Thymocyte compositions and methods of use thereof

Abstract

The present disclosure provides a method for generating and maintaining thymocytes in vitro. The present disclosure also provides compositions and systems of cell populations comprising thymocytes. In one aspect, the present disclosure provides a method for generating a population of thymocytes in vitro by culturing a cell population in the presence of a soluble factor, a mineral, or a combination thereof to induce differentiation or maturation of the cell population into thymocytes, wherein the cell population is optionally engineered to express a cell surface receptor or an intracellular factor, and the cell population comprises one or more cell types selected from the group consisting of pluripotent stem cells (PSCs), definitive endoderm (DE) cells, third pharyngeal pouch endoderm (PPE) cells, and anterior foregut endoderm (AFE) cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 63 / 214,176, filed June 23, 2021, U.S. Provisional Application No. 63 / 256,445, filed October 15, 2021, U.S. Provisional Application No. 63 / 296,250, filed January 4, 2022, and U.S. Provisional Application No. 63 / 321,142, filed March 18, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background information The thymus is a major lymphoid organ that plays a central role in the immune system. The thymic microenvironment provides a unique training ground for the development and maturation of effector cells such as lymphocytes (e.g., T cells). As is well known in the art, complex interactions between thymocytes and effector cells can determine the phenotype and functionality of effector cells. Some thymocyte-effector cell interactions are regulated such that recognition of factors expressed by thymocytes promotes survival of effector cells. In contrast, other thymocyte-effector cell interactions can cause the death of effector cells. By controlling such interactions, the thymus plays a vital role in establishing a repertoire of effector cells that can mount an activated immune response against foreign invaders while establishing tolerance to self.

[0003] There remains a need for improved methods for generating thymocytes and thymocyte products, as well as enriched cell populations of functional thymocytes that can differentiate into functional thymic epithelial cells. Summary of the Invention

[0004] overview The present disclosure provides thymocyte products and methods for making the same. The thymic products of the present disclosure are prepared by (i) generating one or more populations of thymocytes, (ii) extracting and sequencing RNA from each of the populations of thymocytes, (iii) analyzing the RNA sequences to identify subpopulations within each population of thymocytes to generate test thymic maps for each of the populations of thymocytes, (iv) comparing each of the test thymic maps to a reference thymic map, and (v) identifying a thymocyte product by selecting test thymic maps having a subpopulation ratio (SPR) or relative cTEC ratio (RCTR) of about 0.7 to about 1.4. In some embodiments, the cell subpopulations within each thymocyte population are selected from immature thymic epithelial cells (iTEC), cTEC-high cells, cTEC-low cells, Aire+mTEC-high cells, mTEC-low cells, keratinocyte-like mTEC cells, ciliated cells, myelin cells, myoid cells, neuroendocrine cells, and / or tuft / ionocyte cells. In some embodiments, the SPR is about 0.7-1.4. In some embodiments, the SPR is iTEC SPR, cTEC-high SPR, cTEC-low SPR, Aire+mTEC-high SPR, mTEC-low SPR, keratinocyte-like mTEC SPR, ciliated SPR, myelin SPR, myoid SPR, neuroendocrine SPR, and / or tuft / ionocyte SPR. In some embodiments, the SPR is iTEC SPR. As a non-limiting example, the iTEC SPR is 1.

[0005] In some embodiments, the RCTR is about 0.7 to 1.14. As a non-limiting example, the RCTR is 1.

[0006] The thymocyte cell populations of the present disclosure used to prepare the thymocyte product are prepared by differentiation of stem cells.

[0007] In some embodiments, the reference thymus map is generated using a human thymus, for example an adult or fetal thymus.

[0008] In some embodiments, the iTEC subpopulation of the population of thymocytes is a subset of thymocytes that ... 4103), IFI16 (ENSG00000163565), FOXC1 (ENSG00000054598), STAT1 (ENSG00000115415), JUNB (ENSG00000171223), EGFR1, ZFP36 (ENSG00000128016), JUN (ENSG00000177606), FOSB (ENSG00000125740), IER2 (ENSG00000160888), PAX9 (ENSG00000198807), and / or HIF1A (ENSG00000100644).

[0009] In some embodiments, the cTEC subpopulation of the population of thymocytes is / are selected from the group consisting of PSMA3 (ENSG00000100567), FABP5 (ENSG00000164687), APRT (ENSG00000198931), LSM6 (ENSG00000164167), CTSV (ENSG00000136943), SNRPE (ENSG00000182004), ECHS1 (ENSG00000127884), HSPE1 (ENSG00000115541), RAN (ENSG00000132341), TMA7 (ENSG00000232112), TIMM13 (ENSG00000099800), LDHB (ENSG00000111716), ECI1 (ENSG00000111716), IL-1 (ENSG00000111716), IL-2 (ENSG00000111716), IL-3 (ENSG00000111716), IL-4 (ENSG00000111716), IL-5 (ENSG00000111716), IL-6 (ENSG00000111716), IL-8 (ENSG00000111716), IL-9 (ENSG00000111716), IL-11 (ENSG00000111716), IL-12 (ENSG00000111716), IL-13 (ENSG00000111716), IL-14 (ENSG00000111716), IL-15 (ENSG00000 SG00000167969), GCSH(ENSG00000140905), NOP58(ENSG00000055044), MRPL11(ENSG0000017 4547), STOML2 (ENSG00000165283), ING2 (ENSG00000168556), TOMM7 (ENSG00000196683), MRP It may include expression of one or more of S34 (ENSG00000074071), MRPL14 (ENSG00000180992), MRPL57 (ENSG00000173141), IMP3 (ENSG00000177971), MZT2A (ENSG00000173272), and XRCC6 (ENSG00000196419).

[0010] In some embodiments, the keratinocyte-like mTEC subpopulation of the population of thymocytes is a subpopulation of thymocytes that is characterized by the following transcription factors: CD24 (ENSG00000272398), ELF3 (ENSG00000163435), CLDN4 (ENSG00000189143), MAL2 (ENSG00000147676), ASAH1 (ENSG00000104763), TMEM123 (ENSG00000152558), TMBIM6 (ENSG00000139644), LGALS3 (ENSG00000131981-), MYL12B (ENSG0 0000118680), ACADVL(ENSG00000072778), KRT19(ENSG00000171345), SAT1(ENSG00000130066) , RAB25(ENSG00000132698), WFDC2(ENSG00000101443), VAMP8(ENSG00000118640), SPINT1(EN SG00000166145), SERPINB1 (ENSG00000021355), CDH1 (ENSG00000039068), GSN (ENSG000001481 80), SDC4 (ENSG00000124145), MGST2 (ENSG00000085871), CAST (ENSG00000153113), B4GALT1 (ENSG00000086062), PERP (ENSG00000112378), and DMKN (ENSG00000161249).

[0011] The present disclosure provides methods for generating thymocytes in vitro. These methods may include culturing a cell population in the presence of a soluble factor, a mineral, or a combination thereof. Such methods may include differentiation or maturation of the cell population into thymocytes. The cell population may also be engineered to express a cell surface receptor or an intracellular factor. In some embodiments, the cell population may be pluripotent stem cells (PSCs), definitive endoderm (DE) cells, third pharyngeal pouch endoderm (PPE) cells, anterior foregut endoderm (AFE) cells.

[0012] The thymocytes are one or more of immature thymic epithelial cells (iTEC), cTEC-high cells, cTEC-low cells, Aire+mTEC-high cells, mTEC-low cells, keratinocyte-like mTEC cells, ciliated cells, myelin cells, myoid cells, neuroendocrine cells, and tuft / ionocyte cells, or combinations thereof. The soluble factors include, but are not limited to, growth factors, cytokines, transporters, hormones, or combinations thereof. As a non-limiting example, the soluble factors are growth factors such as epidermal growth factor (EGF, ENSG00000138798). The soluble factors are cytokines, such as macrophage inhibitory factor (MIF, ENSG00000240972). The soluble factors may be transporters, such as lactotransferrin (LTF, ENSG00000012223).

[0013] In some embodiments, the cells of the present disclosure are cultured in the presence of a mineral. The mineral is iron, magnesium, calcium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, zinc, chloride, chromium, copper, fluoride, or iodine. In one aspect, the mineral is iron. The cells of the present disclosure are engineered to express a cell surface receptor or intracellular factor for the purpose of generating or maintaining thymocytes. The cell surface receptor is CD74 (ENSG00000019582), integrin beta 1 (ITGB1, ENSG00000150093), and / or epidermal growth factor receptor (EGFR, ENSG00000146648). The intracellular factor is ENO1 (ENSG00000074800).

[0014] The present disclosure also provides methods of maintaining a population of thymocytes in vitro, which may include (i) culturing the population of thymocytes in the presence of a soluble factor or mineral, (ii) culturing the population of thymocytes in the presence of one or more feeder cells, and / or (iii) engineering the population of thymocytes to express a cell surface receptor or an intracellular factor.

[0015] The cells of the present disclosure are cultured in the presence of one or more supporting cells. The supporting cells are endothelial cells, mesenchymal stem cells, macrophages, dendritic cells (DCs), epithelial cells, fibroblasts, stromal cells, adipocytes, fibroblasts, vascular smooth muscle cells (VSMCs), or lymphatic endothelial cells. In some embodiments, the supporting cells are mesenchymal stem cells. In some aspects, the supporting cells are endothelial cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description I. Introduction Thymic epithelial cells are important in T cell differentiation. Thymocytes prepared as described herein can allow the properties of thymus tissue and thymocytes, such as thymus-associated immune function, to be utilized for therapeutic applications. For example, it is known that the decline in immune function associated with aging is caused by changes in the composition and functional capacity of thymocytes. In addition, the thymus itself atrophies or shows senescence due to changes in sex hormones, including androgens and estrogens. The onset of thymic atrophy may begin with the onset of puberty. Thus, regeneration of thymic epithelial cells may provide compositions and methods for alleviating the decline in immune function associated with aging.

