epithelial clusters.

[0045] [Figure 5] This dot plot shows the relative expression levels of Notch signaling ligands, receptors, target genes, and inhibitors in an epithelial subset. The relative size of the dots indicates the proportion of cells in each group expressing the indicated gene.

[0046] [Figure 6] This dot plot shows the relative expression levels of selected genes associated with p53 signaling. The relative size of the dots indicates the proportion of cells in each group that express the indicated gene.

[0047] [Figure 7] The dot plots show the relative expression levels of selected TNF superfamily members (upper panel) and genes associated with Toll-like receptor signaling (lower panel). The relative size of the dots indicates the proportion of cells in each group that express the indicated gene.

[0048] [Figure 8] This diagram illustrates the process by which pluripotent stem cells differentiate into mature thymic epithelial progenitor cells ("mTEPs") in vitro and then into thymic epithelial cells (TECs) in vivo. [Modes for carrying out the invention]

[0049] Current sources of thymic tissue for therapeutic and research purposes have certain technical limitations. Typically, thymic tissue is harvested from cadavers and cultured in vitro for, for example, 12–21 days to remove thymocytes, thereby avoiding graft problems such as graft-versus-host disease (GVHD). The tissue thus obtained must be tested for sterility, screened for infection and quality, and also histologically evaluated before transplantation. Other complications arising from cadaver or donor-derived thymic tissue include the limited availability of donors / tissues. For example, in many cases, donor tissue must come from subjects under 9 months of age, be negative for viruses (e.g., HIV, hepatitis B, hepatitis C, EBV, and / or CMV), and come from donors with a known family history, including no primary relatives with autoimmune diseases. Even then, the risk of GVHD is unavoidable. Furthermore, with current methods, it is virtually impossible to match the HLA type of donor tissue with that of the recipient. Furthermore, current methods for culturing and / or transplanting donor-derived tissue have relatively low success rates, with a failure rate of approximately 30%. The causes contributing to the high failure rate are not always known in all cases and may include complicating factors such as infections occurring in the donor tissue, and other unknown congenital anomalies. For example, see Markert, ML, Devlin, BH, & McCarthy, EA (2010) Thymus transplantation. Clinical Immunology (Orlando, Fla), 135(2), 236-246, which is incorporated herein by reference in its entirety. Therefore, there is a need for novel sources of human thymic tissue, including thymic epithelial progenitor (TEP) cells and thymic epithelial cells (TECs), as well as novel methods for preparing TEPs and TECs, including in vitro methods for producing thymocytes for human transplantation and any other therapeutic applications.

[0050] Improved methods for generating TEP, including generating TEP from stem cells such as pluripotent stem (PS) cells and generating TEP in vitro, are provided herein.

[0051] A method is also provided for generating a population of thymocytes, including TEPs and supporting cells, that replicates the complexity of the endogenous thymic microenvironment in vitro.

[0052] Improved methods for generating TECs are provided herein, including generating TECs from stem cells such as pluripotent stem (PS) cells and generating TECs in vitro.

[0053] A method is also provided for generating a population of thymocytes, including TECs and supporting cells, that replicates the complexity of the endogenous thymic microenvironment in vitro.

[0054] In some embodiments, TEPs are reassembled into a three-dimensional structure that replicates the complexity of the endogenous thymic microenvironment described herein. See, for example, Gill, Jason, et al. “Generation of a complete thymic microenvironment by MTS24+ thymic epithelial cells.” Nature Immunology, 3(7), 635-642, which is incorporated herein by reference in its entirety. See also Park, Jong-Eun, et al. “A cell atlas of human thymic development defines T cell repertoire formation.” Science 367.6480(2020), which is incorporated herein by reference in its entirety.

[0055] Methods and results of comprehensive single-cell transcriptome analysis of human thymic stromal cells, with particular focus on the epithelial compartment, are also provided herein. Ionocytes are identified as an additional subset of medullary epithelial cells, and transcriptome information is provided for rare subsets, including ciliated cells and Schwann cells.

[0056] A subset of postnatal mesenchymal cells was identified as secreting numerous factors associated with WNT signaling, including the non-standard WNT ligand WNT5A and molecules capable of enhancing WNT / Ca2+ signaling (such as RSPO3 and SFRP2). Given that WNT signaling is involved as a key regulator of FOXN1 expression, thymocyte activity, and thymic migration during development, these findings indicate that this subset of mesenchymal cells is a crucial regulator of these processes. In particular, this population also expressed other important regulators of epithelial proliferation and differentiation (e.g., BMP4, IGF1, FGF7, and FGF10).

[0057] Activin A signaling is crucial for TEC maturation, but follistatin, an inhibitor of this pathway, contributes to TEPC accumulation by blocking TEC differentiation. Our findings identify pericytes as the primary source of activin A. These findings further reveal that myosophical cells express high levels of follistatin, providing insight into why conditions such as myasthenia gravis, which affect myosophical cell number, disrupt human TEC differentiation.

[0058] Surprisingly, CFTR+ ionocytes were identified as an additional subset of epithelial cells found in the human thymic medulla. While ionocytes are described as lung epithelium, their presence in the thymus has not been previously reported. Interestingly, lung ionocytes arise from basal cells and also give rise to neuroendocrine and tuft cells. Given that these cell types are also present in the human thymus, found in close proximity to one another within the medulla, and many subsets are associated with Hassall's bodies, it is plausible that similar progenitor cells exist in the thymus. This hypothesis is consistent with reports of thymomas containing neuroendocrine differentiation and thymic carcinomas containing tumor cells with neuroendocrine phenotypes. It is also interesting that myoilike cells arise in different thymic tumors, including different histological variants of thymomas and thymic carcinomas. Tumors exhibiting differentiation of both rhabdomyolike and epithelial cells may also arise in the thymus, suggesting the possibility of a common precursor that gives rise to both epithelial and myoilike cells. The transcriptome data presented in this application, which shows the branching point between neuroendocrine cells and myosoidal cells, as well as the co-expression of epithelial markers and myosoidal cell markers by immunofluorescence in fetal tissue, support this idea.

[0059] The role of Notch signaling in the development of different thymic epithelial subsets has been identified. While Notch signaling has been extensively studied in relation to T cell involvement and epithelial differentiation in other tissues, the role of this pathway in TEC specificization has only recently been reported. In neural and muscle stem cells, high and persistent levels of HES1 can inhibit cell differentiation by antagonizing master regulators of cell fate, such as the preneurial factor ACL1 and the myogenic MYOD1 regulator. Conversely, oscillations in HES1 expression activate stem cell proliferation by driving oscillations in ASCL1 and MYOD1 expression through periodic suppression cycles. Alternatively, when ASCL1 or MYOD1 expression is maintained and HES1 expression and / or activity is inhibited, terminal differentiation is promoted. This mechanism is consistent with our observation that the HES1 inhibitor HES6 is highly expressed in neuroendocrine and myosoidal cells. Therefore, HES6-mediated inhibition of HES1 may enable stable expression of ASCL1 and MYOD1 in progenitor cells that ultimately differentiate into ASCL1+ neuroendocrine cells or MYOD1+ myosomatic cells. Although the mechanism leading to high HES1 expression in progenitor cells is unclear, other signaling pathways such as BMP have been shown to play a crucial role in regulating quiescence by upregulating HES1 in neural stem cells or promoting the degradation of ASCL1 in hippocampal stem cells. Given the evidence that BMP signaling promotes the maintenance of thymic progenitor cells, this pathway may help establish and maintain TEPC quiescence by upregulating HES1 to a level that consistently suppresses the expression of differentiation factors such as ASCL1.

[0060] Finally, the findings of this application provide a valuable database of genes expressed in TECs, enabling a better understanding of the chaotic gene expression in the human thymus. For example, a significant subset of APS-1 antigens is present in AIRE+mTECs, supporting the idea that these genes are AIRE-dependent in humans. Interestingly, the expression of several autoantigens not observed in AIRE+ cells (CYP21A2, CYP17A1, CYP11A1), which are often targeted by autoantibodies in thymoma patients, does not correlate with AIRE expression in thymoma samples, suggesting they are not AIRE gene-dependent. In addition to AIRE+mTECs, other cell types are most likely to be involved in inducing tolerance by providing antigens that can be presented by antigen-presenting cells such as dendritic cells. For example, myosocytes may be involved in inducing immune tolerance to muscle antigens. Indeed, many thymoma patients who typically lack myoioid cells develop myasthenia gravis (MG), a neuromuscular junction autoimmune disease characterized by autoantibodies against acetylcholine receptor (AChR) or other muscle antigens such as titin (TTN). Furthermore, APS-1 patients typically lack detectable autoantibodies against AChR or TTN, suggesting that the expression of these antigens is not entirely AIRE-dependent. Importantly, AChR and TTN expression in this study was significantly higher in myoioid cells compared to AIRE+mTECs, supporting the idea that myoioid cells are the primary source of muscle antigens in the human thymic medulla. Therefore, this analysis provides additional information regarding the regulation of disease-related TSA in the human thymus.

[0061] definition "Pluripotent stem cells" or "pluripotent cells" refer to cells that, under appropriate conditions, have the ability to produce offspring of several different cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can form teratomas. Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic germ stem (EG) cells, induced pluripotent stem (iPS) cells, and adult stem cells. PS cells can be derived from any target organism, including primates such as humans, dogs, cats, mice, horses, pigs, birds, camels, cattle, and sheep.

[0062] Embryonic stem cells, or ES cells, are defined as cells that a) can self-replicate, b) can differentiate to produce all types of cells in an organism, and c) originate from a developing organism or are established ES cell lines derived from a developing organism. ES cells may originate from the inner cell mass of a blastula of a developing organism. ES cells may also originate from blastomeres generated by single blastomeres biopsy (SBB), which involves the removal of a single blastomere from the 8-cell stage of a developing organism. Generally, SBB provides a non-destructive alternative to the isolation of the inner cell mass. The generation of SBB and hES cells from biopsied blastomeres is described in Cell Stem Cell, 2008 Feb7;2(2):113-17. ES cells can be cultured for extended periods while maintaining their ability to differentiate into all types of cells in an organism. In culture, ES cells typically grow as flattened colonies with a large nucleus-to-cytoplasmic ratio, defined boundaries, and a prominent nucleus. Furthermore, ES cells express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but do not express SSEA-1. Examples of methods for generating and characterizing ES cells can be found, for example, in U.S. Patent Nos. 7,029,913, 5,843,780, and 6,200,806, the disclosures of which are incorporated herein by reference.

[0063] "Embryo germ stem cells," "embryo germ cells," or "EG cells" refer to cells derived from germ cells and germ cell progenitor cells that a) can self-replicate, b) can differentiate to produce all types of cells in an organism, and c) can become, for example, primordial germ cells, i.e., sperm and eggs. Embryogery germ cells (EG cells) are thought to have properties similar to those of embryonic stem cells. Examples of methods for generating and characterizing EG cells are described, for example, in U.S. Patent No. 7,153,684, Matsui, Y., et al, (1992) Cell 70:841, Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98:113, Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0064] "Induced pluripotent stem cells" or "iPS cells" mean cells that a) can self-replicate, b) can differentiate to produce all types of cells in an organism, and c) are derived from somatic cells. iPS cells have a morphology similar to ES cells, with a large nucleus-to-cytoplasm ratio, well-defined boundaries, and proliferate as flat colonies with clearly defined nuclei. Furthermore, iPS cells express one or more important pluripotency markers known to those skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. iPS cells can be generated by providing cells with "reprogramming factors," i.e., a cocktail of one or more biologically active factors that act on cells to alter their transcription, thereby reprogramming the cells to be pluripotent. Examples of methods for generating and characterizing iPS cells can be found, for example, in U.S. Patent Applications Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, the disclosures of which are incorporated herein by reference.

[0065] A "somatic cell" refers to any cell in an organism that, without experimental manipulation, does not normally give rise to all types of cells in that organism. In other words, a somatic cell is a fully differentiated cell that does not naturally produce any of the three germ layers of the body: the ectoderm, mesoderm, and endoderm. For example, somatic cells include both neurons and neural progenitor cells, the latter of which can regenerate and naturally give rise to all or part of the cell types of the central nervous system, but cannot give rise to cells of the mesoderm or endoderm lineages.

[0066] The term "cell line" typically refers to a majority or substantially identical population of cells obtained from a single ancestral cell line or from a defined and / or substantially identical population of ancestral cells. Cell lines can be maintained or may be maintained in culture for extended periods (e.g., months, years, or indefinite periods).

[0067] The term "endoderm" refers to the germ layer formed during embryogenesis in animals that gives rise to the digestive tract, airways, endocrine glands and organs, specific structures of the auditory system, and specific structures of the urinary system.

[0068] "Mesoderm" refers to the germ layer formed during embryogenesis in animals that gives rise to muscles, cartilage, bone, dermis, reproductive system, adipose tissue, connective tissue of the intestines, peritoneum, specific structures of the urinary system, mesothelium, notochord, and the structure of the spleen.

[0069] The term "ectoderm" refers to the germ layer formed during embryogenesis in animals, which gives rise to the nervous system, tooth enamel, epidermis, hair, nails, and the inner layer of mucous membrane tissue.

[0070] "Bone morphogenetic proteins" or "BMPs" refer to a family of growth factors that are subfamily members of the transforming growth factor β (TGFβ) superfamily. BMPs (e.g., BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9 / GDF, BMP10, BMP11 / GDF11, BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15 / GDF9B) were first discovered for their ability to induce bone and cartilage formation. BMPs interact with specific receptors on the cell surface called bone morphogenetic protein receptors (BMPRs). BMPR-mediated signaling mobilizes members of the SMAD family of proteins, thereby regulating the transcription of target genes. Of particular interest in the present invention are BMP signaling activators, which can be readily identified by those skilled in the art by any of a number of methods, such as competitive binding assays for binding to BMP or BMP receptors, functional assays, and measurements of enhancement of the activity of downstream signaling proteins, such as nuclear relocalization of SMADs like BR-Smad and transcriptional activation of downstream gene targets known in the art.

[0071] "Transforming Growth Factor Beta," "TGF-s," and "TGFB" refer to TGFB secreted proteins belonging to the subfamily of the Transforming Growth Factor β (TGF) superfamily. TGFB (TGFB1, TGFB2, TGFB3) are multifunctional peptides that regulate proliferation, differentiation, adhesion, and migration in many cell types. Mature peptides can be found as homodimers or as heterodimers with other TGFB family members. TGFB interacts with the Transforming Growth Factor Beta receptor (TGF-R or TGFBR) on the cell surface, and this binding activates signaling pathways directed to MAP kinase, Akt, Rho, and Rac / cdc42, cell structure rearrangement, nuclear localization of SMAD proteins, and regulation of target gene transcription. Of particular interest in the present invention are TGFB signaling inhibitors, which can be readily identified by those skilled in the art by any of a number of methods, such as competitive binding assays for binding to TGFB or the TGFB receptor, or functional assays, such as measuring the suppression of the activity of downstream signaling proteins, including MAPK, Akt, Rho, Rac, and SMADs, such as AR-Smad and others well known in the art.

[0072] "Wnt" refers to a family of highly conserved secretory signaling molecules that play crucial roles in both embryogenesis and tissue maturation. The human Wnt gene family includes at least 19 members (Wnt-1, Wnt-2, Wnt-2B / Wnt-13, Wnt-3, Wnt3a, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-9A / Wnt-14, Wnt-9B / Wnt-15, Wnt-10A, Wnt-10B, Wnt-11, Wnt-16). Wnt proteins regulate cellular activity by binding to the Wnt receptor complex, which contains polypeptides from the Frizzled (Fz) family of proteins and polypeptides from the low-density lipoprotein receptor (LDLR)-related protein (LRP) family of proteins. When activated by Wnt binding, the Wnt receptor complex activates one or more intracellular signaling cascades. These include the standard Wnt signaling pathways, namely the Wnt / planar cell polarity (Wnt / PCP) pathway and the Wnt-calcium (Wnt / Ca2+) pathway.

[0073] Culturing under "non-adherent conditions" means culturing cells under conditions that suppress cell adhesion to the bottom of the container in which they are cultured, such as a tissue culture plate or flask. In certain cases, cells are inherently non-adherent; that is, cells will not adhere to a surface unless their surface is coated with a matrix composition, such as a cell culture medium such as fibronectin, laminin, polyornithine, polylysine, collagen IV, or Matrigel, or a polycarbonate membrane. In certain cases, cells can be kept non-adherent by agitating the culture. For example, cells cultured in suspension can be cultured under non-adherent conditions.

[0074] Culturing under "adhesion conditions" means culturing cells under conditions that promote adhesion to the bottom of the container in which they are cultured, such as a tissue culture plate or flask. In certain cases, simply keeping the culture at rest may induce cells to adhere to the container. In certain cases, the walls of the container to which adhesion is desired may be coated with a composition to which cells can adhere, such as cell culture media such as fibronectin, laminin, polyornithine, polylysine, collagen IV, and Matrigel, and polycarbonate membranes.

[0075] As used herein, “treatment,” “to treat,” and similar terms generally refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and may be therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” includes any treatment of disease in mammals, and includes (a) preventing the onset of the disease in subjects who are predisposed to the disease but have not yet been diagnosed as diseased, (b) inhibiting the disease, i.e., preventing its development, and (c) alleviating the disease, i.e., causing the regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Treatment of an ongoing disease in which the treatment stabilizes or reduces undesirable clinical symptoms in the patient is of particular interest. Such treatment is desirable to be performed before the complete loss of function of the affected tissue. Therapy is preferably administered at the symptomatic stage of the disease, and in certain cases after the symptomatic stage of the disease.

[0076] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, in particular human, that is to be diagnosed, treated, or treated.

[0077] In the context of cell culture, the term “culture medium,” or “cell culture medium,” or “cell culture medium” refers to a cell growth medium suitable for culturing the cells of this disclosure, such as PS cells, DE cells, AFE cells, VPE cells, TEP cells, etc. Examples of cell culture media include Minimum Essential Medium (MEM), Eagle Medium, Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s Modified Eagle Medium, Nutrient Mixture F-12 (DMEM / F12), F10 Nutrient Mixture, Ham’s F10 Nutrient Mixture, Ham’s F12 Nutrient Mixture, Medium 199, RPMI, RPMI 1640, Reduced Serum Medium, Basic Medium (BME), DMEM / F12 (1:1), and combinations thereof. A medium or cell culture medium may be modified by adding one or more additives. The additives may include serum such as fetal bovine serum and / or serum substitutes such as B27, N2, KSR, and combinations thereof, and differentiation factors such as RA receptor activators, nodal, Act-A, Act-B, Wnt family members, BMP signaling activators, TGF-β signaling inhibitors, FGF, Hedgehog signaling inhibitors, and combinations thereof.

[0078] In the context of cells or cell populations, the term "isolated" refers to cells that are in an environment other than their natural environment, such as being separated from the tissues of an organism.

[0079] As used herein, the term "differentiation factor" refers to a drug contained in the culture medium for culturing the cells of this disclosure, which promotes the differentiation of cells from a first cell type to a second cell type.

[0080] As used herein, “expression” and its grammatical equivalent, in the context of markers, refer to the production of a marker and the level or amount of the marker. For example, marker expression in a cell, the presence of a marker, or a cell being positive for a marker means that the marker is expressed at a level similar to that of a positive control. A positive control level may be determined by the level of marker expressed by cells known to have a cell fate associated with the marker. Similarly, the absence of marker expression, or a cell being negative for a marker, means that the marker is expressed at a level similar to that of a negative control. A negative control level may be determined by the level of marker expressed by cells known not to have a cell fate associated with the marker. Therefore, the absence of a marker does not simply mean that the marker is expressed at an undetectable level; in certain cases, a cell may be expressing the marker, but at a level lower than that of a positive control or similar to that of a negative control.

[0081] As used herein, “marker” refers to any molecule that can be measured or detected. For example, markers may include, but are not limited to, nucleic acids, such as gene transcripts, polypeptide products of genes, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules (e.g., molecules with a molecular weight of less than 10,000 amu).

[0082] A “mutant” polypeptide means a biologically active polypeptide, as defined below, that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the native sequence polypeptide. Such mutants include polypeptides in which one or more amino acid residues are added to, or are present in, the N-terminus or C-terminus of the native sequence, about 1 to 40 amino acid residues are deleted and optionally replaced with one or more amino acid residues, and derivatives of the above polypeptides, where the amino acid residues are covalently modified so that the resulting product has amino acids not found in nature. Typically, biologically active mutants have an amino acid sequence that has at least about 90%, at least about 95%, or at least about 99% amino acid sequence identity with the native sequence polypeptide. Mutant polypeptides can be glycosylated naturally or unnaturally; that is, the polypeptide has a glycosylation pattern different from the glycosylation pattern found in the corresponding naturally occurring protein. Mutant polypeptides may have post-translational modifications not found in the native polypeptide.

[0083] As used herein, “analog” or “functional analog” in the context of molecules such as ligands, peptides, and polypeptides refers to a molecule that has similar functional properties but a different structure compared to the naturally occurring form of the molecule. In particular cases, a functional analog may be, for example, a small molecule exhibiting the function of a polypeptide. Any functional analog of the differentiation factors disclosed herein can be used by means of and can be present in the compositions described herein. Such functional analogs are documented in the literature and can also be identified by screening of compound libraries, such as combinatorial compound libraries and peptide libraries.

[0084] The terms “concentrated” or “concentrated” are used interchangeably herein and mean that the yield (fraction) of a certain type of cell is increased by at least 10% compared to the fraction of that type of cell in the initial culture or preparation.

[0085] As used herein, terms such as “thymic microenvironment” and “endogenous thymic microenvironment” refer to the heterogeneous nature of cell types and cell subpopulations, including extracellular factors, and in some cases, supraccellular structures such as organoids found in endogenous thymic tissue, including fetal thymic tissue, postnatal thymic tissue, and / or adult thymic tissue.

[0086] As used herein, the phrase “substantially absent” and its grammatical equivalent in the context of supplements / factors in cell culture media means that the listed supplement or factor is not present in the cell culture medium in an amount that could affect the differentiation of cells present therein. Therefore, this phrase allows for the presence of undetectable or trace amounts of supplements / factors. For example, a medium substantially absent from RAR activators may contain trace amounts of the activator, for example, less than 0.2 μM, less than 0.1 μM, less than 0.02 μM, less than 0.01 μM, less than 0.002 μM, or less than 0.001 μM.

[0087] In vitro generation of thymic epithelial cells Improved methods and compositions for generating thymic epithelial progenitor (TEP) cells are provided. Also provided are improved methods and compositions for generating thymic epithelial cells (TECs). The methods may include culturing a cell population under conditions sufficient to differentiate the cells into TECs. In some embodiments, the methods are carried out in vitro. Methods and compositions described in Bautista, JL, et al. may be useful in this disclosure (Bautista, JL, et al. Nat Commun 12, 1096 (2021) (doi.org / 10.1038 / s41467-021-21346-6, the contents of which are incorporated herein by reference in their entirety).

