Thymic epithelial stem cells

Abstract

The present invention relates to newly identified populations of thymic epithelial stem cells, methods for their isolation, culture and differentiation, and uses of these cells, particularly their use in therapy, the generation of therapeutic thymic constructs, and drug screening.

Description

[Technical field]

[0001] The present invention relates to newly identified populations of thymic epithelial stem cells, methods for their isolation, culture and differentiation, and uses of these cells, particularly their use in therapy, the generation of therapeutic thymic constructs, and drug screening. [Background technology]

[0002] Epithelia are paradigms of tissues that constantly replicate during development, homeostasis, and regeneration. These processes are driven by self-renewing epithelial stem cells (SCs), the only bona fide stem cells capable of extensive proliferation in vitro [1, 2, 3, 4, 5, 6, 7].

[0003] Maintenance of stemness critically depends on crosstalk between SCs and their specialized in vivo microenvironment, also known as the niche. Co-culture with irradiated feeder fibroblasts for stratified epithelial SCs or Paneth cells for intestinal SCs provides evidence of the importance of niche signals for expanding functional SCs for several generations in vitro [8, 9]. With rare exceptions (i.e., LGR5 in intestinal crypt epithelium), most of the “specialized” epithelial SCs in humans do not present unique markers and are defined by multiple phenotypic and functional characteristics [10, 11]. Nevertheless, high expression of TP63 transcription factors (TFs), especially its ΔNTP63α isoform, correlates with epithelial stemness in culture [12, 13, 14]. More recently, a transcriptional signature has been reported for epidermal “holoclones,” keratinocyte clonogenic SCs capable of self-renewal in vitro and in vivo

[15] .

[0004] The thymus stands out among organs for the unique three-dimensional (3D) morphological complexity of its epithelium, and for undergoing progressive atrophy during postnatal life

[16] . The thymus is a major lymphoid organ essential for T cell development and is uniquely required to generate and select a diverse, yet self-tolerant, T cell repertoire during fetal development and early postnatal life. This reflects the critical spatial and temporal interactions of developing thymocytes with the thymic stroma, which is composed of different types of thymic epithelial cells (medullary (m)TECs and cortical (c)TECs), myoid (MC) and neuroendocrine cells (NECs), thymic stromal cells (TICs), endothelial cells (ECs), and hematopoietic subtypes such as dendritic cells (DCs), B cells, and macrophages (Mφs). Hematopoietic cells colonize the epithelial-stromal thymic rudiments during development, facilitating lympho-stromal crosstalk that orchestrates both thymocyte development and epithelial differentiation, as well as morphogenesis. Failure of thymic epithelial specification during development results in congenital thymic insufficiency, leading to severe immunodeficiency and autoimmunity [17, 18]. Embryonic thymic epithelial progenitor cells and their differentiation into cTECs and mTECs have been extensively studied, for example, using lineage tracing, reporter systems or transplantation in mouse models [19, 20].

[0005] In contrast, the postnatal epithelial stem / progenitor cells capable of maintaining thymic homeostasis and the mechanisms that contribute to thymic repair and regeneration remain poorly understood [20, 21, 22, 23, 44]. This gap in understanding has resulted in a paucity of therapeutic strategies to address thymus-related diseases and disorders. Deeper insight into the cellular origins of thymic tissue has the potential to reveal new therapeutic strategies applicable to a wide range of medical conditions, including primary and acquired immune deficiencies, autoimmune diseases, and thymic atrophy or athymus.

[0006] To address this gap in understanding, we performed high-resolution in vivo and in vitro single-cell analyses combined with prospective isolation and differentiation assays that allowed us to identify and characterize stem cells of the postnatal human thymus. We further identified methods for the isolation and culture of these thymic epithelial stem cells (TESCs) and for the downstream differentiation of TESCs into heterogeneous thymic epithelial cell types. Summary of the Invention

[0007] In a first aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being a BCAM pos , CD49F pos , CD90 pos , and CD24 neg It is.

[0008] In a second aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being CD49F pos and CD90 pos It is.

[0009] In a third aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being a BCAM pos It is.

[0010] In a fourth aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being capable of self-renewal ex vivo.

[0011] In a fifth aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell exhibiting long-term proliferation capacity in vitro.

[0012] In a sixth aspect, the present invention provides an isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being capable of differentiating into a cortical thymic epithelial cell and / or a medullary thymic epithelial cell.

[0013] In a seventh aspect, the present invention provides a method for isolating thymic epithelial stem cells from the thymus, the method comprising: (a) obtaining a thymus tissue sample; (b) isolating thymic epithelial cells from the thymic tissue sample to obtain a thymic epithelial cell fraction; (c)BCAM pos CD49F pos CD90 pos CD24 neg and isolating thymic epithelial cells from the thymic epithelial cell fraction to obtain isolated thymic epithelial stem cells.

[0014] In an eighth aspect, the present invention provides a method for culturing thymic epithelial stem cells, comprising the steps of: (a) providing at least one isolated thymic epithelial stem cell; (b) culturing the at least one isolated thymic epithelial stem cell under conditions suitable for the maintenance and proliferation of the at least one isolated thymic epithelial stem cell.

[0015] In a ninth aspect, the present invention provides a method for culturing cortical thymic epithelial cells derived from thymic epithelial stem cells, comprising: (a) providing thymic epithelial stem cells; (b) culturing the thymic epithelial stem cells under conditions suitable for obtaining cortical thymic epithelial cells.

[0016] In a tenth aspect, the present invention provides a method for differentiating isolated thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with the membrane, wherein the at least one isolated thymic epithelial stem cell contacts an upper surface of the membrane; (c) providing a cell culture medium, the cell culture medium being disposed beneath the underside of the membrane.

[0017] In an eleventh aspect, the present invention provides a thymic construct suitable for transplantation into a subject, the thymic construct comprising an isolated thymic epithelial stem cell as described herein.

[0018] In a twelfth aspect, the present invention provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject an isolated thymic epithelial stem cell or thymic construct as described herein.

[0019] In a thirteenth aspect, the present invention provides a method for generating a thymus construct suitable for transplantation into a subject, the method comprising: (a) providing an acellular scaffold; (b) seeding the acellular scaffold with isolated thymic epithelial stem cells as described herein; (c) culturing the seeded scaffold to produce the construct.

[0020] In some embodiments, the isolated thymic epithelial stem cells of the present invention are capable of self-renewal ex vivo.

[0021] In some embodiments, the isolated thymic epithelial stem cells of the present invention exhibit long-term proliferation capacity in vitro. [Brief description of the drawings]

[0022] [Figure 1]Human postnatal thymic epithelial cells (TECs) are highly heterogeneous and contain polykeratin clusters. (A) UMAP plot visualization of thymic epithelial cells color-coded by cell cluster group. (B) UMAP visualization of log10 expression of cytokeratin genes (KRT13, KRT14, KRT15, ​​KRT17) expressed in polykeratin clusters. (C) Mean expression of polykeratin cluster markers identified by linear regression in orange, relative to expression in all other cells. (D) UMAP category feature view plot of polykeratin cluster marker gene log10 expression for: CEBPD, CLU, FN1, IFITM3, TIMP1, VCAM1, CTFG, TAGLN, and CH25H. (E) UMAP category feature view plot and dot plot showing gene categories cortical TEC, (F) mTEC Myo, and (H) and mTEC Neuroendocrine, gene lists defining each of these categories are shown to the left of the plot. (G) UMAP plot showing holoclonal signatures expressed by polykeratinocytes (thymic epithelial stem cells), (I) Category enrichment analysis of DEGs in the polykeratin cluster relative to all other clusters. Each pathway is designated on the Y-axis and -log(FDR) is represented on the X-axis. Color code indicates 10 predominantly upregulated categories in the polykeratin cluster (orange) and downregulated categories in grey. To identify over-represented gene categories, a hypergeometric test was performed on the upregulated and downregulated genes. [Diagram 2]Single-cell trajectory analysis reveals that mTEC and cTEC compartments differentiate from polykeratinocytes. (A) UMAP plot of cells colored according to pseudotime analysis partial trajectories performed with Monocle software: polykeratinocytes differentiate along the trajectory towards mTEC-Myo (left panel) and mTEC-Neuro (middle panel), while polykeratinocytes differentiate towards cortical clusters (right panel), cells. (B, C) Pseudotime heatmap showing the most time-variable genes along single-cell trajectories from polykeratinocytes to mTEC differentiation and mTEC-Neuro (B) and mTEC-Myo (C). Cluster colors and pseudotimes are indicated on top of the X-axis, while categories of variable genes are listed on the Y-axis. (D) Ouija pseudotime heatmap showing trajectories from polykeratinocytes to cTEC clusters. Highlighted genes along the trajectory, and categories of genes are indicated on the left and right of the graph, respectively. Clusters, pseudotime values, and log10 expression are shown in the legend on the right. Cluster colors and pseudotimes are indicated on top of the x-axis. (E) Single-gene log10 expression plots are shown for relevant gene categories, expressed along the Ouija pseudotime trajectory: polykeratin (CLU), cTEC differentiation (ATF3, CCL5) and mature cTEC (CTSV, PRSS16, PSMB11). [Diagram 3]Prospective isolation of mTEC and cTEC polykeratinocyte stem cells. (A) Representative FACS analysis of dissociated and enriched thymocytes for N=5 human postnatal thymi (donors aged 3 days-5 years). cTEC and mTEC cells were gated for CD49Fpos expression and further subdivided and sorted according to CD24 expression. A total of four mTEC populations were isolated: CD49FposCD90posCD24neg, CD49FposCD90negCD24pos, CD49FnegCD90posCD24neg, CD49FnegCD90negCD24pos, and two cTEC populations: CD49FposCD90posCD24neg and CD49FnegCD90intCD24neg. (B) Mean fluorescence intensity quantification of CD90 in CD24pos and CD24neg cells. (C, D) Rhodamine-B staining of each mTEC and cTEC sorted population after two passages in culture: For colony forming efficiency (CFE) assays, 500 cells were seeded in dishes, which were fixed and stained with Rhodamine-B after 12 days in culture. Cells gave rise to colonies of variable size that were considered to be either strongly stained or stained with Rhodamine-B. Different numbers of colonies indicated the diverse clonogenic potential of each population (N=4). [Figure 4]Single-cell analysis demonstrates TEC heterogeneity in vitro and defines thymus-specific cell signatures. (A) UMAP plot visualization of cultured epithelial cells color-coded by cell cluster group for each representative sample. (B) Thymic polykeratin stem cell markers were expressed by cluster 1, present only in thymic cultures: mean expression of cluster 1 genes is plotted against mean expression in all other clusters, with upregulated genes displayed in pink. (C) UMAP plot visualization of marker genes across clusters (log10 expression) shows heterogeneous transcriptional profiles in cultured TEC upregulated hybrid epithelial-mesenchymal signature (THY1, C1), stratification (C2), and keratinization / terminal differentiation (C3) markers. (D, top panel) Representative FACS sorting plots of cultured thymic epithelial cells (N=5). Feederneg / CD49Fpos thymic epithelial cells were sorted for EpCAMneg, EpCAMposCD24neg, and EpCAMposCD24pos (middle and right panels). (D, lower panel) Rhodamine-B staining of EpCAMneg, EpCAMposCD24neg, and EpCAMposCD24pos sorted populations (N=3). 500 sorted cells were plated in dishes for colony forming efficiency (CFE) and the dishes were stained after 12 days of culture. Cells gave rise to colonies of variable size that stained differentially with Rhodamine-B. [Diagram 5]Refractile edge TECs represent thymic stem cells growing in vitro. (A) Schematic diagram visualizing single cell cloning of cultured TECs. (B) Phase contrast images of individual TEC colonies. TECs were classified as refractile edge, scattered / refractile edge and stratified according to their cell morphology and colony pattern. Keratinocytes are classified only as stratified colonies. (C) Immunofluorescence labeling for epithelial and mesenchymal markers on proliferating thymic refractile edge (top left panel), scattered (top right panel), stratified (bottom left panel), and keratinocyte (bottom right panel) colonies. The majority of TECs in the cultures co-expressed cytokeratins (KRT) 5 / 14 (yellow) and KRT8 (cyan). Meanwhile, TE-7 (magenta) was expressed by refractile edge and scattered colonies but not by stratified and keratinocyte colonies (first row of each). In contrast, E-cadherin / CDH1 (magenta) and EpCAM (yellow) were highly expressed by cells in stratified colonies and keratinocytes. CD49F (cyan) was clearly detected in all cells. Nuclei were counterstained with DAPI. N=4, Scale bar, 50 μm. (D) Immunofluorescence staining for CD90 (THY1), IFITM3, and FN1 in proliferating thymic epithelial (KRT5 / 14) colonies. IFITM3 or FN1 (magenta) and CD90 (THY1, cyan) were expressed by refractile margin and scattered colonies, but not by stratified or keratinocyte colonies. FN1 was also detected in mouse feeder cells. N=4, Scale bar, 50 μm. [Figure 6]Polykeratinocytes expanded in vitro differentiate towards both cortical and medullary fates. (A) Immunofluorescence staining of differentiated cTEC and mTEC polykeratinocytes showed differentiation potential towards both cortex (CD205, green) and medulla (KRT5 / 14, red), N=5. (B) Immunostaining of cells upon differentiation (upper panel: low magnification left and high magnification right) and human postnatal thymus 7 mm sections shown in red KRT5-14 and grey KRT10 (lower panel, low magnification left and high magnification right). White arrows indicate KRT7 positive cells in green. (N=5 assays, N=3 human postnatal thymus). (C) RT-qPCR analysis of cultured TECs in 2D expansion (black dots) vs TECs after differentiation: cortical genes in green (CD205, KNCIP3, CD74 and CSTV) and medullary genes in red (CLDN4, MYOG, SOX2 and SYP). Gene expression relative to HPRT housekeeping is shown on the Y-axis, significance: Mann-Whitney test, non-parametric, *p<0.05, **p<0.01, N≧4, mean±SEM. [Figure 7] Schematic workflow for thymic SC differentiation assay from single clones, generated using Biorender.com. Differentiated clones show cells positive for KRT5 (red), cortical cells (LY75-positive, green) on the left, and areas with Hassall's corpuscle (HB) structures positive for KRT10 on the right. Nuclei were counterstained with DAPI. Scale bar, 50 μm (representative images, N=5, independent clones). [Figure 8] Immunofluorescence images of thymic scaffold graft (16 weeks post-implantation, wpt) sections (7 μm) stained for cortical and HB regions: left panel LY75 in green for cortical cells, KRT5 (red) and KRT10 (grey) for medullary cells. Right panel immunofluorescence staining of 16 wpt graft sections: ASCL1 (magenta) and KRT18 (grey) for medullary transitional cells, and KRT18 (grey) SOX2 (cyan) for neuroendocrine cells. Nuclei are counterstained with DAPI. Scale bar, 50 μm (N=8 reassembled scaffolds per time point). [Figure 9]RT-qPCR analysis of thymocytes expanded in 2D expansion (black dots) versus differentiation showing upregulation of cortical genes in green (LY75, KCNIP3, CD74, CSTV, CD274, FOXN1) (top panel) and medulla genes in red (CLDN4, MYOG, SOX2, SYP, ASCL1, CLDN3, SOX11) (bottom panel). RT-qPCR analysis of thymic clones in 2D expansion (black dots) versus post-differentiation thymic clones showing upregulation of cortical genes in green dots (LY75, KCNIP3) and medulla genes in red squares (CLDN4, MYOG, SOX2). Gene expression relative to HPRT housekeeping is shown on the y-axis, significance: Mann-Whitney test, non-parametric, *p<0.05, **p<0.01, N=5, independent clones, mean±SEM. [Figure 10] Polykeratinocytes reside in the subcapsular and perivascular niches in vivo. (A) Low and high magnification images showing immunofluorescent labeling of thymic epithelial cells in human thymus co-stained with anti-ITGA6 (CD49f) antibody (yellow), FN1 (magenta) and EpCAM (cyan). The left panel shows co-staining in the subcapsular region at low and high magnification, and the right panel shows co-staining in the medullary region. Arrows (white) highlight individual triple positive cells. (N=4, human thymus). (B) Immunofluorescent labeling of human postnatal thymus sections with co-staining with anti-KRT15 antibody (yellow), KRT13 (magenta) or KRT8-18 (cyan) at high magnification. The left panel shows co-staining in the subcapsular region, and the right panel shows co-staining in the medulla. Arrows (white) highlight individual cells with triple co-staining (N=4). [Figure 11]Prospective isolation of clonogenic mTECs and cTECs. (A) Volcano plot analysis of freshly sorted clonogenic vs. non-clonogenic thymic epithelial cells. All genes present on the Stem Cell NanoString nCounter Panel-Plus were plotted. Each dot represents one gene. Log fold change values ​​of sorted BCAMpos vs. BCAMneg cells are represented on the x-axis. The y-axis shows the log10 of adjusted p-values. A p-value of 0.05 and a fold change of 2 are shown in grey highlighting the most significantly upregulated (red) and downregulated (blue) genes (N=3, human thymus samples). (B) Single gene expression profile of adhesion molecules and surface markers expressed by BCAMpos and BCAMneg TECs via nCounter NanoString Technologies (N=3, human thymus samples). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The thymus is important for T cell development and the establishment of a vast T cell repertoire required for effective immune function. Thymopoiesis, the process of T cell development within the thymus, is critically dependent on interactions with the thymic stroma, which is composed primarily of thymic epithelial cells and thymic stromal cells. Currently available systems for studying thymopoiesis, and thymic function in general, are hampered by a poor understanding of the origin of thymic epithelial cells. In particular, a population of pluripotent stem cells that give rise to multiple thymic epithelial cell lineages has not been identified so far.

[0024] The present invention describes, for the first time, this new population of thymic epithelial stem cells, a clonogenic population of self-renewing cells that can give rise to multiple different epithelial cell lineages within the thymus, including both cortical and medullary thymic epithelial cell lineages.

[0025] The thymic epithelial stem cells identified by the present inventors are characterized by their unique protein expression patterns and gene signatures. Notably, thymic epithelial stem cells can be prospectively isolated based on the expression (or lack thereof) of surface molecules such as BCAM, CD49F, CD90 and CD24. Thymic epithelial stem cells also uniquely display a "polykeratin" signature, in which cells individually co-express cytokeratin genes that are typically restricted to certain cell types and do not co-express with other cytokeratins.