[0017] II. Composition cell The cells of the present disclosure may include, without limitation, thymocytes, effector cells, pluripotent stem cells, populations thereof, and cells derived therefrom. In some embodiments, the cells of the present disclosure may be derived from pluripotent stem cells. In some embodiments, the thymocytes may be derived from induced pluripotent stem cells.

[0018] In some embodiments, the cells of the disclosure may be autologous, allogeneic, syngeneic, or xenogeneic with respect to a particular individual or subject. In some embodiments, the thymocytes may be autologous, allogeneic, syngeneic, or xenogeneic with respect to the subject who will ultimately benefit from their clinical application. In some embodiments, the cells of the disclosure may be mammalian cells, particularly human cells. The cells may be primary cells or immortalized cell lines. In some aspects, the cells of the disclosure may be prepared or derived from a syngeneic cell source. Any of the cells described herein may be characterized by markers known in the art for the relevant cell type.

[0019] thymocyte The cells of the present disclosure may comprise a population of thymocytes. Thymocytes are cells that have one or more phenotypic or genotypic markers associated with cells derived from the thymus or cells destined to become cells of the thymus. In some embodiments, the population of thymocytes is derived from differentiation of pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. The thymocytes are embryonic, fetal, or adult thymocytes. In some embodiments, the composition of the present disclosure may be a population of thymocytes. In some aspects, the composition of the present disclosure is a thymocyte product.

[0020] In some embodiments, thymocytes are prepared by differentiation of pluripotent stem cells into thymic stem cells by one or more of the following steps: PSCs can be differentiated and / or induced to differentiate into cells resembling definitive endoderm (DE). Definitive endoderm cells can be differentiated and / or induced to differentiate into cells resembling third pharyngeal pouch endoderm (PPE). Definitive endoderm and / or PPE cells can be differentiated and / or induced to differentiate into cells resembling ventral pharyngeal endoderm (AFE). AFE can be differentiated and / or induced to differentiate into cells resembling third pharyngeal pouch endoderm (PPE). Thymic epithelial progenitor cells (TEPCs) can be generated from PPE cells. TECs can be derived from TEPCS. Each of the cell types described herein is characterized by one or more markers. In some embodiments, pluripotent stem cells are associated with increased expression of markers, such as but not limited to OCT4, SOX2, and / or Nanog. In some embodiments, definitive endoderm is associated with increased expression of markers, such as, but not limited to, SOX17, FOXA2, CXCR4, and / or CER1. In some embodiments, anterior foregut cells (AFE) are associated with increased expression of markers, such as, but not limited to, FOXA2, SOX2, and / or PAX9. In some embodiments, third pharyngeal pouch endoderm cells are associated with increased expression of markers, such as, but not limited to, HOXA3, TBX1, PAX9, EYA1, SIX1, PBX1, and / or PAX1. In some embodiments, thymic epithelial progenitor cells are associated with increased expression of markers, such as, but not limited to, FOXN1, K5, K8, and / or HOXA3. In some embodiments, thymocytes are derived from DE cells, third PPE cells, AFE cells, TEPCs, and / or TEC cells.

[0021] To prepare a population of thymocytes of the present disclosure, pluripotent stem cells are cultured and differentiated into definitive endoderm cells. The definitive endoderm cells are further cultured and differentiated into anterior foregut cells. In some embodiments, the anterior foregut cells are cultured and differentiated into pharyngeal endoderm cells. In some embodiments, the pharyngeal endoderm cells are cultured and differentiated into thymocytes, e.g., thymic epithelial cells. In some embodiments, differentiation is performed for about 14 days to about 21 days.

[0022] In some embodiments, the thymocytes are prepared from PSCs. In this regard, the method may include culturing the pluripotent stem cells for a period of time under conditions sufficient to differentiate the pluripotent stem cells into thymocytes. For example, the method may include culturing the pluripotent stem cells in the presence of factors and / or inhibitors that drive differentiation of the PSCs into thymocytes.

[0023] In some embodiments, the method for differentiating PSCs into thymocytes is any method known in the art. The method for differentiating PSCs into thymocytes may include the use of one or more parameters or combinations thereof for differentiation known in the art. The parameters include, but are not limited to, (i) differentiation factors, (ii) differentiation inhibitors, (iii) duration of differentiation, (iv) temperature, (v) substrate, and / or (vi) feeder cells that promote differentiation. Any method or parameters for differentiating PSCs into thymocytes described in the following references may be used herein, including, but not limited to, Parent et al. Cell Stem Cell. 2013 Aug 1;13(2):219-29; Soh et al. Stem Cell Rep. 2014 Vol. 2 j 925-937; Sun et al. Cell Stem Cell. 2013 Aug 1;13(2):230-6; Okabe et al. Cell. Reprog. 2015 Vol 17, No. 5; Su et al. Sci.Rep. 2015 5, 9882; Otsuka et al. Sci Rep2020 10:224, International Patent Publications WO2019060336, WO2020205859, WO2020220040, WO2014134213, WO2010143529, WO2011139628, WO2022076751, WO2014134213, WO2021222297, and Chinese Patent Publication CN201110121243, the entire contents of each of which are incorporated herein by reference.

[0024] In some embodiments, the population of thymocytes may include subpopulations of one or more of the following cell types: iTEC, mTEC, keratinocyte-like mTEC, cTEC-high, cTEC-low, mTEC-low cells.

[0025] In some embodiments, the population of thymocytes may include thymic epithelial cells (TECs). The thymocytes may be or be derived from TECs. In some embodiments, the TECs are derived from differentiation of iPSCs. During embryonic development, TECs are derived from non-hematopoietic cells that are negative for CD45 expression and positive for the epithelial marker EpCAM. The TECs are cortical thymic epithelial cells (cTECs) and / or medullary thymic epithelial cells (mTECs). mTECs are characterized by cytokeratin 5 (K5) and cytokeratin 14 (K14) expression, but have low levels of cytokeratin 8 (K8) expression, whereas cTECs express K8 and K18. In some embodiments, the thymocytes are derived from TECs that express both K5 and K8 (K5+K8+). In some aspects, the K5+K8+ cells are precursors of mTECs and / or cTECs. mTECs may also be positive for cell surface Ulex europaeus agglutinin-1 (UEA-1) expression, but not Ly51 (e.g., UEA-1+Ly51-), whereas cTECs are UEA-1-Ly51+. In some embodiments, thymocytes may be or be derived from mTECs.

[0026] In some embodiments, the population of thymocytes may include medullary thymic epithelial cells (mTECs). In some embodiments, mTECs are derived from differentiation of iPSCs. In some embodiments, mTECs may have high expression of markers, such as, but not limited to, cytokeratin 5, cytokeratin 14, UEA-1, CD80, cathepsin L, and / or cathepsin S.

[0027] In some embodiments, the population of thymocytes may include cortical thymic epithelial cells (cTECs). In some embodiments, the cTECs are derived from differentiation of iPSCs. In some embodiments, the thymocytes may be or be derived from cTECs with high expression of markers, such as but not limited to cytokeratin 8, cytokeratin 18, Ly51, CD205, cathepsin L, and / or thymus-specific serine proteases. As non-limiting examples, the thymocytes may be or be derived from cTECs expressing markers such as CCL25, and / or KRT5. The mTECs may express markers such as CCL19, KRT8, and / or AIRE.

[0028] In some embodiments, the thymocytes may be or be derived from TECs that express one or more markers, such as, for example, FOXN1, PAX9, PAX1, DLIA, ISL1, EYA1, SIX1, IL7, K5, K8, and AIRE.

[0029] Thymocytes can be or be derived from any of the cell types described in Park et al. 2020 Science Vol. 367, Issue 6480, the entire contents of which are incorporated herein by reference. For example, thymocytes can be derived from myoid cells, such as MYOD1 and MYOG-expressing myoid cells (referred to herein as TEC(myo)), and / or NEUROD1, SYP, CHGA-expressing TECs (referred to herein as TEC(neuro)).

[0030] The thymocytes are derived from the cell type described in Bautista et al. 2021 Nat Commun 12, 1096, the entire contents of which are incorporated herein by reference. In some embodiments, the population of thymocytes derived from differentiation of iPSCs is referred to as "cTEC" in Bautista et al. 2021. 低 " cells with lower levels of functional genes (HLA class II) and more KI67 +- Characterized by the inclusion of proliferating cells. Thymocytes are classified as "mTECs" as described by Bautista et al. 2021. 低 " cells and are characterized by expression of CLDN4, lower levels of HLA class II, PSMB11, PRSS16, CCL25, and high levels of the chemokine CCL21.

[0031] In some embodiments, the population of thymocytes derived from differentiation of iPSCs is referred to as "mTEC" as described in Bautista et al. 2021. 高 " cells, characterized by expression of SPIB, AIRE, FEZF2, and higher levels of HLA class II. Thymocytes may be or be derived from keratinocyte-like mTECs as described by Bautista et al. 2021, characterized by expression of KRT1, and / or IVL.

[0032] In some embodiments, the population of thymocytes derived from differentiation of iPSCs may include immature TECs (iTECs) as described by Bautista et al. 2021, which express canonical TEC identity genes, e.g., FOXN1, PAX9, SIX1.

[0033] In some embodiments, the population of thymocytes derived from differentiation of iPSCs may include TECs that express one or more markers, for example but not limited to KRT5, KRT8, AIRE, PSMB11, and / or PRSS16.

[0034] In some embodiments, the population of thymocytes derived from differentiation of iPSCs may include TECs that express one or more markers, for example but not limited to AIRE, CK5, CK8, CXCL12, CCL25, DLL4, and / or HLA-DR.

[0035] In some embodiments, the population of thymocytes derived from the differentiation of iPSCs may include cTEChi (or cTEC-high) cells characterized by the expression of cell surface markers not limited to, for example, CTSV, SLC46A2, HLA-DMA, CXCL12, THY1, ENO1, CALR, ALCAM, ATPIF1, and / or HSPA5.