[0088] The methods described herein provide, for example, the generation of TECs from a cell population including thymic epithelial progenitor (TEP) cells. Therefore, some embodiments require the differentiation of TEP cells into TECs in vitro. In some embodiments, the cell population differentiated into TECs is derived from pluripotent stem (PS) cells. PS cells may be primate PS cells, including human PS cells. In some embodiments, PS cells are or are derived from embryonic stem cells, embryonic germ cells, or induced pluripotent stem cells.

[0089] A method for culturing a cell population under conditions sufficient to differentiate cells into TECs may include culturing the cell population in a medium containing an activator of bone morphogenetic protein (BMP) signaling, an activator of fibroblast growth factor (FGF) signaling, and an inhibitor of transforming growth factor-β (TGF-β) signaling, as described herein.

[0090] A method for culturing a cell population under conditions sufficient to differentiate cells into TECs may include superculturing the cell population for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 6 days under conditions sufficient to differentiate cells into TECs.

[0091] A method for culturing a cell population may further include culturing in a medium containing one or more of the following: keratinocyte growth factor (KGF), heparin, hydrocortisone, and / or triiodo-L-thyronine (T3) supplement, or analogs thereof. As used herein, the term "KGF" refers to FGF7 and its functionally active fragments. As used herein, the term "heparin" refers to heparin and its analogs. As used herein, the term "hydrocortisone" refers to hydrocortisone and its analogs. As used herein, the term "T3 supplement" refers to T3 and its analogs. Analogs include derivatives and molecules that are structurally similar or structurally dissimilar but functionally similar. In certain examples, analogs may exhibit increased potency and / or increased stability compared to a reference molecule.

[0092] A method for culturing a cell population may include superculturing the cell population for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 13 days under conditions sufficient to differentiate the cells into TECs.

[0093] In some embodiments, culturing a cell population under conditions sufficient to differentiate cells into TECs involves culturing the cell population in a medium containing one or more of the insulin-transferrin-selenium (ITS) supplement and / or B27 supplement.

[0094] The methods herein for generating TECs may include culturing a cell population in a liquid medium. Some embodiments provide methods for culturing a cell population to generate TECs in suspension culture such that conditions sufficient for differentiating TECs in vitro include the use of suspension culture conditions.

[0095] This method can be carried out using minimal essential medium (MEM) or Dulbecco's minimal essential medium (DMEM).

[0096] In some embodiments, a method for culturing a cell population under conditions sufficient to differentiate cells into TECs produces a differentiated population of cells containing a subpopulation of TECs. The subpopulation of TECs may include one or more of the following: cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, and / or myoform cells.

[0097] Some embodiments of the methods described herein involve transferring cells to a cell culture medium or gas-liquid interface culture system, such as Matrigel. Cells can be transferred to a cell culture medium or gas-liquid interface culture system, such as Matrigel, at any stage of differentiation.

[0098] Some embodiments of the methods described herein may further include reassembling cells to form a reassembly.

[0099] A method for generating thymic epithelial cells (TECs) is also provided, comprising culturing thymic epithelial progenitor (TEP) cells in suspension culture, optionally transferring the TEP cells to a gas-liquid interface culture system, and differentiating the TEP cells into TECs. In some embodiments, the method may further include transplanting the TEP cells into a target, where the TEP cells differentiate into TECs in vivo. The method may also include transplanting TEC cells containing or not containing TEP cells into a target, where the TEP cells differentiate into TECs in vivo.

[0100] TEP cells and / or TECs can reassemble to form a reassembly. In some embodiments, the reassembly is transplanted into a subject. In some embodiments, the transplanted reassembly contains TEP cells that differentiate into TECs in vivo. In some embodiments, the transplanted reassembly contains TECs prior to transplantation. In some embodiments, the transplanted reassembly contains a subpopulation of TECs prior to transplantation.

[0101] The methods described herein may further include reducing or removing non-epithelial cells from the culture. In some embodiments, the cultured cells are enriched with EPCAM+ cells. In some embodiments, the cultured cells are enriched with EPCAM+ TEP cells. In some embodiments, the cultured cells are enriched with EPCAM+ TEC cells.

[0102] Some embodiments of the methods described herein include adjusting culture conditions to replicate the thymic microenvironment. Some embodiments of the methods described herein include combining cell types in culture to replicate the thymic microenvironment. The thymic microenvironment may be a complex system of cell types similar to the endogenous thymic microenvironment in fetal, postnatal, or adult thymic tissue as described herein.

[0103] In some embodiments, recreating the thymic microenvironment involves culturing TEP cells under conditions sufficient to support the survival of lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

[0104] In some embodiments, replicating the thymic microenvironment involves culturing TEP cells under conditions sufficient to support the differentiation of TECs and their subpopulations. The TEC subpopulations may include one or more of the following: cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, and / or myoform cells.

[0105] In some embodiments, endoderm (DE) cells are cultured in a medium containing a retinoic acid receptor activator, a bone morphogenetic protein (BMP) signaling activator, a fibroblast growth factor (FGF) signaling activator, and a transforming growth factor-β (TGF-β) signaling inhibitor. The BMP signaling inhibitor can be introduced into the medium before the BMP signaling activator. A Wnt signaling inhibitor can be introduced into the medium.

[0106] In some embodiments, anterior foregut endoderm (AFE) cells are produced by culturing DE cells in a medium containing retinoic acid receptor activators, bone morphogenetic protein (BMP) signaling activators, transforming growth factor β (TGF-β) signaling inhibitors, and Wnt signaling inhibitors.

[0107] In some embodiments, ventral pharyngeal endoderm (VPE) cells are produced by culturing AFE cells in a medium containing retinoic acid receptor activators and bone morphogenetic protein (BMP) signaling activators.

[0108] The initiating cells of the methods described herein are, for example, PS cells including embryonic stem cells, embryonic germ cells, or induced pluripotent stem cells, or may be obtained from PS cells. PS cells may be, for example, primate pluripotent stem cell (pPS) cells. PS cells may be human pluripotent stem (hPS) cells.

[0109] Method for generating thymic epithelial progenitor cells in vitro Figure 8 shows a schematic diagram of an exemplary process for differentiating human stem cells into TEP cells in vitro, for example, by culturing them in suspension. This process may further include differentiation into TECs in vivo. In certain embodiments of the process diagrammed in Figure 8, TEP cells are generated using the MEL1-FOXN1-GFP reporter cell line. In some embodiments, the production of TEP cells from PS cells involves four stages of differentiation:

[0110] Stage 1: Culture of PS cells under conditions suitable for DE cell production

[0111] Stage 2: Culture of DE cells under conditions suitable for producing AFE cells

[0112] Stage 3: Culture of AFE cells under conditions suitable for producing VPE cells

[0113] Stage 4: Culture of VPE cells under conditions suitable for TEP cell production

[0114] At least stages 1–3 are performed under cell suspension conditions. In some embodiments, stages 1–4 are performed under cell suspension conditions. Culturing TEP cells in suspension, or performing at least part of the process of culturing TEP cells in suspension culture, allows for the scale-up of TEP cells and TEC preparations. In some embodiments, the methods provided herein include generating thymic epithelial cells (TECs) by fully culturing thymic epithelial progenitor (TEP) cells in suspension, which includes differentiating the TEP cells in suspension. The method may further include transplanting the TEP cells into a subject, e.g., a human subject, and the TEP cells can be further differentiated into TECs in vivo.

[0115] In some embodiments, after Stage 3, VPE cells may be cultured in a cell culture medium such as Matrigel under conditions suitable for producing TEP. In other embodiments, TEP cells and / or TECs may be reassembled, for example, from suspension culture. The cells can be enriched with EPCAM+ cells, thereby depleting non-epithelial cells. The enriched EPCAM+ cells can be reassembled with other supporting cell types such as fibroblasts, endothelial cells, pericytes, and immune cells. Thus, thymic epithelial cells that replicate the complexity of the endogenous thymic microenvironment are produced in vitro according to the methods provided herein. In some embodiments, TEP cells and / or TECs are cultured under conditions sufficient to establish various subsets of TECs in a cell population, including, for example, the subpopulations listed in Table 1. In some embodiments, the methods for producing TEP cells and / or TECs described herein produce a thymic epithelial cell population having diverse subpopulations of epithelial cells similar to those found in human fetal, postnatal, or adult thymic tissue. For example, the method for generating TEP cells and / or TECs described herein produces a population of thymic epithelial cells having subpopulations representing one or more of the subpopulations of thymic epithelial cells shown in Table 1.

[0116] This method may further include reassembling TEP cells and transplanting the reassembled cells. In some embodiments, transplanting the reassembled TEP cells further includes differentiating the TEP cells into TECs in vivo.

[0117] As described herein, the use of two-dimensional cell culture systems can be avoided by culturing TEP cells whole or partially in cell suspension culture. Culturing TEP cells whole or partially in cell suspension culture offers the additional advantage of facilitating in vitro analysis and characterization of TEP cells and cells at various other stages of development into TEP cells. For example, using cell suspension culture allows for easy removal of cell samples at various points in the protocol, facilitating subsequent analysis using flow cytometry and other fluid dynamics-based cytometry analyses and sorting approaches such as fluorescence-activated cell sorting (FACS).

[0118] In some embodiments, stages 1-4 are achieved within 12 days, EPCAM+ cells are enriched, and the production of TECs reassembled with other cell types occurs within 35 days or less, 34 days or less, 33 days or less, 32 days or less, 31 days or less, 30 days or less, 29 days or less, 28 days or less, 27 days or less, 26 days or less, or 26 days or less.

[0119] Culturing at each stage is carried out under culture conditions for a sufficient time to produce the product of that stage, and the product can be characterized by the expression and / or functional characterization of one or more markers, as will be described in more detail below. The culture media for each of these stages are described below.

[0120] The method described herein is intended to be initiated in Stage 1, Stage 2, Stage 3, or Stage 4.

[0121] Stage 1: Culturing PS cells to produce DE cells As described above, a method for generating thymic epithelial progenitor (TEP) cells from PS cells in vitro is provided.

[0122] In certain embodiments, this method involves the differentiation of PS cells into DE cells. PS cells can be differentiated into DE cells by culturing pluripotent stem cells in a medium containing growth factors which may be one or more of Nodal, activin A, and activin B, or their variants or analogs. In certain cases, the medium for culturing PS cells for differentiation into DE cells may contain a combination of activin A and activin B.

[0123] In certain cases, the culture medium for culturing PS cells to induce differentiation into DE cells may contain one or more of Nodal, activin A, and activin B, along with a BMP signaling activator. In certain cases, the culture medium for inducing differentiation from PS cells to DE cells may contain either or both of activin A and activin B, along with a BMP signaling activator, but the culture medium for inducing differentiation from PS cells to DE cells does not require a BMP signaling activator. Therefore, some embodiments of methods for inducing differentiation from PS cells to DE cells utilize a culture medium that does not contain a BMP signaling activator.

[0124] In certain cases, the culture medium for inducing differentiation from PS cells to DE cells may contain Nodal, activin A, activin B, BMP signaling activators, and one or more members of the Wnt family.

[0125] PS cells can be cultured for 1 to 5 days in a differentiation medium containing Nodal, activin A, activin B, BMP signaling activators, and one or more members of the Wnt family.

[0126] In certain cases, PS cells can be cultured to produce DE cells in a differentiation medium containing activin A. In certain cases, PS cells can be cultured to produce DE cells in a differentiation medium containing activin A and activin B. In certain cases, PS cells can be cultured to produce DE cells in a differentiation medium containing activin A, activin B, and BMP4. Culture can be carried out for 1 to 6 days. In certain cases, DE cells are generated from PS cells as described in U.S. Patent No. 8,216,836, which is incorporated herein by reference in whole.

[0127] In certain cases, DE cells can be obtained from PS cells by culturing PS cells in a medium containing one or more of Nodal, activin A, and activin B for a period of 1 to 6 days or longer. In certain cases, culturing PS cells in a medium containing one or more of Nodal, activin A, and activin B may be carried out for 1, 2, 3, 4, 5, or 6 days, thereby generating PS cells.

[0128] In certain cases, DE cells are obtained from PS cells by culturing PS cells in a medium containing one or more of Nodal, activin A, and activin B in combination with Wnt family members for 1 to 5 days, for example, 1, 2, 3, 4, 5, or 6 days. In certain cases, PS cells can be cultured for 1 or 2 days in a medium containing one or more of Nodal, activin A, and activin B in combination with Wnt family members, and then cultured in a medium containing one or more of Nodal, activin A, and activin B, but substantially free of Wnt family members. In certain cases, PS cells can be cultured for one or two days in a medium containing one or more of Nodal, activin A, and activin B in combination with Wnt family members, then cultured in a medium containing one or more of Nodal, activin A, and activin B but substantially free of Wnt family members, and culture without Wnt family members can be carried out for two days, after which retinoic acid receptor activators may be added to the medium, and the culture can be carried out for a further one or two days in the presence of one or more of Nodal, activin A, activin B, and retinoic acid receptor activators.

[0129] In some cases, DE cells obtained by differentiation of PS cells may express specific DE cell markers. For example, DE cells may express one or more DE cell markers such as SOX17, FOXA2 (also known as HNF3B or HNF3), GSC, M1XL1, and CXCR4. Furthermore, DE cells produced by the methods described herein do not express markers of mesoderm cell fate or ectoderm cell fate. Therefore, DE cells do not express Brachyury, MOX1, SOX1, or ZIC1. Furthermore, DE cells produced by the methods described herein do not express markers of the visceral endoderm. For example, DE cells disclosed herein do not express visceral endoderm markers such as SOX7. In certain cases, DE cells produced by the methods disclosed herein are positive for the expression of one or more DE cell markers such as SOXI7, FOXA2, GSC, M1XL1, and CXCR4, and do not express or express at low levels AFP, SPARC, thrombomodulin, and SOX7.

[0130] In certain cases, DE cells can be obtained by differentiating PS cells by culturing PS cells in a medium supplemented with an insulin-transferrin-selenium (ITS) supplement, such as ITS-G100X (Gibco). In such embodiments, ITS may be supplied to the culture medium at concentrations ranging from about 1:10 (v / v) to about 1:10,000 (v / v), or at concentrations greater than about 1:200 (v / v). In certain embodiments, the concentration of ITS in the culture medium is approximately 1:1000 (v / v), approximately 1:900 (v / v), approximately 1:800 (v / v), approximately 1:700 (v / v), approximately 1:600 ​​(v / v), approximately 1:500 (v / v), approximately 1:400 (v / v), approximately 1:300 (v / v), approximately 1:200 (v / v), approximately 1:100 (v / v), or approximately 1:50 (v / v).

[0131] In certain cases, DE cells can be obtained by differentiating PS cells by culturing PS cells in a medium containing a BMP signaling inhibitor. In certain cases, LDN193189 "LDN" can be used as a BMP signaling inhibitor.

[0132] Some embodiments of a method for producing DE cells by culturing PS cells include a Stage 1 of about 4 days, in which PS cells are cultured on day 1 in RPMI medium supplemented with about 0.2% KSR / FBS and about 100 ng / ml and 50 ng / ml of activin A and Wnt3a, respectively; on days 2-3, PS cells are cultured on RPMI medium supplemented with about 0.2% KSR / FBS and about 100 ng / ml and 1:1000 dilution of activin A and ITS, respectively; and on day 4, PS cells are cultured in RPMI medium supplemented with about 100 ng / ml, 0.25 uM and 250 nM of B27 (0.5X or 1:1000 dilution (v / v)) and LDN (or other inhibitors of BMP signaling) instead of KSR and activin A and retinoic acid (or other activators of the retinoic acid receptor (RAR)).

[0133] Stage 2: Culture of DE cells to produce AFE cells As described above, a method for generating thymic epithelial progenitor (TEP) cells in vitro is provided. In certain embodiments, the method comprises culturing endoderm (DE) cells obtained from pluripotent stem cells in a medium containing a retinoic acid receptor activator, a bone morphogenetic protein (BMP) signaling activator, a fibroblast growth factor signaling activator, a Wnt signaling inhibitor, and a transforming growth factor-β (TGF-β) signaling inhibitor (i.e., TGF-β RI kinase inhibitor IV (TGFbi IV) (Calbiochem)) to produce AFE cells.

[0134] Culturing can be carried out for 1 to 6 days or more. For example, DE cells can be cultured for 2-6 days, 1-5 days, 1-3 days, 2-5 days, 2-4 days, 2-3 days, 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0135] In certain embodiments, the culture medium for culturing DE cells to produce TEP cells may not contain activins such as Nodal, activin-A (ActA), or activin-B (ActB).

[0136] AFE cells produced by the methods described herein may express one or more AFE cell markers. For example, AFE cells produced by the methods described herein may express SOX2, FOXA2, and / or HHEX. Furthermore, AFE cells produced by the methods described herein may not express the posterior foregut endoderm marker CDX2.

[0137] In some embodiments, AFE cells can be obtained by culturing DE cells in a medium containing a retinoic acid receptor activator, a bone morphogenetic protein (BMP) signaling activator, a fibroblast growth factor signaling activator, a transforming growth factor-β (TGF-β) signaling inhibitor (i.e., TGF-βRI kinase inhibitor IV (Calbiochem)), a Wnt signaling inhibitor (i.e., IWP2), and ITS.

[0138] Some embodiments of a method for producing AFE cells by culturing DE cells include a Stage 2 of approximately 2 days, on day 1 of Stage 2, DE cells are cultured in DMEM medium supplemented with 0.5X B27, approximately 50 ng / ml BMP4 or other BMP signaling activators, approximately 0.25 μM retinoic acid or other RAR activators, approximately 50 ng / ml FGF8 or other fibroblast growth factors, approximately 2.5 μM TGFbi IV or other TGFβ signaling inhibitors, approximately 5 μM IWP2 or other Wnt signaling inhibitors, and approximately 1:1000 concentration ITS.

[0139] Stage 3: Culture of AFE cells to produce VPE cells In certain embodiments, the production of TEP cells from DE cells may include an intermediate stage in the production of VPE cells from AFE cells by culturing AFE cells as described above.

[0140] Therefore, VPE cells can be produced by culturing AFE cells in a medium containing RA receptor activators, BMP signaling activators, and TGF-β signaling inhibitors, as described above.

[0141] Alternatively, VPE cells can be produced by culturing AFE cells in a medium containing activators of BMP signaling, activators of fibroblast growth factor signaling, and inhibitors of TGF-β signaling. In some embodiments, the medium substantially does not contain one or more of the activators of Wnt signaling or Wnt3a, inhibitors of Hedgehog signaling (e.g., cyclopamine), and activators of retinoic acid signaling.

[0142] AFE cells are cultured in the above medium for approximately 1 to 8 days (for example, 1 to 7 days, 1 to 5 days, 1 to 3 days, 2 to 7 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days) to produce VPE cells.

[0143] VPE cells produced by the methods described herein may express one or more VPE cell markers, such as HOXA3, PAX1, or EYA1.

[0144] Several embodiments of a method for producing VPE cells by culturing AFE cells include a Stage 3 lasting up to 4 days, in which the AFE cells are cultured in DMEM medium supplemented with 0.5X B27, approximately 50 ng / ml of BMP4 or other BMP signaling activators, approximately 50 ng / ml of FGF8 or other fibroblast growth factors, approximately 2.5 μM of TGFbi IV or other TGFβ signaling inhibitors, and approximately 1:200 concentration of ITS. In some embodiments, the medium is not supplemented with Wnt signaling activators or Wnt3a, cyclopamine, or retinoic acid signaling activators. Stage 3 progresses for approximately 1 to 4 days.

[0145] Stage 4: Culture of VPE cells to produce TEP cells A method for producing TEP cells from DE cells produced from PS cells may further include culturing VPE cells produced by culturing AFE cells, and the VPE cell culture is carried out in a medium containing a BMP signaling activator.

[0146] In certain cases, the culture medium for generating thymic epithelial progenitor (TEP) cells from VPE cells produced by culturing AFE cells may contain activators of BMP signaling, fibroblast growth factor (i.e., activators of fibroblast growth factor signaling), and inhibitors of TGFβ signaling.

[0147] In certain cases, the culture medium for generating thymic epithelial progenitor (TEP) cells from VPE cells produced by culturing AFE cells may contain RA receptor activators, BMP signaling activators, Wnt family members, fibroblast growth factors, and Hedgehog signaling inhibitors.

[0148] In certain cases, VPE cells may be cultured in culture medium for approximately 1 to 10 days, during which time they differentiate into TEP cells. In certain cases, VPE cells may be cultured in culture medium for 1 to 10 days (e.g., 1 to 7 days, 1 to 5 days, 1 to 3 days, 2 to 7 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days) during which time they produce TEP cells.

[0149] Some embodiments of a method for producing TEP cells by culturing VPE cells include a Stage 4 lasting up to 4 days, in which VPE cells are cultured in DMEM medium supplemented with 0.5× B27, approximately 50 ng / ml of BMP4 or other activators of BMP signaling, approximately 50 ng / ml of FGF8 or other activators of fibroblast growth factor signaling, approximately 2.5 μM of TGFbi IV or other TGFβ signaling inhibitors, and approximately 1:200 concentration of ITS. Stage 4 progresses for approximately 1 to 4 days.

[0150] TEP cells produced by the methods described herein express TEP cell markers, which are present in TEP cells present in the thymus or thymic tissue, such as the adult human thymus or fetal human thymus. For example, TEP cells produced by the methods described herein may express TEP markers at levels similar to those expressed by cells in the adult or fetal thymus. In certain cases, TEP cells produced by the methods described herein express one or more of FOXN1, HOXA3, EYA1, and EpCAM. In certain cases, TEP cells produced by the methods provided herein express FOXN1 and HOXA3. In certain cases, TEP cells produced by the methods provided herein express FOXN1, HOXA3, PAX1, EpCAM, and EYA1.

[0151] Therefore, a method is provided for generating TEP cells from VPE cells by culturing VPE cells for approximately 1 to 10 days in a medium containing one or more RA receptor activators, BMP signaling activators, Wnt family members, fibroblast growth factors, and Hedgehog signaling inhibitors.