[0026] A method for the isolation of thymic epithelial stem cell populations from thymic tissue is described herein for the first time. Once isolated from the thymus, the described thymic epithelial stem cells can be expanded long-term ex vivo. Thymic epithelial stem cells are multipotent and can differentiate in vitro into multiple specialized thymocytes.

[0027] The described methods for isolating thymic epithelial stem cells, and the use of the cells themselves, provide powerful new research tools for understanding thymic development and for drug screening for agents that may affect, for example, cellular senescence or proliferation. The isolated thymic epithelial stem cell populations also have great potential as therapeutic agents and may prove useful in a variety of applications, particularly those associated with immune dysfunction or other disorders associated with dysfunction or alteration of thymic activity.

[0028] definition The following provides certain definitions of terms, technical means, and embodiments used in this specification.

[0029] As used herein, the term "administration" refers to administration of a composition to a subject. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be bronchial (including by bronchial infusion), buccal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ or tissue (e.g., intrahepatic, intratumoral, peritumoral, etc.), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal infusion), transdermal, vaginal, and intravitreal. Administration can include intermittent dosing. Alternatively, administration can include continuous dosing (e.g., perfusion) for at least a selected period of time.

[0030] In some embodiments, the present invention provides a cell culture composition. For example, a method of isolating and / or culturing thymic epithelial stem cells can provide a population of cultured cells. The population of cultured cells can be stored (e.g., frozen) for future use. In some embodiments, the present invention provides a method of cell banking. The present invention also provides a method of making a cell bank comprising thymic epithelial stem cells. For example, the method of cell banking can include isolating thymic epithelial stem cells from thymic tissue, culturing or expanding the cells to obtain a suitable number of cells, and storing (e.g., freezing) the cells for future use. The method can also include culturing previously isolated thymic epithelial stem cells (e.g., cells previously isolated and stored in a cell bank). A cell bank comprising thymic epithelial stem cells can provide a source for making a pharmaceutical composition. The thymic epithelial stem cells can be used directly from the bank or can be expanded before use.

[0031] As used herein, the term "cell replacement therapy" refers, for example, to thymus removal early in life that is associated with negative outcomes, which replacement can ameliorate.

[0032] As used herein, the term "clonogenic" refers to single cell cloning, i.e., a cell or cells that may have the ability to grow into a colony. The isolated thymic epithelial stem cells of the present invention may be clonogenic cells. For example, as described in the Examples, isolated CD49F pos CD90 pos CD24 neg Thymic epithelial stem cells give rise to growing colonies in vitro.

[0033] Clonogenicity may be assessed by any suitable method described herein or known in the art, for example, by a clonogenic or colony formation assay.

[0034] Cytokeratin Cytokeratins or keratins (KRT) are intermediate filament proteins that define the specific lineage differentiation of simple, stratified, or glandular epithelial cell types in different tissues under physiological conditions. The cells of the present invention surprisingly express multiple cytokeratins and can be referred to as "polykeratin" or "polykeratinocytes". As discussed in the Examples, thymic epithelial stem cells have surprisingly been found to co-express cytokeratins that are typically expressed only by one specific cell type. For example, thymic epithelial stem cells have surprisingly been found to co-express both KRT8 (typically expressed by simple epithelium) and KRT5 (typically expressed by stratified epithelium).

[0035] In some embodiments, the isolated thymic epithelial stem cells express at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 1 cytokeratin gene. In some embodiments, the isolated thymic epithelial stem cells express at least 2 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 3 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 4 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 5 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 6 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 7 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 8 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 9 cytokeratin genes. In some embodiments, the isolated thymic epithelial stem cells express at least 10 cytokeratin genes.

[0036] The cytokeratin or keratin genes expressed by the cells of the invention may include KRT5, KRT8, KRT13, KRT14, KRT15, ​​KRT17, KRT18, and KRT19, or combinations thereof.

[0037] In some embodiments, the isolated epithelial stem cells express KRT5. In some embodiments, the isolated epithelial stem cells express KRT8. In some embodiments, the isolated epithelial stem cells express KRT13. In some embodiments, the isolated epithelial stem cells express KRT14. In some embodiments, the isolated epithelial stem cells express KRT15. In some embodiments, the isolated epithelial stem cells express KRT17. In some embodiments, the isolated epithelial stem cells express KRT18. In some embodiments, the isolated epithelial stem cells express KRT19.

[0038] In some embodiments, the isolated epithelial stem cells express KRT13, KRT18 and KRT15. In some embodiments, the isolated epithelial stem cells express KRT13, KRT18 and KRT17. In some embodiments, the isolated epithelial stem cells express KRT5, KRT14, KRT18 and KRT13. In some embodiments, the isolated epithelial stem cells express KRT5, KRT18 and KRT13. In some embodiments, the isolated epithelial stem cells express KRT14, KRT18 and KRT13. In some embodiments, the isolated epithelial stem cells express KRT14, KRT17 and KRT13. In some embodiments, the isolated epithelial stem cells express KRT14, KRT17 and KRT18. In some embodiments, the isolated epithelial stem cells express KRT15, ​​KRT17 and KRT13. In some embodiments, the isolated epithelial stem cells express KRT15, ​​KRT17 and KRT18.

[0039] In some embodiments, the isolated epithelial stem cells do not express KRT7, KRT1, KRT10, KRT4, KRT16, KRT23, KRT6A, or a combination thereof. In some embodiments, the isolated epithelial stem cells do not express KRT7. In some embodiments, the isolated epithelial stem cells do not express KRT1. In some embodiments, the isolated epithelial stem cells do not express KRT10. In some embodiments, the isolated epithelial stem cells do not express KRT4. In some embodiments, the isolated epithelial stem cells do not express KRT16. In some embodiments, the isolated epithelial stem cells do not express KRT23. In some embodiments, the isolated epithelial stem cells do not express KRT6A.

[0040] Determining the expression of cytokeratin genes can be carried out by any suitable method known in the art and discussed herein.

[0041] As used herein, the term "differentiation" refers to the ability of a dividing cell (e.g., an isolated thymic epithelial stem cell) to change its functional or phenotypic type to give rise to a different cell type.

[0042] It has been surprisingly shown that isolated thymic epithelial stem cells can differentiate into both cortical and medullary thymic epithelial cells, regardless of whether the initial cell was a cortical or medullary thymic epithelial stem cell.

[0043] In some embodiments, the isolated thymic epithelial stem cells can differentiate into medullary thymic epithelial cells. Exemplary medullary thymic epithelial cells (mTECs) include mTEC precursors, neuroendocrine cells, myoid cells, ionocytes, and Hassall's corpuscle region cells. In some embodiments, the mTECs express CLDN3, CLDN4, and / or ASCL1, or a combination thereof.

[0044] In some embodiments, the isolated thymic epithelial stem cells can be differentiated into cortical thymic epithelial cells, including cells expressing CD205, CTSV, FOXN1, KCNIP13, CD274 (PDL-1), or combinations thereof.

[0045] The present invention further provides methods of differentiating thymic epithelial stem cells. In some embodiments, the methods are in vitro methods. In some embodiments, the methods of differentiating isolated thymic epithelial stem cells include: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with the membrane, wherein the at least one isolated thymic epithelial stem cell contacts an upper surface of the membrane; (c) providing a cell culture medium, the cell culture medium being disposed beneath the underside of the membrane.

[0046] The cell culture medium is any cell culture medium suitable for supporting the growth and maintenance of cell cultures in vitro. Suitable cell culture media are known in the art. In some embodiments, the cell culture medium is Pneumacult Maintenance Medium™. In some embodiments, the cell culture medium is Neurocult™ base, e.g., Neurobasal™ medium supplemented with B-27™ Plus Supplement (Gibco, 50X), Gibco B-27™ Plus Supplement (50X), N-2 Supplement (Gibco, 100X), DAPT 10uM. In another example, a suitable cell culture medium includes DMEM-F12 (3:1), penicillin / streptomycin (P / S), 10% serum, insulin, and triiodothyronine. In some embodiments, the cell culture medium is any basal culture medium (e.g., DMEM:F12) that can be supplemented with serum (2-10%) and other supplements suitable for cell survival and proliferation (e.g., growth factors and / or hormones). Alternatively, the basal medium can be combined with supplements that also provide basic nutrients (e.g., serum-free medium).

[0047] In some embodiments, at least one isolated thymic epithelial stem cell is expanded or cultured as described herein prior to differentiation.

[0048] In one aspect, the invention provides a cell culture comprising at least one isolated thymic epithelial stem cell of the invention.

[0049] In some embodiments, the present invention provides a method of differentiating isolated thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with the membrane, wherein the at least one isolated thymic epithelial stem cell contacts an upper surface of the membrane; (c) contacting at least one isolated thymic epithelial stem cell and a membrane with a first cell culture medium; (d) maintaining at least one isolated thymic epithelial stem cell under conditions suitable for proliferation of the isolated thymic epithelial stem cell to obtain an expanded population of thymic epithelial stem cells; (d) removing the first cell culture medium; (e) providing a second cell culture medium, the second cell culture medium being disposed beneath the underside of the membrane; (f) maintaining the expanded population of thymic epithelial stem cells under conditions suitable for differentiation of the expanded population of thymic epithelial stem cells.

[0050] In some embodiments, the first cell culture medium is any medium suitable for supporting the growth of epithelial cells in culture. In some embodiments, the first cell culture medium is a serum-free medium. In some embodiments, the first cell culture medium is a cFAD medium. In some embodiments, the second cell culture medium is Pneumacult Maintenance Medium™. In some embodiments, the second cell culture medium is Neurocult™ based, as described above.

[0051] As used herein, the term "self-renewal" or "ex vivo self-renewal" refers to the ability of a cell, e.g., a thymic epithelial stem cell, to give rise to more cells of the same type. In general, self-renewal indicates that a cell is capable of self-renewal by division. Ex vivo self-renewal refers to cells capable of self-renewal once isolated or otherwise removed from an in vivo environment, e.g., once placed in culture.

[0052] Gene expression The expression of a gene or set of genes can be used to detect, identify or distinguish cell types from each other. The inventors have surprisingly discovered a novel gene expression profile that can distinguish thymic epithelial stem cells from other cell types. This gene expression profile can be used to identify thymic epithelial stem cells and can also be used in methods for isolating and culturing thymic epithelial stem cells.

[0053] In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of EPCAM, CD49F, FN1, TIMP1, IFITM3, VCAM1, BCAM, LIFR, CEPBD, CLU, CCL19, CH25H, COL7A1, CTGF, APOE, FGFR2, BOC, ITGA5, SOX17, YAP1, PTGDS, CD34, VWF, SPARC, CAV-1, EPAS-1, TIMP3, COL4A2, COL5A1, COL6A3, TP63 (e.g., ΔNTP63α), and cMYC, and combinations thereof. In some embodiments, the isolated thymic epithelial stem cells express EPCAM. In some embodiments, the isolated thymic epithelial stem cells express CD49F. In some embodiments, the isolated thymic epithelial stem cells express FN1. In some embodiments, the isolated thymic epithelial stem cells express IFITM3. In some embodiments, the isolated thymic epithelial stem cells express VCAM1. In some embodiments, the isolated thymic epithelial stem cells express BCAM. In some embodiments, the isolated thymic epithelial stem cells express CEPBD. In some embodiments, the isolated thymic epithelial stem cells express CLU. In some embodiments, the isolated thymic epithelial stem cells express CCL19. In some embodiments, the isolated thymic epithelial stem cells express CH25H. In some embodiments, the isolated thymic epithelial stem cells express COL7A1. In some embodiments, the isolated thymic epithelial stem cells express CTGF. In some embodiments, the isolated thymic epithelial stem cells express APOE. In some embodiments, the isolated thymic epithelial stem cells express PTGDS. In some embodiments, the isolated thymic epithelial stem cells express FGFR2. In some embodiments, the isolated thymic epithelial stem cells express BOC. In some embodiments, the isolated thymic epithelial stem cells express ITGA5. In some embodiments, the isolated thymic epithelial stem cells express SOX17. In some embodiments, the isolated thymic epithelial stem cells express LIFR. In some embodiments, the isolated thymic epithelial stem cells express YAP1.In some embodiments, the isolated thymic epithelial stem cells express PTGDS. In some embodiments, the isolated thymic epithelial stem cells express CD34. In some embodiments, the isolated thymic epithelial stem cells express VWF. In some embodiments, the isolated thymic epithelial stem cells express SPARC. In some embodiments, the isolated thymic epithelial stem cells express CAV-1. In some embodiments, the isolated thymic epithelial stem cells express EPAS-1. In some embodiments, the isolated thymic epithelial stem cells express TIMP3. In some embodiments, the isolated thymic epithelial stem cells express COL4A2. In some embodiments, the isolated thymic epithelial stem cells express COL5A1. In some embodiments, the isolated thymic epithelial stem cells express COL6A3. In some embodiments, the isolated thymic epithelial stem cells express TP63. In some embodiments, the isolated thymic epithelial stem cells express ΔNTP63α.

[0054] In some embodiments, the isolated thymic epithelial stem cells express EPCAM, CD49F, and FN1. In some embodiments, the isolated thymic epithelial stem cells express EPCAM, CD49F, and IFITM3. In some embodiments, the isolated thymic epithelial stem cells express CD49F, FN1, and TIMP1. In some embodiments, the isolated thymic epithelial stem cells express COL7A1 and CTGF. In some embodiments, the isolated thymic epithelial stem cells express FN1, IFITM3, and TIMP1. In some embodiments, the isolated thymic epithelial stem cells express EPCAM and CD49F. In some embodiments, the isolated thymic epithelial stem cells express EPCAM and BCAM. In some embodiments, the isolated thymic epithelial stem cells express cytokeratin (KRT) and CD49F. In some embodiments, the isolated thymic epithelial stem cells express cytokeratin (KRT) and BCAM. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TP63 (e.g., ΔNTP63α) and KRT15, ​​or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express cytokeratin (KRT) and TP63. In some embodiments, the isolated thymic epithelial stem cells express cytokeratin (KRT) and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT15 and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT15 and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT15 and BCAM. In some embodiments, the isolated thymic epithelial stem cells express KRT15, ​​BCAM, and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT15, ​​BCAM, and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT8 and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT8 and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT8 and BCAM.In some embodiments, the isolated thymic epithelial stem cells express KRT8, BCAM, and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT8, BCAM, and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT5 and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT5 and ΔNTP63α. In some embodiments, the isolated thymic epithelial stem cells express KRT5 and BCAM. In some embodiments, the isolated thymic epithelial stem cells express KRT5, BCAM, and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT5, BCAM, and TP63. In some embodiments, the isolated thymic epithelial stem cells express KRT5, BCAM, and ΔNTP63α.

[0055] In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1, TIMP1, IFITM3, VCAM1, BCAM, LIFR, CEPBD, CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and TIMP1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and IFITM3, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and VCAM1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and BCAM, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and LIFR, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of FN1 and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and IFITM3 or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and VCAM1 or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and BCAM or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and LIFR or a combination thereof.In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of TIMP1 and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of IFITM3 and VCAM1 or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of IFITM3 and BCAM or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of IFITM3 and LIFR or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of IFITM3 and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of IFITM3 and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of VCAM1 and BCAM or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of VCAM1 and LIFR or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of VCAM1 and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of VCAM1 and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of BCAM and LIFR or a combination thereof.In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of BCAM and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of BCAM and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of LIFR and CEPBD or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of LIFR and CLU or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of CEPBD and CLU or a combination thereof.

[0056] In some embodiments, the thymic epithelial stem cells express one or more cytokeratin genes, as discussed above.

[0057] In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and TIMP1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and IFITM3, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and VCAM1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and BCAM, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and LIFR, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​FN1 and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and IFITM3, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and VCAM1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and BCAM, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and LIFR, or a combination thereof.In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​TIMP1 and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​IFITM3 and VCAM1, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​IFITM3 and BCAM, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​IFITM3 and LIFR, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​IFITM3 and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​IFITM3 and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​VCAM1 and BCAM, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​VCAM1 and LIFR, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​VCAM1 and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​VCAM1 and CLU, or a combination thereof.In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​BCAM and LIFR, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​BCAM and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​BCAM and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​LIFR and CEPBD, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​LIFR and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​LIFR and CLU, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells express at least one gene selected from the group consisting of KRT15, ​​CEPBD and CLU, or a combination thereof.

[0058] In some embodiments, the isolated thymic epithelial stem cells described herein express genes associated with a "holoclonal" signature. A "holoclonal" signature represents an activated / proliferating, long-term proliferating stem cell as defined in the art [6]. In some embodiments, the isolated thymic epithelial stem cells described herein express at least one gene selected from the group consisting of CCNA2, AURKB, FOXM1, ANLN, LMNB1, HMGB2, or a combination thereof. In some embodiments, the isolated thymic epithelial stem cells described herein can be activated. In some embodiments, the activated isolated thymic epithelial stem cells described herein express at least one selected from the group consisting of CCNA2, AURKB, FOXM1, ANLN, LMNB1, HMGB2, or a combination thereof.

[0059] Gene expression can be detected or measured by any suitable method known in the art. For example, gene expression can be measured by polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, RNA-scope, and HiPLEX.

[0060] kit The present invention also provides kits for identifying thymic epithelial stem cells as described herein. In some embodiments, the kits comprise at least one binding molecule that binds to BCAM, CD49F, CD90, or CD24.

[0061] In some embodiments, the kit comprises a binding molecule that binds to BCAM. In some embodiments, the kit comprises a binding molecule that binds to CD49F. In some embodiments, the kit comprises a binding molecule that binds to CD90. In some embodiments, the kit comprises a binding molecule that binds to CD24. In some embodiments, the kit comprises a first binding molecule that binds to BCAM and a second binding molecule that binds to CD49F. In some embodiments, the kit comprises a first binding molecule that binds to BCAM and a second binding molecule that binds to CD90. In some embodiments, the kit comprises a first binding molecule that binds to BCAM and a second binding molecule that binds to CD24. In some embodiments, the kit comprises a first binding molecule that binds to CD49F and a second binding molecule that binds to CD90. In some embodiments, the kit comprises a first binding molecule that binds to CD49F and a second binding molecule that binds to CD24. In some embodiments, the kit comprises a first binding molecule that binds to CD90 and a second binding molecule that binds to CD24. In some embodiments, the kit comprises a first binding molecule that binds BCAM, a second binding molecule that binds CD49F, and a third binding molecule that binds CD90. In some embodiments, the kit comprises a first binding molecule that binds BCAM, a second binding molecule that binds CD49F, and a third binding molecule that binds CD24. In some embodiments, the kit comprises a first binding molecule that binds CD49F, a second binding molecule that binds CD90, and a third binding molecule that binds CD24. In some embodiments, the kit comprises a first binding molecule that binds BCAM, a second binding molecule that binds CD49F, a third binding molecule that binds CD90, and a fourth binding molecule that binds CD24.