[0036] In some embodiments, the population of thymocytes derived from the differentiation of iPSCs may include AIRE+mTEC-high cells characterized by the expression of one or more markers not limited to, for example, LTF, HLA-DRA<CD74, HLA DRB1, HLA-DPA, HLA-DPB1, IL2RG, and / or FCER2.

[0037] In some embodiments, the population of thymocytes derived from the differentiation of iPSCs may include keratinocyte-like mTECs. These cells are called keratinocyte-like because they express genes such as keratinocyte cytoskeleton 1 (KRT1), KRT10, and SPINKS, which are also expressed in keratinocytes of the skin (terminally differentiated keratinocytes). Keratinocyte-like cells also express transcripts that overlap with mTECs, such as AIRE. Therefore, they are called keratinocyte-like mTECs. They are likely precursors that give rise to Hassall's corpuscles, which are unique cells within the human thymus. These cells are also thought to be derived from mTEC precursor cells (Noam Kadouri et al Nature Review Immunology 2020 v20:239. The entire content of which is incorporated herein by reference).

[0038] Pluripotent stem cells (PSCs) In some embodiments, the cells of the present disclosure are derived from pluripotent stem cells.

[0039] Pluripotent stem cells have the ability to give rise to any of the three germ layers, such as endoderm, mesoderm, and ectoderm. Pluripotent stem cells can include stem cells such as embryonic stem cells, nuclear transfer-derived embryonic stem cells, and induced pluripotent stem cells (iPSCs). Pluripotent stem cells can have a stem cell phenotype that includes (i) the ability to self-renew and (ii) pluripotency. Pluripotency-associated genes can include, but are not limited to, Oct-3 / 4, Sox2, Nanog, GDF3, REXI, FGF4, ESGI, DPPA2, DPPA4, hTERT, and SSEAI.

[0040] The cells described herein may be derived from embryonic stem cells. ES cells may include cells that (a) self-renew, (b) differentiate to produce all cell types in an organism, and / or (c) are derived from a developing organism. ES cells may be derived from the inner cell mass of a developing organism's blastula. ES cells may also be derived from blastomeres generated by single blastomere biopsy (SBB), which involves the removal of a single blastomere from the 8-cell stage of a developing organism. ES cells are characterized by expression of markers, such as but not limited to SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and / or alkaline phosphatase. Methods for generating and characterizing ES cells are known in the art and can be found, for example, in U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, U.S. Pat. No. 6,200,806 (the entire contents of each of which are incorporated herein by reference).

[0041] Induced pluripotent stem cells (iPSCs) can also be used to generate the cells of the present disclosure. iPSCs can include cells that have one or more properties, such as, but not limited to, (a) self-renewal, (b) the ability to differentiate to produce all types of cells in an organism, and / or (c) somatic origin. iPSCs can express markers, such as, but not limited to, SSEA3, SSEA4, SOX2, OCT3 / 4, Nanog, TRA160, TRA1818, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, Zpf42. Methods for generating and characterizing iPS cells can be found, for example, in U.S. Patent Publication Nos. US20090047263, US20090068742, US2009191159, US20090227032, US20090246875, and US20090304646, the entire contents of each of which are incorporated herein by reference. In some embodiments, iPSCs are derived from T cells or non-T cells, B cells, or any other cells from peripheral blood mononuclear cells, hematopoietic progenitor cells, or any other somatic cell type.

[0042] In some embodiments, the pluripotent stem cells are derived from adult stem cells. The adult stem cells are obtained from a subject, for example, from the subject's inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood. The PSCs can also include embryonic stem cells from the placenta or umbilical cord, and progenitor cells (e.g., progenitor cells from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, and / or blood).

[0043] supporting cells In some embodiments, the cells of the present disclosure may include or are cultured with support cells that aid in the generation of thymocytes and / or the maintenance of thymocytes in culture. Non-limiting examples of support cells include hematopoietic non-T cell precursors such as macrophages and dendritic cells (DCs), non-hematopoietic cells such as epithelial cells and fibroblasts, stromal cells such as precursors of skeletal tissues, bone, cartilage, hematopoietic support stroma, and components such as adipocytes. In some embodiments, the support cells promote the proliferation, survival, maturation, or function of thymocytes. In some embodiments, the support cells are of mesenchymal origin. In one embodiment, the mesenchymal cells are mesenchymal stem cells.

[0044] Supportive cells are non-immune cells that may be present in the thymic microenvironment, for example, supportive cells are fibroblasts, vascular smooth muscle cells (VSMCs), endothelial cells, and / or lymphatic endothelial cells.

[0045] In some embodiments, the supporting cells are neuroendocrine cells (expressing BEX1, NEUROD1), myosinoid cells (expressing MYOD1, DES), and myelin-positive epithelial cells (expressing SOX10, MPZ) as described in Bautista et al. 2021 Nat Commun 12, 1096 (2021), the entire contents of which are incorporated herein by reference. In some embodiments, the mesenchymal cells are associated with markers such as, but not limited to, LAMA2, LAMA4, PDGFRA, PDGFRB, LUM, CSPG4, COL1A2, COL3A1, IGF1, FGF7, FGF10, FST, BMP4, SFRP2, WNT5A. Mesenchymal cells are positive or negative for one or more of these markers. In some embodiments, the mesenchymal cells are positive for some markers described herein but negative for others.

[0046] In some embodiments, the endothelial cells are associated with one or more markers, for example, but not limited to, VEGFC, PECAM1, APLNR, PROXI, LYVE1, ACKR1, SELE, SELP, FN1, and / or TGFB1. The endothelial cells are positive or negative for one or more of these markers. In some embodiments, the endothelial cells are positive for some of the markers described herein, but negative for others.

[0047] Thymocyte products In some embodiments, the present disclosure provides a thymocyte product. The thymocyte product is prepared by the method described herein. As used herein, "thymocyte product" refers to a population of thymocytes that phenotypically or potently have similarities to the human thymus or a subpopulation thereof and / or are suitable for therapeutic, diagnostic, or research use. The thymic product of the present disclosure is prepared by (i) generating one or more populations of thymocytes, (ii) extracting and sequencing RNA from each of the populations of thymocytes, (iii) analyzing the RNA sequences to identify the subpopulations within each population of thymocytes to generate a test thymic map for each of the populations of thymocytes, (iv) comparing each of the test thymic maps with a reference thymic map, and (v) identifying the thymocyte product by selecting a test thymic map with a subpopulation ratio (SPR) or relative cTEC ratio (RCTR) of about 0.7 to about 1.4. As used herein, SPR refers to the ratio of the proportion of a subpopulation (also referred to herein as a "cell type") of cells in a test thymic map to the proportion of the subpopulation in a reference thymic map. As used herein, cTEC ratio refers to the ratio of the proportion of cTEC high cells to cTEC low cells in a particular thymic map.

[0048] The thymocyte product is prepared by analyzing a thymic map. As used herein, a "thymic map" refers to a molecular profile of a population of thymocytes or thymic tissue. The molecular profile may include a total transcriptional profile of a population of thymocytes or thymic tissue, a subpopulation of cells within said thymocytes or thymic tissue, and / or a transcriptional profile of an identified subpopulation. In some embodiments, a thymic map, as used herein, is a reference thymic map, which refers to a representative map of the human thymus generated by the transcriptional profiles of thymic tissue and known cell types within said thymic tissue. In one embodiment, the reference thymic map may be generated using transcriptional profiling data as described in Bautista et al. 2021. As used herein, a test thymic map refers to a characteristic of a population of thymocytes whose molecular profile is compared to a reference thymic map.

[0049] In one embodiment, the SPR can be 0.70, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.06, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, and / or 1.4. As a non-limiting example, the SPR is 1.

[0050] The RCTR is generated by calculating the cTEC ratio. As used herein, the cTEC ratio refers to the ratio of the proportion of cTEC high cells to cTEC low cells in a particular thymic map (reference or test thymic map). The cTEC ratio may vary between thymic maps, and the RCTR may be 0.70, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.06, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, and / or 1.4. In some embodiments, the RCTR may be about 0.7 to about 1.14. As a non-limiting example, the RCTR may be 1. In some embodiments, cell subpopulations within each of the thymocyte populations can be immature thymic epithelial cells (iTEC), cTEC-high cells, cTEC-low cells, Aire+mTEC-high cells, mTEC-low cells, keratinocyte-like mTEC cells, ciliated cells, myelin cells, myoid cells, neuroendocrine cells, and / or tuft / ionocyte cells. In some embodiments, the SPR can be about 0.7 to about 1.4. In some embodiments, the SPR can be iTEC SPR, cTEC-high SPR, cTEC-low SPR, Aire+mTEC-high SPR, mTEC-low SPR, keratinocyte-like mTEC SPR, ciliated SPR, myelin SPR, myoid SPR, neuroendocrine SPR, and / or tuft / ionocyte SPR. In some embodiments, the SPR can be iTEC SPR. As a non-limiting example, the iTEC SPR can be 1.

[0051] III. Method The present disclosure provides thymocytes, methods of making thymocytes and / or methods of maintaining thymocytes in culture.

[0052] The present disclosure provides methods for generating thymocytes in vitro. These methods may include culturing a cell population in the presence of a soluble factor, a mineral, or a combination thereof. Such methods may include differentiation or maturation of the cell population into thymocytes. The cell population may be engineered to express a cell surface receptor or an intracellular factor. In some embodiments, the cell population may be pluripotent stem cells (PSCs), definitive endoderm (DE) cells, third pharyngeal pouch endoderm (PPE) cells, anterior foregut endoderm (AFE) cells, thymic epithelial progenitor cells (TEPCs), immature thymic epithelial cells (iTECs), thymic epithelial cells (TECs), medullary thymic epithelial cells (mTECs), cortical thymic epithelial cells (cTECs), or a combination thereof.