[0152] In certain embodiments, TEP cells may be generated within approximately 15 days (e.g., 10-15 days, 10-14 days, 10-13 days, 10-12 days, 10-11 days, e.g., 15 days, 14 days, 13 days, 12 days, 11 days, 10 days) from the start of culture of PS cells (e.g., pPS such as primate iPS cells, primate ES cells, human PS, human iPS cells, human ES cells). In certain embodiments, this method includes culturing PS cells for approximately 1-5 days, e.g., 4-5 days, according to the method described herein, to produce DE cells. In certain embodiments, this method further includes culturing DE cells (produced from PS cells) for approximately 1-3 days, e.g., 2-3 days (or up to 4-7 days from the start of culture of PS cells, e.g., up to 5-7 days), according to the method described herein, to generate AFE cells. In certain embodiments, the method further comprises culturing AFE cells (produced from DE cells) according to the method herein for about 1 to 3 days, for example 2 to 3 days (or up to 6 to 10 days from the start of PS cell culture, for example 7 to 9 days) to produce VPE cells. In certain embodiments, the method further comprises culturing VPE cells (produced from AFE cells) according to the method herein for about 1 to 3 days, for example 2 to 3 days (or up to 10 to 15 days from the start of PS cell culture, for example 10 to 12 days or 10 to 11 days) to produce TEP cells.

[0153] The culture methods described herein can be carried out under adherent or non-adherent conditions (e.g., suspension culture). In some embodiments, the cell populations disclosed herein are cultured as adherent culture. In some embodiments, the cell populations disclosed herein are cultured as suspension culture.

[0154] PS cells can originate from any source. In certain cases, PS cells may be embryonic stem cells, embryonic germ cells, and induced pluripotent stem cells. In certain cases, PS cells may be primate pluripotent stem (pPS) cells. In certain cases, pPS cells may be human pluripotent stem (hPS) cells. In certain cases, hPS cells may be human embryonic stem (hES) cells. hPS cells may be induced pluripotent stem (iPS) cells. In certain cases, PS cells may be established stem cell lines. In certain cases, PS cells may be established embryonic stem cell lines. In certain cases, PS cells may be established embryonic stem cell lines, which are derived from blastomeres produced by single blastomeres biopsy (SBB), which involves the removal of a single blastomere from an 8-cell stage organism in development. In certain embodiments, PS cells may be established stem cell lines that do not contain PS cells or ES cells, produced by isolating a human embryo or human blastocyst.

[0155] As described above, cell culture media may contain additives or supplements. In certain cases, cell culture media may not contain serum. In certain cases, cell culture media may not contain serum but may contain serum substitutes such as KSR or B27. The type of cell culture medium and the additives for cell culture media may differ for specific differentiation stages of the cell population.

[0156] In certain embodiments, the culture medium used in the culture method described herein may have reduced or no serum. The serum concentration may range from about 0.05% (v / v) to about 20% (v / v). For example, in certain embodiments, the serum concentration of the medium may be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), and less than about 0.8% (v / v). The percentage may be less than approximately 0.9% (v / v), less than approximately 1% (v / v), less than approximately 2% (v / v), less than approximately 3% (v / v), less than approximately 4% (v / v), less than approximately 5% (v / v), less than approximately 6% (v / v), less than approximately 7% (v / v), less than approximately 8% (v / v), less than approximately 9% (v / v), less than approximately 10% (v / v), less than approximately 15% (v / v), or less than approximately 20% (v / v). In some embodiments, cells are grown in serum-free conditions. In other embodiments, the culture medium used in the culture method described herein may not contain serum, or may contain a serum substitute.

[0157] In further embodiments, the culture medium used in the culture method described herein may include FBS or a knockout serum substitute (KSR). In such embodiments, the KSR or FBS may be provided in the culture medium at a concentration ranging from about 0.1% (v / v) to about 20% (v / v), or at a concentration greater than about 20% (v / v). In a particular mechanism, the concentration of FBS or KSR in the culture medium is approximately 0.1%(v / v), approximately 0.2%(v / v), approximately 0.3%(v / v), approximately 0.4%(v / v), approximately 0.5%(v / v), approximately 0.6%(v / v), approximately 0.7%(v / v), approximately 0.8%(v / v), approximately 0.9%(v / v), approximately 1%(v / v), approximately 2%(v / v), approximately 3%(v / v), approximately 4%(v / v), approximately 5%(v / v), approximately 6%(v / v), approximately 7%(v / v), approximately 8%(v / v), approximately 9%(v / v), approximately 10%(v / v), approximately 15%(v / v), or approximately 20%(v / v).

[0158] In yet another embodiment, the culture medium used in the culture method described herein may contain an insulin-transferrin-selenium (ITS) supplement, for example, ITS-G (100X, Gibco). In such embodiments, ITS may be provided to the culture medium at concentrations ranging from about 1:10 (v / v) to about 1:10,000 (v / v), or at concentrations greater than about 1:200 (v / v). In certain embodiments, the concentration of ITS in the culture medium is approximately 1:1000 (v / v), approximately 1:900 (v / v), approximately 1:800 (v / v), approximately 1:700 (v / v), approximately 1:600 ​​(v / v), approximately 1:500 (v / v), approximately 1:400 (v / v), approximately 1:300 (v / v), approximately 1:200 (v / v), approximately 1:100 (v / v), or approximately 1:50 (v / v).

[0159] In certain cases, RPMI1640 medium may be used for stages 1 and 2, and DMEM / F12 for stages 3 and 4. In certain cases, RPMI1640 medium may be used with increased KSR concentration (0% on day 1 of culture, 0.2% on days 2-3, and 2% on day 4) or supplemented with B27 (0.5× or 1:1000 dilution (v / v) on days 5-7 of culture). In certain cases, DMEM / F12 containing B27 (0.5× or 1:1000 dilution (v / v)) may be used for stages 3 and 4 of culture.

[0160] In some embodiments, the methods described herein produce thymic epithelial cells and cell populations that replicate the complexity of the endogenous thymic microenvironment. In some embodiments, the TEP cells are cultured under conditions sufficient to establish various subsets of TECs in the cell population, including, for example, the subpopulations listed in Table 1. In some embodiments, the methods for generating TEP cells described herein produce thymic epithelial cell populations having diverse subpopulations of epithelial cells similar to those found in human fetal or adult thymic tissue. For example, the methods for generating TEP cells described herein produce thymic epithelial cell populations having subpopulations representing one or more of the subpopulations of thymic epithelial cells shown in Table 1.

[0161] Figure 8 shows a schematic diagram of an exemplary process for differentiating human stem cells into thymic epithelial cells (TECs). In specific embodiments of the process diagrammed in Figure 8, the culture medium and differentiation factors are applied according to the descriptions provided in Table 2. In some embodiments, the production of TECs from PS cells involves culturing TEP cells under conditions suitable for producing TEP cells. TEP cells can be obtained according to the above method, for example, including stages 1-4. In some embodiments, the culture of TEP cells to obtain TECs includes a fifth differentiation stage: Stage 5: Culture of TEP cells under conditions suitable for producing mature TEP (mTEP) cells. mTEPs may produce TECs during in vivo transplantation.

[0162] Stage 5: Culture of TEP cells to produce mTEP cell populations A method for producing TEP cells from DE cells produced from PS cells includes further culturing the TEP cells to produce more mature TEP cells prior to in vivo differentiation into TECs, the further culturing of TEP cells being carried out in a medium containing ITS, T3, KGF, heparin, and / or hydrocortisone. Thus, Stage 5 of the method for producing TEP cells provided herein results in a TEP cell population exhibiting further maturation. For example, during or after Stage 5, the proportion of cells exhibiting the molecular and / or morphological characteristics of TEP cells increases, or the expression level of one or more TEP cell markers described herein increases. Similarly, during or after Stage 5, the proportion of cells exhibiting the molecular and / or morphological characteristics of non-TEP cells decreases, or the expression level of molecular markers of non-TEP cells decreases. In some embodiments, the mTEP cell population contains a higher proportion of cells exhibiting the molecular and / or morphological characteristics of TEP cells compared to the proportion of cells exhibiting the molecular and / or morphological characteristics of TEP cells at the end of Stage 4. For example, during or after Stage 5, the percentage of cells exhibiting the molecular and / or morphological characteristics of TEP cells is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, compared to the percentage of cells exhibiting the molecular and / or morphological characteristics of TEP cells at the end of Stage 4. In some embodiments, the molecular characteristics of the mTEP cell population produced during or after Stage 5 are an increase in the percentage of cells expressing FOXN1. In some embodiments, the molecular characteristics of the mTEP cell population produced during or after Stage 5 are an increase in the level of FOXN1 expression compared to the level of FOXN1 expression in TEP cells produced at the end of Stage 4. For example, FOXN1 expression may increase by approximately 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, or more compared to the level of FOXN1 expression in TEP cells during or after Stage 4.In some embodiments, during or after Stage 5, TEP cells express higher levels of KRT5 compared to the levels of KRT5 expression in TEP cells during or after Stage 4. That is, in some embodiments, KRT5 expression may increase by about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, or more, compared to the levels of FOXN1 expression in TEP cells during or after Stage 4. In some embodiments, during or after Stage 5, TEP cells express higher levels of KRT15 compared to the levels of KRT15 expression in TEP cells after Stage 4. That is, in some embodiments, KRT15 expression may increase by about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, or more, compared to the levels of KRT15 expression in TEP cells during or after Stage 4. In some embodiments, during or after Stage 5, TEP cells express lower levels of DLL1. In other words, in some embodiments, DLL1 expression may decrease by approximately 1 / 1.5×, 1 / 2×, 1 / 2.5×, and 1 / 3× compared to the level of DLL1 expression in TEP cells during or after Stage 4. Thus, Stage 5 results in a cell population with a higher proportion of TEP cells, TEP cells exhibiting increased molecular or morphological characteristics of TEP cells, and a decreased proportion of non-TEP cells and cells exhibiting non-TEP molecular characteristics. While not bound by theory, Stage 5 is thought to involve further maturation of TEP cells, resulting in higher expression levels of TEP cell markers and an increase in the number of cells expressing TEP cell markers.

[0163] In some embodiments, the culture medium used for further culturing of TEP cells may contain an insulin-transferrin-selenium (ITS) supplement, such as ITS-G (100X, Gibco). In such embodiments, ITS may be supplied to the culture medium at concentrations ranging from about 1:10 (v / v) to about 1:10,000 (v / v), or at concentrations greater than about 1:200 (v / v). In certain embodiments, the concentration of ITS in the culture medium is approximately 1:1000 (v / v), approximately 1:900 (v / v), approximately 1:800 (v / v), approximately 1:700 (v / v), approximately 1:600 ​​(v / v), approximately 1:500 (v / v), approximately 1:400 (v / v), approximately 1:300 (v / v), approximately 1:200 (v / v), approximately 1:100 (v / v), or approximately 1:50 (v / v).

[0164] In some embodiments, the culture medium used for further culturing of TEP cells may contain a triiodo-L-thyronine (T3) supplement. In such embodiments, T3 may be provided in the culture medium at concentrations ranging from about 20 nM to about 2,000 nM or at a concentration of about 200 nM. In specific embodiments, the concentration of T3 in the medium is about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 500 nM, or about 1,000 nM.

[0165] In some embodiments, the culture medium used for further culturing of TEP cells may contain keratinocyte growth factor (KGF) / FGF7, or, for example, FGF10, heparin, and other fibroblast growth factor activators, including cortisol, such as hydrocortisone. KGF can be provided in the culture medium at concentrations ranging from 5 ng / ml to 500 ng / ml. In certain embodiments, the concentration of KGF in the culture medium is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml.

[0166] Heparin can be provided in the culture medium at concentrations ranging from approximately 1 μg / ml to approximately 100 μg / ml. In certain embodiments, the concentration of heparin in the medium is approximately 1 ug / ml, approximately 2 ug / ml, approximately 4 ug / ml, approximately 6 ug / ml, approximately 8 ug / ml, approximately 10 ug / ml, approximately 12 ug / ml, approximately 14 ug / ml, approximately 16 ug / ml, approximately 18 ug / ml, or approximately 20 ug / ml.

[0167] Cortisol, such as hydrocortisone, can be supplied to the culture medium at concentrations ranging from about 0.05 ug / ml to about 5 ug / ml. In certain embodiments, for example, the concentration of hydrocortisone in the culture medium is about 0.1 ug / ml, 0.2 ug / ml, 0.3 ug / ml, 0.4 ug / ml, 0.5 ug / ml, 0.6 ug / ml, 0.7 ug / ml, 0.8 ug / ml, 0.9 ug / ml, or about 1.0 ug / ml.

[0168] In certain cases, TEP cells are cultured in culture medium for approximately 1 to 21 days, during which time they further differentiate into TECs in vitro. In certain cases, TEP cells may be cultured in culture medium for 1 to 21 days, for example, approximately 1 to 3 days, 1 to 5 days, 1 to 7 days, 1 to 9 days, 1 to 11 days, 1 to 13 days, 1 to 15 days, 1 to 17 days, 1 to 19 days, or 1 to 21 days. In certain cases, TEP cells are cultured in culture medium for more than 21 days.

[0169] In some embodiments, TEP cells, including mTEP cells produced by the method described herein, are introduced into a subject where they undergo further differentiation into TECs. TEP cells and / or TECs and their subpopulations can be transplanted into a subject requiring TE cells. In certain cases, TEP cells and / or TECs and their subpopulations can be transplanted into a target site of the subject that provides suitable differentiation conditions for the TEP cells and / or TECs and their subpopulations to differentiate into TE cells. Cells can be transplanted by any of the many standard methods in the Art for delivering cells to tissue, e.g., by injecting the cells as a turbidity in a suitable buffer (such as saline, PBS, DMEM, Iskov medium, or a pharmaceutically acceptable carrier), or by providing the cells on a solid support, e.g., a filter such as beads, a mesh filter, or a membrane. In certain cases, TEP cells and / or TECs and their subpopulations can be transplanted into the thymus of a subject. In certain cases, TEP cells and / or TECs can be transplanted under the kidney capsule of a subject.

[0170] TEP and / or TEC produced by the methods described herein express TEC markers, which are present in TECs in the thymus or thymic tissue, such as the adult human thymus or fetal human thymus. For example, TECs produced by the methods described herein may express TEC markers at levels similar to those expressed by cells of the adult or fetal thymus. In specific cases, TEC cells produced by the methods described herein express one or more of the following: CLDN3 and / or CLDN4 (collectively "CLDN3 / 4"), MHC class II genes ("MHCII"), K5 (KRT5), K15 (KRT15), ASCL1, AIRE, IVL, K10 (KRT10), GNB3, K8 (KRT8), CHGA, SOX2, and / or MYOG.

[0171] In some embodiments, the TECs produced by the methods described herein include a cell population comprising, for example, a genetically distinct subpopulation of thymic epithelial cells, including corticothymic epithelial cell (cTEC) lineage cells, pluripotent TEP cells, undifferentiated medullary thymic epithelial cell (mTEC) progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, and / or myoid cells.

[0172] Table 1 shows subsets of thymic epithelial cells and corresponding cell identification markers. In some embodiments, the methods described herein produce populations of TECs comprising genetically distinct subpopulations of thymic epithelial cells, where cTEC lineage cells express one or more of beta5t(PSMB11), CD205(LY75), CCL25, and K8; pluripotent TEP cells express one or more of K5 and K8; undifferentiated mTEC progenitor cells express one or more of CLDN3 / 4 (low levels), MHC class II genes (undetectable to low levels), K5, K15, and ASCL1; immature mTECs express CLDN3 / 4, low levels of MHC class II genes, one or more of K5, K15, and ASCL1; and mature mTECs The cells express CLDN3 / 4, high levels of MHC class II genes, AIRE, and K5; post-AIRE mTECs express CLDN3 / 4, low levels of MHC class II genes, IVL, and K10; tuft cells express CLDN3 / 4, low levels of MHC class II genes, GNB3, and K8; neuroendocrine cells express CLDN3 / 4, undetectable to low levels of MHC class II genes, CHGA, SOX2, and K8; and myosoidal cells express low levels of CLDN3 / 4, undetectable to low levels of MHC class II genes, MYOG, and K8. [Table 1]

[0173] Some embodiments of a method for producing TEC by culturing TEP cells include a multi-day Stage 5, in which TEP cells are cultured in DMEM culture medium supplemented with 0.5X B27 and 50 ng / ml KGF, 1:200 ITS, 10 ug / ml heparin, 0.5 ng / ml hydrocortisone, and 200 nM T3.

[0174] Table 2 provides an example of a cell culture protocol for one embodiment of the method for generating thymic epithelial cells in vitro as described herein. [Table 2]

[0175] differentiation factor The methods and compositions disclosed herein involve the use of various differentiation factors. Examples of differentiation factors used in the methods and compositions disclosed herein are listed below.

[0176] Activators of RA receptors RA receptor activators (RARs) can be molecules capable of activating one or more of the following: RAR, RAR-alpha, RAR-beta, and RAR-gamma. In certain cases, the activator may also be a ligand for the RA receptor. Examples of RA receptor ligands include retinoids such as retinol, retinal, retinoic acid, all-trans retinoic acid, 9-cis-retinoic acid, etretinate, tazarotene, bexarotene, adapalene, TTNPB, DTAB (3-[(4,6-diphenoxy-1,3,5-triazine-2-yl)amino]benzoic acid), or their derivatives or analogues.

[0177] In some embodiments of the methods and compositions described herein, the RA receptor activator is provided to cells in a culture medium at a concentration of at least about 0.01 μM, at least about 0.03 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 1 μM, at least about 1.3 μM, at least about 1.5 μM, at least about 2 μM, at least about 2.3 μM, at least about 2.5 μM, at least about 2.8 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, or at least about 50 μM.

[0178] In certain cases, RA receptor activators may be present at different concentrations at different stages of the TEP cell production process. In certain cases, RA receptor activators may be present at higher concentrations in the culture medium during the production of DE cells (Stage I) and / or AFE cells (Stage 2) than at the concentrations used to produce VPE cells (Stage 3) and / or TEP cells (Stage 4).

[0179] In certain cases, RA receptor activators may be present in the culture medium used to generate DE cells and the culture medium used to generate AFE cells at concentrations of at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 1 μM, at least about 1.3 μM, at least about 1.5 μM, at least about 2 μM, at least about 2.3 μM, at least about 2.5 μM, at least about 2.8 μM, or at least about 3 μM.

[0180] In certain cases, the activator of the RA receptor may be a ligand for the RA receptor. In certain cases, the ligand for the RA receptor may be all-trans retinoic acid (RA). In certain cases, all-trans retinoic acid may be present at a concentration of 0.25 μM in the cell culture medium used to generate DE cells and the cell culture medium used to generate AFE cells.

[0181] In certain cases, ligands for the RA receptor may be present in the culture medium used to generate VPE cells and / or TEP cells at concentrations of at least about 0.01 μM, at least about 0.03 μM, at least about 0.1 μM, or about 0.15 μM. In certain cases, the ligand for the RA receptor may be all-trans retinoic acid (RA). In certain cases, all-trans retinoic acid may be present in the cell culture medium used to generate VPE cells and the cell culture medium used to generate TEP cells at a concentration of 0.1 μM.

[0182] Fibroblast growth factor In certain embodiments of the methods and compositions described herein, one or more differentiation factors of the fibroblast growth factor family, commonly referred herein as “fibroblast growth factor” or “FGF,” may be present in the culture medium used for cell culture. Accordingly, the term “fibroblast growth factor activator” refers to an FGF family protein or a factor that promotes signaling in the FGF pathway. For example, in some embodiments, fibroblast growth factor may be present in the culture medium used to culture cells at concentrations of at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, FGF is present in the cell culture medium at concentrations of 10 ng / ml to 100 ng / ml, such as 20 ng / ml to 100 ng / ml or 30 ng / ml to 100 ng / ml.

[0183] In certain embodiments, FGF may be FGF2, FGF4, FGF7 (also known as KGF or "keratinocyte growth factor"), FGF8a, FGF8b, FGF9, FGF10, or variants thereof.

[0184] In certain embodiments, FGF may be present in the culture medium used for the production of VPE cells and / or TEP cells. In certain embodiments, FGF may be present in the culture medium used for the production of VPE cells, and / or TEP cells may be FGF8 or FGF8b. In certain embodiments, FGF may be present in the culture medium used for the production of VPE cells, and / or TEP cells may be FGF8b present at a concentration of 50 ng / ml.

[0185] In some embodiments of the methods and compositions described herein, the fibroblast growth factor may be FGF7 / KGF, which may be present in the culture medium for cells at concentrations of at least about 10 ng / ml, at least about 20 ng / ml, at least about 30 ng / ml, at least about 40 ng / ml, at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml, or at least about 100 ng / ml. In some embodiments, FGF7 / KGF is present in the cell culture medium at concentrations of 5 ng / ml to 100 ng / ml, for example, 10 ng / ml to 75 ng / ml, or 25 ng / ml to 75 ng / ml.

[0186] Nodal, Activin A, Activin B In some embodiments, one or more differentiation factors, such as Nodal, and / or activin A, and / or activin B, or their variants, or functional analogs, may be present in the cell culture medium at concentrations of at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml, for example, about 10-500 ng / ml, 25 ng / ml-250 ng / ml, and 50 ng / ml-200 ng / ml.

[0187] In some embodiments, one or more differentiation factors, such as Nodal and / or activin A and / or activin B, or their variants or functional analogs, may be present in the culture medium for generating DE cells from PS cells (Stage 1). In certain cases, the culture medium for generating DE cells from PS cells (Stage 1) may contain Act-A at a concentration of 100 ng / ml.

[0188] Functional analogues of Activin-A include IDE1 (2-[6-carboxy-hexanol)-hydrazonomethyl]-benzoic acid) and IDE2 (7-(2-cyclopentylidenehydradyl-7-oxoheptanoic acid), described in Borowial M. et al. Cell Stem Cell 4, 348-358, April; 3, 2009.

[0189] Wnt Family Member In certain embodiments of the methods and compositions described herein, one or more differentiation factors of the Wnt family may be present in the culture medium used for cell culture. For example, in some embodiments, Wnt family members may be present in the culture medium used for cell culture at concentrations of at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, Wnt family members are present in the cell culture medium at concentrations of 5 ng / ml to 100 ng / ml, such as 10 ng / ml to 75 ng / ml or 15 ng / ml to 50 ng / ml.

[0190] In certain cases, Wnt family members may be present at different concentrations at different stages of the process of producing TEP cells. In certain cases, Wnt family members may be present during DE cell generation at lower concentrations than those in the culture medium used to produce TEP cells. In certain cases, Wnt family members may be Wnt3a present at a concentration of 25 ng / ml in the cell culture medium used for PS cell differentiation. In certain cases, Wnt family members may be Wnt3a present at a concentration of 50 ng / ml in the cell culture medium used for AFE cell differentiation and VPE cell differentiation, enabling TEP cell production.