[0062] In some embodiments, the binding molecule is an antibody or a fragment thereof. In some embodiments, the binding molecule is conjugated to or labeled with a detectable agent. In some embodiments, the detectable agent is a fluorophore.

[0063] The kit can be a kit for identifying thymic epithelial stem cells as described herein using fluorescence-activated cell sorting (FACS). Exemplary binding molecules and fluorophores are provided in Table 1 below. [Table 1]

[0064] In some embodiments, the kit comprises instructions for identifying thymic epithelial stem cells as described herein.

[0065] Long-term growth potential Cell culture generally involves the serial passage of cells to expand a cell line or cell population to a certain number of cells. Expansion (i.e., increase in cell number) generally occurs through the division of cells in culture. As used herein, long-term proliferation capacity refers to the capacity of thymic epithelial stem cells to proliferate in vitro for several passages or population doublings before reaching senescence, for example, without losing stem cell phenotype or division capacity.

[0066] In some embodiments, the isolated thymic epithelial stem cells are capable of at least 5 population doublings in vitro. In some embodiments, the isolated thymic epithelial stem cells are capable of at least 10 population doublings in vitro. In some embodiments, the isolated thymic epithelial stem cells are capable of at least 15 population doublings in vitro. In some embodiments, the isolated thymic epithelial stem cells are capable of at least 20 population doublings in vitro. In some embodiments, the isolated thymic epithelial stem cells are capable of at least 25 population doublings in vitro. In some embodiments, the isolated thymic epithelial stem cells are capable of at least 30 population doublings in vitro.

[0067] Treatment method The isolated thymic epithelial stem cells, and related cell culture compositions, pharmaceutical compositions, and thymic constructs described herein may be useful in methods of treating a disease or disorder in a subject.

[0068] In some embodiments, the invention provides a method of treating a disease or disorder in a subject, comprising administering to the subject an isolated thymic epithelial stem cell, cell culture composition, pharmaceutical composition, or thymic construct described herein.

[0069] Surface markers In some embodiments, the cell surface markers or genes discussed herein comprise or consist of sequences defined in the art, e.g., sequence accession databases. In some embodiments, the cell surface markers or genes discussed herein comprise or consist of one or more amino acid sequences set forth in Table 2 below. Exemplary identification of surface markers and further details of information available in the art are set forth in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0070] immunodeficiency The present invention also provides a method for treating immune deficiency in a subject. Immune deficiency refers to a state or manner in which one or more components of the immune response are impaired and unable to effectively resolve infection or disease. Immune deficiency disorders include primary immune deficiencies (PIDs), inherited conditions resulting from genetic mutations, and secondary immune deficiencies (SIDs) in which immune deficiency is acquired as a result of disease or environmental factors (e.g., chemotherapy).

[0071] In some embodiments, the methods comprise administering to a subject an isolated thymic epithelial stem cell as described herein, or a related cell culture composition, pharmaceutical composition, or thymic construct as described herein.

[0072] In some embodiments, the invention provides methods of treating a disease or disorder in a subject, where the disease or disorder is a primary immune deficiency, hi some embodiments, the invention provides methods of treating a disease or disorder in a subject, where the disease or disorder is a secondary immune deficiency.

[0073] Central tolerance dysfunction Central tolerance refers to a mechanism by which the T cell repertoire is selectively modified during development to eliminate T cells with high affinity for self-antigens. A breakdown or dysfunction of central tolerance can result in the development and maintenance of T cells that bind with high affinity to self-antigens and cause autoimmune disease.

[0074] In some embodiments, the invention provides a method of treating a disease or disorder in a subject, where the disease or disorder is a disease or disorder associated with dysfunctional central tolerance.

[0075] autoimmune disease In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED).

[0076] In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is an autoimmune disease. In some embodiments, the autoimmune disease is selected from myasthenia gravis, type 1 diabetes, an autoimmune myopathy, and a connective tissue disease. In some embodiments, the autoimmune disease is myasthenia gravis. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoimmune disease is an autoimmune myopathy. In some embodiments, the autoimmune disease is a connective tissue disease.

[0077] cancer In some embodiments, the invention provides a method of treating a disease or disorder in a subject, where the disease or disorder is cancer, e.g., thymoma, thymic carcinoma, sarcoma, or thymic neuroendocrine tumor. In some embodiments, the cancer is thymoma. In some embodiments, the cancer is thymic carcinoma. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is thymic neuroendocrine tumor.

[0078] thymic atrophy The present invention also provides a method of preventing or reversing thymic atrophy in a subject. Thymic atrophy occurs naturally throughout life by a process of involution in which the thymus undergoes a reduction in thymic mass, loss of thymic structure, and changes in thymic architecture. Thymic atrophy or involution can result in impaired T cell development, including a reduced T cell repertoire and reduced T cell output, which is detrimental to the immune response. Thymic involution may affect immune function, especially in the elderly

[24] . In addition, thymic atrophy may also occur after cancer treatment, or in conditions such as infection or pregnancy

[24] . The present invention provides a method of preventing or reversing thymic atrophy in a subject, comprising administering to the subject an isolated thymic epithelial stem cell as described herein, or a cell culture composition, pharmaceutical composition, or thymic construct as described herein.

[0079] athymic The present invention also provides a method for treating athymus in a subject, and a method for treating athymic-related or athymic-caused disease or disorder. Congenital athymus is a fatal condition because the absence of T cells leads to severe primary immune deficiency (PID), which results in opportunistic infections that cause early death. DiGeorge syndrome (deletion in chromosome 22) is the first and most frequent cause of athymus, but Foxn1 loss of function (nude phenotype, thymic epithelial failure) or Tbx1 mutations have been reported in some cases. In addition, there are new reports of congenital immune deficiencies that may be associated with thymic stromal dysfunction.

[0080] In some embodiments, the present invention provides a method of treating athymus in a subject, comprising administering to the subject an isolated epithelial stem cell, cell culture composition, pharmaceutical composition, or thymic construct described herein.

[0081] In some embodiments, the subject referred to in the methods described herein is a mammal. In some embodiments, the subject is a human.

[0082] The present invention also encompasses isolated thymic epithelial stem cells for use in the therapeutic methods described above and herein. The present invention also encompasses isolated thymic epithelial stem cells, pharmaceutical compositions, or cell culture compositions comprising thymic constructs for use in the methods described herein. For example, the present invention also provides isolated thymic epithelial stem cells as described herein for use in a method of treating a disease or disorder in a subject.

[0083] Drug Screening Methods The present invention also provides methods of drug screening using the thymic epithelial stem cells described herein. As previously explained, isolated thymic epithelial stem cells are capable of self-renewal and long-term proliferation in vitro, which makes them a suitable model for screening a wide range of drugs or agents, for example drugs or agents that affect proliferation, pluripotency, senescence, or differentiation.

[0084] Many studies have shown that thymic involution is reversible. Several approaches to enhance proliferation have been proposed, including factors that can target IL15, IL22, KGF, IGF, or RANKL signaling. However, these conclusions are either based on experiments in mice with uncertain relevance to human biology, or extrapolated from clinical data with potentially confounding complexities. The thymic epithelial stem cell populations (TECSPs) described herein have demonstrated regenerative capacity in vitro and have utility as a complementary, unbiased approach to reverse thymic involution. As an example, TECSP can be challenged with well-characterized small molecules (i.e., small molecules with significant target annotations

[25] ). Cells can be fixed and immunostained at optimized time points to reveal a range of phenotypic outputs. In some embodiments, these may include Ki67+Hoechst+ (proliferation), p16 and gH2AX (senescence), AIRE or Fzf2 (functional medulla), and FOXN1 or b5T (functional cortex). Thymic epithelial stem cells can be imaged, for example, using the Opera-Phenix automated microscope platform. Image analysis can be performed using the Harmony-image package. Drug screens as described herein can include evaluation of multiple parameters leading to further analysis of specific hits, including the magnitude of any drug effect and any dose-dependent effects of the drug / small molecule.

[0085] In some embodiments, thymic epithelial stem cells may be used to screen for drugs that increase proliferation or proliferation markers, where proliferation markers are any genes or molecules expressed by cells undergoing proliferation. In some embodiments, thymic epithelial stem cells may be used to screen for drugs that decrease proliferation or proliferation markers. Proliferation markers may include PCNA, MKI67, AURKB, and / or TP63 (e.g., ΔNTP63α). Cell cycle analysis may also be performed by FACS using viability dyes.

[0086] In some embodiments, thymic epithelial stem cells may be used to screen for drugs that increase cellular senescence or senescence markers, where a senescence marker is any gene or molecule expressed by senescent cells. In some embodiments, thymic epithelial stem cells may be used to screen for drugs that decrease cellular senescence or senescence markers. Drug screening as described herein may include screening for drugs that can activate or inhibit cell signaling pathways such as the p16-pRB axis or the p53-p21 axis.

[0087] In some embodiments, thymic epithelial stem cells can be used to screen for drugs that induce or increase the expression of functional markers, which are any genes or molecules expressed by cells associated with a particular cell function or type. For example, functional markers can include FOXN1, which generally indicates cortical thymic epithelial cells. In some embodiments, thymic epithelial stem cells can be used to screen for drugs that prevent or reduce the expression of functional markers. Other functional markers include AIRE, PSMB11, TBATA, PRSS16, and FEZF2.

[0088] In some embodiments, the methods of drug screening described herein may use isolated thymic epithelial stem cells or related cell culture compositions, pharmaceutical compositions, or thymic constructs.

[0089] Methods for isolating thymic epithelial stem cells The present invention provides methods for isolating or obtaining thymic epithelial stem cells. In some embodiments, the present invention provides methods for isolating thymic epithelial stem cells from the thymus, e.g., for isolating or obtaining thymic epithelial stem cells from a sample of thymic tissue. In some embodiments, the thymus is a human thymus and the thymic epithelial stem cells are human thymic epithelial stem cells.

[0090] In some embodiments, the method includes obtaining a thymus tissue sample. Alternatively, the method may include providing a thymus tissue sample previously obtained from the subject.

[0091] The method may include isolating thymic epithelial cells from a thymic tissue sample to obtain a thymic epithelial cell fraction.

[0092] In some embodiments, the method may include dissociating the thymus tissue sample to obtain single cells. Dissociating the thymus tissue sample may include treating the tissue sample with one or more enzymes. Exemplary enzymes that may be used in the method include, for example, collagenase D, dispase II, DNAse I, and combinations thereof.

[0093] In some embodiments, the method may include isolating single cells from the thymus tissue sample to obtain a single cell fraction.

[0094] In some embodiments, the method may include substantially depleting a single cell fraction of hematopoietic cells. In some embodiments, the method may include substantially depleting a single cell fraction of CD45+ cells. In some embodiments, the method may include substantially depleting a single cell fraction of red blood cells. In some embodiments, the method may include substantially depleting a single cell fraction of CD235+ cells. In some embodiments, the method may include substantially depleting a single cell fraction of hematopoietic cells and red blood cells. In some embodiments, the method may include substantially depleting a single cell fraction of CD45+ and CD235+ cells. Depletion of CD45+ and / or CD235+ cells may be accomplished by any suitable method known in the art, such as antibody-based depletion methods, magnetic separation, immunopanning, and others.

[0095] In some embodiments, the method may include isolating cells expressing an epithelial cell marker from the cell fraction described herein to obtain a thymic epithelial cell fraction. In some embodiments, the method may include isolating EPCAM+ cells from the cell fraction described herein to obtain a thymic epithelial cell fraction. Isolating EPCAM+ cells may include labeling or staining cells for EPCAM. Any suitable method known in the art for labeling or staining cells may be used. Isolating EPCAM+ cells may include separating or sorting cells labeled or stained for EPCAM by methods known in the art, for example, FACS. In some embodiments, isolating thymic epithelial stem cells may include isolating cells expressing an epithelial cell marker from the cell fraction described herein to obtain a thymic epithelial cell fraction. pos Cells and EPCAM neg CD205 pos This involves sorting the cells.

[0096] In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD90, CD24, BCAM, or a combination thereof. Any suitable method known in the art for labeling cells may be used. In some embodiments, the method may include labeling thymic epithelial cells for CD49F. In some embodiments, the method may include labeling thymic epithelial cells for CD90. In some embodiments, the method may include labeling thymic epithelial cells for CD24. In some embodiments, the method may include labeling thymic epithelial cells for BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD49F and CD90. In some embodiments, the method may include labeling thymic epithelial cells for CD49F and CD24. In some embodiments, the method may include labeling thymic epithelial cells for CD49F and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD90 and CD24. In some embodiments, the method may include labeling thymic epithelial cells for CD90 and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD90 and CD24. In some embodiments, the method may include labeling thymic epithelial cells for CD90 and BCAM.

[0097] In some embodiments, the method may include labeling thymic epithelial cells for CD24 and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD90, and CD24. In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD90, and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD24, and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD90, CD24, and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD90, CD24, and BCAM. In some embodiments, the method may include labeling thymic epithelial cells for CD49F, CD90, CD24, and BCAM.

[0098] In some embodiments, the method comprises: pos , CD49F pos , CD90 pos or CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are BCAM, BCAM-derived thymic epithelial cells, or a combination thereof to obtain isolated thymic epithelial stem cells. pos In some embodiments, the method may include isolating thymic epithelial cells that are CD49F pos In some embodiments, the method may include isolating thymic epithelial cells that are CD90 pos In some embodiments, the method may include isolating thymic epithelial cells that are CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos and CD49F pos In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos and CD90 pos In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos and CD24 negIn some embodiments, the method may include isolating thymic epithelial cells that are CD49F pos and CD90 pos In some embodiments, the method may include isolating thymic epithelial cells that are CD49F pos and CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are CD90 pos and CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos , CD49F pos , and CD90 pos In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos , CD49F pos , and CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos , CD90 pos , and CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are CD49F pos , CD90 pos , and CD24 neg In some embodiments, the method may include isolating thymic epithelial cells that are BCAM-positive. pos , CD49F pos , CD90 pos , and CD24 neg The method may include isolating thymic epithelial cells which are

[0099] In some embodiments, the method for isolating thymic epithelial stem cells from the thymus comprises: a. obtaining a thymus tissue sample; b.BCAM pos CD49F pos CD90 pos CD24 neg and isolating thymic epithelial cells from the thymic tissue sample to obtain isolated thymic epithelial stem cells.

[0100] In some embodiments, the method for isolating thymic epithelial stem cells from the thymus comprises: a. obtaining a thymus tissue sample; b. isolating thymic epithelial cells from the thymic tissue sample to obtain a thymic epithelial cell fraction; c.BCAM pos CD49F pos CD90 pos CD24 neg and isolating thymic epithelial cells from the thymic epithelial cell fraction to obtain isolated thymic epithelial stem cells.

[0101] Generally, the step of separating or isolating cells (e.g., from other cell types or other tissues) in the methods described herein may be carried out by any suitable method known in the art, for example, by using magnetic beads, and / or fluorescence activated cell sorting (FACS).

[0102] How to culture Methods for culturing thymic epithelial stem cells - Patents.com The present invention provides a method for culturing thymic epithelial stem cells. In some embodiments, the method comprises providing at least one isolated thymic epithelial stem cell and culturing the at least one isolated thymic epithelial stem cell under conditions suitable for the maintenance and proliferation of the at least one isolated thymic epithelial stem cell, the maintenance and proliferation having their usual meaning in the art.

[0103] In some embodiments, the present invention provides a method of culturing thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with at least one feeder cell; (c) contacting the at least one isolated thymic epithelial stem cell and the at least one support cell with a cell culture medium.

[0104] In some embodiments, at least one feeder cell is a feeder cell. In some embodiments, at least one feeder cell or feeder cell is any cell suitable for supporting the growth of epithelial cells in culture. In some embodiments, at least one feeder cell or feeder cell is a fibroblast. In some embodiments, at least one feeder cell or feeder cell is a mouse fibroblast. In some embodiments, at least one feeder cell or feeder cell is a sublethally irradiated mouse fibroblast. In some embodiments, at least one feeder cell or feeder cell is a sublethally irradiated mouse fibroblast 3T3-J2.

[0105] In some embodiments, the cell culture medium is any medium suitable for supporting the growth of epithelial cells in culture. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is a cFAD medium. In some embodiments, the cFAD medium comprises DMEM, F-12 Nutrient Mix, fetal bovine serum (FBS), antimicrobial agents, hydrocortisone, cholera toxin, triiodothyronine (T3), and insulin. In some embodiments, the cell culture medium is a cFAD medium, which comprises 3:1 DMEM 1× and F-12 Nutrient mix, 10% FBS, 1% penicillin and streptomycin (100×), hydrocortisone (0.4 μg / ml), cholera toxin (10 -10 M), triiodothyronine (2 × 10 -9 M), and insulin (5 μg / ml). Other suitable culture media are known in the art.

[0106] In some embodiments, the method of culturing isolated thymic epithelial stem cells includes contacting isolated thymic epithelial stem cells and / or at least one support cell with a growth promoting agent. In some embodiments, the growth promoting agent is a growth factor. The cells may be contacted with the growth factor once, or more than once, or repeatedly, for example, every 3 days. In some embodiments, the growth factor may be selected from the group consisting of human epidermal growth factor (hEGF), keratinocyte growth factor (KGF), IL-22, BMP4, RANKL, or a sex steroid inhibitor. Other suitable growth promoting agents and growth factors are known in the art.

[0107] In some embodiments, the present invention provides a method of culturing thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with at least one sublethally irradiated mouse fibroblast; (c) contacting at least one isolated thymic epithelial stem cell and at least one sublethally irradiated mouse fibroblast cell with cFAD cell culture medium.

[0108] Suitable conditions for maintaining thymic epithelial stem cells and / or support cells in culture are known in the art. For example, the cells can be maintained at a temperature of about 30°C to about 40°C, about 35°C to about 39°C, about 36°C to about 38°C, or at a temperature of about 37°C.

[0109] In some embodiments, isolated thymic epithelial stem cells can be cultured until they reach confluence or subconfluence. In some embodiments, thymic epithelial stem cells can be cultured (e.g., maintained in culture) for a certain number of population doublings (PD). A population doubling, as known in the art, is the total number of times cells in a given population have doubled during in vitro culture.