[0053] In some embodiments, the thymocytes can be immature thymic epithelial cells (iTECs). In some aspects, the iTECs can express at least one cell surface receptor, wherein the at least one cell surface receptor can be IGKC (ENSG00000211592), ANAX2, LIMA1 (ENSG00000050405), or EGFR (ENSG00000146648). In some embodiments, the iTECs can express at least one intracellular factor, wherein the at least one intracellular factor is selected from the group consisting of ASCL1 (ENSG00000139352), HES1 (ENSG00000114315), JUND (ENSG00000130522), FOS (ENSG00000170345), ARID5B (ENSG00000150347), IRF1 (ENSG00000125347), MAFB (ENSG00000204103), IFI16 (ENSG00000163565). , FOXC1 (ENSG00000054598), STAT1 (ENSG00000115415), JUNB (ENSG00000171223), EGFR1, ZFP36 (ENSG00000128016), JUN (ENSG00000177606), FOSB (ENSG00000125740), IER2 (ENSG00000160888), PAX9 (ENSG00000198807), or HIF1A (ENSG00000100644).

[0054] Soluble factors include, but are not limited to, growth factors, cytokines, transporters, hormones, or combinations thereof.

[0055] As a non-limiting example, the soluble factor can be a growth factor, such as epidermal growth factor (EGF, ENSG00000138798).

[0056] The soluble factor can be a cytokine, for example, macrophage inhibitory factor (MIF, ENSG00000240972).

[0057] The soluble factor may be a transporter, for example, lactotransferrin (LTF, ENSG00000012223).

[0058] In some embodiments, the cells of the present disclosure can be cultured in the presence of a mineral. The mineral can be iron, magnesium, calcium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, zinc, chloride, chromium, copper, fluoride, or iodine. In one aspect, the mineral can be iron.

[0059] The cells of the present disclosure can be engineered to express cell surface receptors or intracellular factors for the purpose of generating or maintaining thymocytes. The cell surface receptor can be CD74 (ENSG00000019582), integrin beta 1 (ITGB1, ENSG00000150093), and / or epidermal growth factor receptor (EGFR, ENSG00000146648).

[0060] The intracellular factor can be ENO1 (ENSG00000074800).

[0061] The present disclosure also provides methods for maintaining a population of thymocytes in vitro, which may include (i) culturing the population of thymocytes in the presence of a soluble factor or mineral, (ii) culturing the population of thymocytes in the presence of one or more feeder cells, and / or (iii) engineering the population of thymocytes to express a cell surface receptor or an intracellular factor.

[0062] The cells of the present disclosure may be cultured in the presence of one or more supporting cells. The supporting cells may be endothelial cells, mesenchymal cells, macrophages, dendritic cells (DCs), epithelial cells, fibroblasts, stromal cells, adipocytes, fibroblasts, vascular smooth muscle cells (VSMCs), or lymphatic endothelial cells. In some embodiments, the supporting cells may be mesenchymal cells. In some aspects, the supporting cells may be endothelial cells.

[0063] Preparation and maintenance of thymocytes In some embodiments, the disclosure provides methods for preparing one or more cells or cell types described herein. In some embodiments, the cells may be thymocytes.

[0064] A series of data accumulated in public databases provides single-cell transcriptomes of primary human and mouse thymus (see Bautista et al. 2021 Nat Commun 12, 1096; Kernfeld, et al. Immunity. 2018 Jun 19;48(6):1258-1270.e6; Zeng et al. Immunity. 2019 Nov 19;51(5):930-948.e6, the entire contents of each of which are incorporated herein by reference). This provides a rich source of material for identifying factors that promote differentiation and / or maturation of the cells of the present disclosure into thymocytes. By analysis of scRNA sequencing data, the present disclosure identifies potential factors and / or supporting cells that may promote and / or maintain the thymocyte phenotype.

[0065] In some embodiments, the cells of the present disclosure may be isolated from an organism. In some embodiments, the organism may be a mammal. The mammalian cells may be isolated from human, rodent, porcine, and / or bovine sources. The human source of the cells of the present disclosure may be autologous or allogeneic. In some embodiments, tissues containing the cells of the present disclosure may be harvested and used intact for the applications described herein. The cells of the present disclosure may be obtained from embryos, fetuses, and adult organisms. In some aspects, the organism may be a living or cadaveric organism.

[0066] The cells described herein may be derived from other cell types. As a non-limiting example, the cells of the present disclosure may be derived from pluripotent stem cells (PSCs). In some embodiments, the cells of the present disclosure may be derived from progenitor cells. In some embodiments, the cells of the present disclosure may be derived from the differentiation of PSCs and / or progenitor cells.

[0067] In some embodiments, thymocytes may be prepared from PSCs. In this regard, the method may include culturing pluripotent stem cells for a period of time under conditions sufficient to differentiate the pluripotent stem cells into thymocytes. For example, the method may include culturing the pluripotent stem cells in the presence of factors and / or inhibitors that drive the differentiation of PSCs into thymocytes. Methods for differentiating PSCs into thymocytes are known in the art. Methods for differentiating PSCs into thymocytes may include the use of one or more parameters or combinations thereof for differentiation known in the art. The parameters include, but are not limited to, (i) differentiation factors, (ii) differentiation inhibitors, (iii) duration of differentiation, (iv) temperature, (v) substrate, and / or (vi) feeder cells that promote differentiation. Any method or parameters for differentiating PSCs into thymocytes described in the following references may be used herein: Parent et al. Cell Stem Cell. 2013 Aug 1;13(2):219-29; Soh et al. Stem Cell Rep. 2014 Vol. 2 j 925-937; Sun et al. Cell Stem Cell. 2013 Aug 1;13(2):230-6; Okabe et al. Cell. Reprog. 2015 Vol 17, No. 5; Su et al. Sci.Rep. 2015 5, 9882; Otsuka et al. Sci Rep 2020 10:224, International Patent Publications WO2019060336, WO2020205859, WO2020220040, WO2014134213, WO2010143529, WO2011139628, and Chinese Patent Publication CN201110121243, the entire contents of each of which are incorporated herein by reference.

[0068] soluble factors The factors described herein can be used to induce maturation, fate specification, and / or help maintain a particular cellular state of the cells of the present disclosure. Soluble factors can include proteins or peptides that can bind to cell surface molecules or can be taken up by cells. Uptake of soluble factors by cells can occur by passive diffusion, transporters, and / or endocytosis. Soluble factors can include growth factors, hormones, transporters, and cytokines. When provided to a cell, the soluble factors can generate a signal to the cell. The signal can cause one or more cellular actions, such as survival, proliferation, and differentiation.

[0069] In some embodiments, the soluble factor can be a ligand.

[0070] In some embodiments, the soluble factor may be a growth factor. The growth factor may be epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), granulocyte-macrophage colony-stimulating factor (GMCSF), granulocyte colony-stimulating factor (GCSF), transforming growth factor (TGF), growth stimulating factor (MSF), erythropoietin, thrombopoietin (TPO), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), GDF, neurotrophin, sarcoma growth factor (SGF), and / or growth / differentiation factor (GDF).

[0071] As a non-limiting example, the growth factor can be epidermal growth factor (EGF, ENSG00000138798).

[0072] In some embodiments, the soluble factor can be a cytokine. Any cytokine known in the art can be used. As a non-limiting example, the cytokine can be produced by thymocytes or effector cells in vivo. Yan F, et al. Mol Med Rep16: 7175-7184, 2017 provides cytokines produced by T cells or thymocytes that can be useful in the present disclosure (the entire contents of which are incorporated herein by reference). As a non-limiting example, the cytokine can be macrophage inhibitory factor (MIF, ENSG00000240972).

[0073] In some embodiments, the soluble factor can be a transporter protein, which can bind to a second companion protein or a mineral to facilitate its transfer from one location to another. Transfer can occur from the extracellular environment into a cell, or from one location to another within an organism. In some embodiments, the transporter protein can be a mineral transporter protein. As a non-limiting example, the transporter protein can be lactotransferrin (LTF, ENSG00000012223).

[0074] mineral In some embodiments, the cells of the present disclosure may be prepared or cultured in the presence of a mineral. The mineral may be iron, magnesium, calcium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, zinc, chloride, chromium, copper, fluoride, or iodine. In some embodiments, the mineral may be iron, or a salt or derivative thereof.

[0075] Cell Surface Receptors The soluble factor or ligand interacts with a ligand-specific counterpart receptor, and together they can transmit a message or signal to a cell to have a particular function or phenotype. In some embodiments, the cell can be a thymocyte. The cell surface receptor can be a G protein-coupled receptor, an enzyme-coupled receptor, and an ion channel-linked receptor.

[0076] Non-limiting examples of cell surface receptors include CD74 (ENSG00000019582), integrin beta 1 (ITGB1, ENSG00000150093), and / or epidermal growth factor receptor (EGFR, ENSG00000146648). In one embodiment, the cell surface receptor can be CD74. CD74 has been shown to regulate thymocyte cellularity and maturation by activating NF-kappa B (Wang et al. FASEB J. 2021 May;35(5):e21535, the entire contents of which are incorporated herein by reference).

[0077] intracellular factor The methods of the present disclosure may include culturing a cell population described herein with one or more intracellular factors. An intracellular factor may be any protein or peptide present in the cytoplasm or nucleus.

[0078] In some embodiments, the intracellular factor is a cytoplasmic intracellular factor. As a non-limiting example, the intracellular factor can be enolase 1 (ENO1, ENSG00000074800). In other aspects, the intracellular factor can be a factor present in the nucleus. For example, the factor can be a transcription factor or a transcription-associated factor. Non-limiting examples of intracellular nuclear factors can include NFKB1, PAX1, PRDX15, PSIP1, AIRE, DLX5, FEZF2, LTF, SPIB.