[0191] In certain cases, members of the Wnt family may be inducers of standard Wnt signaling. In certain embodiments, Wnt family members may be Wnt3a or its variants that mediate standard Wnt signaling. In certain cases, Wnt family members may be Wnt / betacatenin pathway agonists, such as glycogen synthase kinase 3 beta (GSK3b) inhibitors or casein kinase 1 (CK1) inhibitors. Non-limiting examples of Wnt agonists include β-catenin-encoding DNA (e.g., naked DNA encoding β-catenin, plasmid expression vectors encoding β-catenin, viral expression vectors encoding β-catenin), β-catenin polypeptides, one or more Wnt / β-catenin pathway agonists (e.g., Wnt ligands, DSH / DVL-1, -2, -3, LRP6N, WNT3A, WNT5A, and WNT3A, 5A), and one or more glycogen synthase kinase 3β (GSK3β) inhibitors (e.g., lithium chloride (LiCl), pluvaranol A, oromoucin, alsterpaulon, kaempaulon, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadione Azole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxoasiadiazolidine-3-thion (OTDZT), (2'Z,3'E)-6-bromoindilbine-3'-oxime (BIO), α-4-dibromoacetophenone (i.e., tau protein kinase I (TPKI) inhibitor), 2-chloro-1-(4,5-dibromothiophen-2-yl)-ethanone, N-(4-methoxybenzyl )-N'-(5-nitro-1,3-thiazole-2-yl)urea (AR-A014418), indirubin-5-sulfonamide; indirubin-5-sulfonic acid (2-hydroxyethyl)-amideindirubin-3'-monoxime; 5-iodoindirubin-3'-monoxime; 5-fluoroindirubin; 5,5'-dibromoindirubin; 5-nitroindirubin; 5-chloroindirubin;5-methylindilbine, 5-bromoindilbine, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thion (OTDZT), (2'Z,3'E)-6-bromoindilbine-3'-oxime (BIO), α-4-dibromoacetophenone (i.e., tau protein kinase I (TPKI) inhibitor), 2-chloro-1-(4 This includes 5-dibromo-thiophene-2-yl)-etanone, (vi)N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazole-2-yl)urea (AR-A014418), H-KEAPPAPPQSpP-NH2 (L803) and Myr-N-GKEAPPAPPQSpPNH2 (L803-mts), one or more antisense RNAs or siRNAs that specifically bind to u8K3βmRNA, and one or more casein kinase 1 (CK1) inhibitors (e.g., antisense RNAs or siRNAs that specifically bind to CK1mPvNA).

[0192] In certain embodiments of the methods described herein, Wnt signaling inhibitors are used. Wnt signaling inhibitors include factors that inhibit the Wnt signaling pathway, such as Dickkopf (DKK) family proteins, Wnt inhibitor-1 (WIF-1), and secreted Frizzled-associated protein (sFRP). In some embodiments, small molecule inhibitors of Wnt signaling, such as IWP2, are used.

[0193] Activators of BMP signaling In certain embodiments of the methods and compositions described herein, one or more differentiation factors, such as BMP signaling activators, may be present in the culture medium used for cell culture. For example, in some embodiments, BMP signaling activators may be present in the culture medium used for cell culture at concentrations of at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In some embodiments, the BMP signaling activator is present in the cell culture medium at concentrations of 5 ng / ml to 100 ng / ml, such as 10 ng / ml to 75 ng / ml or 25 ng / ml to 75 ng / ml.

[0194] In certain embodiments, the BMP signaling activator may be BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9 / GDF, BMP10, BMP11 / GDF11, BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15 / GDF9B, and their variants. In certain embodiments, the BMP signaling activator may be BMP4 or its variants or functional analogs.

[0195] BMP signaling inhibitors In certain embodiments of the methods and compositions described herein, BMP signaling inhibitors may be present in the culture medium for cell culture. BMP signaling inhibitors may be present at concentrations of at least approximately 25 μM, at least approximately 50 μM, at least approximately 75 μM, at least approximately 100 μM, at least approximately 125 μM, at least approximately 150 μM, at least approximately 150 μM, at least approximately 175 μM, at least approximately 200 μM, at least approximately 225 μM, at least approximately 250 μM, at least approximately 275 μM, at least approximately 300 μM, at least approximately 325 μM, at least approximately 350 μM, at least approximately 375 μM, at least approximately 400 μM, at least approximately 425 μM, at least approximately 450 μM, at least approximately 475 μM, or at least approximately 500 μM, for example, 100 μM to 150 μM, 150 μM to 200 μM, 200 μM to 250 μM, 250 μM to 300 μM, or 300 μM to 350 μM.

[0196] In certain embodiments, the BMP signaling inhibitor is an antibody or fragment thereof that binds to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9 / GDF2, BMP10, BMP11 / GDF11, BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15, Smad factor, Smad1, Smad5, Smad8, Smad4, Smad6, Smad7, BMP receptor, BMPR-1A(ALK3), BMPR-1B(ALK6), ActR-1A(ALK2, ACVR1), BMPR-2, ActR-2A(ACVR2A), ActR-2B(ACVR2B). In some embodiments, noggin is used as a BMP signaling inhibitor. In certain embodiments, the BMP signaling inhibitor may be a small molecule inhibitor. In certain cases, the BMP signaling inhibitor may be LDN193189 ("LDN"). Generally, the BMP signaling inhibitors used in the methods and compositions disclosed herein do not inhibit Nodal, activin, and / or retinoic acid receptor signaling.

[0197] In certain embodiments, LDN may be present in the culture medium for cell culture at a concentration of about 250 nM. In some embodiments, LDN may be present at concentrations of at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, at least about 200 nM, at least about 225 nM, at least about 250 nM, at least about 275 nM, at least about 300 nM, at least about 325 nM, at least about 350 nM, at least about 375 nM, at least about 400 nM, at least about 425 nM, at least about 450 nM, at least about 475 nM, or at least about 500 nM, for example, 100 nM to 150 nM, 150 nM to 200 nM, 200 nM to 250 nM, 250 nM to 300 nM, or 300 nM to 350 nM.

[0198] TGF-β signaling inhibitors In certain embodiments of the methods and compositions described herein, TGF-β signaling inhibitors may be present in the culture medium for cell culture. Inhibitors of TGF-β signaling may be present at concentrations of at least approximately 0.01 μM, at least approximately 0.03 μM, at least approximately 0.1 μM, at least approximately 0.2 μM, at least approximately 0.25 μM, at least approximately 0.28 μM, at least approximately 0.3 μM, at least approximately 1 μM, at least approximately 1.3 μM, at least approximately 1.5 μM, at least approximately 2 μM, at least approximately 2.3 μM, at least approximately 2.5 μM, at least approximately 2.8 μM, at least approximately 3 μM, at least approximately 3.5 μM, at least approximately 4 μM, at least approximately 4.5 μM, at least approximately 5 μM, at least approximately 10 μM, at least approximately 20 μM, at least approximately 30 μM, at least approximately 40 μM, or at least approximately 50 μM, for example, 0.5 μM to 50 μM, 1 μM to 25 μM, or 1 μM to 10 μM.

[0199] In certain embodiments, the TGF-β signaling inhibitor may be an antibody or fragment thereof that binds to TGF-β1, TGF-2, TGF-3, TGF-β receptor I and / or II. In certain embodiments, the TGF-β signaling inhibitor may be a small molecule inhibitor. In certain cases, the TGF-β signaling inhibitor may be LY364947 (SD208), SM16, SB-505124, ALK5 inhibitor II, or SB-431542. In some embodiments, the TGF-β signaling inhibitor is TGF-β RI kinase inhibitor IV (Calbiochem). Generally, the TGF-β signaling inhibitors used in the methods and compositions disclosed herein do not inhibit Nodal, Activin, and / or BMP signaling.

[0200] In certain embodiments, TGFbi IV may be present in the culture medium for cell culture at a concentration of approximately 2.5 μM. In some embodiments, TGFbi IV is present in the culture medium for cells at concentrations of at least about 0.01 μM, at least about 0.03 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 1 μM, at least about 1.3 μM, at least about 1.5 μM, at least about 2 μM, at least about 2.3 μM, at least about 2.5 μM, at least about 2.8 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, or at least about 50 μM, for example, 0.5 μM to 50 μM, 1 μM to 25 μM, or 1 μM to 10 μM.

[0201] Hedgehog signaling inhibitors In certain embodiments of the methods and compositions described herein, inhibitors of hedgehog signaling may be present in the culture medium for cell culture. Hedgehog signaling inhibitors may be present at concentrations of at least approximately 0.01 μM, at least approximately 0.03 μM, at least approximately 0.1 μM, at least approximately 0.2 μM, at least approximately 0.25 μM, at least approximately 0.28 μM, at least approximately 0.3 μM, approximately 1 μM, at least approximately 1.3 μM, at least approximately 1.5 μM, at least approximately 2 μM, at least approximately 2.3 μM, at least approximately 2.5 μM, at least approximately 2.8 μM, at least approximately 3 μM, at least approximately 3.5 μM, at least approximately 4 μM, at least approximately 4.5 μM, at least approximately 5 μM, at least approximately 10 μM, at least approximately 20 μM, at least approximately 30 μM, at least approximately 40 μM, or at least approximately 50 μM, for example, 0.05 μM to 5 μM, 0.01 μM to 2.5 μM, 0.05 μM to 1 μM, or 0.1 μM to 1 μM.

[0202] In certain embodiments, the Hedgehog (Hh) signaling inhibitor may be an inhibitor of the Sonic Hedgehog (Shh) signaling, the Desert Hedgehog homolog (Dhh) signaling, and / or the Indian Hedgehog homolog (Ihh) signaling. In certain cases, the Hedgehog signaling inhibitor may be an inhibitor of the Sonic Hedgehog signaling. In certain cases, the Hedgehog signaling inhibitor may be a small molecule. In certain cases, the Hedgehog signaling inhibitor may be a small molecule such as CU 61414, IPI-926, (Salidegib), IPI-269609, cyclopamine, bismodegib, or erythmodegib, or their derivatives and analogs.

[0203] Additional factors that guide thymocyte development In some embodiments, soluble factors or other factors can be used in the methods herein to direct the fate of thymocytes, such as thymic epithelial cells or other cell types within the thymic microenvironment, or their development in cell culture. Certain factors may be present in the culture medium for cells to support or promote the development of specific subsets of thymocytes, including, for example, epithelial cells, mesenchymal cells, pericytes, vascular arterial endothelial cells, vascular venous endothelial cells, lymphatic endothelial cells, erythrocytes, immune cells, and / or mesothelial cells (see Table 3). In some embodiments, for example, cTEC-hi, cTEC-lo, immature TEC, mTEC-lo, AIRE+mTEC-hi may be present in the culture medium for cells to support or promote the development of specific subsets of epithelial cells, including keratinocyte-like mTECs, neuroendocrine cells, myosocytes, or myelin cells (see Table 5).

[0204] Examples of factors that may be added to the culture medium to direct thymocyte development and / or support specific subsets of thymocytes in culture include factors related to WNT signaling, e.g., WNT5A (ENSG000001142510), WNT6 (ENSG00000115596), ROR1 (ENSG00000185483), ROR2 (ENSG00000169071), RYK (ENSG00000163785), FRZB (ENSG00000162998), RSPO1 (ENSG00000169218), RSPO3 (EN Factors related to BMP signaling, e.g., BMP4 (ENSG00000125378), BMP5 (ENSG00000112175), and / or FST (ENSG00000134363), factors related to TGF beta signaling, e.g., TGFB1 (ENSG00000105329), TGFBR2 (ENSG00000163513), CXCL12 (ENSG000001075) 62), and / or CCL21 (ENSG00000137077), factors related to IGF signaling, e.g., IGF1R (ENSG00000105329), factors related to the FGF signaling pathway, e.g., FGFR2 (ENSG00000066468), and / or FGF7 / KGF (ENSG00000140285), factors related to NOTCH signaling, e.g., NOTCH1 (ENSG00000148400), NOTCH2 (ENSG00000134250), NOTCH3 (ENSG00000074181), HES1(ENSG00000114315), HES6(ENSG00000144485), DLL4(ENSG00000128917), JAG2(ENSG00000184916), JAG1(ENSG00000101384), HES2(ENSG00000069812), HES4(ENSG00000188290), HEY1(ENSG00000164683), NRARP(ENSG00000198435), DLK1(ENSG00000185559), and / or DLK2(ENSG00000171462),TNF receptors (RANK / TNFRSF11A (ENSG00000141655), CD40 (ENSG00000101017), LTBR (ENSG0000011132), TNFRSF4 (ENSG00000186827), TNFRSF9 (ENSG00000049249), LTB (ENSG00000227507), and / or CD70 (ENSG00000125726)) or their ligands, factors related to p53 signaling, e.g., PERP (ENSG00000112378), SFN (ENSG00000175793), CT This includes SD (ENSG00000117984), CDKN2A (ENSG00000147889), and / or CDKN2B (ENSG00000147883), and / or TOLL-like receptors, such as TLR1 (ENSG00000174125), TLR2 (ENSG00000137462), TLR3 (ENSG00000164342), TLR4 (ENSG00000136869), TLR5 (ENSG00000187554), TLR6 (ENSG00000174130), and / or TLR10 (ENSG00000174123).

[0205] Evaluation of cell population generation In certain cases, cell populations cultured according to the methods disclosed herein may be monitored to assess the cellular changes induced by the culture in order to characterize the produced cell population (e.g., during the stages of the culture methods disclosed herein). In certain embodiments, the production of TECs, including various subpopulations of DE cells, AFE cells, VPE cells, TEP cells, and / or TECs, may be assessed by determining the expression of markers characteristic of these cell populations.

[0206] In certain cases, the expression of a particular marker is determined by detecting the presence or absence of the marker. Alternatively, the expression of a particular marker can be determined by measuring the level at which the marker is present in cells of a cell culture or cell population. In such processes, the measurement of marker expression can be qualitative or quantitative. One method for quantifying the expression of a marker produced by a marker gene is by using quantitative PCR (Q-PCR). Methods for performing Q-PCR are well known in the art. Other methods known in the art can also be used to quantify marker gene expression. For example, the expression of a marker gene product can be detected by using an antibody specific to the marker gene product of interest. In certain processes, it is possible to determine the expression of a marker gene characteristic of a target cell population, as well as the absence of significant expression of a marker gene characteristic of PS cells and other cell types.

[0207] The development of DE cells can be monitored by measuring the expression of the SOX17 gene. Therefore, endoderm cells produced by the process described herein express the SOX17 marker gene and thereby produce the SOX17 gene product. DE cells produced by the method described herein also express the Foxa2 gene. Other endoderm markers include CXCR4, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, and CRIP1. Because endoderm cells express the SOX17 marker gene at higher levels than the SOX7 marker gene, which is characteristic of primitive endoderm and visceral endoderm, in certain cases, the expression of both SOX17 and SOX7 can be monitored. In other embodiments, the expression of both the SOX17 marker gene and the OCT4 marker gene, which are characteristic of hESCs, can be monitored. Furthermore, since endoderm cells express the SOX17 marker gene at higher levels than the AFP, SPARC, or thrombomodulin™ marker genes, the expression of these genes can also be monitored.

[0208] Accordingly, in some embodiments described herein, the expression of the SOX17 marker and / or CXCR4 marker in endoderm cells or cell populations is at least about 2 to at least about 10,000 times higher than the expression of the SOX17 marker and / or CXCR4 marker in non-endoderm cells or cell populations, such as pluripotent stem cells. In other embodiments, the expression of the SOX17 marker and / or CXCR4 marker in endoderm cells or cell populations is at least about 4 times higher, at least about 6 times higher, at least about 8 times higher, at least about 10 times higher, at least about 15 times higher, at least about 20 times higher, at least about 40 times higher, at least about 80 times higher, at least about 100 times higher, at least about 150 times higher, at least about 200 times higher, at least about 500 times higher, at least about 750 times higher, at least about 1000 times higher, at least about 2500 times higher, at least about 5000 times higher, at least about 7500 times higher, or at least about 10,000 times higher than the expression of the SOX17 marker and / or CXCR4 marker in non-endoderm cells or cell populations, such as pluripotent stem cells.

[0209] Markers and methods for identifying DE cells or cell populations are described in US8,216,836, which is incorporated herein by reference in its entirety.

[0210] As described above, monitoring of AFE cell generation can be performed by determining SOX2 expression. Monitoring of VPE cell generation can be performed by determining HOXA3, PAX9, or EYA1 expression. Monitoring of TEP cell generation can be performed by determining FOXN1, HOXA3, EYA1, PAX9, and EpCAM.

[0211] In certain cases, monitoring the generation of TECs, including DE cells, AFE cells, VPE cells, TEP cells, and / or various subpopulations of TECs described herein, may be carried out by performing functional analysis of the cells of interest. For example, TEP cells produced by the methods described herein may be functional. Functional TEP cells may generate thymic epithelial cells in vivo or in vitro. In certain cases, functional TEP cells produced by the methods disclosed herein may produce functional TE cells that support T cell development in vivo or in vitro.

[0212] Functional analysis of cells may include, for example, the identification of marker gene expression. The expression of cell-identification marker genes can be assessed by detecting the mRNA encoding the marker, or by detecting all or part of the marker gene product polypeptide.

[0213] In some embodiments, the DE cell identification markers include SOX17 and / or FOXA2.

[0214] In some embodiments, the AFE cell identification markers include SOX2 and / or FOXA2.

[0215] In some embodiments, the VPE cell identification markers include PAX9 and / or SIX1.

[0216] In some embodiments, TEP cell identification markers include HOXA3, EYA1, EPCAM, K5+, K8+, FOXN1, P63, PAX9, and / or SIX1. For example, TEP cells produced according to the method herein can be identified by an increase of approximately 1×, approximately 1.5×, approximately 2×, approximately 2.5×, approximately 3×, or more in FOXN1 expression levels compared to starting cells, PS cells, DE cells, AFE cells, and / or VPE cells. TEP cells produced according to the method herein may also have increased KRT5 and / or KRT15 expression levels compared to expression levels in starting cells, PS cells, DE cells, AFE cells, and / or VPE cells. Furthermore, TEP cells produced according to the method described herein may have reduced DLL1 expression levels by approximately 1 / 1.5×, 1 / 2×, 1 / 2.5×, and 1 / 3× compared to the expression levels of DLL1 in starting cells, PS cells, DE cells, AFE cells, and / or VPE cells.

[0217] In some embodiments, TEC identification markers include EPCAM, K8+ or K5+, FOXN1, P63, PAX9, SIX1, and / or MHC class II genes.

[0218] Useful cell identification markers for evaluating subsets of thymic epithelial cells are listed below and are shown in Table 1.

[0219] In some embodiments, cTEC cell lines are identified by the expression of β5t(PSMB11), CD205(LY75), CCL25, and / or K8.

[0220] In some embodiments, pluripotent TEP cells are identified by the expression of K5 and / or K8.

[0221] In some embodiments, undifferentiated mTEC progenitor cells are identified by the expression of CLDN3 / 4 (low), MHC class II genes (negative to low), K5, K15, and / or ASCL1.

[0222] In some embodiments, immature mTECs are identified by the expression of CLDN3 / 4, MHC class II genes (low), K5, K15, and / or ASCL1.

[0223] In some embodiments, mature mTECs are identified by the expression of CLDN3 / 4, MHC class II genes (high), AIRE, and / or K5.

[0224] In some embodiments, post-AIRE mTECs are identified by the expression of CLDN3 / 4, MHC class II genes (low), IVL, and / or K10.

[0225] In some embodiments, tuft cells are identified by the expression of CLDN3 / 4, MHC class II genes (low), GNB3, and / or K8.

[0226] In some embodiments, neuroendocrine cells are identified by the expression of CLDN3 / 4, MHC class II genes (negative to low), CHGA, SOX2, and / or K8.

[0227] In some embodiments, muscle-like cells are identified by the expression of CLDN3 / 4 (low), MHC class II genes (negative to low), MYOG, and / or K8.

[0228] In certain cases, this method does not involve monitoring the generation of DE cells, AFE cells, VPE cells, and / or TEP cells.

[0229] Cell population enrichment, separation, and / or purification A target cell population, such as DE cells, AFE cells, VPE cells, TEPs, and / or TECs containing subpopulations of thymic epithelial cells produced by any of the methods described herein, can be enriched, isolated, and / or purified by using affinity tags specific to such cells. Examples of affinity tags specific to the target cells or cell population include antibodies, ligands, or other binders specific to marker molecules such as polypeptides that are present on the cell surface of the target cells but substantially absent on other cell types that may be found in cell cultures produced by the methods described herein.

[0230] Methods for producing antibodies and using them for cell isolation are known in the art, and such methods can be carried out for use with the antibodies and cells described herein. In one process, antibodies that bind to a marker expressed by a target cell population are attached to magnetic beads and then bound to target cells in an enzymatically treated cell culture to reduce cell-cell and substrate adhesion. The cell / antibody / bead complex is then exposed to a magnetic field used to separate bead-bound endoderm cells from unbound cells. Once the target cells are physically separated from other cells in culture, the antibody binding is broken, and the cells are replated into a suitable tissue culture medium.

[0231] Additional methods may also be used to obtain the desired concentrated, isolated, or purified cell population. For example, in some embodiments, an antibody against a marker expressed by the cells of interest is incubated in a cell culture containing the cells of interest that have been treated to reduce cell-cell and substrate adhesion. The cells are then washed, centrifuged, and resuspended. The cell suspension is then incubated with a secondary antibody, such as a FITC-conjugated antibody that can bind to the primary antibody. The cells are then washed, centrifuged, and resuspended in buffer. The cell suspension is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). Antibody-bound cells are collected separately from cells not bound to the marker-specific antibody, thereby separating the cells of interest. If necessary, the separated cell composition may be further purified by using another affinity-based method or by performing an additional round of sorting using the same or different markers specific to the cells of interest.

[0232] In certain cases, target cells such as DE cells, AFE cells, VPE cells, TEP cells, TECs, and / or subpopulations of TECs are concentrated, isolated, and / or purified from other cell types after the PS cell culture has been induced for differentiation. It will be understood that the above concentration, isolation, and purification procedures may be used for such cultures at any stage of differentiation.

[0233] In addition to the procedures described above, the target cells, e.g., TEP cells, TECs, and / or subpopulations of TECs, may also be isolated by other techniques for cell isolation. Furthermore, the target cells may also be enriched or isolated by serial subculturing under growth conditions that promote the selective survival or selective proliferation of the target cells.