[0110] The isolated thymic epithelial stem cells to be cultured can be any type of thymic epithelial stem cell. In some embodiments, the isolated thymic epithelial stem cells are cortical thymic epithelial stem cells, e.g., cells that express CD205. In some embodiments, the isolated thymic epithelial stem cells are cells that express CD205. pos EPCAM neg In some embodiments, the isolated thymic epithelial stem cells are medullary thymic epithelial stem cells, e.g., cells that do not express CD205. In some embodiments, the isolated thymic epithelial stem cells are medullary thymic epithelial stem cells, e.g., cells that do not express CD205. neg EPCAM pos It is.

[0111] As stated elsewhere, any type of thymic epithelial stem cell (e.g., cortical or medullary) can give rise to differentiated thymic epithelial cells of both cortical and medullary lineages, regardless of whether the initially isolated thymic epithelial stem cell expressed cortical or medullary markers when isolated. It should also be understood that the protein expression of isolated thymic epithelial stem cells can change over time and in culture. For example, an isolated thymic epithelial stem cell that expresses CD205 when initially isolated from thymic tissue can lose or reduce expression of CD205 in culture. Such changes in protein or gene expression do not necessarily affect the self-renewal capacity of the cells or give rise to multiple differentiated cell types, i.e., they do not generally affect stemness.

[0112] Any of the culture methods described herein may also be applied to bulk thymocyte fractions, such as single cell fractions obtained after dissociation of thymic tissue, without isolating thymic epithelial stem cells from other cell types. Similarly, the culture methods described herein may also be applied to a population of thymic epithelial cells, including at least one thymic epithelial stem cell. In some embodiments, such a population of thymic epithelial cells may be selected from the group consisting of EPCAM, EPCAM-1, EPCAM-2, EPCAM-3, EPCAM-4, EPCAM-5, EPCAM-6, EPCAM-7, EPCAM-8, EPCAM-9, EPCAM-10, EPCAM-11, EPCAM-12, EPCAM-13, EPCAM-14, EPCAM-15, EPCAM-16, EPCAM-17, EPCAM-18, EPCAM-19, EPCAM-21, EPCAM-22, EPCAM-23, EPCAM-24, EPCAM-25, EPCAM-25, EPCAM-25, EPCAM-26, EPCAM-27, EPCAM-28, EPCAM-29, EPCAM-31, EPCAM-32, EPCAM-33, EPCAM-34, EPCAM-35, EPCAM-35, EPCAM-35, EPCAM-35, EPCAM-35, EPCAM-41, EPCAM-42, EPCAM-43, EPCAM-44, EPCAM-45, EPCAM-45, EPCAM-55, EPCAM-15, EPCAM-16, EPCAM-17, EPCAM-18, EPCAM-25, EPCAM-19, EPCAM-25, EPCAM-26, EPCAM-35, EPCAM-27, EPCAM-36, pos and / or EPCAM neg / CD205 pos The antibody may be obtained by isolating a cell which is

[0113] Any of the culture methods described herein can further include a step of separating or isolating the thymic epithelial stem cells from other cell types, such as feeder cells.

[0114] Methods for culturing cortical thymic epithelial cells The present invention also provides a method for culturing cortical thymic epithelial cells derived from thymic epithelial stem cells. The inventors have surprisingly found that culturing thymic epithelial stem cells under certain conditions promotes the differentiation of thymic epithelial stem cells into cortical thymic epithelial cells. Culturing thymic epithelial stem cells under certain conditions can also promote the proliferation of cortical thymic epithelial cells compared to other conditions. In particular, the inventors have surprisingly found that conditions of low oxygen tension promote cortical differentiation and support the proliferation of cortical thymic epithelial cells. Without wishing to be bound by theory, it is believed that lower oxygen tension in culture may reproduce the lower vascular density (and therefore lower perfusion) in the cortex of the thymus compared to the medulla.

[0115] Thus, in some embodiments, the present invention provides a method for culturing cortical thymic epithelial cells derived from thymic epithelial stem cells, comprising: (a) providing thymic epithelial stem cells; (b) culturing the thymic epithelial stem cells under conditions suitable for obtaining cortical thymic epithelial cells.

[0116] In some embodiments, the method comprises culturing the thymic epithelial stem cells at a low oxygen tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 0.5% to about 10%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 1% to about 9%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 2% to about 8%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 3% to about 7%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 4% to about 6%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 0.5%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 1%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 2%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 3% O2 ​​tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 4% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 5% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 6% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 7% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 8% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 9% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 10% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 11% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 12% O2 tension.

[0117] In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 0.5% to about 15%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 1% to about 14%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 2% to about 13%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 3% to about 12%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 4% to about 11%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 5% to about 10%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 6% to about 9%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 7% to about 8%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 0.5% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 1% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 2% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 3% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 4% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 5% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 6% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 7% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 8% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 9% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 10% CO2 tension.

[0118] In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 1% to about 9%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 2% to about 8%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 3% to about 7%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 4% to about 6%.

[0119] Also within the scope of the present invention are cortical thymic epithelial cells obtainable by the methods described herein.

[0120] Methods for culturing medullary thymic epithelial cells The present invention also provides a method for culturing medullary thymic epithelial cells derived from thymic epithelial stem cells. The inventors have surprisingly found that culturing thymic epithelial stem cells under certain conditions promotes the differentiation of thymic epithelial stem cells into medullary thymic epithelial cells. Culturing thymic epithelial stem cells under certain conditions can also promote the proliferation of medullary thymic epithelial cells compared to other conditions. In particular, the inventors have surprisingly found that conditions of atmospheric oxygen tension promote medullary differentiation and support the proliferation of medullary thymic epithelial cells. Without wishing to be bound by theory, it is believed that higher oxygen tension in cultures can reproduce the higher vascular density (and therefore increased perfusion) in the medulla of the thymus compared to the cortex. Notwithstanding the above, it is also noted that thymic epithelial stem cells can also be cultured at low O2 tension to provide medullary thymic epithelial cells.

[0121] Thus, in some embodiments, the present invention provides a method for culturing medullary thymic epithelial cells derived from thymic epithelial stem cells, comprising: (a) providing thymic epithelial stem cells; (b) culturing the thymic epithelial stem cells under conditions suitable to obtain medullary thymic epithelial cells.

[0122] In some embodiments, the method includes culturing the thymic epithelial stem cells under conditions of atmospheric oxygen tension. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 0.5% to about 10%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 10% to about 30%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 11% to about 29%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 12% to about 28%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 13% to about 27%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 14% to about 26%. In some embodiments, the method includes culturing the thymic epithelial stem cells at an O2 tension of about 15% to about 25%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 16% to about 24%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 17% to about 23%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 18% to about 22%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 19% to about 21%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 10%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 11%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 12%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 13%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at an O2 tension of about 14%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 15% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 16% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 17% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 18% O2 tension.In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 19% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 20% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 21% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 22% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 23% O2 ​​tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 24% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 25% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 26% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 27% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 28% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 29% O2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 30% O2 tension.

[0123] In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 0.5% to about 15%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 1% to about 14%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 2% to about 13%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 3% to about 12%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 4% to about 11%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 5% to about 10%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 6% to about 9%. In some embodiments, the method includes culturing the thymic epithelial stem cells at a CO2 tension of about 7% to about 8%. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 0.5% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 1% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 2% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 3% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 4% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 5% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 6% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 7% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 8% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 9% CO2 tension. In some embodiments, the method comprises culturing the thymic epithelial stem cells at about 10% CO2 tension.

[0124] In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 10% to about 30%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 11% to about 29%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 12% to about 28%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 13% to about 27%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 14% to about 26%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 15% to about 25%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 16% to about 24%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 17% to about 23%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 18% to about 22%. In some embodiments, the method comprises culturing thymic epithelial stem cells at a CO2 tension of about 5% to about 10% and an O2 tension of about 19% to about 21%. In some embodiments, the methods include culturing thymic epithelial stem cells at about 5% to about 10% CO tension and about 20% O tension.

[0125] Also within the scope of the present invention are medullary thymic epithelial cells obtainable by the methods described herein.

[0126] form The thymic epithelial stem cells according to the present invention can also be identified by their different cell morphology in culture, such as their shape, structure, morphology, size, and organization. The inventors have surprisingly discovered that thymic epithelial stem cells in culture can be distinguished from other cell types based on their morphology. The inventors have found that cultured (i.e., in vivo grown, isolated cells) thymic epithelial stem cells exhibit a "refractile border" morphology, in which the cells are highly motile instead of adhering to each other. Thymic epithelial stem cells with a refractile border morphology can weakly stain with rhodamine B in vitro. Thymic epithelial stem cells with a refractile border morphology can express or co-express the above markers (e.g., TE-7, THY1, VIM, FN1, TIMP1, IFITM3).

[0127] pluripotency As used herein, the term "pluripotency" refers to the ability of a cell, usually a stem cell, to self-renew by division and develop into multiple specialized differentiated cell types. Pluripotent cells can give rise to terminally differentiated cells. In the present invention, the isolated thymic epithelial stem cells are generally pluripotent. The isolated thymic epithelial stem cells described herein can retain multilineage differentiation potential in vitro.

[0128] In particular, the thymic epithelial stem cells described herein can give rise to both cortical and medullary differentiated thymic cell types, regardless of whether the thymic epithelial stem cells are of cortical or medullary origin. For example, isolated cortical thymic epithelial stem cells (e.g., cells that express CD205 and / or have low levels of EPCAM expression) are generally capable of giving rise to both cortical and medullary differentiated cells.

[0129] Pharmaceutical Compositions As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in an amount of unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. The pharmaceutical composition may be formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., subcutaneous, intramuscular, intravenous, intratumoral, or epidural injections, as a sterile solution or suspension, or sustained release formulation; topical application, e.g., as a cream, ointment, or controlled release patch, or spray applied to the skin, lungs, or buccal cavity; and those compatible with vaginal, rectal, sublingual, ophthalmic, transdermal, nasal, pulmonary, and other mucosal surfaces.

[0130] In some embodiments, the pharmaceutical compositions described herein may include a pharma- ceutically acceptable carrier, diluent, or excipient. As used herein, the term "pharma-ceutically acceptable" as applied to a carrier, diluent, or excipient used to formulate a composition disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0131] Protein expression and surface markers The term "surface marker" refers to molecules such as proteins expressed on the surface of cells. The pattern of protein expression and the presence of certain surface markers can be used to identify and isolate the thymic epithelial stem cells of the present invention and to distinguish them from other cell populations. Protein expression and cell surface markers can be detected by any suitable method. For example, protein expression can be detected by immunohistochemistry (IHC), immunocytochemistry (ICC), magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), mass cytometry or proteomics.

[0132] In some embodiments, the thymic epithelial stem cells express BCAM. In some embodiments, the thymic epithelial stem cells express CD49F. In some embodiments, the thymic epithelial stem cells express CD90. In some embodiments, the thymic epithelial stem cells do not express CD24. In some embodiments, the thymic epithelial stem cells express BCAM and CD49F. In some embodiments, the thymic epithelial stem cells express CD49F and CD90. In some embodiments, the thymic epithelial stem cells express BCAM and CD90. In some embodiments, the thymic epithelial stem cells express BCAM and CD49F and do not express CD24. In some embodiments, the thymic epithelial stem cells express CD49F and CD90 and do not express CD24. In some embodiments, the thymic epithelial stem cells express BCAM and CD90 and do not express CD24. In some embodiments, the thymic epithelial stem cells express BCAM and CD90 and do not express CD24. In some embodiments, the thymic epithelial stem cells express BCAM, CD49F and CD90. In some embodiments, thymic epithelial stem cells express BCAM, CD49F and CD90, and do not express CD24.

[0133] The expression of a surface marker or the level of an expressed protein may be described in positive or negative terms. A cell may be negative for a particular marker if the expression level of that marker is equivalent or comparable to that of a negative control, e.g., a cell known not to express that particular marker. For example, a cell may be described as negative for a marker if the detectable level of that marker corresponds to a fluorescence intensity signal that has a distribution equivalent to one of the negative controls on a fluorescence log plot. A negative control may be an unlabeled sample (e.g., a sample that does not contain a detectable label) or a compensation control that is a cell stained with all fluorophores minus one of the target proteins, also known as Fluorescence Minus One (FMO). A negative control may be used to set the PMT voltage value. A cell may be positive for a particular marker if the expression level of that marker is increased compared to a control, e.g., a negative control. A cell may be positive for a particular marker if the expression level of the particular marker is increased by at least 10% compared to a negative control, e.g., increased by at least 20%, increased by at least 30%, increased by at least 40%, increased by at least 50%, increased by at least 60%, increased by at least 70%, increased by at least 80%, increased by at least 90%, or increased by at least 100% compared to a negative control. A cell may be positive for a particular marker if the expression level of the particular marker is comparable to a positive control (i.e., a marker known to be expressed on the same cells).

[0134] The amount, degree or level of expression of surface marker or expressed protein may also change. For example, the cell may show a low level of expression of the surface marker. The cell may show a high level of expression of the surface marker. The cell may show an intermediate level of expression of the surface marker. The expression level of the surface marker may be defined according to whether the surface marker is expressed above or below a threshold level. The expression level of the surface marker may be determined by any suitable method known in the art. For example, the expression level of the surface marker may be determined by FACS.

[0135] In some embodiments, a cell has an intermediate level of expression of a surface marker if it expresses the marker at a level 1-2 log (i.e., 10-100 fold) higher than a negative control (e.g., a cell known not to express the surface marker). In some embodiments, a cell has a high level of expression of a surface marker if it expresses the marker at a level 1-2 log (i.e., 10-100 fold) higher than a cell expressing the surface marker at an intermediate level (e.g., a cell as defined above) compared to a negative control (e.g., a cell known not to express the surface marker), or if it expresses the marker at a level 3-4 log (i.e., 100-1000 fold) higher. Methods for determining expression thresholds are known in the art.

[0136] In some embodiments, thymic epithelial stem cells express high levels of CD49F (CD49F high In some embodiments, thymic epithelial stem cells exhibit high levels of expression of CD90 (CD90 high In some embodiments, the thymic epithelial stem cells are pos and CD49F high In some embodiments, the thymic epithelial stem cells are BCAM pos and CD90 high In some embodiments, the thymic epithelial stem cells are CD49F high and CD90 high In some embodiments, the thymic epithelial stem cells are BCAM pos, CD49F high , and CD90 high In some embodiments, the thymic epithelial stem cells are BCAM pos , CD49F high , and CD24 neg In some embodiments, the thymic epithelial stem cells are BCAM pos , CD90 high and CD24 neg In some embodiments, the thymic epithelial stem cells are CD49F high , CD90 high , and CD24 neg In some embodiments, the thymic epithelial stem cells are BCAM pos , CD49F high , CD90 high , and CD24 neg It is.

[0137] In some embodiments, the thymic epithelial stem cells express EPCAM, FN1, IFITM3, TIMP1, or a combination thereof. In some embodiments, the thymic epithelial stem cells express EPCAM. In some embodiments, the thymic epithelial stem cells express FN1. In some embodiments, the thymic epithelial stem cells express IFITM3. In some embodiments, the thymic epithelial stem cells express TIMP1. In some embodiments, the thymic epithelial stem cells express FN1 and IFITM3. In some embodiments, the thymic epithelial stem cells express FN1 and TIMP1. In some embodiments, the thymic epithelial stem cells express IFITM3 and TIMP1. In some embodiments, the thymic epithelial stem cells express FN1, IFITM3, and TIMP1.

[0138] In some embodiments, the isolated thymic epithelial cells may express proteins equivalent to or encoded by the genes described herein.

[0139] sample As used herein, the terms "sample" or "biological sample" (used interchangeably) refer to a sample that is typically obtained or derived from a biological source of interest (e.g., a tissue, or organism, or cell culture) as described herein. The source of interest may be an organism, such as an animal or a human. The sample may include biological tissue or fluid.

[0140] The methods described herein relate to isolating thymic epithelial stem cells from the thymus, particularly from a thymic tissue sample. In some embodiments, the thymus or thymic tissue sample is a human thymus or thymic tissue sample. In some embodiments, the thymus or thymic tissue sample is a non-human mammalian thymus or thymic tissue sample. In some embodiments, the thymus or thymic tissue sample is a mouse thymus or thymic tissue sample.

[0141] In some embodiments, the method is performed on a sample obtained from the subject, e.g., a human subject, at an earlier time point. In other embodiments of the invention, the method may include obtaining a sample from the subject using any suitable method.

[0142] In some embodiments, the thymic epithelial stem cells are human cells. In some embodiments, the thymic epithelial stem cells are from a non-human mammal. In some embodiments, the thymic epithelial stem cells are from a mouse.

[0143] thymus gland The thymus is the central organ for the development of mature self-tolerant T cells that recognize peptide antigens in the context of self major histocompatibility (MHC) antigens. The requirement for self MHC molecules to present antigens is referred to as MHC restriction. Athymic individuals do not have the organ to generate normal numbers of MHC-restricted T cells and are therefore immunodeficient.

[0144] The thymus is composed of immature T cells called thymocytes, and lining cells called epithelial cells that help thymocytes develop. Normally developing T cells react appropriately with the body's MHC immune receptors (called positive selection) and do not react to the body's proteins (called negative selection). The thymus is largest and most active during the neonatal and prepubertal periods. By the early teens, the thymus begins to decrease in size and activity, and thymic tissue is gradually replaced by fatty tissue. Nevertheless, some T cell development continues throughout adulthood.

[0145] Abnormalities of the thymus can result in reduced numbers of T cells and autoimmune diseases such as autoimmune polyendocrine syndrome type 1 and myasthenia gravis. These are often associated with cancer of the tissue of the thymus, called thymoma, or tissue arising from immature lymphocytes such as T cells, called lymphoma. Removal of the thymus is called thymectomy.

[0146] The thymus consists of two lobes fused in the middle and surrounded by a capsule with blood vessels running into it. The lobes are composed of a cell-rich outer cortex and a less dense inner medulla. The lobes are divided into lobules 0.5-2 mm in diameter, between which radial insertions extrude from the capsule along septa.

[0147] The cortex is composed primarily of thymocytes and epithelial cells. Thymocytes, immature T cells, are supported by a network of finely branched epithelial reticular cells that is continuous with a similar network in the medulla. This network forms an adventitia to blood vessels that enter the cortex through septa near their junction with the medulla. Other cells are also present in the thymus, including macrophages, dendritic cells, and small numbers of B cells, neutrophils, and eosinophils.