[0079] Analysis of thymocyte populations Thymocyte populations derived from differentiation of stem cells may be analyzed by one or more methods described herein. In some embodiments, the thymocyte population may include one or more of the following cell types, including but not limited to cTEC-high, cTEC-low, immature TEC, mTEC-low, Aire+mTEC-high, keratinocyte-like mTEC, neuroendocrine cells, myoid cells, myelin cells. Each of the thymic cell types may be associated with one or more markers. In some embodiments, cTEC-high cells may be associated with expression of markers, including but not limited to PSMB11, PRSS16, and CCL25. In some embodiments, cTEC-low cells may be associated with expression of markers, including but not limited to PSMB11, PRSS16, CCL25 (low levels of HLA class II, PSMB11, PRSS16, CCL25, and increased KI67+ proliferating cells). In some embodiments, immature TEC cells may be associated with expression of markers such as, but not limited to, FOXN1, PAX9, SIX1, lacking cTEC / mTEC functional genes. In some embodiments, mTEC-low cells may be associated with expression of markers such as, but not limited to, CLDN4, low levels of HLA class II, high levels of the chemokine CCL21. In some embodiments, Aire+mTEC high cells may be associated with expression of markers such as, but not limited to, SPIB, AIRE, FEZF2, high levels of HLA class II. In some embodiments, keratinocyte-like mTEC cells may be associated with expression of markers such as, but not limited to, KRT1, IVL. In some embodiments, neuroendocrine cells may be associated with expression of markers such as, but not limited to, BEX1, NEUROD1. In some embodiments, myoid cells may be associated with expression of markers such as, but not limited to, MYOD1, DES. In some embodiments, myelin cells may be associated with expression of markers such as, but not limited to, MPZ.As used throughout this description, reference to a "low" or "lower" level of expression of a marker gene in a cell subpopulation means about 1.5-fold lower, about 2-fold lower, about 2.5-fold lower, about 3-fold lower, about 3.5-fold lower, about 4-fold lower, about 4.5-fold lower, or about 5-fold lower, or less than 5-fold lower than the average expression level of the marker gene across the entire cell population. Similarly, throughout this description, reference to a "high" or "higher" level of expression of a marker gene in a cell subpopulation means about 1.5-fold higher, about 2-fold higher, about 2.5-fold higher, about 3-fold higher, about 3.5-fold higher, about 4-fold higher, about 4.5-fold higher, or about 5-fold higher, or more than 5-fold higher than the average expression level of the marker gene across the entire cell population.

[0080] In some embodiments, CellRouter may be used to perform quality control, cell cycle, and mitochondrial content regression (to account for potential confounding effects of cell cycle and stressed cells), followed by variable gene identification, dimensionality reduction (UMAP=Uniform Manifold Approximation and Projection) and clustering, as described in da Rocha et al. Nature Communications. 2018 Mar 1;9(1):892, the entire contents of which are incorporated herein by reference. CellRouter may be used to identify transcriptional clusters within thymocyte populations. In some embodiments, single cells and clusters may be visualized in UMAP space. Several lineage standard marker genes may be used to assign cell type identity to each transcriptional cluster.

[0081] In some embodiments, thymocyte scRNA-seq data from organisms in vitro or obtained from organisms in vivo can be compared. Large single-cell transcriptome datasets using various techniques contain batch-specific systematic variations that pose challenges to data integration. Methods of batch correction can include regression-based batch correction (da Rocha et al. Nature Communications. 2018 Mar 1;9(1):892, the entire contents of which are incorporated herein by reference) and harmonization-based batch correction (Korsunsky et al. Nature Methods; 16, pages 1289-1296 (2019)).

[0082] In some embodiments, batch correction can be performed using Single Cell Net, Symphony (Kang, JB, et al. Nat Commun 12, 5890 (2021), the entire contents of which are incorporated herein by reference), and Label Transfer (as implemented in the package Seurat (Stuart et al., 2019, Cell 177, 1888-1902, the entire contents of which are incorporated herein by reference)).

[0083] IV. Definition Expression: As used herein, "expression" and its grammatical equivalents, in the context of a marker, refers to the production of a marker as well as the level or amount of the marker. For example, the expression of a marker in a cell or the presence of a marker or a cell being positive for a marker refers to the expression of the marker at a level similar to a positive control level. The positive control level may be determined by the level of the marker expressed by a cell known to have a cell fate associated with the marker. Similarly, the absence of expression of a marker or a cell being negative for a marker refers to the expression of the marker at a level similar to a negative control level. The negative control level may be determined by the level of the marker expressed by a cell known not to have a cell fate associated with the marker. Thus, the absence of a marker does not simply mean that the expression of the marker is at an undetectable level, but rather that in certain cases, a cell may express the marker, but the expression may be low compared to the positive control or at a level similar to that of the negative control.

[0084] Effector cell: As used herein, "effector cell" refers to any cell or cell type that acquires the ability to execute, initiate or propagate a signal or cell death trigger upon contact or proximity to a thymocyte. "Contact or proximity" refers to sufficient spatiotemporal proximity to allow cell-intrinsic or cell-extrinsic (e.g., cell-cell) signaling or other communication or interaction.

[0085] Lymphocyte: As used herein, "lymphocyte" encompasses the meaning and use as one of ordinary skill in the art would understand the term, and further refers to a type of immune cell derived from bone marrow that is found in lymphatic tissue or blood. In some embodiments, lymphocytes mature in the thymus gland.

[0086] Negative: As used herein, the term "negative" (sometimes abbreviated as "-"), when used herein in reference to expression of an indicated cell marker, means that the cells do not express the indicated cell marker at detectable levels.

[0087] Positive: As used herein, the term "positive" (sometimes abbreviated as "+"), when used herein in reference to expression of an indicated cell marker, means that a cell expresses the indicated cell marker at a detectable level, and can include, for example, expression at a low (but detectable) level as well as expression at a high (high) level.

[0088] Pre-T cell: As used herein, "pre-T cell" refers to a lymphocyte that can mature or differentiate into a T cell.

[0089] Soluble Factor: As used herein, "soluble factor" refers to any protein or peptide that can bind to a cell surface molecule or be taken up by a cell. Uptake by a cell can be by passive diffusion, transporters, and / or endocytosis.

[0090] Subpopulation Ratio (SPR): As used herein, SPR refers to the ratio of the percentage of a thymocyte subpopulation (also referred to herein as a "cell type") in a test thymic map to the percentage of a thymocyte subpopulation in a reference thymic map. In some embodiments, the SPR can be iTEC SPR, cTEC-high SPR, cTEC-low SPR, Aire+mTEC-high SPR, mTEC-low SPR, keratinocyte-like mTEC SPR, ciliary SPR, myelin SPR, myoid SPR, neuroendocrine SPR, or tuft / ionocyte SPR.

[0091] cTEC ratio and relative cTEC ratio (RCTR): As used herein, cTEC ratio refers to the ratio of the proportion of cTEC high cells to cTEC low cells in a particular thymic map (reference or test thymic map). RCTR refers to the ratio of the cTEC ratio in a test thymic map to the cTEC ratio in a reference thymic map.

[0092] Thymocyte or origin or lineage: As used herein, "thymocyte or thymic origin or thymic lineage" refers to a cell that has one or more phenotypic or genotypic markers associated with a cell derived from the thymus or a cell that is destined to become a cell of the thymus. As used herein, the thymus can be an embryonic, fetal or adult thymus.

[0093] Thymocyte cell product: As used herein, "thymocyte product" refers to a population of thymocytes that phenotypically or functionally bears similarity to the human thymus or a subpopulation thereof and / or is suitable for therapeutic, diagnostic, or research use.

[0094] Thymic Map: As used herein, "thymic map" refers to the molecular profile of a population of thymic cells or thymic tissue. The molecular profile may include the entire transcriptional profile of a population of thymic cells or thymic tissue, a subpopulation of thymic cells or cells within the thymic tissue, and / or a transcriptional profile of an identified subpopulation. In some embodiments, a thymic map, as used herein, may be a reference thymic map, which refers to a representative map of the human thymus generated by transcriptional profiles of thymic tissue and known cell types within the thymic tissue. In one embodiment, the reference thymic map may be generated using transcriptional profiling data as described in Bautista et al. 2021. As used herein, a test thymic map refers to the characteristics of a population of thymic cells whose molecular profile is compared to a reference thymic map.

[0095] Variant: The term "variant" as used in reference to a biomolecule (e.g., a training factor or an end factor) refers to a biomolecule that is related to or derived from a parent molecule. A variant may be, for example, a modified, truncated, mutated, homologous, or otherwise altered version of the parent molecule. The term variant can be used to describe either a polynucleotide or a polypeptide.

[0096] The details of one or more embodiments of the present disclosure are described in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the present description. In this description, the singular form includes the plural form unless the context indicates otherwise. Unless otherwise defined, all technical and scientific terms used in this description have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present description controls.

[0097] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0098] Example 1. Single-cell RNA sequencing analysis of thymocytes We performed an analysis of scRNA-seq data from 19-day-old fetal and 25-year-old adult thymuses published in Bautista, J. et al. (Single-cell transcriptional profiling of human thymic stroma reveals novel cellular heterogeneity in the thymic medulla. Nat Commun 12, 1096 (2021), the entire contents of which are incorporated herein by reference).

[0099] Quality control, cell cycle, and mitochondrial content regressions (to account for potential confounding effects of cell cycle and stressed cells) were performed using CellRouter, followed by variable gene identification, dimensionality reduction (UMAP) and clustering, as described in da Rocha et al. Nature Communications. 2018 Mar 1;9(1):892, the entire contents of which are incorporated herein by reference. CellRouter identified 24 transcriptional clusters. Single cells and clusters were visualized in UMAP space. Several lineage canonical marker genes were used to assign cell type identity to each transcriptional cluster. Heatmaps were generated showing the average expression of each gene within each cluster.