[0234] Using the methods described herein, a cell population or cell culture can be enriched to the cells of interest, e.g., TEP cells, TECs, and / or subpopulations of TECs, by at least about 2 to about 1000 times compared to the unenriched cell population produced. For example, in some embodiments, DE cells, and / or AFE cells, and / or VPE cells, and / or TEP cells, and / or TECs and their subpopulations can be enriched by at least about 5 to about 500 times compared to the untreated cell population or cell culture. In other embodiments, the cells of DE cells, and / or AFE cells, and / or VPE cells, and / or TEP, and / or TECs and their subpopulations can be enriched by at least about 10 to about 200 times compared to the untreated cell population or cell culture. In yet another embodiment, DE cells, and / or AFE cells, and / or VPE cells, and / or TEP cells, and / or TECs and their subpopulations can be enriched by at least about 20 to about 100 times compared to the untreated cell population or cell culture. In yet another embodiment, DE cells, and / or AFE cells, and / or VPE cells, and / or TEP, and / or TEC cells and their subpopulations can be enriched at least about 40 to about 80 times compared to an untreated cell population or cell culture. In a particular embodiment, DE cells, and / or AFE cells, and / or VPE cells, and / or TEP, and / or TEC cells and their subpopulations can be enriched at least about 2 to about 20 times compared to an untreated cell population or cell culture.

[0235] Genotypic characteristics of the cell populations disclosed herein Using single-cell RNA sequencing (scRNA-seq), we comprehensively profiled the human thymic stroma across multiple stages of life. Analysis of developmental factors expressed by stromal cells identified mesenchymal and pericyted cells as potential key regulators of TEC cell fate and differentiation. Subclustering of epithelial cells also revealed previously uncharacterized markers of TEC subsets, revealing the presence of ionocytes, ciliated cells, and myelin-positive cells as novel populations in the human thymic medulla. Expression of tissue-specific antigens associated with human autoimmune diseases mapped to different subsets of TECs, highlighting the potential contribution of specific cell types to the induction of human immune tolerance. (See also Park, Jong-Eun, et al. “A cell atlas of human thymic development defines T cell repertoire formation.” Science 367.6480 (2020), which is incorporated herein by reference in its entirety.) These analyses provide crucial information about how the vast diversity of cell types constituting the thymic microenvironment is established and how this heterogeneity contributes to the induction of human immune tolerance.

[0236] To fairly define cellular heterogeneity in the human thymic microenvironment, we mapped the transcriptome of individual stromal cells using scRNA-seq. Non-lymphoid stromal cells from embryonic, postnatal, and adult tissues were analyzed to investigate cell fate specialization and TEC maturation processes at different developmental time points. These studies identified novel candidate pathways modulating TEC fate determination, revealed previously uncharacterized TEC markers, and exposed the presence of distinct mTEC subsets (AIRE+, keratinocyte-like, CCL21+, and tufts). Furthermore, ciliated cells, myelin+ neurons, and CFTR+ ionocytes were identified as novel subsets of epithelial cells present in the human thymic medulla that lack known mouse counterparts. To better understand how immune tolerance is established in the human thymus, we analyzed the expression of disease-related genes in the epithelial compartment. The results of the scRNA-seq analysis described herein yield novel subsets of marker genes and genotypic characteristics of cell populations produced by the methods disclosed herein, including TEP cells, TECs, and subpopulations of TECs.

[0237] When derived from isolated PS cells or established PS cell lines, the cell populations of this disclosure can be characterized as descendants of the original cells or cell lines. Therefore, the cell populations possess the same genome as the cells from which they originate. This means that, in addition to any karyotype changes, the chromosomal DNA is more than 98% identical (e.g., at least 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.9%, or greater) between the PS cells and the cell populations derived therefrom. Cell populations of this disclosure treated by recombination methods involving the introduction of transgenes or the knockout of endogenous genes are still considered to possess the same genome as the lineage from which they originate, since all unmanipulated genetic elements are preserved. The cell populations and PS cells of this disclosure can be identified as having the same genome by standard genetic techniques. If the cell populations are obtained from undifferentiated lines during normal mitosis, ownership of the same genome can also be inferred.

[0238] In certain industrial applications, this characteristic is a valuable feature of the cell populations of this disclosure. In particular, since PS cells can be proliferated as needed and differentiated into more cell populations of this disclosure, the availability of the original PS cells provides a further supply of genetically matched differentiated cell populations. Furthermore, PS cells can differentiate into other therapeutically important lineages.

[0239] The technology described in this application enables the production of a large population of cells that share the same genome by proliferating cells before and after differentiation.

[0240] Theoretically, populations of 10^8, 10^10, or 10^12 cells are possible. Such large populations are usually divided into separate containers suitable for further culture, drug screening, or therapeutic administration.

[0241] Certain embodiments of this disclosure include origin cells (undifferentiated PS cell lines, or intermediate populations, e.g., DE cells, AFE cells, VPE cells, TEP cells, or TEC cells) combined with one or more populations of differentiated cells having the characteristics of these subpopulations. The populations may be in the same container, in separate containers in the same facility, or in two different locations. Undifferentiated and differentiated cells may exist simultaneously or at different time points, such as when differentiating an undifferentiated cell culture into TEP cells, as described herein.

[0242] Compositions and systems containing cell populations cell composition This disclosure provides a composition comprising a differentiated cell population. The composition may comprise differentiated TECs, which are derived from PS cells and comprise one or more subpopulations of TECs, including cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-positive cells, and myosoidal cells.

[0243] Compositions are also provided that include reassembled thymic epithelial progenitor (TEP) cells in combination with one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

[0244] Compositions are also provided that include reassembled thymic epithelial cells (TECs) in combination with one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

[0245] The compositions provided herein can be produced according to the methods described herein.

[0246] Compositions comprising TECs provided herein further comprise subpopulations of TECs, including one or more cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, and / or myoform cells. Thus, compositions comprising thymic epithelial cells that replicate the complexity of the endogenous thymic microenvironment are provided. In some embodiments, compositions of TECs comprising various subsets of TEP cells and / or TECs comprise, for example, the subpopulations listed in Table 1. In some embodiments, compositions comprise populations of thymic epithelial cells having diverse subpopulations of epithelial cells similar to those found in human fetal, postnatal, or adult thymic tissue. For example, compositions comprising TEP cells and / or TECs described herein comprise subpopulations representing one or more subpopulations of thymic epithelial cells shown in Table 1.

[0247] The composition may further contain reassembled TEP cells and / or TECs.

[0248] Cell compositions produced by the methods described herein include cell cultures comprising isolated TEP cells and cell populations enriched in isolated TEP cells, isolated TECs and cell populations enriched in isolated TECs. In certain cases, cell compositions comprising isolated TEP cells and / or TECs may further comprise one or more of the following: RA receptor activators, BMP signaling activators, Wnt family members, fibroblast growth factors, and hedgehog signaling inhibitors.

[0249] In general, the TEP cells present in the systems, cell populations, and compositions described herein are functional. In certain embodiments, the TEP cells are functional and, under appropriate conditions, further differentiate into TE cells in vivo or in vitro. The functional activity of TEP cells can be evaluated by any of the methods described herein or by any method recognized in the art, for example, as described in Inami Y. et al, Immunology and Cell Biology (2011) 89, 314-321; Lai L. and Jin J., Stem Cells 2009 Dec; 27(12): 3012-20; Lai L. et al, Blood. 2011 Sep 22; 118(12): 3410-8. For example, functional TEP cells may further mature upon transplantation into functional TE cells that support T cell development.

[0250] TECs produced according to the method described herein contain subpopulations of epithelial cells including one or more subpopulations listed in Table 1, thereby enabling the reproduction of the biological complexity of endogenous thymic tissue.

[0251] In certain embodiments, the TEP cells of the Disclosure present in the systems, cell populations, and compositions described herein express one or more TEP cell markers present in TEP cells present in the thymus or thymic tissue, such as the adult human thymus or the fetal human thymus. For example, TEP cells produced by the methods described herein may express TEP markers at levels similar to those expressed by TEP cells in the adult or fetal thymus. In certain cases, the TEP cells of the Disclosure express one or more of FOXN1, HOXA3, EYA1, GCM2, and EpCAM. In certain cases, TEP cells produced by the methods provided herein express FOXN1. In certain cases, TEP cells produced by the methods provided herein express HOXA3. In certain cases, TEP cells produced by the methods provided herein express FOXN1 and HOXA3. In certain cases, TEP cells produced by the methods provided herein express FOXN1, HOXA3, and EpCAM. In certain cases, TEP cells produced by the methods provided herein express FOXN1, HOXA3, and EYA1. In certain cases, TEP cells produced by the methods provided herein express FOXN1, HOXA3, PAX1, EpCAM, and EYA1. In certain cases, TEP cells provided herein do not express at significant levels marker genes characteristic of mature TECs, such as HLA-DRA (MHC class II molecule) and AIRE. Detection of the expression of one or more of FOXN1, HOXA3, EYA1, GCM2, and EpCAM can be achieved according to methods known in the art, such as the methods discussed herein.

[0252] Therefore, the TEP cells of this disclosure express one or more of the markers provided herein and are functional.

[0253] As described herein, the TEP cells of this disclosure may be mammalian, such as primate TEP cells, including human TEP cells.

[0254] In certain embodiments, the TECs and / or subpopulations present in the systems, cell populations, and compositions described herein express one or more TEC markers, which are present in TECs present in the thymus or thymic tissue, such as the adult human thymus or fetal human thymus. For example, TECs produced by the methods described herein may express TEC markers at levels similar to those expressed by TEC cells in the adult or fetal thymus. In certain cases, the TECs of the disclosure express one or more of β5t(PSMB11), CD205(LY75), CCL25, K8, K5, K10, K15, CLDN3 / 4, MHCII, ASCL1, AIRE, IVL, GNB3, CHGA, and / or SOX2. In certain cases, TECs produced by the methods provided herein express β5t(PSMB11). In certain cases, the TEC produced by the method provided herein expresses CD205 (LY75). In certain cases, the TEC produced by the method provided herein expresses CCL25. In certain cases, the TEC produced by the method provided herein expresses K8. In certain cases, the TEC produced by the method provided herein expresses K5. In certain cases, the TEC produced by the method provided herein expresses K10. In certain cases, the TEC produced by the method provided herein expresses K15. In certain cases, the TEC produced by the method provided herein expresses CLDN3 / 4. In certain cases, the TEC produced by the method provided herein expresses an MHC class II gene. In certain cases, the TEC produced by the method provided herein expresses ASCL1. In certain cases, the TEC produced by the method provided herein expresses AIRE. In certain cases, the TEC produced by the method provided herein expresses IVL. In certain cases, the TEC produced by the method provided herein expresses GNB3. In certain cases, the TEC produced by the method provided herein expresses CHGA.In certain cases, the TEC produced by the method provided herein expresses SOX2.

[0255] Therefore, the TECs of this disclosure express one or more of the markers provided herein, i.e., Table 1 and / or Table 2, and they are functional.

[0256] As described herein, the TECs of this disclosure may be mammalian, for example, primate TEP cells such as human TECs.

[0257] The cell compositions produced by the described methods include isolated VPE cells and cell cultures containing cell populations enriched with VPE cells. In certain cases, the cell composition containing VPE cells may contain one or more of the following: RA receptor activators, BMP signaling activators, Wnt family members, fibroblast growth factors, and Hedgehog signaling inhibitors. In certain cases, the cell composition containing VPE cells may contain one or more of the following: RA receptor activators, BMP signaling activators, TGF-β signaling inhibitors, Wnt family members, fibroblast growth factors, and Hedgehog signaling inhibitors. Generally, VPE cells present in a cell population can be differentiated into TEP cells when cultured according to the methods disclosed herein.

[0258] The cell compositions produced by the described methods include cell cultures containing AFE cells and cell populations enriched with AFE cells. In specific cases, the cell composition containing AFE cells may contain one or more of the following: activators of the RA receptor, activators of BMP signaling, inhibitors of TGF-β signaling, Wnt family members, fibroblast growth factors, and inhibitors of Hedgehog signaling. Generally, AFE cells present in a cell population can be differentiated into VPE cells and TEP cells when cultured according to the methods disclosed herein.

[0259] The cell compositions produced by the described method include cell cultures containing DE cells and cell populations enriched with DE cells. In certain cases, the cell composition containing DE cells may contain one or more of the following: RA receptor activators, BMP signaling activators, and TGF-β signaling inhibitors. In certain cases, the cell composition containing DE cells may contain one or more of the following: RA receptor activators, Nodal, Act-A, and Act-B. Generally, DE cells present in a cell population can differentiate into AFE cells, VPE cells, and TEP cells when cultured according to the methods disclosed herein.

[0260] The cell compositions produced by the described method include isolated TEP cells and cell cultures containing TEP cell-enriched cell populations. In certain cases, the cell composition containing TEP cells may include one or more of the following: RA receptor activators, BMP signaling activators, Wnt family members, fibroblast growth factors, and hedgehog signaling inhibitors.

[0261] The cell composition produced by the described method includes cell cultures containing isolated TECs and TEC-enriched cell populations and subpopulations thereof.

[0262] In some embodiments, a cell composition comprising the cells of the Disclosure (e.g., TEC, TEP cells, or VPE cells, or AFE cells, or DE cells) can be produced in which at least about 50% to 80% of the cells in culture are cells of interest. The differentiation methods described herein result in the conversion of pluripotent cells to cells of interest in amounts of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%.

[0263] For example, by using affinity reagents that bind to target cells, a substantially pure population of target cells can be recovered in embodiments where isolation of target cells is used.

[0264] Some embodiments described herein relate to cell compositions comprising at least about 5% to at least about 95% of the target cells. In some embodiments, the cell culture or cell population comprises mammalian cells. In preferred embodiments, the cell culture or cell population comprises human cells. For example, certain specific embodiments relate to cell compositions comprising human cells, wherein at least about 5% to at least about 95% of the human cells are TECs. Other embodiments relate to cell compositions comprising human cells, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more than 90% of the human cells are TECs.

[0265] The cell composition produced by the above method and the composition therefrom can be evaluated using the markers and methods described herein and methods known in the art.

[0266] The cell composition produced by the above method and the composition therefrom can be concentrated, isolated or purified using the methods described herein and methods known in the art.

[0267] The cell compositions provided herein may be pharmaceutical compositions comprising a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include saline, buffers, diluents, expanders, salts, stabilizers, solubilizers, cell culture media, and other materials well known in the art. In some embodiments, the formulations do not contain detectable DMSO (dimethyl sulfoxide).

[0268] For general principles regarding the pharmaceutical formulation of cell compositions, readers should refer to *Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy*, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996, and *Cell Transplantation for Neurological Disorders*, TB Freeman et al. eds., Humana Press, 1998. Cells are packaged in devices or containers suitable for distribution or clinical use, and, where necessary, accompanied by information regarding cell storage, use as a pharmaceutical to treat clinical conditions, or other valuable purposes.

[0269] A first in vitro cell population comprising primate (e.g., human) pluripotent stem cells and a second in vitro cell population comprising offspring of a portion of the first in vitro cell population, the offspring being TEP cells and / or TECs as described herein.

[0270] Therefore, the TEP cells and / or TECs in the second in vitro cell population are functional and may express the markers provided herein.

[0271] The first and second in vitro cell populations may exist simultaneously or at different times. The first and second in vitro cell populations may exist in the same container or in different containers.

[0272] In certain cases, the first in vitro cell population may be pPS cells, DE cells, AFE cells, or VPE cells, and the second in vitro cell population may be TEP cells and / or TEC cells, which are descendants of pPS cells, DE cells, AFE cells, or VPE cells.

[0273] In certain cases, the first in vitro cell population may be DE cells, and the second in vitro cell population may be AFE cells, where AFE cells are descendants of DE cells.

[0274] The first in vitro cell population could be DE cells, and the second in vitro cell population could be VPE cells, which are descendants of DE cells.

[0275] The first in vitro cell population could be AFE cells, and the second in vitro cell population could be VPE cells, which are descendants of AFE cells.

[0276] Furthermore, this specification provides first, second, and third in vitro cell populations, the first of which may be AFE cells, the second of which may be VPE cells, and the third of which may be TEP cells, where VPE cells are descendants of AFE cells and TEP cells are descendants of VPE cells.

[0277] Furthermore, this specification provides first, second, third, and fourth in vitro cell populations, the first of which may be DE cells, the second of which may be AFE cells, the third of which may be VPE cells, and the fourth of which may be TEP cells, where AFE cells are descendants of DE cells, VPE cells are descendants of AFE cells, and TEP cells are descendants of VPE cells.

[0278] First, second, third, fourth, and fifth in vitro cell populations are also provided herein, the first cell population may be pPS cells, the second cell population may be DE cells, the third cell population may be AFE cells, the fourth cell population may be VPE cells, and the fifth cell population may be TEP cells, where DE cells are descendants of pPS cells, AFE cells are descendants of DE cells, VPE cells are descendants of AFE cells, and TEP cells are descendants of VPE cells.

[0279] Some embodiments of the cell compositions described herein include one or more cell populations comprising a population of DE cells, a population of AFE cells, a population of VPE cells, a population of TEP cells, and / or a population of TEC, or a subpopulation thereof.

[0280] System Also provided herein is a system for efficiently generating primate TEP cells and / or TEC and subpopulations thereof for use in the research and preparation of pharmaceutical compositions for the treatment of a subject in need of treatment with TEP cells.

[0281] The systems of the present disclosure include a set or combination of cells that exist at any point during manufacture, distribution, or use. The cell set includes any combination of two or more cell populations described in the present disclosure, and may combine differentiated pPS-derived cell types (e.g., TEC and / or subpopulations thereof, TEP cells, VPE cells, AFE cells, DE cells, by way of example and not limitation) with undifferentiated pPS cells or other differentiated cell types to share the same genome. Each cell type within the set can be packaged together or separately in the same facility or different locations, simultaneously or at different times, under the control of the same corporate entity or different corporate entities that share the same corporate or business relationship, in the same container or separate containers.

[0282] In certain embodiments, a differentiated cell population is provided as part of a system for generating TEP cells and / or TEC and subpopulations thereof. The TEP cells and / or TEC and subpopulations of the system have the functional and phenotypic characteristics provided herein (e.g., expression of TEP cell markers) and are descendants of primate pluripotent stem (pPS) cells. In other words, the TEP cells and / or TEC and subpopulations of the system are produced by the differentiation of pPS cells.

[0283] [[ID=十七]] In an exemplary embodiment, a system of components for generating TEP cells and / or TECs and their subpopulations may include a lineage of undifferentiated human PS cells and a population of TEP cells and / or TECs and their subpopulations differentiated therefrom, wherein the TEP cells and / or TECs and their subpopulations express one or more TEP cell markers, such as those provided herein.

[0284] A system of components for generating TEP cells and / or TECs and their subpopulations may include a population of human AFE cells and TEP cells, and / or its subpopulation of TECs differentiated therefrom, wherein the TEP cells and / or TECs and their subpopulations express one or more TEP cell markers, such as those provided herein.

[0285] A system of components for generating TEP cells and / or TECs and their subpopulations may include human VPE cells and cell populations of TEP cells and / or TECs and / or subpopulations thereof, wherein the TEP cells and / or TECs and their subpopulations express one or more TEP cell markers, such as those provided herein.

[0286] A system of components for generating TEP cells and / or TECs and their subpopulations may include a cell population of human PS cells and DE cells differentiated therefrom, wherein the DE cells express one or more DE cell markers, such as those provided herein.

[0287] A system of components for generating TEP cells and / or TECs and their subpopulations may include a cell population of human PS cells and AFE cells differentiated therefrom, wherein the AFE cells express one or more AFE cell markers, such as those provided herein.

[0288] A system of components for generating TEP cells and / or TECs and their subpopulations may include a cell population of human PS cells and VPE cells differentiated therefrom, wherein the VPE cells express one or more VPE cell markers, such as those provided herein.

[0289] A system of components for generating TEP cells and / or TECs and their subpopulations includes human PS cells, a cell population of DE cells differentiated from PS cells, a cell population of AFE cells differentiated from DE cells, a cell population of VPE cells differentiated from AFE cells, and a cell population of TEP cells and / or TECs and their subpopulations differentiated from AFE cells, wherein the cell population expresses one or more markers typical of a particular cell, such as those described herein.

[0290] The cell populations of TEP cells and / or TECs and their subpopulations in the systems and compositions described herein may contain at least 10% to 95% or more of TEP cells and / or TECs and their subpopulations (e.g., 15% to 90%, 20% to 80%, 50% to 70%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%).

[0291] The cell population of VPE cells in the systems and compositions described herein may contain at least 10% to 95% or more VPE cells (e.g., 15% to 90%, 20% to 80%, 50% to 70%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%).

[0292] The AFE cell population of the systems and compositions described herein may contain at least 10% to 95% or more AFE cells (e.g., 15% to 90%, 20% to 80%, 50% to 70%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%).

[0293] The cell population of DE cells in the systems and compositions described herein may contain at least 10% to 95% or more DE cells (e.g., 15% to 90%, 20% to 80%, 50% to 70%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%).

[0294] Use of cell populations Cell population for screening The cells of this disclosure can be used to screen for agents (such as small molecules, peptides, polynucleotides, etc.) or environmental conditions (such as culture conditions or operations) that affect the characteristics of PS cells, DE cells, AFE cells, VPE cells, TEP cells, and / or TECs and their subpopulations as described herein.

[0295] For example, PS cells, DE cells, AFE cells, and / or VPE cells (undifferentiated or initiation into the differentiation paradigm) are used to screen for factors that promote maturation to TEP cells, or factors that promote the proliferation and maintenance of TEP cells, and / or factors of TECs and their subpopulations in long-term culture. For example, candidate differentiation or growth factors are tested by adding them to cells in different wells, and the resulting phenotypic changes are then determined according to desired criteria for further culture and use of the cells. This may lead to improved induction and culture methods for generating DE cells, AFE cells, VPE cells, TEP cells, and / or TECs.

[0296] Other screening methods described herein relate to testing pharmaceutical compositions for potential adverse effects on TEP cells and / or TECs. This type of screening is suitable not only when a compound is designed to have a pharmacological effect on TEP cells and / or TECs and their subpopulations themselves, but also for testing TEP cell / TE cell-related side effects of compounds designed for primary pharmacological effects elsewhere.

[0297] Other screening methods relate to the use of TEP cells and / or TECs and their subpopulations to measure the effects of small molecule agents that may affect the immune system. For this purpose, cells can be combined with test compounds in vitro to determine the effects of the compounds on TEP cells and / or TECs and their subpopulations.

[0298] The general principles of drug screening are described in U.S. Patent No. 5,030,015 and in the textbook In vitro Methods in Pharmaceutical Research, Academic Press 1997. The evaluation of the activity of a candidate pharmaceutical compound generally involves combining the differentiated cells of the present invention with the candidate compound, either alone or in combination with other drugs. The researcher determines the changes in cell morphology, markers, or functional activity resulting from the compound (compared to cells treated with untreated cells or a negative control compound) and correlates the effect of the compound with the observed changes.

[0299] TEP cells and / or TEC and its subpopulations in clinical treatment Cell populations containing TEP cells, such as cell populations enriched in TEP cells, and purified TEP cells and / or TEC and its subpopulations produced by the methods described herein can be used in many clinical applications.

[0300] In certain embodiments, the TEP cells and / or TEC and its subpopulations produced using the methods provided herein can be used to generate a functional thymic epithelium (TE) in a subject that needs it.