[0148] In the medulla, the network of epithelial cells is coarser than in the cortex, and the number of lymphoid cells is relatively low. Concentric nests called Hassall's corpuscles (also called thymic corpuscles) are formed by aggregation of medullary epithelial cells. These are concentric layered whorls of epithelial cells that increase in number throughout life. They are remnants of epithelial ducts that grow from the third pharyngeal pouch of the embryo to form the thymus.

[0149] thymus construct As used herein, the term "thymic construct" refers to an anatomical phenocopy of a native thymus reconstituted in vitro. In general, the isolated thymic epithelial stem cells described herein can phenocopy the unique 3D epithelial network of the thymus and rearrange the scaffold to generate a thymic construct. The thymic construct is functional and can establish a functional microenvironment that supports the development of human T cells from lymphoid precursors in vitro and from hematopoietic stem cells in vivo. In some embodiments, the thymic construct supports the development of mature T cells in vivo after transplantation into humanized immunodeficient mice. In some embodiments, the thymic construct can be considered an artificial organ, in particular an artificial thymus.

[0150] In some embodiments, the invention provides a thymic construct suitable for transplantation into a subject, the thymic construct comprising an isolated thymic epithelial stem cell or cell culture composition described herein. The thymic construct described herein may be suitable for use in a method of treating a disease or disorder in a subject, for example, a method of treating athymic or thymic atrophy.

[0151] In some embodiments, the present invention provides a method of generating a thymus construct suitable for transplantation into a subject, comprising: (i) providing an acellular scaffold; (ii) seeding the acellular scaffold with isolated thymic epithelial stem cells as described herein; (iii) culturing the seeded scaffold to produce the construct.

[0152] In some embodiments, the acellular scaffold can be a decellularized tissue scaffold or a synthetic scaffold. Such scaffolds and the methods for producing them are known in the art. For example, WO0214480 describes several scaffold categories: (1) non-degradable synthetic polymers, (2) degradable synthetic polymers, (3) non-human collagen gels that are non-porous, (4) non-human collagen meshes that are processed to desired porosity, and (5) decellularized tissues.

[0153] Acellular scaffolds typically do not include cells or cellular components, however, it will be appreciated that, for example, if a scaffold is used from a biological source, e.g., a decellularized scaffold, it is possible that some cells may remain on the scaffold after decellularization, e.g., as discussed below.

[0154] In one embodiment, the scaffold is an artificial scaffold, which may be a synthetic or natural polymer scaffold.

[0155] Other synthetic scaffolds may be proteinaceous in nature, for example consisting primarily of purified proteins such as collagen. Non-synthetic scaffolds may also be proteinaceous in nature, or consist primarily of collagenous extracellular matrix (ECM) from tissue. The scaffold may be a 3D printed scaffold, which may include any of the aforementioned materials.

[0156] Preferably, the scaffold is a decellularized (biological) matrix.

[0157] In some embodiments, the scaffold comprises an acellular thymus scaffold.

[0158] In a preferred embodiment, the acellular thymus scaffold is an acellular whole thymus scaffold, preferably a whole thymus that has been decellularized, and in a preferred embodiment, the resulting construct is a reconstituted thymus.

[0159] In some embodiments, the step of seeding the acellular scaffold with isolated thymic epithelial stem cells comprises seeding the acellular scaffold with a population of isolated thymic epithelial cells. In some embodiments, the population of isolated thymic epithelial cells comprises thymic epithelial stem cells. In some embodiments, the isolated thymic epithelial cells for seeding the acellular scaffold are substantially all thymic epithelial stem cells.

[0160] In some embodiments, the acellular scaffold is seeded with other thymic cells in addition to thymic epithelial stem cells. Other suitable thymic cells include hematopoietic stromal cells, such as thymic stromal cells, endothelial cells, fibroblasts, hematopoietic progenitor cells, mesenchymal cells, and dendritic cells.

[0161] Methods for generating thymic constructs are generally performed in vitro, although it will be appreciated that further cell proliferation and / or differentiation and generation of the construct may occur following implantation in vivo. Preferably, therefore, generation of the construct is performed in vitro until a construct is generated that is sufficiently populated with sufficiently differentiated thymic epithelial cells to permit successful implantation into a subject.

[0162] Further cell proliferation in and / or on the scaffold can then occur, for example, after implantation.It will therefore be understood that the scaffold does not need to be completely colonized with seeded cells to be useful for implantation into a subject.For example, the scaffold can have areas that are free of seeded cells, for example, the scaffold can have seeded cells over at least 70%, 80%, 90%, 95%, or at least 99% of its surface.

[0163] The cells used in the method are typically autologous, i.e. originating from or derived from the intended recipient of the tissue or organ construct produced by the method of the invention. However, cells for use in the method may also be allogeneic, i.e. obtained from or derived from a subject that is not the recipient of the tissue or organ construct to be produced. In addition, xenogeneic cells, i.e. cells derived from a different species to the recipient of the tissue / organ construct, may also be used. The cells may also be generated from pluripotent stem cells or induced pluripotent stem cells.

[0164] Aspects and embodiments described herein with the term "comprising" may include other features or steps within the scope. It is understood that aspects and embodiments described as "comprising" also describe aspects and embodiments in which the term "comprising" is replaced by the term "consisting essentially of" or "consisting of."

[0165] The phrase "selected from the group comprising" wherever they occur in this specification may be replaced with the phrase "selected from the group consisting of" and vice versa.

[0166] It is also understood that the present application discloses all combinations of any of the above aspects and embodiments with each other unless the context requires otherwise.Similarly, the present application discloses all combinations of preferred and / or optional features alone or together with any of the other aspects, unless the context requires otherwise.

[0167] The present invention will now be further illustrated by the following examples, which are meant to serve to assist the skilled artisan in practicing the present invention and are not intended to limit the scope of the invention in any way, with reference to the drawings. EXAMPLES

[0168] Materials and Methods Human tissue Postnatal thymuses were donated by patients (3 days old–82 years old). Written informed consent was obtained from the patients’ parents or legally authorized representatives under ethical approval. Human fetal livers were provided by the Joint MRC / Wellcome Trust Human Developmental Biology Resource (HDBR) under informed ethical consent with ethical approval from the Research Tissue Bank.

[0169] Isolation of thymic epithelial cells (TEC) and thymic epithelial stem cells (TESC) Thymus tissue fragments were dissociated into single cells by enzymatic treatment (0.4 mg / mL collagenase D (Roche), 0.6 mg / mL dispase II (Roche), 40 μg / mL DNAse I (Roche)) for approximately 30–45 min using a Gentle MACS instrument (Miltenyi). After dissociation, the supernatant was collected, filtered through a cell strainer (100 μm), centrifuged at 1200 rpm for 5 min, and cells were counted with trypan blue (SIGMA-ALDRICH) to assess viability. A portion of the total dissociated cell suspension was used for culture (see chapter below), while the other was depleted of CD45+ and CD235+ cells by staining them with biotinylated antibodies, then incubated with magnetic negative beads (Magnisort SAV negative beads, Invitrogen) and terminated in a magnet (STEMCELL Technologies) for 10 min. The flow-through fraction was collected and repassed through the magnet at least three times. The final enriched fraction (CD45-CD235-) was stained for surface markers to isolate epithelial cells EpCAM, CD205, and further stained for CD49F, CD90, CD24, and BCAM. Cells were sorted using a FACS Aria III instrument (BD) and sorted events were placed in culture or lysed in RTL for transcriptome analysis or processed for 10 single cell sequencing.

[0170] Thymic epithelial cell (TEC) and thymic epithelial stem cell (TESC) culture Thymic epithelial cells derived from dissociation and / or sorting were plated on a layer of sublethally irradiated mouse fibroblasts (3T3-J2) as previously described (J. G. Rheinwald & H. Green (1975), Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell, 1975 Nov;6(3):331-43). These cells were cultured in a 3:1 mixture of DMEM1X (Gibco) and F-12 Nut Mix (Gibco), supplemented with 10% fetal bovine serum (SIGMA-ALDRICH and Gibco), 1% penicillin and streptomycin (100x, Sigma), hydrocortisone (0.4 μg / ml, Calbiochem), cholera toxin (10 -10 M, Sigma), triiodothyronine (T3) (2 × 10 -9 The cells were maintained in culture in cFAD medium composed of 50 mM Sigma and insulin (5 μg / ml, SIGMA-ALDRICH). All reagents were filtered through a 0.22 μm strainer. All dissociated and sorted thymic epithelial cultures EpCAM+ were maintained in a 37°C incubator in an atmosphere of 6% CO2 and 20% O2, while CD205 pos EpCAM neg Fractions were grown at 37°C in an atmosphere of 6% CO2 and 5% O2. Human epidermal growth factor (hEGF, 10 ng / ml, PeproTech) was added to the cultures after 3 days of incubation and then every other day. Thymic epithelial cells were cultured at 2000-6000 cells / cm. 2 When subconfluent, epithelial cells were harvested using TrypLe express (Gibco) at 37°C for 3–5 min, blocked with medium, spun down at 1200 rpm, and counted.

[0171] Colony formation efficiency assay Colony formation efficiency assays or plating efficiency were performed as per other routes. A specific number of cells (e.g., 300-500 cells for bulk cultures (all dissociated); 500-1500 cells for sorted cells) were plated by serial dilution in MW6 or 60 mm dishes previously plated with lethally irradiated 3T3-J2 cells. On days 4 and 8, cultures were supplemented with hEGF (10 ng / ml, PeproTech). After 12 days of culture, cells were fixed with 4% paraformaldehyde (PFA, SIGMA-ALDRICH a) for 10 min and stained with rhodamine B (1%, SIGMA-ALDRICH) for 15 min. Dishes were washed with tap water and dried at room temperature.

[0172] Single cell cloning was performed as follows: thymic epithelial cells were trypsinized and counted as above. Once a single cell suspension was obtained, serial dilutions were performed to plate one single cell in each well into 48-well plates pre-coated with a layer of sublethally irradiated mouse fibroblasts (3T3-J2).

[0173] Coverslip immunophenotyping TECs were seeded on glass coverslips pre-seeded with irradiated 3T3-J2 at a density of 1200-2500 cells / cm2 per well (12-well plate) and cultured for up to 7 days. Cultured cells were then fixed with 4% paraformaldehyde (PFA, SIGMA-ALDRICH) for 10 min, washed twice with PBS, and kept at 4 °C until immunofluorescence (IF) staining. Quality control of primary cell cultures included mycoplasma PCR screening, STR authentication to confirm unique profiles, and KarioStat™ arrays (ThermoFisher, catalog no. 905403) to screen for possible chromosomal abnormalities.

[0174] Thymic epithelial cell differentiation assay After collection, thymic epithelial cells were cultured at 400–800 cells / mm 2TECs were plated on the membrane of thincert cell culture inserts for well plates (Greiner Bio) with a density of 100 μg / well. TECs on the membrane were cultured in cFAD until day 2, when they were exposed to air, and maintained in medium only under the insert for the remainder of the culture. Cells were differentiated in either Pneumocult maintenance medium (Stem cells technologies) or Neurocult Base (Gibco) supplemented with B-27™ supplement (50×, serum-free Gibco), N-2 supplement (100×, Gibco), and DAPT (gamma γ-secretase inhibitor) 10 uM was added during the differentiation phase. From day 2 onwards, medium was changed every other day. For immunofluorescence and transcriptome analysis (qPCR), differentiation was stopped at several time points up to 30 days. For IF, membranes were washed with PBS and then fixed with 4% PFA (SIGMA-ALDRICH) for 10 min. After fixation, membranes were washed twice in PBS and either stored at 4° C. or used immediately for immunostaining. Alternatively, membranes were covered, washed with BL buffer (Promega) for RNA harvesting, and stored at −80° C. until extraction.

[0175] Flow cytometry analysis Single cell suspensions were stained with ad-hoc antibody mixes in Hanks Balanced Salt Solution (HBSS, Life Technologies) supplemented with 2% FBS (Life Technologies) for 30 min on ice. Live and dead cells were distinguished using DAPI (SIGMA-ALDRICH) or Zombie Live-Dead dye (Invitrogen). FACS phenotyping was performed using a Fortessa X-20 instrument (BD Bioscience) and FlowJo™ software.

[0176] In vivo assay: Implantation of thymic rat scaffolds into NSG-nude mice All animal procedures were in accordance with ethical approval and UK Home Office Project License (PPL) PP9619702. NOD.Cg-Prkdcscid.Il2Rγctm1Wjl (NSG) and NOD.CgFoxn1<em>1Dvs.Prkdcscid.Il2Rγctm1Wjl (NSG-Nude, stock number: 026263) were obtained from the Jackson Laboratory, re-derived, and maintained in the Biological Resources Facility at The Francis Crick Institute.

[0177] CD and Winster ham rats were purchased from Charles River Laboratories. Rat thymus angiomicrosurgery, perfusion and decellularization were performed as described

[44] .

[0178] Fetal liver (FL) CD34+ were isolated from human fetal liver samples at 18 weeks post conception (wpc). Tissue digestion was performed at 37°C using an enzyme solution (0.1 U / mL collagenase A (Roche), 0.8 U / mL dispase II (Gibco), and 100 ug / ml DNase I (Roche) in RPMI, 2% FBS, and 1% penicillin / streptomycin. Cells were pelleted and processed for ammonium chloride red blood cell lysis. FL mononuclear cells (MNC) were magnetically sorted for CD34 positivity using the EasySep™ human CD34 positive selection kit II according to the manufacturer's instructions (STEMCELL Technologies).

[0179] NSG-nude and NSG (8-12 weeks old) mice were sublethally irradiated with 2.25 Gy from a 137 Cs source (IBL637 gamma irradiator). For primary engraftment, 200k purified fetal liver (18wpc) CD34+ cells were injected intravenously per mouse. Engraftment of human cells in mouse bone marrow was assessed at the time of sacrifice. Scaffold repopulation was achieved as follows: cell suspensions in cFAD medium of TECs and thymic stromal cells (TICs) were injected into decellularized rat thymus scaffolds using an insulin syringe (Terumo, 29.5G) (2M cells, 5:1 ratio in 100 μl each per lobe) and cultured in cFAD for 5 days. huFoetal liver CD34+ (18wpc) (230,000) ± VeraVec (100,000) cells were cultured in 50ul volume of co-culture medium (DMEM 1x (Gibco), 10% FBS (SIGMA-ALDRICH), 1% penicillin and streptomycin (100x, SIGMA-ALDRICH), triiodothyronine (T3) (2x10-9 M SIGMA-ALDRICH), insulin (5 μg / ml, SIGMA-ALDRICH), and cytokines (interleukin-7, 5 ng / mL (PeproTech), stem cell factor 5 ng / mL (PeproTech), and FLT3-L (5 ng / mL, PeproTech)) were injected the day before implantation (day 6). Subcutaneous implantation of the scaffolds was performed in NSG and NSG-nude mice 4 to 5 weeks after CD34+ injection as described previously. Mice were selected 10 and 16 weeks post-transplant (wpt) after implantation. We performed subcutaneous implantation in three humanized NSG-nude and three humanized NSG mice: all mice showed bone marrow reconstitution. Mice were implanted with four repositioned scaffolds each, resulting in a total of 24 repositioned scaffolds, of which 20 were recovered.

[0180] RNA isolation and RT-qPCR Cultured cells were harvested for gene expression analysis with either BL+TG-buffer from the ReliaPrep™ kit (Promega) or Trizol TRI Reagent (SIGMA-ALDRICH) according to the manufacturer's instructions. Precipitated and dried RNA was resuspended in nuclease-free water (Qiagen). RNA concentration was measured using a Nanodrop1000 (ThermoScientific). RNA was converted to cDNA using the GoScript™ Reverse Transcriptase Kit (Promega) according to the manufacturer's protocol. cDNA concentration was adjusted to 10 ng / μl. Quantitative (q)PCR was performed using a QuantStudio3 real-time PCR system (Applied Biosystems) with low ROX and Taqman qPCR probes (Integrated DNA Technology in MicroAmp Fast Optical 96-well reaction plates (Applied Biosystems) using a PCR master mix (PrecisionPLUS-R-Primerdesign Ltd).

[0181] Single-cell RNA sequencing - 10x genomics of fresh thymus tissue and cultured cells Trypsinized cells and FACS sorted events were resuspended in a final volume of 50 μl of HBSS+0.04% BSA solution.

[0182] Cell numbers were confirmed using an Eve automated cell counter (NanoEnTek). Where possible, appropriate volumes for 10,000 cells were adjusted with nuclease-free water. Reverse transcription and library construction were prepared by following the Chromium Single Cell 3' Reagent v3 protocol (10x Genomics) according to the manufacturer's recommendations. All complementary DNA syntheses were performed using 12 amplification cycles, and final cDNA yields ranged from approximately 3ng / μl to 15ng / μl. 10x Genomics sequencing libraries were constructed as described and sequenced on an Illumina HiSeq 4000.

[0183] Bioinformatics data analysis of single-cell data 10x FASTQ files were aligned with the CellRanger toolkit (10x Genomics, version 5.0.0) toolkit to the Ensembl human GRCh38 reference transcriptome. To identify mouse feeder cells in in vitro experiments, the in vitro dataset was also aligned to the combined human GRCh38 and mouse mm10 reference transcriptomes. Mouse feeder cells were filtered from the total cell population.

[0184] Individual samples were assessed using the Seurat R-package (version 4.0.5) (Stuart et al., 2019) and filtered based on the percentage of the following mitochondrial genes: in vivo samples: Epip6<20%; Epip7<20%; Epip10<30%; Epip14<30%; all in vitro culture samples were filtered below 20% and Seurat nFeature RNA parameter filtering was performed at nFeature RNA>200 for all in vivo samples and >750 for all in vitro samples. Individual samples from in vitro and in vivo experiments were integrated using the canonical correlation analysis method of the Seurat R-package.

[0185] For the in vivo dataset, we used selected EpCAM low CD205 pos For (cortex), 3872 cells were sorted, and EpCAM high CD205 neg For (medulla), 4935 cells, cTEC CD49f pos 1349 cells for mTEC and mTEC CD49f pos For , 1,414 cells were profiled.

[0186] For the in vitro data set, we profiled 2798 and 1871 cells for sorted cTEC-polykeratin, 3463 and 3430 cells for sorted mTEC-polykeratin, 3463 and 3430 cells for bulk TEC cultures, and 5509 cells for skin keratinocyte cultures after performing QC and mouse feeder layer read removal. For the second in vitro data set, we profiled two additional replicates of thymic epithelial cells (5968 and 3876 cells), one of skin keratinocytes (7633 cells), and one of basal airway cells (4237 cells) after performing QC and mouse feeder layer read removal.