[0100] Five mesenchymal cell (MC) states were identified (termed MC1, MC2, MC3, MC4, and MC5). Further unbiased analysis of cell-cell communication identified mesenchymal-epithelial interactions, e.g., between MC2 and mTEC.

[0101] The cell-cell communication / interaction was visualized as a heat map to analyze the degree of interaction between different cells. An unbiased analysis of cell-cell communication identified mesenchymal-epithelial interactions, especially between MC2 and mTEC. Cells designated Endo1 and Endo3 appeared to interact with specific TEC subsets. Identification of such interactions may facilitate the identification of candidate interacting and regulatory genes.

[0102] Endothelial cells were found to strongly interact with TEC cells, especially mTECs, prioritizing ligand-receptor interactions that lead to signaling pathways reported to be important for the development of thymic epithelial cells and cell types potentially expressing the ligand. One such example is the EGF receptor (EGFR) pathway in cTECs and mTECs. These data highlight the potential utility of using EGF in thymocyte culture. Cell type expressed ligands were paired with cell type expressed receptors and analyzed. Strong interactions between endothelial cells and TEC cells, especially mTECs, were identified. Receptors such as EGFR, ITGB1 were identified in the analysis.

[0103] Further prioritization of ligand-receptor interactions identified signaling molecules and networks that may be important for the development of thymic epithelial cells (e.g., MIF and CD74) and potential effectors (e.g., CD74 to LTF). We investigated the enrichment of cell type-specific gene signatures in gene sets predicted to be controlled by transcriptional regulators reported based on gene regulatory network analysis using activity scores.

[0104] Matching of receptors and downstream effectors identified putative signaling molecules and networks that mediate the receptors' activity. Transcriptional regulators that respond to specific cell surface receptors that may be important for TEC development were also identified. For example, LTF and ENO-1 were identified that respond to CD74. Eno-1 (enolase) is known to be important for T cell metabolism. LTF (lactoferrin) is involved in iron transport. These findings suggest the utility of iron supplementation in TECs cultured in vitro. As MIF (macrophage inhibitory factor) is a known ligand for CD74, this finding indicates the utility of adding MIF in inducing differentiation of one or more cell types into thymocytes and / or maintaining thymocytes.

[0105] Example 2. Comparison of fetal and adult thymus Comparison of the transcriptional profiles of young (or fetal, as referred to herein) (19 weeks) versus older (25 years) thymuses (or thymi) showed strikingly different profiles. The transcriptional profile of the young thymus showed multiple subpopulations including mTEC high, cTEC high, mTEC low, cTEC low, iTEC, neuroendocrine, endothelial cells (Endo1, Endo2, Endo3, Endo4, Endo5), mesenchymal cells (MC1, MC2, MC3, MC4, MC5), and pericytes (pericyte-1, pericyte-2, pericyte-3). In contrast, the transcriptional profile of the older thymus showed subpopulations of endothelial, epithelial, pericyte, and mesenchymal cells. The abundance of epithelial cells was significantly decreased in the adult thymus (25 years) compared to the fetal thymus (19 weeks). It is believed that there are immature TECs among the epithelial cells as well. Endothelial cell subsets and some pericyte subsets appeared to be preserved in the thymus between 19 weeks and 25 years of age, whereas mesenchymal cell subsets appeared to differ between ages. These data indicate that thymic deficiency occurs even at age 25 years.

[0106] Example 3. Transcriptional Regulatory Factors Using an algorithm to track cell differentiation trajectories, we demonstrated distinct dynamics of mTEC vs. cTEC development from initial iTECs. Each of the trajectories highlighted specific prominent gene networks, signaling molecules, and regulators that may contribute to differentiation into either mTECs or cTECs. The dynamic patterns of prioritized transcriptional regulators showed distinct iTEC-to-cTEC differentiation patterns compared to iTEC-to-mTECs.

[0107] Example 4. Comparison of scRNA-seq datasets of thymus tissue and iPSC-derived thymocytes We compared thymus tissue scRNA-seq data from Bautista, J. et al. Nat Commun 12, 1096 (2021) with thymus tissue scRNA-seq data from Park et al. 2020 Science Vol. 367, Issue 6480 (the entire contents of which are incorporated herein by reference). Large single-cell transcriptome datasets using various techniques contain batch-specific systematic variations that pose challenges to data integration. We did not account for differences arising from batch variations and clustered the data by age of thymus tissue (fetal vs. adult) rather than cell type. Two methods of batch correction were performed: regression-based batch correction (da Rocha et al. Nature Communications. 2018 Mar 1;9(1):892, the entire contents of which are incorporated herein by reference) and harmony-based batch correction (Korsunsky et al. Nature Methods; 16, pages 1289-1296 (2019)). These data showed that compared with regression-based batch correction, harmonization-based batch correction was more effective at clustering data between the two studies.

[0108] To quantify the cellular similarity among the different populations in fetal and adult thymus identified in the scRNA-seq data in Bautista et al. and Park et al., we employed the mean classification score (MCS).

[0109] Using cell types from Park et al. (herein referred to as the "Teichmann study"), machine learning models were trained to identify cell types using single-cell gene expression profiles and the SingleCellNet algorithm (Tan Y, et al. Cell Syst. 2019 Aug 28;9(2):207-213.e2, the entire contents of which are incorporated herein by reference). As such, these models represent the identity of a cell type given its transcriptome. These models were then queried to identify what transcriptomic similarities (if any) exist between cell types in the dataset included in Bautista et al. (herein referred to as the "parent" dataset). To facilitate visualization, an average classification of scores was calculated. Each single-cell subpopulation from the parent dataset has a score, and a heatmap shows the average score of previously annotated cell types from the parent data. Classification scores range from 0 to 1, with 0 indicating no similarity and 1 indicating high similarity. Subpopulations described in the Teichmann dataset were identified with high MCS in the parent dataset. The observed conservation of cell types identified after batch correction between both studies represents an important step in the harmonization of published thymic tissue scRNA-seq datasets, and insights gained from these datasets will be used to characterize thymic populations derived from pluripotent stem cell differentiation.

[0110] scRNA sequencing was performed on thymocytes differentiated from pluripotent stem cells. Seven cell subpopulations were identified from these TEP derivatives. The scRNA-seq data from in vitro derived thymocyte derivatives was compared to the scRNA-seq dataset in Bautista et al., and the average classification score was calculated. This comparison provides insight into the cell populations present within the in vitro derivatives and their correlation with cell types present in thymic tissue in vivo. Heatmaps based on the average classification scores revealed that the populations in the TEP derivatives had high similarity to neuroendocrine cells (populations 5, 3, and 4) and less similarity to keratinocyte-mTEC-like cells (populations 1, and 2 and populations 6, 7), cTEC-low, ionocytes, and myelin cells. The enrichment of neuroendocrine cells in the populations is likely due to the presence of only 45% definitive endoderm cells, and differentiation into neuroendocrine cells may be promoted by SMAD inhibition during differentiation of definitive endoderm into the anterior foregut. Neuroendocrine cells may be neural cells that develop a neuroendocrine phenotype. The presence of keratinocyte-like mTECs in the derived material is desirable. TEP induction conditions may be adjusted and culture conditions that support enrichment of iTECs as determined by comparison of scRNAseq data may be desirable.

[0111] Regarding keratinocyte-like cells (populations 1, 2) that resemble mTECs, this population was identified in Bautista et al. 2021 and, based on the analysis herein, may be equivalent to mTEC III described in Park et al. 2020. The cells are called keratinocytes because they express genes such as keratin cytoskeleton 1 (KRT1), KRT10, and SPINKS, which are also expressed in skin keratinocytes (terminally differentiated keratinocytes). Keratinocyte-like cells also express transcripts that overlap with mTECs, such as AIRE. Therefore, they are called keratinocyte-like mTECs. They are likely to be the precursors that give rise to Hassall's corpuscles, which are unique cells in the human thymus. These cells are also thought to be derived from mTEC progenitors (Noam Kadouri et al Nature Review Immunology 2020 v20:239, the entire contents of which are incorporated herein by reference). Keratinocyte-like populations 1 and 2 also appear to share some similarities with tuft / ionocyte cells (Miller C et al 2018 Nature 559:627, the entire contents of which are incorporated herein by reference). Thymic tuft cells have been shown to localize adjacent to keratinocyte-like (Kadouri N, et al. Nat Rev Immunol. 2020 Apr;20(4):239-253, the entire contents of which are incorporated herein by reference).

[0112] Example 5. Thymocyte surface receptors and transcriptional regulators One of the goals of the comparative analysis of scRNA sequences is to identify the precursors or progenitors of the cells that ultimately give rise to TEP cells during differentiation of pluripotent stem cells in vitro. The existence of "bipotent" progenitor cells, also called iTEC cells, that give rise to both mTEC and cTEC populations has been speculated in the art but has not been conclusively identified. Computational tracking of cell connectivity performed herein indicates that iTEC populations can further be classified as "cTEC" cells. 高 " and "mTEC 高 "cTEC" cells develop into 低 " and "mTEC 低"The analysis suggests that thymocytes may give rise to "cells that are specifically expressed in the thymus." To aid in the recognition of thymocyte subpopulations, surface markers and transcription factors that are preferentially expressed in the various subpopulations were identified. In particular, the analysis identified surface markers and transcription factors for two groups of iTEC cells. Cell surface markers include IGKC (ENSG00000211592) for iTEC-1, and ANAX2, LIMA1 (ENSG00000050405), and EGFR (ENSG00000146648) for iTEC-2. The transcriptional regulators for iTEC-1 were ASCL1 (ENSG00000139352), HES1 (ENSG00000114315), JUND (ENSG00000130522), FOS (ENSG00000170345), ARID5B (ENSG00000150347), IRF1 (ENSG00000125347), MAFB (ENSG00000204103), IFI16 (ENSG00000163565), and FOXCI (ENSG00000170345). ENSG0000054598), STAT1 (ENSG00000115415) for iTEC-2, and JUNB (ENSG00000171223), EGFR1, ZFP36 (ENSG00000128016), JUN (ENSG00000177606), FOSB (ENSG00000125740), IER2 (ENSG00000160888), PAX9 (ENSG00000198807), and HIF1A (ENSG00000100644) for iTEC-3.