[0301] Subjects that need TEP cells and / or TEC and its subpopulations can be subjects having genetic and / or developmental defects that result in a decrease or undetectability of thymic function. In certain cases, the subject may have DiGeorge syndrome or complete DiGeorge syndrome. Complete DiGeorge syndrome is a fatal condition in which thymic function in infants is not detected. The TEP cells and / or TEC and its subpopulations of the present disclosure can be used for the treatment of infants with complete DiGeorge syndrome. For example, infants with complete DiGeorge syndrome can be treated using the TEP cells and / or TEC and its subpopulations of the infant according to the transplantation techniques described in Markert M.L. et al., Blood. 2003 Aug 1;102(3):1121-30. Epub 2003 Apr 17.

[0302] In certain embodiments, TEP cells and / or TECs and their subpopulations produced using the methods provided herein may be used in thymic regeneration therapy.

[0303] TEP cells and / or TECs and their subpopulations can be transplanted into subjects requiring TE cells. In specific cases, TEP cells and / or TECs and their subpopulations can be transplanted into target sites of subjects that provide appropriate differentiation conditions for TEP cells and / or TECs and their subpopulations to differentiate into TE cells. Cells can be transplanted by any of the many standard methods in the art for delivering cells to tissue, e.g., by injection as a suspension in a suitable buffer (such as saline, PBS, DMEM, Iskov medium, or a pharmaceutically acceptable carrier), or by providing cells on a solid support such as a filter (such as beads or a mesh filter) or a membrane. In specific cases, TEP cells and / or TECs and their subpopulations can be transplanted into the thymus of a subject. In specific cases, TEP cells and / or TECs can be transplanted under the kidney capsule of a subject.

[0304] In certain cases, subjects requiring TEP cell and / or TEC transplantation may be those requiring increased enhancement or restoration of thymic function. In certain cases, subjects may have significantly degenerated thymus due to aging. In certain cases, subjects may have severely degenerated thymus due to radiation exposure. In certain cases, subjects may have severely degenerated thymus due to chemotherapy.

[0305] In certain cases, thymic tissue or thymic epithelial cell populations produced according to the method herein can be used to develop T lymphocytes for therapeutic use. In some embodiments, thymic tissue or thymic epithelial cell populations produced according to the method herein can be used to support the development of diverse self-tolerant peripheral T cell pools.

[0306] In certain cases, thymic tissue or thymic epithelial cell populations produced according to the methods described herein can be used to promote and / or manipulate immune tolerance in a variety of therapeutic applications, such as the treatment of autoimmune conditions including type 1 diabetes. In some embodiments, methods can be provided for promoting, reducing, or manipulating immune tolerance in human subjects using thymic tissue or thymic epithelial cell populations produced according to the methods described herein. For example, the AIRE+mTEC described herein may be involved in inducing tolerance by providing antigens that can be presented by antigen-presenting cells such as dendritic cells. For example, myoiroid cells may be involved in inducing immune tolerance to muscle antigens. For example, many thymoma patients who typically lack myoiroid cells develop myasthenia gravis (MG), a neuromuscular junction autoimmune disease characterized by autoantibodies against acetylcholine receptor (AChR) or other muscle antigens such as titin (TTN). On the other hand, APS-1 patients typically do not have detectable autoantibodies against AChR or TTN, suggesting that the expression of these antigens is not entirely dependent on AIRE. Importantly, AChR and TTN expression has been shown to be much higher in myoiform cells compared to AIRE+mTECs, supporting the idea that myoiform cells are the primary source of muscle antigens in the human thymic medulla. Therefore, autoimmune tolerance can be manipulated, i.e., regulated, by dose-setting the relative presence of subpopulations of thymic epithelial cells present in the compositions herein using the methods provided herein. Alternatively, the methods provided herein could establish a roadmap for directing the development of thymic epithelial cells based on the presence of specific factors and combinations of factors present in thymic tissue, enabling novel interventions for correcting affected or deficient thymic tissue in vivo. Thus, these findings provide methods for regulating disease-associated tissue-specific antigens in the human thymus.

[0307] In some embodiments, thymic tissue or thymic epithelial cell populations produced according to the methods described herein can be used to repair or replenish a target thymic tissue defect. In some embodiments, the compositions and methods described provide therapeutic compositions and methods that may help establish and maintain healthy thymic tissue throughout the course of disease or aging.

[0308] TEPs generated from patient-specific induced pluripotent stem (iPS) cell lines can also be used as a tool to model human diseases.

[0309] In certain cases, the TEP produced by the method described herein can be genetically modified to express the protein of interest.

[0310] The present invention can be further described by the following non-limiting embodiments. [Examples]

[0311] A method for producing TEP and TEC is described, for example, in International Patent Application Publication No. 2014 / 134213, which is incorporated herein by reference in its entirety.

[0312] method Thymus tissue acquisition Human fetal thymic tissue was obtained from specimens at 19–23 weeks of gestation, following guidelines from a protocol approved by the UCSF Institutional Research Board (UCSFIRB) in the Department of Obstetrics, Gynecology and Reproductive Sciences at San Francisco General Hospital. Pediatric tissue was obtained from patients undergoing corrective cardiac and thoracic surgery, following a protocol approved by the UCSF Human Research Protection Program Institutional Review Board. Human adult thymic tissue was obtained from deceased organ donors who consented to the study at the time of organ acquisition for clinical transplantation, through an IRB-approved research protocol by Donor Network West, an organ procurement organization in Northern California. All donors were free from chronic diseases and cancer, and were negative for hepatitis B / C and HIV.

[0313] Organizational preparation Thymic tissue placed in RPMI (ThermoFisher) containing 100 μg / ml DNase I (Roche) was cut into small pieces using scissors. The tissue pieces were transferred to gentleMACS C tubes (Miltenyi) containing 10 ml of RPMI containing DNase. The gentleMACS program m_spleen_02 was run three times. Thymic fragments were separated from the supernatant, which was enriched with thymocytes, by centrifugation. The remaining fragments were returned to a C tube containing fresh RPMI containing DNase before running program m_spleen_01. The supernatant was removed and replaced with 10 ml of digestion medium containing 100 μg / ml DNase I and 100 μg / ml Liberase® (Sigma-Aldrich) in RPMI. The tubes were placed in a 37°C water bath and the fragments were crushed every 5 minutes to aid mechanical digestion. After 30 minutes, the tubes were briefly centrifuged to pellet any undigested fragments, and the supernatant was discarded. Fresh digestion medium or accumax (STEMCELL Technologies) was added to the remaining fragments, and digestion was repeated for a further 15-30 minutes until most of the fragments were digested. The supernatant from this second round of digestion was transferred to a tube containing cold MACS buffer (0.5% BSA, 2 mM EDTA in PBS) to stop the enzymatic digestion. If necessary, a third enzymatic digestion was performed on the remaining fragments for a further 5-10 minutes using accumax. The cells were pooled with the supernatant from the previous digestion and passed through a 40 μm filter (Falcon). Some samples were treated with 2 ml of ACK lysis buffer (Lonza) for 5 minutes before interstitial concentration.

[0314] Concentration of interstitial cells For fetal and postnatal tissues, single cells from digested tissues were resuspended in MACS buffer containing 10 μM ROCK inhibitor Y-27632 (Tocris). Immune cells were depleted using human CD45 MicroBeads (Miltenyi) according to the manufacturer's instructions with the following modification: 5 μL of CD45 MicroBeads per 10^7 total cells were added instead of 20 μL. An LD column was used for depletion, and the CD45-negative fraction was collected in MACS buffer. Stromal cells from adult thymus were enriched using fluorescence-activated cell sorting (FACS). Blocking was performed with human Fc Receptor Binding Inhibitor Monoclonal Antibody (eBioscience), followed by staining for 20 minutes with human-specific antibodies against EPCAM (Clone 1B7, eBioscience) and CD45 (Clone HI30, Biolegend). After staining, cells were washed and resuspended in FACS buffer containing DAPI. Cells were sorted using BD FACS Aria II. Pregating was first performed on live cells based on DAPI staining.

[0315] Single-cell RNA sequencing and computer analysis Single cells were captured using a 10× Chromium microfluidics system (10× Genomics). Cells were encapsulated, and barcoded cDNA libraries were prepared using a single-cell 3' mRNA kit (v2 or v3, 10× Genomics). Single-cell libraries were sequenced using NovaSeq6000. Cell barcodes were demultiplexed using the Cell Ranger software pipeline (10× Genomics, version 2.0.0, 2.1.1, or 3.0.2), reads were mapped to the human genome (GRCh38), transcriptomes were created using the STAR aligner, and gene count vs. cell matrices were produced. Single-cell data analysis was performed using SCANPY (Wolf, FA, Angerer, P. & Theis, FJSCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15-5 (2018)). Cell-specific gene count matrices from all samples were concatenated into a single matrix. 200–5000 genes were detected, and cells expressing less than 10% of mitochondrial genes, as well as genes expressed in 3 or more cells, were retained (a total of 68008 cells with an average count of 4769 per cell). Counts were logarithmically transformed, and total counts per cell were normalized. The dataset was filtered for highly variable genes (minimum mean = 0.0125, maximum mean = 3, and variance 0.5 per gene), and variances caused by changes in mitochondrial gene expression and cell cycle-dependent gene expression were excluded from the regression. BBKNN (Polanski, K. et al. BBKNN: Fast Batch Alignment of Single Cell Transcriptomes. Bioinformatics 36, 411. (2019)) was applied to correct for donor-specific effects. K nearest neighbor graphs were created (n_neighbors=15), and clustering was performed using the Leiden algorithm with a resolution of 0.5.The clustering results were visualized using Uniform Manifold Approximation and Projection (UMAP).

[0316] Pseudo-time analysis and gene scoring Pseudo-time analysis was performed using RNA Velocity (LaManno, G. et al. RNA velocity of single cell. Nature 560, 494-498 (2018)). Spliced ​​and unspliced ​​expression matrices were generated using a standard verocyte pipeline. The following steps were performed using the scVelo package (Bergen, V., Lange, M., Peidli, S., Wolf, FA & Theis, F. Generalizing RNA velocity to transient cell states through dynamical modeling. bioRxiv820936 (2019)). The matrix was size-normalized to the median of all mRNA molecules across all cells. Genes were selected based on a threshold of a minimum of 20 expression counts for both spliced ​​and unspliced ​​mRNA. The top 2000 highly variable genes were retained for further downstream analysis. The nearest neighbor graph was calculated with 30 neighbors based on the normalized gene expression matrix from the original analysis. Velocity estimates were calculated using the standard scVelo pipeline, and the resulting velocity graphs were projected onto a previously generated UMAP using SCANPY. TSA and APS-1 scores were calculated using the scanpy.tl.score_genes function, which calculates the average expression of a set of genes by subtracting the average expression of a reference set of randomly selected genes (Satija, R., Farrell, JA, Gennert, D., Schier, AF & Regev, A. Spatial reconstruction of single-cell gene expression data. Nature Biotechnology 33, 495-502 (2015)). TSA genes were identified using data from the GNF Mouse Gene Atlas reported by Sansom, SNet al. Genome Res. 24, 1918-1931 (2014).The APS-1 gene was selected based on its association with autoantibodies in APS-1 patients (Constantine, GM & Lionakis, MS Lessons from primary immunodeficiencies, Autoimmune regulator and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Immunol. Rev. 287, 103-120 (2019)). The mean expression levels of the scored genes were visualized using UMAP.

[0317] Immunofluorescence staining and imaging For immunofluorescence, tissue was fixed with 4% paraformaldehyde, washed with PBS, and incubated overnight in 30% (w / v) sucrose (Sigma-Aldrich) in PBS. The tissue was embedded in an optimal cutting temperature compound (Tissue-Tek) and stored at -80°C before sectioning in a cryostat (Leica). Slides were briefly rehydrated with PBS, then permeabilized and blocked in a CAS block (ThermoFisher) containing 0.2% TritonX-100 (Sigma-Aldrich), followed by primary antibody staining overnight at 4°C. Secondary antibody staining was performed at room temperature for 1 hour, if necessary. Sections were washed with PBS-Tween 0.1% before mounting in a ProLong Diamond Antifade Mountant (ThermoFisher). Images were acquired using an Apotome microscope (Zeiss). The following antibodies were used: KRT8-Alx647 (clone EP1628Y, ab192468, Abcam), KRT5Alx488 (clone EP1601Y, ab193894, Abcam), KRT15 (clone EPR1614Y, ab52816, Abcam), ASCL1 (ab74065, Abcam), AIRE (14-9534-82, eBioscience), SOX2 (AF2018, R&DSystems), KRT10-Alx647 (clone EP1607IHCY, ab194231, Abcam).

[0318] immunohistochemistry Tissue was fixed with 4% paraformaldehyde (ThermoFisher), washed with PBS, and embedded in paraffin. Antigen recovery was performed on rehydrated tissue by boiling sections in Antigen Recovery Citra Solution (Biogenex). Sections were blocked for 30 minutes at room temperature using CAS-Block (ThermoFisher) containing 0.2% Triton X-100 (Sigma-Aldrich), and then incubated overnight at 4°C with the primary antibody. Staining with biotinylated secondary antibody was performed at room temperature for 1 hour. Slides were prepared using ABC kits (Vector Labs) and DAB kits (Vector Labs) and counterstained with hematoxylin. The following antibodies were used: anti-CFTR (clone #24-1, R&D Systems), anti-desmin (clone D33, Dako), and anti-synaptophysin (clone Snp88, Biogenex).

[0319] mouse The Rosa26-CAG-stopflox-tdTomato and Ascl1-cre-ERT2 (Kim, EJ, Ables, JL, Dickel, LK, Eisch, AJ & Johnson, JEAscl1(Mash1) defines cells with long-term neurogenic potential in subgranular and subventricular zones in adult mouse brain. PLoS ONE 6, e18472 (2011)) mice were obtained from Jackson Laboratory (JAX#007914 and #012882). The ADIG mouse has been previously described (Gardner, JMet al. Deletional Tolerance Mediated by Extrathymic Aire-Expressing Cells. Science 321, 843-847 (2008)). Mice were maintained in a pathogen-free animal facility at the University of California, San Francisco (UCSF) in accordance with guidelines established by the Institutional Animal Care and Use Committee (IACUC) and the Laboratory Animal Resource Center. All experimental procedures were approved by the UCSF Laboratory Animal Resource Center. Mice aged 12–15 weeks were used for strain tracking experiments. Tamoxifen (Sigma-Aldrich) was dissolved in corn oil (Sigma-Aldrich) and administered as a single 4 mg oral gastric tube feeding using a flexible plastic feeding tube (Instech).

[0320] Flow cytometry and antibodies For lineage tracking experiments, single-cell suspensions were prepared as described above (Miller, CNet al. Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development. Nature 559, 627-631 (2018)). Briefly, mouse thymuses were isolated, fat removed, and transferred to DMEM (UCSF Cell Culture Facility) containing 2% FBS (Atlanta Biologics) on ice. The thymuses were minced with a razor blade, and up to four thymuses were pooled into a single digest. The tissue fragments were transferred to 15 ml tubes and briefly vortexed in digestion medium (DMEM containing 2% FBS, 100 μg / ml DNase I, and 100 μg / ml liberase™). After the fragments settled, the medium was removed and replaced with fresh digestion medium. The tubes were transferred to a 37°C water bath, and the fragments were digested by crushing them with a glass Pasteur pipette every 6 minutes. At 12 minutes, the tubes were briefly rotated to pellet any undigested fragments, and the supernatant was transferred on ice to 20 ml of 0.5% BSA (Sigma-Aldrich) and 2 mM MEDTA (TekNova) in PBS (MACS buffer) to stop enzymatic digestion. This was repeated twice, or until no tissue fragments remained, for a total of three 12-minute digestion cycles. The single-cell suspension was then pelletized and washed once with MACS buffer. Stromal cells were concentrated using density gradient centrifugation with a three-layer Percoll gradient (GE Healthcare) with specific gravities of 1.115, 1.065, and 1.0. Cells isolated from the Percoll-light fraction between the 1.065 and 1.0 layers were resuspended in MACS buffer and counted. Next, the cells were incubated with Live / Dead Fixable Blue Dead Cell Stain (ThermoFisher) in PBS at 4°C for 20 minutes, followed by blocking with anti-mouse CD16 / CD32 (24G2) (UCSF Hybridoma Core Facility) and 5% normal rat serum at 4°C for 20 minutes. The cells were then washed with FACS buffer and stained for surface markers at 4°C for 30–45 minutes.Flow cytometry data were collected using an LSRII Flow Cytometer (BD Biosciences) housed in the UCSF Single Cell Analysis Center and analyzed using FlowJo software (TreeStar Software). The following antibodies were used: CD11c (N418), CD45 (30-F11), EpCAM (G8.8), and I-Ab (25-9-17). Antibodies were purchased from Abcam, BioLegend, BD Biosciences, eBioscience, or Miltenyi.

[0321] Example 1. Single-cell profiling of human thymus-derived stromal cells To identify the distinct cell types constituting the human thymic microenvironment, single-cell RNA sequencing (scRNA-seq) was performed on stromal cells isolated from fetal, postnatal, and adult thymic tissue (Figure 1). Figure 1 herein is also described as Figure 1a in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6), the entirety of which is incorporated herein by reference. Stromal cells were obtained by enzymatic digestion of thymic tissue followed by depletion of CD45-positive immune cells using magnetic beads (MACS), or by fluorescence-activated cell sorting (FACS) purification of CD45-negative cells. These procedures enriched both EpCAM+ / CD45-epithelial cells and EpCAM- / CD45-non-epithelial stromal cells. Cells isolated from two fetal samples (19 and 23 weeks of gestation), two postnatal samples (6 days and 10 months), and one adult sample (25 years old) were analyzed using scRNA-seq.

[0322] Specifically, single cells were captured using a 10×Chromium microfluidics system (10×Genomics). Cells were encapsulated, and barcoded cDNA libraries were prepared using a single-cell 3' mRNA kit (v2 or v3; 10×Genomics). The single-cell libraries were sequenced using NovaSeq6000. Cell barcodes were demultiplexed using the Cell Ranger software pipeline (10×Genomics, versions 2.0.0, 2.1.1, or 3.0.2), reads were mapped to the human genome (GRCh38), transcriptomes were created using the STAR aligner, and gene count versus cell matrices were produced. Single-cell data analysis was performed using SCANPY (Wolf, FA, Angerer, P. & Theis, FJSCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15-5 (2018)). The cell-specific gene count matrices for all samples were concatenated into a single matrix. 200–5000 genes were detected, and cells expressing less than 10% of mitochondrial genes, as well as genes expressed in 3 or more cells, were retained (a total of 68008 cells with an average count of 4769 per cell). Counts were logarithmically transformed, and total counts per cell were normalized. The dataset was filtered for highly variable genes (minimum mean = 0.0125, maximum mean = 3, and variance 0.5 per gene), and variances caused by changes in mitochondrial gene expression and cell cycle-dependent gene expression were excluded from the regression. BBKNN (Polanski, K. et al. BBKNN: Fast Batch Alignment of Single Cell Transcriptomes. Bioinformatics 36, 411. (2019)) was applied to correct for donor-specific effects. K-neighbor graphs were created (n_neighbors=15), and clustering was performed using the Leiden algorithm with a resolution of 0.5.An unsupervised, graph-based clustering strategy was employed, and 12 stromal clusters were identified using known markers (Table 3). Epithelial clusters were identified using common epithelial markers EPCAM and KRT8, and FOXN1, PSMB11, LY75, CLDN4, AIRE, IVL, NEUROD1, and MYOD1 were identified as markers for specific subsets. [Table 3]

[0323] Table 4 lists stromal cell markers useful for identifying cell types within the human thymus. An ensemble gene identifier (ENSG00000######) is provided for each marker. [Table 4]

[0324] Studies in animal models have shown that the neural crest, mesenchymal cells, and endothelial cells are important in establishing the thymic microenvironment that supports thymogenesis through the production of soluble factors and intercellular interactions (Kuratani, S. & Bockman, DEAnat. Rec. 228, 185-190 (1990), Jenkinson, WE, Jenkinson, EJ & Anderson, GJ Exp. Med. 198, 325-332 (2003), Neves, H., Dupin, E., Parreira, L. & Le Douarin, NM Developmental Biology 361, 208-219 (2012), Sun, L. et al. Sci Rep 5, 14871 (2015), Wertheimer, T. et al. Sci Immunol 3, eaal2736 (2018)). However, the function and cell type specificity of these soluble factors in human thymus development are not well understood. To define the signaling provided by human stromal cells, we evaluated the expression of soluble factors in the WNT, BMP, TGF-beta, IGF, and FGF signaling pathways. These have been described as important for regulating TEC development and function.(Lepletier,A.et al.Cell Rep 27,3887-3901.e4(2019), Balciunaite,G.et al.Nature Immunology 3,1102-1108(2002),Gordon,J.,Patel,SR,Mishina,Y.& Manley,NRDevelopmental Biology 339,141-154(2010), Patel,SR,Gordon,J.,Mahbub,F.,Blackburn,CC& Manley,NRGene Expression Patterns 6,794-799(2006),Bleul,CC& Boehm,TJImmunol.175,5213-5221(2005),Barsanti,M.et al.Eur.J.Immunol.47,291-304(2017), Swann,JB,Krauth,B.,Happe,C.& Boehm,T.Sci Rep 7,8492(2017), Tsai,PT,Lee,RA&Wu,H.Blood 102,3947-3953(2003). The results revealed that mesenchymal cells express many ligands and regulators of these important pathways, including WNT5A, RSPO3, SFRP2, IGF1, and FGF10 (Figure 2). Figure 2 in this specification is also described as Figure 1f in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6), which is incorporated herein by reference in its entirety. In particular, BMP4, FGF7 (also known as KGF), and the secreted WNT inhibitor Frizzled Related Protein FRZB are expressed more frequently in postnatal and adult mesenchymal cells compared to fetal mesenchymal cells, suggesting that TEC differentiation and proliferation are differently regulated over time by mesenchymal factors.

[0325] Most endothelial cells expressed TGFB1 and TGFBR2, but arterial and lymphatic subsets had high levels of chemokines known to promote the homing of hematopoietic progenitor cells to the thymus (CXCL12 or CCL21), suggesting that these chemokines are secreted by endothelial cells and, for example, regulate the migration of hematopoietic progenitor cells. Cell subsets also expressed WNT2B, WNT5A, RSPO3, BMP4, and IGF1. Endothelial cells also expressed extracellular matrix and adhesion molecules such as fibronectin (FN1) and LGALS3 (Figure 2).