[0187] Differential gene expression and trajectory analysis Differential gene expression analysis between various single-cell populations was performed using the glmGamPoi R-package version 1.2.0 (Ahlmann-Eltze and Huber, 2021). Unbiased trajectory analysis was performed using the monocle3 R-package version 0.2.2. Marker gene-directed trajectory analysis was performed using the Ouija R-package version 0.99.1

[40] .

[0188] Gene expression profile nCounter analysis The multiplexed NanoString nCounter™ Stem Cell Panel, enriched with a custom Stem Plus panel, was used as an expression assay for profiling of 780+25 (thymus) human genes (NanoString Technologies, Inc., Seattle, WA, USA). The assay was performed according to the manufacturer's protocol. In summary, crude cell lysates were used as input material and sorted TECs (CD205 pos BCAM pos , CD205 neg BCAM neg , EpCAM pos BCAM pos and EpCAM pos BCAM neg) were lysed in RLT lysis buffer (Qiagen) at 2,000–10,000 cells / sample.

[0189] Samples were snap frozen on liquid nitrogen and stored at -80°C. mRNA expression was measured on a NanoString nCounter™ MAX system in a final volume of 15 μl by using 2 μl of cell lysate mixed with 3' biotinylated capture probe / capture probe+ and 5' reporter probe / reporter probe+ tagged with fluorescent barcode. Probes and target transcripts were hybridized overnight at 65°C for 22 hours as recommended by the manufacturer. Data were collected on an nCounter digital analyzer (NanoString™) and imported into nSolver analysis software v4.0 (www.nanostring.com) for data quality checks, background thresholding, and normalization. The quality of the run for each sample was checked by quality control considering the 6 spike-in RNA positive controls and 8 negative controls present in the panel, FOVs (field of view per sample) were counted and binding density was confirmed.

[0190] Gene expression data were normalized in two steps: (a) by using all 12 housekeeping genes present in the panel, and (b) by adjusting the number of cells / sample to a total of 2000 cells for each sample. Background levels were determined by the average count of 8 negative control probes + 2 standard deviations. Samples containing less than 50% of probes above background or with imaging or positive control linearity flags were excluded from further analysis. Probes with raw counts below background in all samples were excluded from differential expression analysis to avoid false positive results. For differential gene expression analysis, we followed a procedure previously described

[26] . NanoString count matrices were normalized using the RUVSeq R package, and differential gene expression was performed using the R-package DESeq2.

[0191] histology Human thymus samples were fixed in 4% PFA (2 h to overnight) and processed for either cryo- or paraffin embedding. For cryo-embedding, fixed tissues were equilibrated in sucrose 25% and embedded in OCT compound (VWR). Cryosections (thickness, 7 μm) were cut on a Leica Cryostat3050. For paraffin embedding, a Leica PelorisII tissue processor and a Sakura Tissue-Tech embedding station were used. Paraffin sections (thickness, 3-5 μm) were generated using a ThermoFisher rotary microtome.

[0192] Frozen or paraffin sections were stained with hematoxylin-eosin using an automated station (Tissue-Tek Prisma) to verify the histology of each tissue and then used for immunohistochemical analysis.

[0193] immunostaining OCT-embedded tissue sections or coverslips fixed in 4% PFA were directly blocked and simultaneously permeabilized using a solution of 5% normal donkey serum (NDS, Jackson 19 Immuno Research) in PBS containing 0.5% TritonX (TritonTMX-100, SIGMAALDRICH). Paraffin-embedded samples underwent a heat-inactivated antigen retrieval process in Cytrate buffer (Sigma-Aldrich) pH 6.0 before blocking. Tissue sections / coverslips were incubated with primary antibodies 5% NDS, 0.01% TritonTMX solution overnight at 4°C. Secondary antibodies were incubated for 45 min at room temperature (RT). Nuclei were counterstained with Hoechst 33432 (10-6M) or DAPI present in Fluoroshield™ mounting medium (Abcam).

[0194] Example 1 - Identification and characterization of thymic epithelial "polykeratin" cell clusters Single-cell RNA sequencing (scRNA-seq) makes it possible to define the cellular heterogeneity of the organ microenvironment. However, a complete characterization of the thymic stroma is influenced by several factors, including the limited number of epithelial cells (<0.02%) that can be efficiently isolated from an organ where the majority of cellularity (>99%) is represented by developing thymocytes [27, 28, 29, 44].

[0195] To identify all epithelial cells in the postnatal thymus at high resolution, we performed a multi-step analysis of EpCAM-derived stromal cells after several rounds of enrichment for stromal cells. neg CD205 pos (cortex) and EpCAM pos CD205 negIndependent scRNA-sequencing analysis of the cortex and medulla populations sorted based on their chromosome (medulla) was performed. All thymic epithelial cells (TECs) were visualized in a UMAP plot (homogeneous manifold approximation and projection) with cortical cell (cTEC) clusters in light / dark green and medulla cell (mTEC) clusters in pink / red (Figure 1A). Independent scRNA-seq of each of the cTEC and mTEC sorted populations allowed us to define a specific cluster common to the cortex and medulla, which we named "polykeratin", visualized in yellow / orange in the UMAP plot (Figure 1A). In this cluster, cells expressed multiple cytokeratins (KRTs), intermediate filament proteins that define specific lineage differentiation of monolayer, stratified, or glandular epithelial cell types in different tissues under physiological conditions. The expressed keratins include KRT5, KRT8, KRT13, KRT14, KRT15, ​​KRT17, KRT18, and KRT19 (Figure 1B). Notably, polykeratinocytes co-expressed KRTs that in other tissues are associated with either proliferating SCs (e.g., KRT15) or differentiated layers (e.g., KRT13) (Figure 1B). They also co-expressed KRTs that are normally found only in monolayered (e.g., KRT8 / 18) or stratified (e.g., KRT5 / 14) epithelia and that define cTECs and mTECs, respectively, in the mature thymus. Nevertheless, some KRTs that characterize other differentiated cell types, e.g., KRT7 (lung ionocytes) and KRT1 / KRT10 (upper layers of the epidermis), were expressed only by thymic specialized differentiation clusters, i.e., ionocytes (Io) and keratinized Hassall's corpuscle regions (HB) (Figure 1A). Thus, the KRT profile of polykeratinocytes was broad but not completely promiscuous.

[0196] We further investigated the transcriptional profile of this cluster and defined, in addition to KRT, a signature atypical for epithelial cells that was visualized in the average gene expression scatter plot and feature view UMAP (Fig. 1C, D). Notably, transcripts of several genes encoding extracellular matrix (ECM) proteins or molecules for anchoring to the ECM (including FN1, TIMP1, VCAM1) provided evidence that polykeratinocytes can generate components of their own niche. In addition, we noted the expression of genes related to inflammation and, more recently, stemness, such as CLU, CEBPD, and IFITM3 (Fig. 1C, D) [30, 31, 32].

[0197] Another small cluster common to the cortex and medulla was characterized by genes associated with active proliferation, besides poly-KRT (Figure 1A). We hypothesized that this may represent an "activated" state of polykeratinocytes, and that polykeratinocytes and polykeratinocyte-proliferating cells may represent putative stem / progenitor cells of the postnatal thymus. Clonogenicity is a concept established for epidermal keratinocytes, where "holoclones" represent activated / proliferating, long-term proliferating stem cells [6]. Given that a "holoclonal" signature (including CCNA2, AURKB, FOXM1, ANLN, LMNB1, HMGB2) was recently reported

[15] , we investigated whether it was expressed by polykeratinocytes. Indeed, this signature was detected in the polykeratinocyte-proliferating cluster, consistent with these cells representing thymic stem cells activated / proliferating in vivo (Figure 1G).

[0198] Example 2 - Cortical and medulla cell heterogeneity defines new functional clusters As a first step to pursue this hypothesis, we dissected all other clusters, both in the cortex and medulla, to assign their identity and gene signatures. We found four cortical clusters (cTEC-I, cTEC-II, cTEC-III, and cTEC-IV) that were clearly identified by differentiated cortical cell signatures (e.g., TBATA, PRSS16, CD74, CTSV, KCNIP3) as shown in the feature view UMAP and dot plot (Figure 1E). cTEC-I-III confirmed the cortical clusters previously described by us and others [44, 27, 28], while the identification of the fourth cTEC-IV cluster reflected the higher level of resolution of this study. When we determined the marker genes for cTEC-IV, we observed that these differentiated cTECs expressed a signature of recently activated neurons developing synaptic plasticity (i.e., EGR1, ARC, JUN, FOS, ATF3)

[33] . This signature may reflect a specific functional state of cortical cells, which also express CD274 (Figure 1E)

[34] , confirming in addition to the traditional cortical function genes FOXN1, TBATA, and CD205. CD274 (PD-L1) has been reported to be widely upregulated in thymic tumors [35, 36]. Its expression in specialized differentiated subpopulations of healthy cTECs highlights the role of PD-L1 in controlling the activation of immature thymocytes undergoing positive selection.

[0199] In the medulla, we defined a signature of seven differentiated and specialized cells that we grouped into five categories: myoid cells (mTEC-Myo), a progressively mature population with molecules and transcription factors of smooth, skeletal, and cardiac cells (Figure 1F); two major groups of neuroendocrine cells (mTEC-NeuroI / II), characterized by the expression of GKAP1, HIGD1B, NEUROD1, NHLH1, NLRP1, SOX11, STMN2; and mTEC-NeuroIII / IV, characterized by the expression of IRX2, ATOH1, SOX2, PAX2, POU4F3, CCER2, S100A1 (Figure 1H). These clusters represent two major polyendocrine cell types with novel signatures that define the endocrine compartment of the thymus at the single cell level.

[0200] Ionocytes were defined by, among others, CFTR, FOXI1, and KRT7, reflecting the CFTR-expressing lung ionocyte population.

[37] Finally, we defined HB cluster cells characterized by expression of AIRE, FEZF2, KRT1, KRT6A, and KRT10, which are important for tolerance induction.

[0201] In addition to the polykeratinoid, cortical, and medulla specialized clusters, we identified "transition" clusters in the UMAP plot that arose from polykeratinoids toward either medulla or cortical differentiated cells (Figure 1A). We investigated whether these new clusters could drive differentiation toward some mature fate and thus share premedullary and precortical signatures, respectively.

[0202] Example 3 - Multilineage differentiation of thymic epithelial polykeratinocytes We performed pseudotime analysis using two independent methods. Pseudotime UMAP plots obtained using the Monocle algorithm highlighted distinct trajectories from the polykeratin cluster toward either cTEC-I / III or mTEC-Myo and mTEC-Neuro differentiated cells passing through two transition clusters (Figure 2A). We therefore studied which genes were progressively upregulated in these clusters. Feature view UMAP plots for selected genes including KCNIP3, SCX1, and IFIT3 showed that they were upregulated in the cTEC transition cluster, driving cTEC fate, as well as myoid cells (mTEC-Myo) and neuroendocrine cells (mTEC-Neuro-I / II, mTEC-Neuro-III / IV) derived from the transitioning mTEC cluster, characterized by ASCL1 and CLDN3, CLDN4 upregulation. Upregulation of the transcription factor ASCL1, which plays a key role in activating neural pathways [38, 39], was an unexpected finding in a differentiated epithelial population.

[0203] Pseudotime heatmaps depicted in more detail the genes involved in major differences across the trajectory towards specialized differentiation clusters. mTEC-Neuro (Figure 2B) and mTEC-Myo (Figure 2C) heatmaps displayed several successive steps of differentiation with progressive gene down- and up-regulation through transition clusters. Interestingly, the fate of cTECs was determined by the coordinated up-regulation of functional cortical genes that remained stably expressed in the mature cortex.

[0204] We found that CD24 was expressed in all mTEC differentiation clusters (HB region, ionocytes, mTEC-transitional, mTEC-Neuro, and mTEC-Myo) and its medulla-specific expression was confirmed by immunohistochemistry.

[0205] In addition, when we investigated the main sources of biological variation by principal component analysis (PCA), we observed that polykeratinocytes were separated from the clusters that were multidirectionally differentiated towards all specialized differentiation clusters, including the HB region, cTEC I-IV, and mTEC-Myo and mTEC-Neuro, which was also supported by the categorical cluster markers displayed in the PCA plot.

[0206] Thus, these data, obtained by complementary bioinformatic analyses, support the fact that the common polykeratinous epithelial population of the cortex and medulla represents multipotent multilineage stem cells of the postnatal thymus.

[0207] The Ouija algorithm

[40] allowed us to retrospectively check the accuracy of unsupervised pseudotime and infer how cTEC fate was acquired from a set of genes. Interestingly, cTEC fate was determined by the coordinated upregulation of functional cortical genes that remained stably expressed in the mature cortex, as shown by the pseudotime heatmap (Figure 2D), further supported by selected single gene plots, e.g., PSMB11, PRSS16, and CTSV (Figure 2E). Concurrently, polykeratin genes such as CLU were progressively downregulated, while others, e.g., ATF3, CCL5, were transiently expressed in the transition cluster and then downregulated as the cells acquired cTEC identity (Figure 2E). Thus, the acquisition of cTEC fate appeared to be determined by the synchronous activation of cortical transcription factors and marker genes (e.g., KCNIP3, SCX, PSMB11, CTSV, PRSS16) that established both differentiation commitment and traits related to cortical function. Taken together, our data strengthen the conclusion that the polykeratinous epithelial population represents a stem cell of the postnatal thymus common to the cortex and medulla and with a remarkably high pleiotropic, multilineage potency.

[0208] Example 4 - Polykeratinocytes can be prospectively isolated and are clonogenic TECs To corroborate that polykeratinocytes are thymic stem cells, we set out to achieve high-resolution scRNA-seq datasets for each cTEC and mTEC subpopulation that are positive for the surface marker CD49F (ITGA6), which has been reported to enrich for epithelial progenitor cells in various tissues [41, 42]. In the UMAP dot plots, red dots represent mTEC-CD49F pos The green dot cells are cTEC-CD49F pos Clustering analysis of these two populations revealed that polykeratin and polykeratin proliferation signatures were associated with mTEC-CD49F pos were more abundant in cTEC-CD49F pos and mTEC-CD49F pos In addition to polykeratin, cTEC-CD49F pos also contained differentiated cells of the cTEC-IV cluster, whereas mTEC-CD49F pos The cells also contained cells of the mTEC-Myo and mTEC-ionocyte clusters. Aiming to define a panel of surface molecules to purify the polykeratinocyte subclusters for prospective isolation, we considered that differentiated cells expressed CD24, whereas polykeratinocytes did not (Figure 3B). We proceeded with multiple enrichment steps for stromal cells and analyzed the cells by FACS for CD24 and CD90 (THY1), the latter a surface molecule that we previously reported to be expressed by TECs with an epithelial-mesenchymal hybrid phenotype

[44] . Thus, as shown by mean fluorescence intensity (MFI) quantification, the cortical (CD205 pos EpCAM neg ) and medulla (CD205 neg EpCAM pos ) Cells were further subdivided based on the expression levels for CD49F, CD24, and also CD90 (Figure 3A) (Figure 3B).

[0209] These findings led us to adopt the following selection strategy: two cortical populations were selected using CD49F pos CD90 pos CD24 neg and CD49F neg CD90 int CD24 neg The four medullary populations were isolated as pos CD90 pos CD24 neg ;CD49F pos CD90 neg CD24 pos ;CD49F neg CD90 int CD24 neg ;CD49F neg CD90 neg CD24 pos All of these fractions, when assessed independently for clonogenicity in culture, were CD49F pos CD90 pos CD24 neg Only CD49F cells, regardless of their medullary or cortical origin, gave rise to growing colonies (Figures 3C, 3D). neg CD90 int CD24 neg The mTEC fraction gave rise to a small number of colonies that could not be subcultured (Figure 3D). pos CD90 pos CD24 neg defined thymic epithelial clonogenic cells with long-term proliferation potential to both the cortex and medulla, which we refer to here as cTEC and mTEC clonogenic cells, given that their surface molecules excluded cells of differentiated clusters.

[0210] Example 5 - scRNA sequencing of TECs in culture defines a thymus-specific signature To investigate whether clonogenic epithelial cells retain the polykeratin trait in culture, we performed scRNA sequencing of cells grown in vitro. We performed scRNA-seq on two biological replicates for each culture type: clonogenic cTECs, mTECs, and TECs isolated without prospective isolation, also known as bulk cultures (Figure 4A,B). Such an analysis allowed us to address whether clonogenic cells differ according to the compartment of origin (cortex or medulla) and / or whether they differ according to the isolation method. We also included one sample of clonogenic stem cells from the epidermis that have long-term regenerative capacity

[15] . The epidermal bulk culture was used as a comparison to the thymic culture. Cells were expanded and subconfluent cultures were harvested 5 days after plating and processed for 10x genome single-cell sequencing. We profiled 2796 and 4297 cells for sorted cTEC-polykeratin, 2455 and 4297 cells for sorted mTEC-polykeratin, 5155 and 4787 cells for bulk TEC cultures, and 9478 cells for epidermal bulk cultures. All samples were further processed for subclustering to exclude mouse feeder cells.

[0211] All thymic cultures, independent of their derivation, showed comparable profiles in UMAP plots with the identification of three major cell populations (Figure 4A). Clustering analysis showed that a thymus-specific (C1) cluster emerged, but thymocytes and epidermal cells shared the majority of the cluster (Figure 4A, 4B). The thymus-specific C1 cluster highlighted atypical epithelial signatures expressed by polykeratinocytes in vivo (i.e., FN1, TIMP1, IFITM3, VCAM1) in addition to CD90 (THY1) (Figure 4C), which is consistent with the epithelial-mesenchymal hybrid phenotype described above

[44] . Clusters C2 and C3 were common to TEC and epidermal keratinocyte cultures and expressed markers of stratified and keratinized epithelium. Interestingly, cluster C1 (CD90 pos, purple dots in the feature view plot) are layered cluster C2 (EpCAM pos ), followed by keratinocyte cluster C3 (CD24 pos ) did not express markers that were progressively expressed by the IL-1β-lactamase (Figure 4D).

[0212] We therefore set out to define the surface molecular profile of TEC clusters in vitro. We analyzed by FACS thymic cultures stained with the surface markers used above for prospective cell subset isolation: EpCAM, CD49F, CD90 (THY1), and CD24. The cortical surface protein CD205 (LY75) was not included in the panel, as it was not expressed by cultured expanded cells, either as transcript or protein. We excluded mouse 3T3-J2 feeder cells by staining with feeder-PE antibody. Cultured TECs expressed CD49F pos Feeder-PE neg Surprisingly, most of the thymic epithelial cells in culture were identified as EpCAM pos All cells from mTEC-sorted cells downregulated EpCAM. Most were positive for CD90, consistent with a hybrid epithelial-mesenchymal phenotype. In contrast, CD24 downregulated EpCAM. pos It was highly expressed by a subpopulation of cells (Figure 4D). Conversely, all skin keratinocytes expressed CD90 neg EpCAM pos and expressed CD24.