[0113] Example 6. Cell identity analysis workflow The aim of this study was to develop a workflow using algorithms to determine the molecular similarity of iPSC-derived thymocytes to the reference cell atlases published in Bautista et al. 2021 and Park et al. 2020. The analysis of iPSC-derived thymocytes is often challenging due to substantial heterogeneity and asynchronous cellular behavior during directed differentiation of iPSCs into target cell types. Since the algorithms are built on different assumptions and may be favorable to capture some aspects of the data while overlooking others, it is important to employ orthogonal and complementary approaches to determine the molecular similarity of in vitro iPSC-derived thymocytes to their in vivo counterparts. The workflow used SingleCellNet, Symphony (Kang, JB, et al. Nat Commun 12, 5890 (2021), the entire contents of which are incorporated herein by reference), and Label Transfer (as implemented in the package Seurat (Stuart et al., 2019, Cell 177, 1888-1902), the entire contents of which are incorporated herein by reference). The algorithms were used to classify cells from iPSC-derived thymocytes (SingleCellNet, which maps iPSC-derived cells to a reference UMAP and classifies cells based on class labels derived from a training dataset (Symphony)) and transfer the labels learned from the training dataset to individual cells in the query dataset.

[0114] The workflow was applied to thymocyte populations differentiated from iPSC cells (also referred to herein as "iPS-derived thymocytes"). Two iPSC-derived thymocyte population samples were used: 2-179 and Exp21 iPS-TEP. First, the Symphony algorithm was applied to the projection of the query cell (sample 2-179) and the reference UMAP cell atlas generated by Bautista et al. 2021, such that the query and training cells were located in similar positions in the UMAP space (UMAP = uniform manifold approximation and projection). Cell identity was also determined using this approach based on specific criteria. Consistent with the SingleCellNet analysis, Symphony also assigned the query cell to a neuroendocrine state. However, the keratinocyte-like similarity that determines whether the majority of iPSC-derived cells from sample 2-179 are cTEC-low or cTEC-high was not captured by Symphony. We also used a Label Transfer approach (available in the software package Seurat), in which class labels (cell types) learned from a training dataset, e.g., Bautista et al. 2021, were transferred to query cells, e.g., samples 2-179 and 4-191. After UMAP analysis of iPSC sample 2-179 (query), labels were transferred from the reference to the query cells. Symphony and Seurat analyses showed good agreement regarding the assignment of neuroendocrine cell populations to cTEC-low or cTEC-high cell populations.

[0115] Next, a comparison was established by analyzing sample Exp21 iPS-TEP using Symphony, and also showing previous results from SingleCellNet. The Symphony analysis showed an increased number of neuroendocrine cells in comparison to the same analysis of sample 2-179 (consistent with the SingleCellNet results), but a higher number of cTEC-low cells. According to these predictions, the number of cTEC-high cells is lower in this sample, but myelin and myoid cells are substantially more abundant, which is partially consistent with the SingleCellNet analysis. In addition, an i-TEC population was detected by analyzing Exp21 iPS-TEP using Symphony.

[0116] The percentage frequency of different cell types between thymocyte-derived material (experiment 21) and query was compared with the frequency of cell types identified in primary human thymus in Bautista et al. 2021, i.e., the reference. This comparison revealed that the percentage of neuroendocrine cells was higher in the query compared to the reference. Aire+mTECs were only present in the reference and not in the query. In contrast, the frequency of iTEC and cTEC-rich populations in the query was very similar to the frequency of iTECs in the reference. Thus, the frequency of cell types identified in TEPs derived from iPS cells was comparable to that identified in primary human thymus tissue. The number of cells of each cell type in the query and reference is shown in Table 1.

[0117] Table 1. Cell counts in queries and references TIFF2024523557000001.tif81134

[0118] Example 7. Recapitulation of thymic diversity in iPSC-derived thymocytes The algorithms CellRouter and Symphony were used to generate a reference single-cell atlas of epithelial cells from the human thymus (also referred to herein as the "reference thymus map" as described in Bautista et al. 2021). iPSC-derived thymocytes were mapped onto the reference single-cell atlas, and the iPSC-derived thymocytes were classified based on molecular similarity to their in vivo counterparts in the human thymus to generate a test thymus map. As used herein, "molecular similarity" may be defined as a classification score calculated by a supervised machine learning algorithm called a classifier, where a score closer to 0 means that the given cell has a low probability of resembling a particular cell type, and a score closer to 1 means that the given cell has a high probability of resembling their in vivo counterparts in the human thymus.

[0119] This approach demonstrated that in vitro protocols for differentiating thymic epithelial cells from iPSCs (e.g., Experiment 21 and Experiment 23) are better at recapitulating the cell type diversity of the human thymus (data not shown). By examining the proportions of iPSC-derived cell types across samples, Experiments 21 and 23 revealed that they contained cells transcriptionally similar to key cell types in the thymus, including cTECs (including cTEC-high and cTEC-low), subsets of mTECs, immature TECs, and other cell types (see Table 2 below).

[0120] Table 2. Proportions of different cell types derived from iPSC cells. TIFF2024523557000002.tif83167

[0121] Subpopulation ratios (SPRs) were calculated for the samples in Table 2, and the results are shown in Table 3. As used herein, SPR refers to the ratio of the percentage of a subpopulation (also referred to herein as a "cell type") in the test thymic map to the percentage of the cell subpopulation in the reference thymic map. In some embodiments, the SPR can be iTEC SPR, cTEC-high SPR, cTEC-low SPR, Aire+mTEC-high SPR, mTEC-low SPR, keratinocyte-like mTEC SPR, ciliary SPR, myelin SPR, myoid SPR, neuroendocrine SPR, or tuft / ionocyte SPR. The value of iTEC SPR in experiment 21 / 22 was close to 1, suggesting that the percentage of iTECs in this experiment was similar to the percentage of iTECs in the human thymus (reference thymic map).

[0122] The cTEC ratio and relative cTEC ratio (RCTR) were calculated for the samples listed in Table 2, and the results are shown in Table 4. As used herein, cTEC ratio refers to the ratio of the proportion of cTEC high cells to cTEC low cells in a particular thymic map. RCTR refers to the ratio of the cTEC ratio of the test thymic map to the reference thymic map. The value of RCTR in experiment 23 was close to 1, suggesting that the cTEC ratio in this experiment was similar to the cTEC ratio in the human thymus (reference thymic map).

[0123] Table 3. SPR of thymocyte-derived substances TIFF2024523557000003.tif97164

[0124] Table 4. RCTR of thymocyte-derived products TIFF2024523557000004.tif71143

[0125] Specifically for experiment 23, the analysis showed that iPSC-derived thymocytes were primarily similar to cTECs, iTECs, keratinocyte-like mTECs, and neuroendocrine cells from a transcriptional standpoint.

[0126] We performed UMAP and clustering analysis of iPSC-derived thymocytes. This analysis revealed the presence of 16 unbiased transcriptional clusters. Cluster-specific gene signatures were calculated using the algorithm CellRouter, which identified overlaps of these signatures with gene expression signatures derived from scRNA-seq analysis of the human thymus (described in Bautista et al. 2021). Heatmaps show the statistical significance of such enrichment. Cluster 16-specific genes were significantly enriched in the neuroendocrine gene signature from human thymus, whereas cluster 11 had an overlapping gene program with keratinocyte-like mTECs, and clusters 1, 4, and 5 showed significant signature overlap with cTECs. Taken together, these results indicated that transcriptional programs are shared between iPSC clusters and in vivo cell types in the human thymus. To identify putative cell types represented in each transcriptional cluster, we integrated clustering and Symphony analysis. This analysis explored how Symphony-predicted cell types are distributed across transcriptional clusters (Table 5).

[0127] Table 5. Proportion of different cell types in different clusters TIFF2024523557000005.tif85165

[0128] The majority of cells in cluster 16 were classified by Symphony as neuroendocrine cells, clusters 1, 3, 4, 5, and 14 contained high proportions of cells classified as cTEC, and cluster 6 contained primarily cells classified as cTEC-low. These data indicate that Symphony predictions can be used to guide the identification of cell types from transcriptional clusters, a step that is often employed in exploratory data analysis.

[0129] To identify markers associated with neuroendocrine cells, we analyzed cell markers expressed within cluster 16. Using CellRouter, we analyzed cell surface markers specifically expressed by cluster 16 in iPSC-derived thymocytes. Cell surface markers for neuroendocrine cells included PDPN (ENSG00000162493), CNTN2 (ENSG00000184144), NCAM1 (ENSG00000149294), CXCR4 (ENSG00000121966), NGFR (ENSG00000064300), L1CAM (ENSG00000198910), CNTNAP2 (ENSG00000174469), ANK3 (ENSG00000151150), NTRK1 (ENSG00000198400), and / or SLC1A2 (ENSG00000110436). Using these cell surface markers, neuroendocrine cells can be depleted from iPSC-derived thymocyte populations to generate cell populations enriched for other thymic cell types, e.g., cTECs, and / or cell populations that are not limited to iTECs.