[0326] Epithelial cells and mesothelial cells were enriched with BMP7 and many WNT ligands (WNT4, WNT5A, WNT6, WNT7B, WNT10A, WNT10B), and mesothelial cells also expressed many WNT signaling modulators (WNT2B, RSPO1, RSPO3, SFRP2, SFRP5) and BMP4.

[0327] Regarding pericytes, they expressed FRZB, as well as WNT6, BMP5, and FGF7. The gene encoding the activin A subunit (INHBA), recently shown to be important for TEC differentiation, was expressed almost exclusively by pericytes. In contrast, the activin antagonist follistatin (FST) (Lepletier, A. et al. Cell Rep 27, 3887-3901.e4 (2019)), which promotes TEPC maintenance and inhibits differentiation, was found primarily in subsets of adult mesenchymal and epithelial cells.

[0328] Therefore, human thymic mesenchymal cells, endothelial cells, and pericytes express many factors important for TEC development. These results reveal a previously unappreciated role of pericytes in regulating TEC differentiation through the secretion of activin signaling regulators. Overall, the results indicate that a variety of distinct cell types support the thymic microenvironment. Epithelial cells, mesenchymal cells, pericytes, endothelial cells, erythrocytes, immune cells, and mesothelial cells, including their subgroups, are present and support the human thymic microenvironment and / or express factors that support the human thymic microenvironment.

[0329] Example 2. Profiling of human thymic epithelial cells To further define heterogeneity within the epithelial compartment, three epithelial superclusters (Table 3, epithelium-1, -2, and -3) were divided into nine distinct subclusters (Table 5). [Table 5]

[0330] As used throughout this specification, reference to “low” or “lower” levels of marker gene expression in a cell subpopulation means that it is approximately 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times lower, or less than 5 times lower, than the average expression level of the marker gene in the entire cell population. For example, a cTEC-low subpopulation may have lower PSMB11 expression levels compared to the average PSMB11 levels of a cTEC-high subpopulation, meaning that a cTEC-low subpopulation has PSMB11 expression levels approximately 1.5 to 5 times lower than a cTEC-high subpopulation.

[0331] Similarly, throughout this specification, references to “high” or “higher” levels of marker gene expression in a cell subpopulation mean that these levels are approximately 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times higher than the average expression level of the marker gene in the entire cell population. For example, the mTEC-low subpopulation may have higher levels of CCL21 expression compared to the average CCL21 levels of other epithelial populations, meaning that the mTEC-low subpopulation has CCL21 expression levels approximately 1.5 to 5 times higher compared to other epithelial populations such as mTEC-high.

[0332] Subclusters were annotated based on combinations of known TEC markers and a list of differentially expressed genes including FOXN1, PAX9, SIX1, HLA-DQB1, PSMB11, CLDN4, CCL21, SPIB, AIRE, FEZF2, IVL, NEUROD1, MYOD1, MPZ, and MKI67. Two subclusters expressed genes characteristic of cTEC (PSMB11, PRSS16, CCL25). Cells in the cTEC-low subcluster expressed lower levels of functional genes (HLA class II, PSMB11, PRSS16, CCL25) and contained more KI67+ proliferating cells. Genes characteristic of mTECs were detected in three subclusters corresponding to mTEC-low (CLDN4, low levels of HLA class II), mTECs-high (SPIB, AIRE, FEZF2, high levels of HLA class II), and keratinocyte-like mTECs (KRT1, IVL). Cells within the mTEC-low subcluster expressed high levels of the chemokine CCL21, reminiscent of the CCL21-expressing mTEC-lo / jTEC population reported in mice (Lkhagvasuren, E., Sakata, M., Ohigashi, I. & Takahama, Y. The Journal of Immunology 190, 5110-5117 (2013), Onder, L. et al. Eur. J. Immunol. 45, 2218-2231 (2015), Miragaia, R. J et al. Sci Rep 8, 685 (2018), Michel, C. et al. The Journal of Immunology 199, 3488-3503 (2017)). One subcluster of cells was identified as immature TECs. These cells expressed standard TEC identity genes (FOXN1, PAX9, SIX1) but lacked functional genes characteristic of cTEC or mTEC. These immature cells were found in all samples and may represent progenitor cells not involved in any particular lineage or cells that have lost their differentiated phenotype. Three additional subclusters were identified as neuroendocrine (BEX1, NEUROD1), myoloid cells (MYOD1, DES), and myelin + epithelial cells (MPZ).

[0333] Example 3. Identification of a novel TEC marker Additional markers for the TEC subset were further analyzed, with a particular focus on immature TECs. ZBED2, a zinc finger protein transcription factor with no mouse counterpart recently linked to the maintenance of the basal state of human keratinocytes, was identified as a gene highly expressed in immature TECs and cTECs. Genes regulating TGF-β signaling, such as TDGF1 (also known as CRIPTO), CTGF, and IGF signaling regulators (IGFBP5 and IGFBP6), were also enriched within immature TECs and cTECs. Furthermore, the atypical cadherin gene (CDH13) was identified as a potentially useful cell surface marker for separating immature TECs from other TEC subsets in combination with cTEC surface markers. Two subpopulations expressing different markers (immature TEC-1 and immature TEC-2) were identified (Figure 3). Figure 3 in this specification is also described as Figure 3c in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6), the entirety of which is incorporated herein by reference. Interestingly, the expression of several genes enriched with immature TEC-2 (IGFBP5, NNMT, MAOA, DPYS, FKBP5, GLUL) was significantly higher in adult cells compared to fetal and postnatal tissues.

[0334] While the expression of many cytokeratins has been widely studied and used as markers for specific TEC subsets, the expression of KRT15 in the thymus has not been reported until now. This cytokeratin is particularly interesting because it is found in hair follicles, esophageal epithelium, and pluripotent progenitor cell populations of the small intestine (Lyle, S. et al. J. Cell. Sci. 111 (Pt 21), 3179-3188 (1998), Giroux, V. et al. J. Clin. Invest. 127, 2378-2391 (2017), Giroux, V. et al. Stem Cell Reports 10, 1947-1958 (2018)). KRT15 was highly expressed in mTEC-low cells, but was also detected in immature TECs, and its expression increased over time. Immunofluorescence revealed that KRT8+ / KRT5+ cells (potentially indicating immature TECs) found in the corticomedulosine junction expressed low levels of KRT15, ​​while KRT15-high cells were observed in the medulla, co-expressing KRT5 and likely indicating low CCL21+mTEC levels (data not shown). Most TECs isolated from adult tissue expressed combinations of KRT8, KRT5, and KRT15.

[0335] In addition to immature TEC markers, genes enriched with mTEC-low (GABRA5, LYPD1), mTEC-high (CLEC7A, MARCO, FXYD2, FXYD3, IL4I1, CHI3L1, CD70 / CD27L, TNFRSF9), or keratinocyte-like mTEC (FXYD3, IL1RN, LYPD2) were identified (Figure 4). Figure 4 herein is also described as Figure 4a in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6), which is incorporated herein by reference in its entirety. In particular, some of the genes that indicate AIRE+ cells encode cell surface proteins that may allow for the selection of this population from the human thymus. The preneurial basic helix-loop-helix (bHLH) transcription factor Achaete-scute complex 1 (ASCL1) was also enriched in cTEC-high, mTEC-low, AIRE+mTEC-high subsets, as well as in multiple epithelial subsets including neuroendocrine cells (data not shown). The role of this chromatin remodeling factor is well characterized in the brain, where it is expressed during the division of neural progenitor cells and promotes the proliferation, specialization, and differentiation of neural progenitor cells into neurons (Bertrand, N., Castro, DS & Guillemot, F. Nat. Rev. Neurosci. 3, 517-530 (2002), Castro, D. Set al. Genes & Development 25, 930-945 (2011)). ASCL1 has also been shown to play a role in the development of neuroendocrine cells in the lung (Borges, M. et al. Nature 386, 852-855 (1997)). ASCL1 expression in the fetal and postnatal thymic medulla was confirmed by immunofluorescence, but expression was also observed in the fetal cortex, not just in postnatal tissue (data not shown). Co-expression of ASCL1 and KRT15 was detected in a subset of medullary cells, likely representing low CCL21+mTEC-, while KRT15-negative cells may represent high AIRE+mTEC-. The presence of cells co-expressing ASCL1 and AIRE was confirmed by immunofluorescence staining (data not shown).ASCL1 target genes such as INSM1, DLL3, HES6, ST3GAL5, LYPD1, and POU4F1 were detected by TEC, suggesting ASCL1 activity rather than expression as part of an indiscriminate gene expression program.

[0336] To evaluate whether the ASCL1 transcription factor also marks the pool of thymic progenitor cells, in vivo gene lineage tracking experiments were performed using the fluorescent reporter system Ascl1creERT2 (Rosa26CAGstopflox-tdTomato). In this model, tamoxifen treatment permanently labels ASCL1-expressing cells and their offspring, allowing for differentiation between long-lived progenitor cells (labeled cells are seen long after Cre induction) and transit-amplified cells (reporter-expressing cells decrease over time). These mice were also crossed with the Aire GFP reporter line (Gardner, J Met al. Science 321, 843-847 (2008)) to facilitate quantification of Aire expression by flow cytometry. Mice were injected with a single dose of tamoxifen, and thymuses were harvested 36 hours and 5 weeks post-tamoxifen to analyze fluorescent reporter expression. 19% of TECs were labeled with Ascl1 strain traces after 36 hours, but this number decreased to 5% after 5 weeks. The proportion of Aire-expressing cells labeled with Ascl1 strain traces also decreased over time, suggesting that cells maintaining the Aire+mTEC-high pool do not express Ascl1. Therefore, these data suggest that Ascl1+mTEC-low and Ascl1+mTEC-high cells are likely to be replenished from the Ascl1-negative cell pool.

[0337] Example 4. Lineage determination within the thymic epithelial compartment To better understand the relationships between different epithelial subsets, we performed a pseudo-temporal analysis using RNA velocity (Yano, M. et al. Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance. Journal of Experimental Medicine 205, 2827-2838 (2008)). Considering both spliced ​​and unspliced ​​mRNA counts, this method, which estimates how mRNA levels change over time, can be used to predict the potential direction of transitions between cellular states. Because mouse TEC development differs significantly between embryonic and postnatal tissues, fetal, postnatal, and adult samples were analyzed separately. The analysis showed that low cTECs resulted in high cTECs in both fetal and postnatal tissues. The analysis also suggested that low cTECs may lead to immature TECs in fetal samples, but in postnatal tissues, this relationship appears to be reversed, with immature TECs reversing to low cTECs. Immature TECs were recovered from adult tissues, while cTECs were barely detectable. This suggests that the accumulation of immature TECs impairs functional cTECs in older tissues. This analysis predicts that in fetal tissues, high AIRE+mTECs precede both low CCL21+mTECs and low keratinocyte-like mTECs, while high AIRE+mTECs appear to lead to keratinocyte-like mTECs only in postnatal tissues. Therefore, low CCL21+mTECs may arise from AIRE+mTECs in fetal tissues.

[0338] Analysis confirmed that the epithelial compartment is composed of many different subsets. Analysis of differentially expressed genes across all epithelial clusters confirmed that the Notch target gene HES1 and the Notch inhibitor HES6 were enriched in mTEC-low and neuroendocrine / myosoidal cell clusters, respectively. Considering the crucial role of Notch signaling in regulating lineage selection in other tissues, the expression of genes involved in this pathway, including ligands, receptors, target genes, and inhibitors, was investigated. DLL4 and JAG2, key ligands that promote Notch1-dependent T cell specialization and maturation, were major ligands expressed in cTECs, while JAG1 was detected in immature TECs and mTECs (Figure 5). Figure 5 herein is also described as Figure 5b in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6, the entire figure of which is incorporated herein by reference). Importantly, three of the receptors (NOTCH1, NOTCH2, NOTCH3) and some of their target genes (HES1, HES2, HES4, HEY1, and NRARP) were expressed at higher levels in mTECs, indicating that Notch signaling is more active in mTECs than in cTECs. The Notch inhibitor DLK1 was also detected in cTECs, while DLK2 was detected in immature TECs. This suggests that Notch signaling is reduced in these cells. Finally, the expression of HES6, a negative regulator of HES1, was considerably higher in neuroendocrine and myoioid cells, indicating that Notch activity is actively blocked in these epithelial subsets. These results suggest that, in addition to its crucial role in T cell specificity, the regulation of Notch signaling influences the cell fate outcomes of epithelial compartments. Therefore, factors of the Notch signaling pathway can be used to direct and / or confirm thymocyte differentiation.

[0339] To further define TEC specificity, we analyzed pathways that have been shown to regulate the development of CCL21+mTEC-low and AIRE+mTEC-high in mice. Maintenance of mouse AIRE+mTEC-high cells is mediated by TNF receptor superfamily signaling, including NF-κB (RANK), CD4039-42, and osteoclast differentiation inhibitor (OPG), which acts as a decoy receptor for RANKL43, while CCL21+mTEC-low is dependent on LTBR signaling. Expression of subsets of these receptors was detected in CCL21+mTEC-low and keratinocyte-like mTEC (LTBR), mTEC-high (RANK / TNFRSF11A, OPG / TNFRSF11B), or mTEC-low and mTEC-high (CD40), suggesting that similar pathways may control the differentiation of human mTEC-high cells. Interestingly, other TNF receptors were also found in mTECs-high (TNFRSF4(OX40) and TNFRSF9(4-1BB)), while several TNF ligands were found in mTECs-low (LTB) or mTECs-high (LTB and CD70). Given the importance of LTBR signaling in the development of CCL21+mTECs, FEZF2+mTECs44, and keratinocyte-like mTECs, the observation that mTECs-low and mTECs-high express ligand LTB suggests that, in addition to signaling from thymocytes, the mTEC compartment itself can regulate the differentiation of these TEC subsets. In summary, the analysis revealed a novel regulator of cell fate commitment in the epithelial compartment. Tuft cells, ionocytes, and ciliated cells are present in the human thymic medulla.

[0340] Genes associated with p53 signaling (PERP, SFN, CTSD, CDKN2A, 302CDKN2B) (Figure 6), and many TNF superfamily members (Figure 7, upper panel) and Toll-like receptors (TLR1-6, TLR10) (Figure 7, lower panel) were identified as being upregulated in keratinocyte-like mTECs. Figure 6 herein is also described as Figure 5e in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (https: / / doi.org / 10.1038 / s41467-021-21346-6), the entirety of which is incorporated herein by reference). The upper and lower panels of Figure 7 in this specification are also described in Bautista, JL, et al. Nat Commun 12, 1096 (2021) (also described as Figures 5d and 5f in https: / / doi.org / 10.1038 / s41467-021-21346-6, the contents of which are incorporated herein by reference in their entirety). The data suggest a keratinocyte-like / post-AIRE mTEC subset with high levels of p53 activity dependent on the p53 signaling pathway. With respect to Toll-like receptors, this pathway may regulate the differentiation of mTEC-high cells into involucrin-+ post-AIRE cells. In summary, the findings reveal important information about the processes of cell fate determination in the epithelial compartment, and the factors involved in these pathways may be used to direct and / or confirm thymocyte differentiation.

[0341] To better understand the heterogeneity of the medullary compartment, characterize rare medullary epithelial subsets, and gain insights into the relationship between mTECs and other epithelial subsets, mTECs, neuroendocrine cells, myoiform cells, and myelin-expressing cells were reclustered and eight clusters were obtained with increased resolution. The newly identified subclusters are shown in Table 6. [Table 6]

[0342] A previously unreported population of ciliated cells (positive for ATOH1, GFI1, LHX3, and FOXJ1) was identified. Myelin-positive cells were found to closely resemble Schwann cells (SOX10, MPZ, MBP, and S100A1). Clusters containing cells with characteristics typical of chemosensible tuft cells (GNB3, TRPM5, GNAT3, PLCB2, OVOL3, and POU2F3) recently identified in mouse thymus were also identified (see Miller, CN et al. Nature 559, 627-631 (2018) and Bornstein, C. et al. Nature 559, 622-626 (2018)). Furthermore, cells with characteristics reminiscent of ionocyte populations present in lung tissue (FOXI1, ASCL3, CFTR, CLCNKB) were identified (see Montoro, DT et al. Nature 560, 319-324 (2018), Plasschaert, LW et al. Nature 560, 377-381 (2018)). Ciliated cells and ionocytes were found in all samples, but myelin+ was detected only in fetal and adult samples. The presence of ionocytes in the thymus was confirmed by immunofluorescence against KRT8+ / CFTR+ cells in the human thymic medulla of both fetal and postnatal tissues (data not shown). Ionocytes were found as isolated cells within the medulla or as part of Hassall's bodies. In addition to ionocytes and thymic tuft cells, subsets of neuroendocrine cells and myosophical cells were also detected in close proximity to Hassall's bodies (data not shown). Therefore, these data indicate that Hassall bodies have a more heterogeneous and complex structure than previously recognized. Analysis of differentially expressed genes among medullary cells confirmed that the transcription factor SOX2 is highly expressed in ciliated cells. Furthermore, SOX2 expression was detected in mTECs such as keratinocytes, neuroendocrine cells, and myelin+ cells.

[0343] The role of this transcription factor in the postnatal thymus has not been reported. Immunofluorescence analysis confirmed that SOX2 is expressed in Hassall bodies and a small number of isolated cells scattered throughout the medulla (data not shown). SOX2 staining was observed in KRT8+ cells and cells expressing KRT5 and / or KRT10, confirming expression in different subsets of medullary epithelial cells.

[0344] In summary, single-cell profiling of medullary epithelial cells revealed the existence of a new cell type in the thymic medulla, further highlighting the complexity of human thymic stroma cells.

[0345] Example 5. Characterization of tissue-specific antigen expression by human TEC. While it is clear that loss-of-function mutations in the human AIRE gene cause multi-organ autoimmune diseases known as autoimmune polyendocrine candidiasis ectodermal dystrophy (APECED) or autoimmune polyglandular syndrome type 1 (APS-1) (Finnish-German APECED Consortium. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat. Genet. 17, 399-403 (1997), Nagamine, K. et al. Nat. Genet. 17, 393-398 (1997)), direct evidence of TSA expression in human AIRE+mTEC is lacking. To assess whether AIRE+mTEC cells express higher levels of TSA than other epithelial subsets, TSA scores were calculated by averaging the expression of a list of tissue-specific genes and subtracting the mean expression of a reference set of genes (summarized by Sansom, SNet al. Genome Res. 24, 1918-1931 (2014)). Results were visualized using UMAP (data not shown). This analysis confirmed that TSA expression is particularly enriched in human AIRE+mTEC-high cells compared to other epithelial subsets. Using a similar approach, the expression of antigens known to induce autoantibodies in APS-1 patients was analyzed (Constantine, GM & Lionakis, MSImmunol. Rev. 287, 103-120 (2019)), which are predicted to be human AIRE-dependent genes. This analysis confirmed that APS-1-related genes are enriched in AIRE+mTEC cells. Furthermore, visualization of individual genes using UMAP revealed which antigens were most frequently detected in AIRE+ / post-AIRE keratinocyte-like mTECs (IL6, IL17F, IL22, CASR, TG, TPO, BPIFB1, DDC, TPH1, ATP4A, ATP4B, HDC, TGM4, TH).In contrast, other genes were primarily expressed in neuroendocrine clusters or myelin+ clusters (DEFA5, SOX10, MPZ). Therefore, this analysis provides new evidence that AIRE-expressing cells play a crucial role in the development of APECED / APS-1 disease.

[0346] To better understand the role of TEC subsets in inducing immune tolerance in humans, we analyzed the expression of antigens that have been shown to play a role in organ-specific autoimmune diseases. Antigens associated with type 1 diabetes (T1D), such as insulin (INS) and IAPP, were rarely detected in AIRE+ / post-AIRE cells, while IA-2 (PTPRN) was detected in a small number of neuroendocrine cells. Therefore, these data support the possibility that insulin expression in the human thymus, as in the mouse thymus, is AIRE-dependent. In contrast, genes encoding acetylcholine receptor (CHRNA1), muscle antigen titin (TTN), and MUSK, associated with the neuromuscular autoimmune disease myasthenia gravis, were found primarily in subsets of neuroendocrine and myosoidal cells (data not shown). These data indicate that different sources of disease-associated antigens exist in the human thymic medulla, increasing the likelihood that multiple subsets of epithelial cells are involved in inducing immune tolerance.

[0347] Example 6. Transplantation of reassembled thymus Purified MTS24+ embryonic TEC (R2 fraction) was reassembled and transplanted subcapsularly into the kidney capsule. After 8 weeks, the cells formed a structured, functional thymus with clearly separated cortical and medullary regions (MTS10+) and blood vessels (CD31). See Gill, Jason, et al. “Generation of a complete thymic microenvironment by MTS24+ thymic epithelial cells.” Nature immunology 3.7(2002):635-642, which is incorporated herein by reference in its entirety.

[0348] Purified CD205+ / CD40-embryonic TEC cells were reassembled and transplanted subcapsularly into the kidney capsule. After 6–8 weeks, the cells formed a structured thymus expressing Aire, with clearly separated cortical and medullary (ERTR5) regions. See Baik, Song, et al. “Generation of both cortical and Aire+ medullary thymic epithelial compartments from CD205+ progenitor cells.” European Journal of Immunology 43.3(2013):589–594, the entire report of which is incorporated herein by reference. Purified CD205+CD40-E15TEC (typically 1 × 10⁶ 5 Reassembled thymic organ cultures (RTOCs) were prepared using cells. Intact RTOCs were transplanted subcapsularly into the kidneys of wild-type mice and harvested after 6–8 weeks. RTOCs were prepared in a 1:1 ratio from a mixture of thymic stroma in the presence of 2'-deoxyguanosine (dGuo) and CD4+8+ thymocytes or CD4+3-LTi cells.

[0349] Prior to kidney capsule transplantation, cells from different populations were sorted and injected into the fetal thymus. Four weeks later, the sorted cells (GFP+) co-expressed markers for cortical TEC (DEC205) and mTEC (Aire). See Kahlia, et al. “Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus.” Cell reports 8.4(2014):1198-1209, which is incorporated herein by reference in its entirety. RTOC was performed as recently described in Seech, Natalie, et al. “Double positive thymocytes select mucosal-associated invariant T cells.” The Journal of Immunology 191.12(2013):6002-6009, which is incorporated herein by reference in its entirety. In short, E14.5 thymic lobes were enzymatically digested and reassembled with a GFP+ adult TEC subset in a 5:1 ratio. Typically, 7.5 × 10^4 to 1 × 10^5 adult TECs were added per RTOC. The reassembled tissue was incubated overnight (18 hours) at 37C (5% CO2) before transplantation under the kidney capsule of B6 adult male recipients. Grafts were harvested between 1 and 12 weeks and either enzymatically digested for FACS analysis or flash-frozen for immunofluorescence (IF).