[0213] We demonstrated that clonogenic TECs express thymus-specific EpCAM, which is characterized by an atypical polykeratin signature. neg It was concluded that this represents a subpopulation.

[0214] Example 6 - Polykeratinocytes in culture display "refractile edge" morphology To further investigate the nature of thymus-specific clonogenic cells, we performed single-cell clonal analysis of TECs at limiting dilution (Figure 5A). This allowed us to classify TECs based on distinct colony morphology and Rhodamine-B staining. Rhodamine-B is a dye used to assess epithelial cell keratinization in cultured cells that correlates with its intensity

[43] . Phase contrast images of individual colonies highlighted morphological heterogeneity: we defined "refractile border" colonies composed of cells with a refractile border, we named "refractile border / scattered" colonies as containing cells that are highly motile instead of adhering to each other, and another morphology was termed "stratified" due to similarity to the colony morphology of cultured stratified epithelium, e.g., epidermal keratinocytes (Figure 5B). Colonies that displayed proliferation and stacked differentiation were named "arrested" as colonies in which keratinocytes had arrested. When we analyzed them by immunocytochemistry, expression of EpCAM with additional epithelial and mesenchymal markers such as KRT5, KRT8, E-cadherin (CDH1) and TE-7, we noticed that all TEC morphologies corresponded to epithelial cells, since they co-expressed KRT5 / KRT8 (Figure 5C). However, CDH1 and EpCAM stained only colonies with a "stratified" morphology, whereas TE-7 stained only "refractile edge" and "refractile edge / scattered" colonies (Figure 5C). Based on the co-expression of TE-7 mesenchymal proteins with KRT, we concluded that our previously described motile, hybrid epithelial-mesenchymal phenotype

[44] corresponds to "refractile edge" colonies.

[0215] Therefore, we studied the expression of marker genes that identified polykeratin clusters in vivo, and thymus-specific C1 clusters in vitro. We stained these colonies for newly identified polykeratin-specific proteins such as FN1, IFITM3, and TIMP1. The results showed expression of these proteins only in the "refractile border" morphology, but not in "stratified" colonies of either the thymus or epidermis (Figure 5D). In addition, FACS analysis of cultured TECs revealed that EpCAM neg CD90 pos Only in this fraction did we show expression of another polykeratin-specific marker, VCAM1.

[0216] Next, we expanded single clones to study the hierarchical relationships of each cell type. The lightly Rhodamine B stained colonies corresponded to refractile border cell morphology, whereas the stratified clones stained strongly with Rhodamine B. These results confirmed that single cells with "refractile border" colony-forming ability were able to generate all morphotypes upon subculture, whereas "stratified" only generated "stratified" and terminally differentiated colonies. Thus, EpCAM neg The properties of the "refractile edge" cells were consistent with those expected of a multipotent clonal thymic population in culture and corresponded to the cell type identified as the thymus-specific C1 cluster in our scRNA-seq dataset.

[0217] Example 7 - Polykeratinocyte stem cells retain multilineage differentiation potential in vitro The above examples describe complementary bioinformatic analyses of scRNA-seq datasets showing that polykeratinocytes were capable of multilineage differentiation in vivo. To determine whether in vitro expanded polykeratinocyte SCs retained multilineage differentiation potential, we developed an assay that favors TEC differentiation, which we named "tissoid", obtained by seeding only one expanded epithelial cell type without support from other stromal or hematopoietic cells. "tissoid" is equivalent to the "organoid" assay for assessing lineage differentiation of expanding stem cells, but the expansion occurs in 2D instead of 3D.

[0218] Proliferated polykeratin TECs were seeded on the membrane at high density for 2 days until confluence. The growth medium was then replaced with differentiation medium, which was changed every other day for at least 14 days and up to 21 days. Differentiated cells were then fixed for immunohistochemistry (IHC) or their lysates were processed for RT-qPCR analysis. Cortical (CD205 pos KRT5 neg KRT14 neg ) and medulla (CD205 neg KRT5 pos KRT14 pos ) Differentiation was achieved independently of the cell type (cTEC or mTEC polykeratinocytes) that initiated the culture, and medullary and cortical regions were clearly distinguishable and mutually exclusive (Figure 6A). Notably, cultured cells were able to generate mTEC HB-like regions (KRT10+), few ionocytes (KRT7+), similar to the medullary regions of native thymus (Figure 6B). The medullary fate was further confirmed by upregulation of mTEC transient cluster (ASCL1, CLDN3, CLDN4), neuroendocrine (SOX2, SOX11, SYP) and myoid (MYOG) cell lineage genes, whereas the cortical fate was further confirmed by upregulation of CTSV, FOXN1, CD74, CD274 and KCNIP3 genes in differentiated cultures compared to the same stem cells in growth conditions (Figure 6C).

[0219] To conclusively demonstrate the inherent pluripotency of cTEC and mTEC polykeratins, we expanded single clones and assessed their progeny by the same assays described above. All clones that could be expanded demonstrated multilineage differentiation potential that gave rise to multiple medullary and cortical fates (Figure 7).

[0220] Example 8 - Polykeratinocyte stem cells retain multilineage differentiation potential in vivo Using a whole organ thymic reconstitution assay we developed previously, we assessed whether multilineage differentiation of clonogenic SCs could also be achieved in vivo

[44] . In this assay, clonogenic TECs (as defined above) were injected into acellular thymic scaffolds together with cultured thymic stromal cells (TICs) and subsequently transplanted into humanized NSG and NSG-nude (athymic) mice, where they became vascularized and, if functional, were able to attract hematopoietic progenitor cells from the reconstituted bone marrow. Grafts were harvested at 10 and 16 weeks post-transplant (wpt). Thymic reconstitution is characterized by the progressive maturation of the stroma and its compartmentalization into cortical and medullary regions, whereas the seeding of hematopoietic progenitor cells followed by the development of thymocytes (CD3+ cells) demonstrated proper function of the reconstituted organ. Indeed, thymocyte repopulation increased from 10 wpt to 16 wpt. Thymic reconstitution by expanded polykeratin TECs phenotypically recapitulated the organization of native thymic tissue, with the stroma compartmentalized into cortical and medullary regions, including HB formation, and the medullary progenitor cells (ASCL1 + ) and mature neuroendocrine cells (SOX2 + ) (Figure 8).

[0221] Thus, we can conclude that, similar to polyKRT in vivo, clonogenic polykeratin TECs retain pluripotency after isolation and expansion in vitro and have the capacity to reconstitute multiple thymic compartments even from a single clone. These are similar to the defining criteria for human pluripotent SCs that have been shown to be capable of self-organization and organ reconstitution. We conclude that clonogenic polykeratin stem cells retained pluripotency upon isolation and expansion in vitro.

[0222] Example 9 - The subcapsular and perivascular spaces represent the in vivo niche of polykeratinocytes Noting the expression of ECM-related genes by polykeratinocytes, we investigated their protein expression in the human postnatal thymus with the aim of localizing them in vivo. We performed immunohistochemistry on thymus sections for EpCAM, CD49F (ITGA6), KRT13, KRT14, KRT15, ​​KRT17, KRT18, and the above-mentioned polykeratin-specific proteins FN1, IFITM3, and TIMP1.

[0223] Both medulla and cortex regions were screened and imaged by confocal microscopy. Triple colocalization of EpCAM, CD49F, FN1 or IFITM3 and CD49F, FN1 and TIMP1 was detected in rare cells in the subcapsular region and scattered cells in the medulla (Figure 10A). Polykeratinocytes displayed strong signals for CD49F, and we further confirmed their epithelial nature by EpCAM immunostaining, which was bright in the medulla and dimmed in the cortex, along with their FACS profile. Importantly, they coexpressed the novel markers IFITM3 and FN1 (Figure 10A). In addition, triple colocalization of cytokeratins (KRT13, KRT18, and KRT15 or KRT17) displayed similar expression patterns in both the subcapsular and medulla regions (Figure 10B).

[0224] Next, we investigated the expression pattern of the TP63 transcription factor (TF) and its isoform ΔNTP63α, which is expressed in the basal layer of the epidermis and has previously been associated with stemness in various epithelia, including those of the thymus [12, 14]. Our data showed that the TP63 antibody (which recognizes all TP63 isoforms) had a broader expression pattern in the thymus with some brightly stained cells in both the cortex and medulla that colocalized with KRT17 and KRT18. The ΔNTP63α isoform had a more restricted expression pattern with a distribution that mirrored that of polykeratinocytes.

[0225] The ECM, and in particular the basal layer, represents a key component of the epithelial stem cell niche

[45] . To define the spatial distribution of the basal layer in the postnatal human thymus, we performed 3D reconstruction of confocal images of 300 μm thick sections for FN1, one of the major ECM components (also produced by polykeratinocytes). FN1 immunostaining clearly defined the subcapsular and perivascular spaces within the cortex and medulla, thus suggesting that the basal layer may represent the in vivo niche for thymic SCs.

[0226] We examined our scRNA-seq dataset and found that basal cell adhesion molecule (BCAM) is one of the genes that defines the polykeratin signature. BCAM is a receptor for the basal layer ECM glycoprotein laminin-A5 (LAMA5) [46, 47], and we confirmed its expression within the same region of polykeratinocytes. Therefore, we set out to isolate BCAM-expressing cells using our sequential enrichment and sorting strategy. BCAM pos cTEC(EpCAM neg CD205 pos ) and BCAM pos mTEC(EpCAM pos CD205 neg ) was selected, resulting in long-term growing cultures.

[0227] We conclude that polykeratinocytes, located in the subcapsular and perivascular niches in vivo, are long-lived thymic stem cells.

[0228] Example 10 - BCAM as a specific marker for clonogenic thymic stem cells We set out to purify both cortical and medullary polykeratinocytes based on the expression levels of the newly validated basal cell adhesion molecule BCAM. We dissociated whole thymic tissue into single cells and purified cortical (CD205 pos EpCAM low ) and medulla (CD205 neg EpCAM high ) Staining for surface markers and BCAMpos and BCAM neg and further subdivided into TECs ( Fig. 11A ).

[0229] The present inventors attempted to isolate BCAM-expressing cells by FACS and found that BCAM-expressing cells were expressed in both the cortex and medulla. pos We found that only BCAM cells were clonogenic and gave rise to epithelial colonies that could be subcultured and expanded (Figure 11B). In full agreement with the spatial phenotype described above, our flow cytometry studies also confirmed that the medulla contained a greater proportion of clonogenic cells (Figure 11A). Freshly isolated clonogenic (BCAM) pos ) and specialized non-clonogenic (BCAM neg ) TEC, sorted cells were subjected to nCounter® automated analysis (NanoString) allowing multiplex gene expression profiling.

[0230] Of note, clonogenic BCAM pos When freshly isolated, the cells retained some of their cortical or medullary identity reflected by differential expression of compartment-specific genes such as cortical CTSV, FOXN1, PSMB11, SCX, LY75, and medullary CCL21, EpCAM, CLDN3, and CLDN4, respectively. Notably, however, volcano plots displaying differentially expressed genes (DEGs) between clonogenic and non-clonogenic TECs provided independent confirmation of polykeratin gene expression in the clonogenic fraction, together with BCAM and ITGA6 adhesion molecules, using an independent method (Figure 11A,B).

[0231] conclusion Our data reveal that the postnatal thymus harbors true epithelial stem cells (SCs) with multilineage differentiation potential. These cells are characterized by hierarchical differentiation, where pluripotent stem cells give rise to distinct progenitors that represent intermediate stages of differentiation, including cortical differentiated cells that upregulate CD205, CTSV, FOXN1, and KCNIP3. With regard to the medulla fate, we observed a “transition” cluster of cells that upregulates CLDN3 and CLDN4, previously reported to be expressed in mTEC-restricted progenitors

[48] . These cells also upregulate ASCL1, a basic helix-loop-helix (BHLH) transcription factor involved in regulating neuroendocrine cell development in several organs (e.g., lung and intestine).

[0232] Indeed, our thymic epithelial SCs are multipotent and do not express most of the genes previously associated with a progenitor phenotype (e.g., FOXN1 or CLDN3 / 4). Instead, they are defined by gene expression signatures that reflect their upstream and sustain multiple functions, their ability to resist stress and injury, and their presence in “strategic” regions that may sense tissue tension / modification (subcapsular) and metabolite supply (perivascular). Our data support a new model in which postnatal SCs can be isolated from both cortical and medullary compartments based on common features and surface molecules. In vivo, they give rise to specialized differentiated cell types in both the cortex and medulla, including those that are not clearly epithelial in nature, including neuroendocrine and myoid cells, previously thought to be of unknown origin

[27] . The discovery of SCs with multilineage differentiation and the presence of different types of progenitors may be important for understanding the high heterogeneity of thymic epithelial tumors (i.e., thymomas and thymic carcinomas), which are often also associated with autoimmunity

[49] .

[0233] Remarkably, once isolated, thymic SCs can proliferate extensively, similar to stem cells in constantly regenerating tissues such as the epidermis. This seems paradoxical considering that the epidermis is characterized by high cell turnover and is completely replaced every 3 weeks throughout life, whereas the thymic epithelium is not actively proliferating and the thymus itself undergoes regressing with a progressive decrease in functional output throughout postnatal life.

[0234] In addition to sharing several important properties, such as a remarkable in vitro proliferation capacity, thymic epithelial SCs displayed several unique traits that provide new and important insights into epithelial stem cell biology.

[0235] First, they co-express several cytokeratins (KRTs) that individually define a specific lineage of simple, stratified, or glandular epithelium, and thus thymic SCs display a unique polykeratin signature that has been described among epithelia so far. This reflects a promiscuous, yet regulated trait for proteins such as KRTs that are typically known for their specificity and lineage determination [50, 51]. This may support the plasticity of epithelial stem cells (the ability to change or increase their potency during differentiation) that has been reported for thymic epithelial cells in the transplantation context

[52] .

[0236] Second, thymic epithelial stem cells constitutively express several genes involved in immune and inflammatory responses, such as CLU, CEBPD, and IFITM3. For example, IFITM3 likely represents an increased resistance of these cells to viral infection, a property that has been observed in other stem cells

[53] . Furthermore, IFITM3 may have roles beyond its classical antiviral activity, such as regulating epithelial-mesenchymal transition (EMT), a process important in tumor development and cancer progression

[54] . Indeed, its constitutive expression in unstimulated epithelial stem cells is striking compared to its tightly regulated expression in infected or inflamed tissues or non-epithelial stromal cells [54, 55, 56]. Nevertheless, we note that in vivo, the thymus is a unique site of constitutive type I IFNα expression that may contribute to IFITM3 regulation

[57] .

[0237] Third, thymic multipotent SCs localize to subcapsular and perivascular microenvironments where they contribute to the niche by generating extracellular matrix (ECM), and they also express ECM-binding proteins such as CD49F, which are important for their polarization and adhesion to the basal layer. This may reflect a structural difference from classical epithelia, where single or multiple epithelial layers organize on a basal layer that separates them from the underlying mesenchyme. The 3D meshwork of the thymus is in fact characterized by interconnections between epithelial and stromal cells, creating a unique microenvironment for thymocyte migration and development. It is noteworthy that in this context, TECs also express mesodermal markers such as TE-7, vimentin, and CD90, making them an atypical epithelium with a high capacity for motility

[44] . The importance of epithelial adhesion to specific ECM for the maintenance of stemness is further demonstrated by the ability of proteins such as FN1 to inhibit human keratinocyte terminal differentiation

[58] . Thus, although the niche ECM appears to share molecular features among different epithelia (including the thymus), the unity of thymic epithelial SCs is expressed by their ability to produce secreted proteins (e.g., FN1, TIMP1) that are produced by their own niche ECM and by the supporting matrix primarily in other organs.

[0238] Finally, thymic SCs can be prospectively isolated from different anatomical compartments based on the expression of surface molecules such as EpCAM, CD49F, CD90, and CD24. Alternatively, BCAM expression can be used to isolate clonogenic thymic stem cells. Once seeded in culture, they are activated and expand extensively in vitro, where they retain their polykeratinous characteristics and their capacity for multilineage differentiation independent of their origin or isolation method. Importantly, this supports the view that the capacity for thymic homeostasis and regeneration resides in the same (polykeratinous) population. These multipotent thymic-specific stem cells are easily identifiable by their characteristic refractile border morphology in culture, and they share the same polykeratinous characteristics identified in vivo independent of their isolation method. This has important consequences for their future use in cell replacement therapy (i.e., transplantation in athymic patients), as minimal manipulation during isolation reduces stress-induced senescence

[58] and facilitates clinical protocols. Bulk culture is in fact the current method for the isolation of both epidermal and limbal SCs in clinical use [59, 60].

[0239] Exploring how to control the activation and differentiation capacity of thymic epithelial SCs in diseased or aged thymus in vivo may provide a means to regenerate and / or rejuvenate thymic function in some disease situations, especially in the elderly. By studying how polykeratinocytes and their niches change during progressive thymic atrophy and how they respond to exogenous factors both in vivo and in vitro, we can study the mechanisms of thymic involution and design new strategies to increase thymic output, for example to increase vaccination responses in vulnerable subjects or improve immune responses against cancer.

[0240] Equivalents and Scope Those skilled in the art will appreciate that the present invention is defined by the appended claims, and not by the specific embodiment examples or other descriptions contained herein.

[0241] Similarly, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0242] Unless otherwise defined above, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry described herein, and the techniques thereof, are well known and commonly used in the art or in accordance with manufacturer's specifications.