[0130] Example 8: Overlapping gene expression patterns between iPSC-derived thymocytes and the human thymus We investigated gene expression programs that are potential sources of molecular similarity between iPSC cells and cell types in the human thymus. From the heatmap, we extracted overlapping gene sets between iPSC-derived thymocyte clusters and cell types in the human thymus and plotted their average expression levels over the expression levels in the human thymus. This analysis showed that genes expressed in iPSC cluster 1 and cTEC-high in the human thymus are indeed more highly expressed in this cell type compared to all other cell types (see Table 6 for a list of genes and associated ENSEMBL gene identifiers). Also, the gene expression program shared between cluster 11 and the keratinocyte-like mTEC population is indeed more highly expressed in keratinocyte-like TECs from the human thymus (see Table 7 for a list of genes and associated ENSEMBL gene identifiers). Similarly, the gene program shared between cluster 16 and neuroendocrine cells is evident, where canonical neuroendocrine markers were identified in iPSC cells, e.g., NEUROD1, supporting the validity of our approach (see Table 8 for a list of genes and associated ENSEMBL gene identifiers). Taken together, this analysis demonstrated molecular similarities between iPSC-derived TEPs and their in vivo counterparts.

[0131] Table 6. Genes expressed in the overlap between iPSC-thymocyte cluster 1 and the cTEC-high group in the human thymus TIFF2024523557000006.tif159128

[0132] Table 7. Genes expressed in the overlap between iPSC-thymocyte cluster 11 and the keratinocyte-like mTEC population in the human thymus TIFF2024523557000007.tif159130

[0133] Table 8. Genes expressed in the overlap between iPSC-thymocyte cluster 16 and neuroendocrine cell populations in the human thymus TIFF2024523557000008.tif176129

[0134] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not limited to the above specification, but is as set forth in the appended claims.

[0135] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless indicated to the contrary or clear from the context. A claim or specification containing "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which a plurality, or the entire group member, is present in, employed in, or otherwise relevant to a given product or process.

[0136] It should also be noted that the term "comprising" is intended to be open, allowing for, but not requiring, additional elements or steps to be included. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0137] When ranges are specified, the endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood as contemplated in different embodiments of the present disclosure to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0138] Furthermore, it should be understood that any particular embodiment of the present disclosure that is within the prior art may be expressly excluded from any one or more claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient, any method of manufacture, any method of use, etc.) may be excluded from any one or more claims, regardless of the reason, whether related to the existence of prior art or not.

[0139] The words that have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0140] While the present disclosure has been described in some detail and with respect to certain described embodiments, the present disclosure should not be limited to such details or embodiments, or to particular embodiments, but rather should be interpreted against the appended claims in a manner that provides the broadest possible interpretation of such claims in view of the prior art and thus effectively encompasses the intended scope of the present disclosure.

Claims

**Claim 1** a) generating one or more populations of thymocytes; b) extracting and sequencing RNA from each of said populations of thymocytes; c) analyzing the RNA sequenced in b) to identify subpopulations within each population of thymocytes to generate a test thymic map for each of said populations of thymocytes; d) comparing each of said test thymic maps to a reference thymic map; and e) identifying a thymocyte product by selecting a test thymic map having a subpopulation ratio (SPR) or relative cTEC ratio (RCTR) of about 0.7 to 1.4 A thymocyte product produced by the method. **Claim 2** The thymocyte product according to claim 1, wherein said subpopulations within each population of thymocytes comprise one or more of immature thymic epithelial cells (iTECs), cTEC-high cells, cTEC-low cells, Aire+mTEC-high cells, mTEC-low cells, keratinocyte-like mTEC cells, ciliated cells, myeloid cells, myoid cells, neuroendocrine cells, and tuft / ionocyte cells. **Claim 3** The thymocyte product according to claim 1, wherein said SPR is about 0.7 to 1.

4. **Claim 4** The thymocyte product according to claim 3, wherein said SPR is an iTEC SPR, a cTEC-high SPR, a cTEC-low SPR, an Aire+mTEC-high SPR, an mTEC-low SPR, a keratinocyte-like mTEC SPR, a cilia SPR, a myeloid SPR, a myoid SPR, a neuroendocrine SPR, and a tuft / ionocyte SPR. **Claim 5** The thymocyte product according to claim 1, wherein said iTEC SPR, said cTEC-high SPR, said cTEC-low SPR, said Aire+mTEC-high SPR, said mTEC-low SPR, said keratinocyte-like mTEC SPR, said cilia SPR, said myeloid SPR, said myoid SPR, said neuroendocrine SPR, and said tuft / ionocyte SPR are about 0.7 to 1.

4. **Claim 6** The thymocyte product according to claim 5, wherein said SPR is an iTEC SPR and said iTEC SPR is 1. **Claim 7** The thymocyte product according to claim 1, wherein said RCTR is about 0.7 to 1.

4. **Claim 8** The thymocyte product according to claim 1, wherein said one or more populations of thymocytes are prepared by differentiation of stem cells. **Claim 9** The thymocyte product according to claim 1, wherein said reference thymic map is created from one or more human thymuses. **Claim 10** The thymocyte product according to claim 9, wherein the human thymus is selected from a fetal human thymus, a postnatal human thymus, an adolescent thymus, an adult human thymus, or a combination thereof.

11. The thymocyte product according to claim 2, wherein the subpopulation includes iTEC, and the iTEC includes the expression of one or more of IGKC (ENSG00000211592), ANAX2, LIMA1 (ENSG00000050405), EGFR (ENSG00000146648), ASCL1 (ENSG00000139352), HES1 (ENSG00000114315), JUND (ENSG00000130522), FOS (ENSG00000170345), ARID5B (ENSG00000150347), IRF1 (ENSG00000125347), MAFB (ENSG00000204103), IFI16 (ENSG00000163565), FOXC1 (ENSG00000054598), STAT1 (ENSG00000115415), JUNB (ENSG00000171223), EGFR1, ZFP36 (ENSG00000128016), JUN (ENSG00000177606), FOSB (ENSG00000125740), IER2 (ENSG00000160888), PAX9 (ENSG00000198807), and / or HIF1A (ENSG00000100644).

12. The thymocyte product according to claim 2, wherein the subpopulation includes cTEC, and the cTEC includes the expression of one or more of PSMA3 (ENSG00000100567), FABP5 (ENSG00000164687), APRT (ENSG00000198931), LSM6 (ENSG00000164167), CTSV (ENSG00000136943), SNRPE (ENSG00000182004), ECHS1 (ENSG00000127884), HSPE1 (ENSG00000115541), RAN (ENSG00000132341), TMA7 (ENSG00000232112), TIMM13 (ENSG00000099800), LDHB (ENSG00000111716), ECI1 (ENSG00000167969), GCSH (ENSG00000140905), NOP58 (ENSG00000055044), MRPL11 (ENSG00000174547), STOML2 (ENSG00000165283), ING2 (ENSG00000168556), TOMM7 (ENSG00000196683), MRPS34 (ENSG00000074071), MRPL14 (ENSG00000180992), MRPL57 (ENSG00000173141), IMP3 (ENSG00000177971), MZT2A (ENSG00000173272), and / or XRCC6 (ENSG00000196419).

13. The thymocyte product according to claim 2, wherein the subpopulation includes keratinocyte-like mTECs, and the keratinocyte-like mTECs express one or more of CD24 (ENSG00000272398), ELF3 (ENSG00000163435), CLDN4 (ENSG00000189143), MAL2 (ENSG00000147676), ASAH1 (ENSG00000104763), TMEM123 (ENSG00000152558), TMBIM6 (ENSG00000139644), LGALS3 (ENSG00000131981-), MYL12B (ENSG00000118680), ACADVL (ENSG00000072778), KRT19 (ENSG00000171345), SAT1 (ENSG00000130066), RAB25 (ENSG00000132698), WFDC2 (ENSG00000101443), VAMP8 (ENSG00000118640), SPINT1 (ENSG00000166145), SERPINB1 (ENSG00000021355), CDH1 (ENSG00000039068), GSN (ENSG00000148180), SDC4 (ENSG00000124145), MGST2 (ENSG00000085871), CAST (ENSG00000153113), B4GALT1 (ENSG00000086062), PERP (ENSG00000112378), and / or DMKN (ENSG00000161249).

14. A method for generating a population of thymocytes in vitro, comprising: culturing a cell population in the presence of a soluble factor, a mineral, or a combination thereof to induce differentiation or maturation of the cell population into thymocytes wherein the cell population is optionally engineered to express a cell surface receptor or an intracellular factor, and wherein the cell population comprises one or more cell types selected from the group consisting of pluripotent stem cells (PSCs), definitive endoderm (DE) cells, pharyngeal pouch endoderm (PPE) cells, and anterior foregut endoderm (AFE) cells. The method.

15. ​ The method of claim 14, wherein the population of thymocytes comprises one or more subpopulations of immature thymic epithelial cells (iTECs), cTEC-high cells, cTEC-low cells, Aire+ mTEC-high cells, mTEC-low cells, keratinocyte-like mTEC cells, ciliated cells, myeloid cells, myoid cells, neuroendocrine cells, and tuft / ionocyte cells.

16. A method for maintaining a population of thymocytes in vitro, comprising: a) culturing the population of thymocytes in the presence of a soluble factor or a mineral; b) culturing the population of thymocytes in the presence of one or more feeder cells; and / or c) engineering the population of thymocytes to express a cell surface receptor or an intracellular factor The method comprising one or more of the above.

17. The method of claim 16, wherein the population of thymocytes is engineered to express a cell surface receptor or an intracellular factor.

18. The method of claim 16, wherein the population of thymocytes is cultured in the presence of one or more feeder cells.

19. The method of claim 18, wherein the feeder cells are one or more of endothelial cells, mesenchymal stem cells, macrophages, dendritic cells (DCs), epithelial cells, fibroblasts, stromal cells, adipocytes, vascular smooth muscle cells (VSMCs), or lymphatic endothelial cells.

20. The method of claim 16, wherein the population of thymocytes comprises one or more subpopulations selected from the group consisting of thymic epithelial progenitor cells (TEPCs), immature thymic epithelial cells (iTECs), thymic epithelial cells (TECs), medullary thymic epithelial cells (mTECs), and cortical thymic epithelial cells (cTECs).