[0350] Example 7. Implantation of hESC-derived TEP into NSG mice Luciferase-enhanced hESCs (e.g., MEL1-INS-GFP or MEL1-FOXN1-GFP cells) were differentiated into TEPs on Matrigel according to the protocol described above and subsequently transplanted subcapsularly into the kidneys of NSG mice. hESC survival was quantified over time in mice using bioluminescence. Kidneys were harvested after 4–6 weeks. TEPs showed good viability in vivo after 4–6 weeks. Overgrowth of non-epithelial cell types was observed.

[0351] Example 8. Enrichment and reassembly of EPCAM+ cells To reduce the overgrowth of non-epithelial cells in TEP transplanted in vivo, EPCAM+ cells were enriched and reassembled before transplantation. EPCAM+ cells were enriched using magnetic bead-based purification. The purified cell population was identified by detecting the TEP cell identification marker FOXN1. Reassembly was achieved in two ways: using an air-to-liquid approach, which involved culturing cells on a porous membrane covering liquid growth medium, and using AggreWell plates.

[0352] Example 9. Incorporation of other cell types into TEP reassemblies To reproduce the complexity of the thymic microenvironment in TEP reassemblies, different types of supporting cells found in the human thymic microenvironment were identified using single-cell RNA sequencing (scRNA-seq). Fetal and pediatric thymic tissues were compared. Fetal or postnatal human thymic tissue was enzymatically digested, and CD45+ cells were depleted using magnetically activated cell sorting (MACS). Single-cell RNA sequencing was performed on non-immune cells, and cell types based on marker gene expression were determined in human fetal thymuses at 19 and 23 weeks of gestation, and in human thymuses at 8 days and 10 months of postnatal age. Identified cell types included lymphatic endothelium, vascular endothelium, epithelium (including cTECs and mTECs), immune cells, mesenchymal cells, pericytes, and erythrocytes. A roadmap of human thymic development was depicted from the RNA-seq output as follows: From cTEC-lineage cells, pluripotent TEP cells were derived; from pluripotent TEP cells, undifferentiated mTEC progenitor cells; from undifferentiated mTEC progenitor cells, immature mTEC cells; from immature mTEC cells, mature TEC cells; and from mature TEC cells, post-Aire mTEC cells. Tuft cells and other TEC subsets were identified.

[0353] Different cell combinations were reassembled using hESC-TEP and transplanted into humanized mice according to the protocol outlined above. In short, purified EPCAM+ cells were reassembled with other supporting cell types (e.g., fibroblasts, endothelial cells, immune cells). Thymus-deficient NSG-FOXN1 null or thymectomy mice were subjected to sublethal radiation and injection of human CD34+ human stem cells. The reassembled cells were transplanted subcapsularly into the kidney. Mice were monitored for the presence of human T cells in peripheral blood. Grafts were collected and analyzed for the presence of human thymocytes and TECs by flow cytometry and immunofluorescence.

[0354] Example 10. In vitro culture of thymic epithelial cells from pluripotent stem cells Thymic epithelial cells are cultured in vitro according to the protocols described above, as well as in Table 2 and Figure 2. Briefly, on day 1, pluripotent stem (PS) cells are cultured in RPMI medium containing 0.2% KSR / FBS in the presence of activin A (approximately 100 ng / ml) and Wnt3a (approximately 50 ng / ml). On days 2-3, the medium is replaced with RPMI containing 0.2% KSR / FBS, activin A (approximately 100 ng / ml), and ITS (1:1000 dilution). On day 4, the medium is replaced with RPMI containing B27 (0.5X), activin A (approximately 100 ng / ml), retinoic acid (approximately 0.25 μM), and LDN (approximately 250 nM). On days 5-6, replace the culture medium with DMEM medium containing B27 (0.5X), BMP4 (approx. 50 ng / ml), retinoic acid (approx. 0.25 μM), FGF8 (approx. 50 ng / ml), TGFβi IV (approx. 2.5 μM), IWP2 (approx. 5 μM), and ITS (1:1000). On days 7-12, replace the culture medium with DMEM medium containing B27 (0.5X), BMP4 (approx. 50 ng / ml), FGF8 (approx. 50 ng / ml), TGFβi IV (approx. 2.5 μM), and ITS (1:200). From day 13 onward, the culture medium is replaced with DMEM medium containing B27 (0.5X), KGF (approximately 50 ng / ml), ITS (1:200), heparin (approximately 10 ug / ml), hydrocortisone (approximately 0.5 ug / ml), and T3 (approximately 200 nM).

[0355] Cells are analyzed by FACS or MACS at one or more stages of differentiation to evaluate, sort, and / or enrich based on the expression of one or more marker genes.

[0356] Equivalents and range Those skilled in the art can recognize or confirm, by ordinary experimentation alone, many equivalents to the specific embodiments of the present invention described herein. The scope of the present invention is not limited to the above specification, but is as set forth in the appended claims.

[0357] In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or made clear from the context. Claims or specifications containing “or” between one or more members of a group are considered satisfied if one, two or more, or all of the group members are present in, used in, or otherwise related to a given product or process, unless otherwise specified in a contradictory or otherwise clearly not made clear from the context. The present invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise related to a given product or process. The present invention includes embodiments in which multiple, or all, group members are present in, used in, or otherwise related to a given product or process.

[0358] It should also be noted that the term “contains” is intended to be open and allows for additional elements or steps, but does not require their inclusion. Therefore, wherever the term “contains” is used herein, the term “consisting of” is also encompassed and disclosed.

[0359] If a range is specified, it includes the endpoint. Furthermore, unless otherwise indicated, or unless it is obvious from the context and the understanding of those skilled in the art, the values ​​expressed as a range may be any specific value or subrange within the range described in different embodiments of the invention, up to one-tenth of the lower limit unit of the range, unless the context clearly indicates otherwise.

[0360] Furthermore, it should be understood that any particular embodiment of the present invention that falls within the scope of the prior art may be expressly excluded from any one or more claims. Such embodiments are considered to be known to those skilled in the art, and therefore may be excluded even if the exclusion is not expressly stated herein. Any particular embodiment of the composition of the present invention (e.g., any antibiotic, therapeutic or active ingredient, any manufacturing method, any method of use, etc.) may be excluded from any one or more claims for any reason whatsoever, whether or not it relates to the existence of the prior art.

[0361] It should be understood that the terms used are descriptive, not restrictive, and can be modified within the scope of the appended claims without departing from the true scope and intent of the invention in a broader aspect.

[0362] This disclosure includes the following embodiments. Embodiment 1 A method for generating thymic epithelial progenitor (TEP) cells in vitro, The cell population is cultured in a first medium containing an activator of bone morphogenetic protein (BMP) signaling, an activator of fibroblast growth factor (FGF) signaling, and an inhibitor of transforming growth factor-β (TGF-β) signaling. A method comprising further culturing the cell population to induce further maturation of the TEP cells in vitro, wherein the further culturing comprises culturing the cell population in a second medium containing keratinocyte growth factor (KGF), heparin, and hydrocortisone. Embodiment 2 The method according to Embodiment 1, wherein the second culture medium further comprises a triiodo-L-thyronine (T3) supplement. Embodiment 3 The method according to Embodiment 1 or 2, wherein the second culture medium further comprises an insulin-transferrin-selenium (ITS) supplement. Embodiment 4 The method according to any one of Embodiments 1 to 3, wherein the first and / or second culture medium further comprises a B27 supplement. Embodiment 5 The method according to any one of Embodiments 1 to 4, wherein the cell population comprises one or more of the following: endoderm (DE) cells, anterior foregut endoderm (AFE) cells, ventral pharyngeal endoderm (VPE) cells, and TEP cells. Embodiment 6 The method according to any one of Embodiments 1 to 4, wherein the further culture of the cell population for inducing further maturation of the TEP cells in vitro comprises further culturing the cell population for up to 14 days. Embodiment 7 The method according to any one of Embodiments 1 to 6, wherein the further culture of the cell population for inducing further maturation of the TEP cells in vitro comprises transferring the cell population to an extracellular matrix-based medium such as Matrigel. Embodiment 8 The method according to any one of Embodiments 1 to 6, wherein the first and / or second culture medium is a liquid culture medium and the culture conditions include suspension culture. Embodiment 9 The method according to Embodiment 8, wherein the first and / or second culture medium is minimal essential medium or Dulbecco's minimal essential medium (DMEM). Embodiment 10 The method according to any one of Embodiments 1 to 9, further comprising transplanting the TEP cells, wherein the further culture of the cell population to induce further maturation of the TEP cells in vitro results in the formation of a subpopulation of thymic epithelial cells (TECs) after in vivo transplantation. Embodiment 11 The method according to Embodiment 10, wherein the subpopulation of TECs includes one or more of the following: cortical TEC (cTEC) lineage cells, pluripotent TEP cells, undifferentiated medullary TEC (mTEC) progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells. Embodiment 12 The method according to any one of Embodiments 1 to 11, further comprising transferring the cell population to a gas-liquid interface culture system before transplantation. Embodiment 13 The method according to any one of embodiments 1 to 12, further comprising reassembling the cells before transplantation to form a reassembly. Embodiment 14 The method according to any one of embodiments 1 to 13, further comprising reducing or eliminating non-epithelial cells from a culture of differentiated TEP cells. Embodiment 15 The method according to Embodiment 14, comprising enriching EPCAM+ TEP cells. Embodiment 16 The method according to any one of Embodiments 1 to 15, comprising adjusting culture conditions or combining cell types during culture to reproduce the thymic microenvironment. Embodiment 17 The method according to Embodiment 16, comprising culturing TEP cells under conditions sufficient to support the survival of lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof, in order to recreate the thymic microenvironment. Embodiment 18 The method according to Embodiment 16, comprising culturing TEP cells under conditions sufficient to support the differentiation of TECs and their subpopulations, thereby recreating the thymic microenvironment. Embodiment 19 The method according to Embodiment 18, wherein the TEC and its subpopulations include cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells. Embodiment 20 The method according to any one of Embodiments 1 to 19, wherein the method comprises culturing endoderm (DE) cells in the first medium containing a retinoic acid receptor activator, a BMP signaling activator, an FGF signaling activator, and a TGF-β signaling inhibitor. Embodiment 21 The method according to Embodiment 20, wherein the DE cells are differentiated in a first cell culture medium containing a BMP signaling inhibitor before the first medium containing the BMP signaling activator. Embodiment 22 The method according to Embodiment 20 or 21, wherein a Wnt signaling inhibitor is introduced into the first culture medium. Embodiment 23 The method according to any one of Embodiments 20 to 22, wherein the method comprises culturing anterior foregut endoderm (AFE) cells produced by the culture of the DE cells, wherein the culture of the AFE cells is carried out in the first medium containing a BMP signaling activator, a FGF signaling activator, and a TGF-β signaling inhibitor. Embodiment 24 The method according to Embodiment 23, wherein the method comprises culturing ventral pharyngeal endoderm (VPE) cells produced by the culture of the AFE cells, and the culture of the VPE cells is carried out in the first medium containing a BMP signaling activator, an FGF signaling activator, and a TGF-β signaling inhibitor. Embodiment 25 The method according to Embodiment 23 or 24, wherein the first culture medium is not supplemented with retinoic acid receptor signaling activators. Embodiment 26 The method according to any one of Embodiments 1 to 25, wherein the initiating cells are obtained from pluripotent stem (PS) cells. Embodiment 27 The method according to Embodiment 26, wherein the PS cells are embryonic stem cells, embryonic germ cells, or induced pluripotent stem (iPS) cells. Embodiment 28 The method according to Embodiment 26 or 27, wherein the PS cells are primate pluripotent stem cell (pPS) cells. Embodiment 29 The method according to any one of embodiments 26 to 28, wherein the PS cells are human pluripotent stem (hPS) cells. Embodiment 30 A method for generating TEP cells in vitro, comprising culturing a cell population including anterior foregut endoderm (AFE) cells in a first cell culture medium containing a BMP signaling activator, an FGF signaling activator, and a TGF-β signaling inhibitor, thereby producing TEP cells. Embodiment 31 The method according to Embodiment 30, wherein the first cell culture medium substantially does not contain at least one of the activators of retinoic acid receptor signaling, activators of Wnt signaling, and inhibitors of Hedgehog signaling. Embodiment 32 A method for generating TEP cells in vitro, comprising: culturing a cell population including endoderm (DE) cells in a first cell culture medium containing a BMP signaling activator, a retinoic acid receptor signaling activator, a FGF signaling activator, a TGF-β signaling inhibitor, and a Wnt signaling inhibitor to produce a cell population including AFE cells; and culturing the AFE cell population in a cell culture medium containing a BMP signaling activator, a FGF signaling activator, and a TGF-β signaling inhibitor, but substantially free of a Wnt signaling inhibitor, to produce TEP cells. Embodiment 33 The method according to any one of embodiments 30 to 32, wherein the first cell culture medium further comprises an insulin-transferrin-selenium (ITS) supplement. Embodiment 34 The method according to any one of embodiments 30 to 33, wherein the first cell culture medium further comprises B27. Embodiment 35 The method according to any one of embodiments 30 to 34, wherein the first cell culture medium comprises minimal essential medium or Dulbecco's minimal essential medium (DMEM). Embodiment 36 The method according to any one of embodiments 30 to 35, further comprising culturing the TEP cells in a second cell culture medium containing KGF and T3 under conditions sufficient to induce further differentiation of the TEP cells and generate a mature TEP cell population. Embodiment 37 A composition comprising a differentiated population of TEP cells produced according to the method described in any one of Embodiments 1 to 36. Embodiment 38 A composition comprising reassembled thymic epithelial progenitor (TEP) cells differentiated from PS cells, wherein the composition further comprises one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof. Embodiment 39 A composition comprising TEC differentiated from PS cells, further comprising one or more of the following: cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTEC, mature mTEC, post-AIRE mTEC, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells. Embodiment 40 A composition comprising reassembled thymic epithelial cells (TECs) differentiated from PS cells, further comprising one or more cell types selected from lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof. Embodiment 41 The composition according to Embodiment 40, wherein the reassembled TEC comprises a subpopulation including one or more of the following: cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells. Embodiment 42 a. Factors related to WNT signaling, b. Factors related to BMP signaling, c. Factors related to TGF beta signaling, d. Factors related to IGF signaling, e. Factors related to FGF signaling, f. Factors related to NOTCH signaling, g. TNF receptor, Factors related to h.p53 signaling, and / or i. Toll-like receptors The method according to any one of embodiments 1 to 29, 30 or 31, or 32 to 36, further comprising introducing one or more of the above into a culture medium to induce the development of one or more subpopulations of thymic epithelial cells. Embodiment 43 The method according to Embodiment 42, wherein the introduction into the culture medium includes introducing one or more soluble factors and / or introducing cells expressing one or more factors. Embodiment 44 The thymic microenvironment is a. Factors related to WNT signaling, b. Factors related to BMP signaling, c. Factors related to TGF beta signaling, d. Factors related to IGF signaling, e. Factors related to FGF signaling, f. Factors related to NOTCH signaling, g. TNF receptor, Factors related to h.p53 signaling, and / or i. Toll-like receptors The method according to any one of embodiments 16 to 18, characterized by the presence of cells expressing one or more of the following. Embodiment 45 a. Factors related to WNT signaling include WNT5A, WNT6, ROR1, ROR2, RYK, FRZB, RSPO1, RSPO3, SFRP2, and / or SFRP5. b. Factors related to BMP signaling include BMP4, BMP5, and / or FST, c. Factors related to TGF beta signaling include TGFB1, TGFBR2, CXCL12, and / or CCL21. d. Factors related to IGF signaling include IGF1R, e. Factors related to FGF signaling include FGFR2 and / or FGF7 / KGF, f. Factors associated with NOTCH signaling include NOTCH1, NOTCH2, NOTCH3, HES1, HES6, DLL4, JAG2, JAG1, HES2, HES4, HEY1, NRARP, DLK1, and / or DLK2. g. The TNF receptor comprises RANK / TNFRSF11A, CD40, LTBR, ​​TNFRSF4, TNFRSF9, LTB, and / or CD70. Factors related to h.p53 signaling include PERP, SFN, CTSD, CDKN2A, and / or CDKN2B. i. Toll-like receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, and / or TLR10. The method according to any one of embodiments 42 to 44. While the present invention has been described in some detail and to some extent with respect to several described embodiments, the present invention should not be limited to such details or embodiments or specific embodiments, but should be interpreted with respect to the appended claims in consideration of the prior art to provide the broadest possible interpretation of such claims and thus effectively encompass the intended scope of the present invention.

Claims

1. A method for generating mature thymic epithelial progenitor (mTEP) cells in vitro, This method includes inducing the maturation of a population of thymic epithelial progenitor (TEP) cells in vitro by culturing them in a culture medium containing keratinocyte growth factor (KGF), heparin, and hydrocortisone. The method for obtaining the TEP cell population is as follows: (i) differentiating a cell population including embryonic endoderm (DE) cells into anterior foregut endoderm (AFE) cells; (ii) differentiating the AFE cells into ventral pharyngeal endoderm (VPE) cells; and differentiating the VPE cells into TEP cells.

2. The method according to claim 1, wherein the culture medium further comprises a triiodo-L-thyronine (T3) supplement.

3. The method according to claim 1 or 2, wherein the culture medium further comprises an insulin-transferrin-selenium (ITS) supplement.

4. The method according to any one of claims 1 to 3, wherein the culture medium further comprises a B27 supplement.

5. The method according to any one of claims 1 to 4, wherein inducing the maturation of the TEP cells into mTEP cells in vitro by culturing the population of TEP cells comprises culturing the population of TEP cells for up to 14 days.

6. The method according to any one of claims 1 to 5, wherein inducing the maturation of the TEP cells into mTEP cells in vitro by culturing the population of TEP cells comprises transferring the population of TEP cells to an extracellular matrix-based medium.

7. The method according to any one of claims 1 to 5, wherein the culture medium is a liquid medium and the culture conditions include suspension culture.

8. The method according to claim 7, wherein the culture medium is minimal essential medium or Dulbecco's minimal essential medium (DMEM).

9. The method according to any one of claims 1 to 8, wherein the method is for producing a transplantation composition comprising the mTEP cells, the transplantation comprising transplanting the mTEP cells to a target, and inducing the maturation of the TEP cells to mTEP cells in vitro by culturing the population of TEP cells, thereby generating a population of thymic epithelial cells (TECs) after in vivo transplantation.

10. The method according to claim 9, wherein the population of TECs includes one or more of the following: cortical TEC (cTEC) lineage cells, pluripotent TEP cells, undifferentiated medullary TEC (mTEC) progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells.

11. The method according to any one of claims 1 to 10, further comprising transferring the population of mTEP cells to a gas-liquid interface culture system before transplantation.

12. The method according to any one of claims 1 to 11, further comprising reassembling the mTEP cells before transplantation to form a reassembly.

13. The method according to any one of claims 1 to 12, further comprising reducing or eliminating non-epithelial cells from a culture of differentiated mTEP cells.

14. The method according to claim 13, comprising enriching EPCAM+ TEP cells.

15. The method according to any one of claims 1 to 14, comprising adjusting culture conditions or combining cell types during culture in order to reproduce the thymic microenvironment.

16. The method according to claim 15, wherein recreating the thymic microenvironment involves culturing TEP cells under conditions sufficient to support the survival of lymphatic endothelial cells, vascular endothelial cells, immune cells, mesenchymal cells, pericytes, erythrocytes, or combinations thereof.

17. The method according to claim 15, comprising culturing mTEP cells under conditions sufficient to support the differentiation of a population of TECs, thereby recreating the thymic microenvironment.

18. The method according to claim 17, wherein the population of TECs includes cTEC lineage cells, pluripotent TEP cells, undifferentiated mTEC progenitor cells, immature mTECs, mature mTECs, post-AIRE mTECs, tuft cells, neuroendocrine cells, ionocytes, ciliated cells, myelin-expressing cells, and / or myosoidal cells.

19. The method according to any one of claims 1 to 18, wherein the method comprises culturing endoderm (DE) cells in a first medium containing a retinoic acid receptor activator, a BMP signaling activator, an FGF signaling activator, and a TGF-β signaling inhibitor.

20. The method according to any one of claims 1 to 19, wherein the cell population including the DE cells is obtained from pluripotent stem (PS) cells.

21. The method according to claim 20, wherein the PS cells are embryonic stem cells, embryonic germ cells, or induced pluripotent stem (iPS) cells.

22. The method according to claim 20 or 21, wherein the PS cells are primate pluripotent stem cells (pPS) cells.

23. The method according to any one of claims 20 to 22, wherein the PS cells are human pluripotent stem (hPS) cells.

24. a. Factors related to WNT signaling, b. Factors related to BMP signaling, c. Factors related to TGF-beta signaling, d. Factors related to IGF signaling, e. Factors related to FGF signaling, f. Factors related to NOTCH signaling, g. TNF receptor, h. Factors related to p53 signaling, and / or i. Toll-like receptors The method according to any one of claims 1 to 23, further comprising introducing one or more of the above into a culture medium to induce the development of one or more subpopulations of thymic epithelial cells.

25. The method according to claim 24, wherein the introduction into the culture medium includes introducing one or more soluble factors and / or introducing cells expressing one or more factors.

26. The thymic microenvironment is a. Factors related to WNT signaling, b. Factors related to BMP signaling, c. Factors related to TGF-beta signaling, d. Factors related to IGF signaling, e. Factors related to FGF signaling, f. Factors related to NOTCH signaling, g. TNF receptor, h. Factors related to p53 signaling, and / or i. Toll-like receptors The method according to any one of claims 15 to 17, characterized by the presence of cells expressing one or more of the following.

27. a. Factors related to WNT signaling include WNT5A, WNT6, ROR1, ROR2, RYK, FRZB, RSPO1, RSPO3, SFRP2, and / or SFRP5. b. Factors related to BMP signaling include BMP4, BMP5, and / or FST, c. Factors related to TGF-beta signaling include TGFB1, TGFBR2, CXCL12, and / or CCL21. d. Factors related to IGF signaling include IGF1R, e. Factors related to FGF signaling include FGFR2 and / or FGF7 / KGF, f. Factors related to NOTCH signaling include NOTCH1, NOTCH2, NOTCH3, HES1, HES6, DLL4, JAG2, JAG1, HES2, HES4, HEY1, NRARP, DLK1, and / or DLK2, g. The TNF receptor comprises RANK / TNFRSF11A, CD40, LTBR, ​​TNFRSF4, TNFRSF9, LTB, and / or CD70. h. Factors related to p53 signaling include PERP, SFN, CTSD, CDKN2A, and / or CDKN2B. i. Toll-like receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, and / or TLR10, The method according to any one of claims 24 to 26.

28. The method according to claim 6, wherein the extracellular matrix-based culture medium is Matrigel.

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