[0243] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0244] The invention is also described in the following numbered clauses: 1. An isolated thymic epithelial stem cell, comprising BCAM pos , CD49F pos , CD90 pos , and CD24 neg An isolated thymic epithelial stem cell. 2. Isolation of thymic epithelial stem cells: BCAM pos , CD49F high , CD90 high , and CD24 neg 2. The isolated thymic epithelial stem cell according to clause 1, which is 3. The isolated thymic epithelial stem cell of clause 1, wherein the isolated thymic epithelial stem cell expresses at least one cytokeratin gene. 4. The isolated thymic epithelial stem cell of clause 1 or 2, wherein at least one cytokeratin gene is selected from the group consisting of KRT5, KRT8, KRT13, KRT14, KRT15, ​​KRT17, KRT18, and KRT19. 5. The isolated thymic epithelial stem cell of any one of clauses 1 to 3, wherein the isolated thymic epithelial stem cell expresses KRT13, KRT18, and KRT15. 6. The isolated thymic epithelial stem cell of any one of clauses 1 to 3, wherein the isolated thymic epithelial stem cell expresses KRT13, KRT18, and KRT17. 7. The isolated thymic epithelial stem cell of any one of clauses 1 to 3, wherein the isolated thymic epithelial stem cell does not express KRT7, KRT1, KRT10, KRT4, KRT16, KRT23, or KRT6A. 8. The isolated thymic epithelial stem cell of any one of clauses 1-6, wherein the isolated thymic epithelial stem cell further expresses at least one selected from the group consisting of EPCAM, CD49F, FN1, TIMP1, IFITM3, VCAM1, CEPBD, CLU, CCL19, CH25H, COL7A1, CTGF, APOE, FGFR2, BOC, ITGA5, SOX17, LIFR, YAP1, PTGDS, CD34, VWF, SPARC, CAV-1, EPAS-1, TIMP3, COL4A2, COL5A1, COL6A3, TP63 (e.g., ΔNTP63α) and cMYC. 9. The isolated thymic epithelial stem cell of any one of clauses 1 to 7, wherein the isolated thymic epithelial stem cell further expresses EPCAM, CD49F, and FN1. 10. The isolated thymic epithelial stem cell of any one of clauses 1 to 7, wherein the isolated thymic epithelial stem cell further expresses EPCAM, CD49F, and IFITM3. 11. The isolated thymic epithelial stem cell of any one of clauses 1-7, wherein the isolated thymic epithelial stem cell further expresses CD49F, FN1, and TIMP1. 12. The isolated thymic epithelial stem cell of any one of clauses 1 to 11, wherein the isolated thymic epithelial stem cell further expresses COL7A1 and CTGF. 13. The isolated thymic epithelial stem cell of any one of clauses 1-12, wherein the isolated thymic epithelial stem cell expresses at least one selected from the group consisting of CCNA2, AURKB, FOXM1, ANLN, LMNB1, HMGB2, or a combination thereof. 14. An isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being capable of self-renewal ex vivo. 15. An isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being clonogenic. 16. An isolated thymic epithelial stem cell, which exhibits long-term proliferation capacity in vitro. 17. The isolated thymic epithelial stem cell according to clause 16, wherein the isolated thymic epithelial stem cell is capable of at least 15 population doublings in vitro. 18. An isolated thymic epithelial stem cell, the isolated thymic epithelial stem cell being capable of differentiating into a cortical thymic epithelial cell and / or a medullary thymic epithelial cell. 19. Thymic epithelial stem cells are cortical thymic epithelial cells and express CD205 pos KRT5 neg KRT14 neg 2. The isolated thymic epithelial stem cell of any one of the preceding clauses, which is 20. Thymic epithelial stem cells are medullary thymic epithelial cells and express CD205 neg CK5 pos KRT14 pos 2. The isolated thymic epithelial stem cell of any one of the preceding clauses, which is 21. The isolated thymic epithelial stem cell of any one of clauses 1 to 20, wherein the thymic epithelial stem cell is pluripotent. 22. The isolated thymic epithelial stem cell of any one of clauses 1 to 21, wherein the isolated thymic epithelial stem cell is a human cell. 23. A method for isolating thymic epithelial stem cells from the thymus, comprising: (a) obtaining a thymus tissue sample; (b) isolating thymic epithelial cells from the thymic tissue sample to obtain a thymic epithelial cell fraction; (c)BCAM pos CD49F pos CD90 pos CD24 neg and isolating thymic epithelial cells from the thymic epithelial cell fraction to obtain isolated thymic epithelial stem cells. 24. Step (b) is (i) dissociating a thymus tissue sample to obtain single cells; (ii) isolating single cells from the thymus tissue sample to obtain a single cell fraction; (iii) substantially depleting the single cell fraction of hematopoietic cells and / or red blood cells; (iv) isolating EPCAM+ cells to obtain a thymic epithelial cell fraction. 25. Step (c) is (i) labeling thymic epithelial cells for CD49F, CD90, CD24, and BCAM; (ii) BCAM pos CD49F pos CD90 pos CD24 neg and separating the cells from the thymic epithelial cell fraction to obtain isolated thymic epithelial stem cells. 26. (a) obtaining a thymus tissue sample; (b) dissociating the thymus tissue sample to obtain single cells; (c) isolating single cells from the thymus tissue sample to obtain a single cell fraction; (d) substantially depleting the single cell fraction of hematopoietic cells and / or red blood cells; (e) isolating thymic epithelial cells from the single cell fraction; (f) labeling thymic epithelial cells for CD49F, CD90, CD24, and BCAM; (g) BCAM pos CD49F pos CD90 pos CD24 neg 26. The method for isolating thymic epithelial stem cells from the thymus according to any one of clauses 23 to 25, comprising a step of isolating thymic epithelial cells to obtain isolated thymic epithelial stem cells. 27. A method for isolating thymic epithelial stem cells from the thymus described in any one of clauses 24 to 26, wherein the step of dissociating the thymic tissue sample comprises treating the thymic tissue sample with one or more enzymes. 28. The step of isolating thymic epithelial cells is carried out by EPCAM pos 28. A method for isolating thymic epithelial stem cells from the thymus according to any one of clauses 23 to 27, comprising isolating the cells. 29. BCAM pos CD49F pos CD90 pos CD24 neg 29. A method for isolating thymic epithelial stem cells from the thymus according to any of clauses 23 to 28, wherein the step of isolating the epithelial cells is carried out using fluorescence-activated cell sorting (FACS). 30. A cell obtainable by the method according to any one of clauses 23 to 29. 31. A method for culturing thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) culturing the at least one isolated thymic epithelial stem cell under conditions suitable for the maintenance and proliferation of the at least one isolated thymic epithelial stem cell. 32. (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with at least one feeder cell; (c) contacting the at least one isolated thymic epithelial stem cell and the at least one support cell with a cell culture medium. 33. The method of clause 31 or 32, wherein at least one support cell is a feeder cell. 34. The method of clause 33, wherein the feeder cells are mouse fibroblasts. 35. The method of clause 33 or 34, wherein the feeder cells are sublethally irradiated mouse fibroblasts. 36. The method according to any one of clauses 31 to 35, wherein the cell culture medium is a cFAD medium. 37. The method of any of clauses 31 to 36, comprising contacting at least one isolated thymic epithelial stem cell and / or at least one feeder cell with a proliferation-promoting agent. 38. The method of culturing thymic epithelial stem cells according to clause 31, wherein the isolated thymic epithelial stem cells of step (a) are cortical thymic epithelial stem cells. 39. Cortical thymic epithelial stem cells express CD205 pos EPCAM neg 39. The method for culturing thymic epithelial stem cells according to Clause 38, 40. The method of culturing thymic epithelial stem cells according to clause 31, wherein the isolated thymic epithelial stem cells of step (a) are medullary thymic epithelial stem cells. 41. Medullary thymic epithelial stem cells express CD205 neg EPCAM pos 41. The method according to claim 40, 42. An isolated population of thymic epithelial stem cells, obtainable by a method according to any one of clauses 23 to 41. 43. The isolated population of thymic epithelial stem cells according to clause 42, wherein the thymic epithelial stem cells display a refractile border morphology in culture. 44. A method for culturing cortical thymic epithelial cells derived from thymic epithelial stem cells, comprising: (a) providing thymic epithelial stem cells; (b) culturing thymic epithelial stem cells under conditions suitable for obtaining cortical thymic epithelial cells. 45. The method of claim 44, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 1% to about 9%. 46. ​​The method of clause 44 or 45, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 4% to about 6%. 47. A method for culturing medullary thymic epithelial cells derived from thymic epithelial stem cells, comprising: (a) providing thymic epithelial stem cells; (b) culturing thymic epithelial stem cells under conditions suitable for obtaining medullary thymic epithelial cells. 48. The method of claim 47, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 10% to about 30%. 49. The method of clause 47 or 48, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 20%. 50. The method of any of clauses 44-49, wherein step (b) comprises culturing the thymic epithelial stem cells at a carbon dioxide (CO2) tension of about 5% to about 10%. 51. A cortical thymic epithelial cell obtainable by a method according to any one of clauses 44 to 46. 52. Medullary thymic epithelial cells, obtainable by the method according to any one of clauses 47 to 50. 53. A method for differentiating isolated thymic epithelial stem cells, comprising: (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with the membrane, wherein the at least one isolated thymic epithelial stem cell contacts an upper surface of the membrane; (c) providing a cell culture medium, wherein the cell culture medium is disposed beneath the underside of the membrane. 54. (a) providing at least one isolated thymic epithelial stem cell; (b) contacting at least one isolated thymic epithelial stem cell with the membrane, wherein the at least one isolated thymic epithelial stem cell contacts an upper surface of the membrane; (c) contacting at least one isolated thymic epithelial stem cell and a membrane with a first cell culture medium; (d) maintaining at least one isolated thymic epithelial stem cell under conditions suitable for proliferation of the isolated thymic epithelial stem cell to obtain an expanded population of thymic epithelial stem cells; (d) removing the first cell culture medium; (e) providing a second cell culture medium, the second cell culture medium being disposed beneath the underside of the membrane; (f) maintaining the expanded population of thymic epithelial stem cells under conditions suitable for differentiation of the expanded population of thymic epithelial stem cells. 55. A cell culture composition comprising an isolated thymic epithelial stem cell according to any one of clauses 1 to 22. 56. A pharmaceutical composition comprising an isolated thymic epithelial stem cell according to any one of clauses 1 to 22 and a pharma- ceutically acceptable carrier. 57. A thymic construct suitable for transplantation into a subject, comprising an isolated thymic epithelial stem cell according to any one of clauses 1 to 22 or a cell culture composition according to clause 55. 58. A method of treating a disease or disorder in a subject, comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1 to 22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 59. The method of claim 58, wherein the disease or disorder is a primary immune deficiency. 60. The method according to clause 58, wherein the disease or disorder is a disease or disorder associated with dysfunctional central tolerance. 61. The method according to clause 58, wherein the disease or disorder is autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). 62. The method according to clause 58, wherein the disease or disorder is an autoimmune disease. 63. The method of clause 62, wherein the autoimmune disease is selected from myasthenia gravis, type 1 diabetes, autoimmune myopathy, and connective tissue disease. 64. The method according to clause 58, wherein the disease or disorder is cancer. 65. The method according to clause 64, wherein the cancer is a thymoma, thymic carcinoma, sarcoma, or a neuroendocrine tumor of the thymus. 66. A method of preventing or reversing thymic atrophy in a subject, comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1-22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 67. A method of treating an immune deficiency in a subject, comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1 to 22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 68. A method of treating athymus in a subject, comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1-22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 69. A method of regenerating thymic tissue in a subject, the method comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1 to 22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 70. A method of increasing, restoring or regenerating a population of circulating T cells in a subject, the method comprising administering to the subject an isolated thymic epithelial stem cell according to any one of clauses 1 to 22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56 or a thymic construct according to clause 57. 71. Use of an isolated thymic epithelial stem cell according to any one of clauses 1 to 22 in cell replacement therapy. 72. A method for generating a thymus construct suitable for transplantation into a subject, comprising: (a) providing an acellular scaffold; (b) seeding the acellular scaffold with the isolated thymic epithelial stem cells according to any one of clauses 1 to 22; (c) culturing the seeded scaffold to produce the construct. 73. A method of drug screening comprising the use of an isolated thymic epithelial stem cell according to any one of clauses 1 to 22, a cell culture composition according to clause 55, a pharmaceutical composition according to clause 56, or a thymic construct according to clause 57. 74. A kit for identifying thymic epithelial stem cells according to any one of clauses 1 to 22, comprising means for identifying one or more cell markers selected from the list consisting of BCAM, CD49F, CD90 and CD24. 75. The kit according to clause 74, wherein the kit comprises binding molecules specific for BCAM, CD49F, CD90, and CD24. 76. An isolated thymic epithelial stem cell, comprising BCAM pos An isolated thymic epithelial stem cell. 77. The isolated thymic epithelial stem cell according to clause 76, wherein the isolated thymic epithelial stem cell expresses at least one cytokeratin gene. 78. The isolated thymic epithelial stem cell of clause 77, wherein at least one cytokeratin gene is selected from the group consisting of KRT5, KRT8, KRT13, KRT14, KRT15, ​​KRT17, KRT18, and KRT19. 79. The isolated thymic epithelial stem cell of any one of clauses 76 to 78, wherein the isolated thymic epithelial stem cell expresses KRT13, KRT18, and KRT15. 80. The isolated thymic epithelial stem cell of any one of clauses 76-79, wherein the isolated thymic epithelial stem cell expresses KRT13, KRT18, and KRT17. 81. The isolated thymic epithelial stem cell of any one of clauses 76 to 80, wherein the isolated thymic epithelial stem cell does not express KRT7, KRT1, KRT10, KRT4, KRT16, KRT23, or KRT6A. 82. The isolated thymic epithelial stem cell of any one of clauses 76-81, wherein the isolated thymic epithelial stem cell further expresses at least one selected from the group consisting of EPCAM, CD49F, FN1, TIMP1, IFITM3, VCAM1, CEPBD, CLU, CCL19, CH25H, COL7A1, CTGF, APOE, FGFR2, BOC, ITGA5, SOX17, LIFR, YAP1, PTGDS, CD34, VWF, SPARC, CAV-1, EPAS-1, TIMP3, COL4A2, COL5A1, COL6A3, TP63 (e.g., ΔNTP63α) and cMYC.

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Claims

1. Isolated thymic epithelial stem cells, wherein the stem cells are BCAMpos, CD49Fpos, CD90pos, and CD24neg.

2. (i) The isolated thymic epithelial stem cells express at least one cytokeratin gene, and optionally, the at least one cytokeratin gene is selected from the group consisting of KRT5, KRT8, KRT13, KRT14, KRT15, ​​KRT17, KRT18, and KRT19, or a combination thereof. (ii) The isolated thymic epithelial stem cells further express at least one selected from the group consisting of EPCAM, CD49F, FN1, TIMP1, IFITM3, VCAM1, CEPBD, CLU, CCL19, CH25H, COL7A1, CTGF, APOE, FGFR2, BOC, ITGA5, SOX17, LIFR, YAP1, PTGDS, CD34, VWF, SPARC, CAV-1, EPAS-1, TIMP3, COL4A2, COL5A1, COL6A3, TP63 (e.g., ΔNTP63α) and cMYC, or combinations thereof, and / or (iii) The isolated thymic epithelial stem cells according to claim 1, wherein the isolated thymic epithelial stem cells express at least one selected from the group consisting of CCNA2, AURKB, FOXM1, ANLN, LMNB1, HMGB2, or a combination thereof.

3. Isolated thymic epithelial stem cells that are capable of self-renewal ex vivo.

4. Isolated thymic epithelial stem cells that exhibit long-term proliferative capacity in vitro.

5. The isolated thymic epithelial stem cells according to claim 4, wherein the isolated thymic epithelial stem cells are capable of population duplication at least 15 times in vitro.

6. Isolated thymic epithelial stem cells capable of differentiating into cortical thymic epithelial cells and / or medullary epithelial cells.

7. (i) (a) The thymic epithelial stem cells are cortical thymic epithelial cells, and are CD205posKRT5negKRT14neg, or (b) The thymic epithelial stem cells are medullary thymic epithelial cells, and are CD205negKRT5pos and / or KRT14pos, (ii) The thymic epithelial stem cells are pluripotent and / or (iii) The isolated thymic epithelial stem cells are human cells. Isolated thymic epithelial stem cells according to any one of claims 1 to 6.

8. A method for isolating thymic epithelial stem cells from the thymus, (a) Obtain a thymic tissue sample, (b) Isolating thymic epithelial cells from the thymic tissue sample to obtain a thymic epithelial cell fraction, (c) A method comprising isolating BCAMposCD49FposCD90posCD24neg thymic epithelial cells from the thymic epithelial cell fraction to obtain isolated thymic epithelial stem cells.

9. A method for isolating thymic epithelial stem cells from the thymus according to claim 8, wherein the step of isolating BCAMposCD49FposCD90posCD24neg epithelial cells is carried out using fluorescence-activated cell sorting (FACS).

10. A method for culturing thymic epithelial stem cells, (a) To provide at least one isolated thymic epithelial stem cell, (b) A method comprising culturing the at least one isolated thymic epithelial stem cell under conditions suitable for the maintenance and proliferation of the at least one isolated thymic epithelial stem cell.

11. The method for culturing thymic epithelial stem cells according to claim 10, wherein the isolated thymic epithelial stem cells in step (a) are corticothymic epithelial stem cells.

12. The method for culturing thymic epithelial stem cells according to claim 10, wherein the isolated thymic epithelial stem cells in step (a) are medullary thymic epithelial stem cells.

13. A method for culturing corticothymic epithelial cells derived from thymic epithelial stem cells, (a) To provide thymic epithelial stem cells, (b) A method comprising culturing the thymic epithelial stem cells under conditions suitable for obtaining cortical thymic epithelial cells.

14. The method according to claim 13, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 1% to about 9%.

15. The method according to claim 13 or 14, wherein step (b) comprises culturing the thymic epithelial stem cells at an oxygen (O2) tension of about 4% to about 6%.

16. A method for differentiating isolated thymic epithelial stem cells, (a) To provide at least one isolated thymic epithelial stem cell, (b) Contacting the at least one isolated thymic epithelial stem cell to a membrane, wherein the at least one isolated thymic epithelial stem cell is in contact with the upper surface of the membrane. (c) A method for providing a cell culture medium, wherein the cell culture medium is placed below the lower surface of the membrane.

17. A thymic construct suitable for transplantation into a subject, comprising isolated thymic epithelial stem cells according to any one of claims 1 to 6.

18. A composition containing isolated thymic epithelial stem cells according to any one of claims 1 to 6 for treating a disease or disorder in a subject.

19. The thymic structure according to claim 17 for treating a disease or disorder in a subject.

20. A method for generating a thymic construct suitable for transplantation into a subject, (a) A step of providing a cell-free scaffold, (b) The step of seeding isolated thymic epithelial stem cells according to any one of claims 1 to 6 onto the cell-free scaffold, (c) A method comprising the step of culturing the seeded scaffold to produce the structure.

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