Immortalized intestinal telocytes

Abstract

The present invention relates to an immortalized telocyte cell line, to methods for its preparation which include immortalizing intestine-derived cells labeled with a Foxll -driven reporter gene, and uses of the cell line including for supporting organoid growth.

Description

[0001] IMMORTALIZED INTESTINAL TELOCYTES

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to immortalized telocytes. More specifically, the invention relates to cell lines produced from immortal intestinal telocytes, which are capable of supporting stem cell function in vitro.

[0004] BACKGROUND OF THE INVENTION

[0005] Telocytes, unique mesenchymal cells characterized by the expression of the transcription factor Foxll, have recently been recognized as crucial for maintaining intestinal homeostasis. Historically, telocytes were described primarily through electron microscopy as special interstitial cells present in the stroma of many organs. They are distinct from fibroblasts due to their exceptionally long cellular extensions, known as telopodes, which can extend for hundreds of microns from the cell body. These telopodes vary in width, and feature dilated regions called podoms, which contain mitochondria, caveolae, lipid rafts used for signal transduction, and elements of endoplasmic reticulum (ER). Through these extensions, telocytes form intricate 3D networks with other telocytes and diverse cell types, including epithelial cells, blood and lymphatic vessels, immune cells, fibroblasts, myofibroblasts and nerve bundles. While they are present in many organs, the essential role of telocytes in maintaining homeostasis was first explored in the intestine.

[0006] Three-dimensional tissue culture techniques and advanced growth factor supplementation have enabled the establishment of patient-derived organoid models for a wide range of human normal and tumor tissues (Drost, J. and H. Clevers, (2018) Nat Rev Cancer 18(7): p. 407-418). There are various successful protocols for growing organoids, but until now, all organoid culture protocols are dependent on the supplementation of the medium with specific factors.

[0007] The properties of telocytes make it a desirable material for the establishment of immortalized cell lines, which would be capable of preserving their many functions including supporting stem cells and cellular differentiation. Until now, no such cell line has been established, and the combination of such a cell line with organoids has not yet been achieved. Organoids derived from both healthy and tumor tissues are routinely grown with fibroblasts (usually human foreskin derived fibroblasts) but this is done with a regular organoid growth medium including growth factor supplementation.

[0008] Accordingly, there is still a need for developing telocyte cell lines, that preserve their many functions including supporting stem cells and cellular differentiation. SUMMARY OF INVENTION

[0009] The following embodiments are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0010] In some embodiments, there is provided an immortalized telocyte cell line including telocytes including DNA encoding Simian virus 40 (SV40) large T antigen.

[0011] In some embodiments, the telocytes are derived from intestine. In some embodiments, the telocytes are mouse telocytes or human telocytes.

[0012] In some embodiments, the telocytes express Foxll.

[0013] In some embodiments, at least a portion of the telocytes express platelet-derived growth factor receptor alpha (PDGFRa). In some embodiments, the portion of telocytes expressing PDGFRa is at least about 50%.

[0014] In some embodiments, the immortalized telocyte cell line includes a population expressing PDGFRa at a high level and a population expressing PDGFRa at a low level, and wherein the high expression level is at least about 2 fold higher compared to the low expression level.

[0015] In some embodiments, at least a portion of the telocytes expresses a gene selected from matrix metalloproteinase 10 (MMP10) and alpha-smooth muscle actin (aSMA). In some embodiments, the portion of telocytes expressing a gene selected from MMP10 and aSMA is at least about 10%.

[0016] In some embodiments, at least a portion of the telocytes express a gene selected from Glioma-associated oncogene homolog 1 (Glil), Gremlin 1, R-spondin 3 (Rspo3), Noggin, Wnt2b, Wnt5a, bone morphogenic proteins 1 (BMP1) and 4 (BMP4), bone morphogenetic protein receptor type-lB (BMPR1B), Chordin, Bambi, Wnt4, chondroitin sulfate proteoglycan 4 (CSPG4), tenascin-C (TNC), and Delk I.

[0017] In some embodiments, the immortalized telocyte cell line further includes subpopulations which differ in expression levels of at least one gene selected from SRY-box 2 (Sox2), Wnt5a, doublecortin-like kinase 1 (Dclkl), and CD34.

[0018] In some embodiments, the telocytes display morphological features including at least one cellular projection having a length of at least about 10 microns. In some embodiments, the at least one cellular projection includes multivesicular bodies, caveolae, rough endoplasmic reticulum (rER), mitochondria, and / or extracellular vesicles.

[0019] In some embodiments, the telocytes express a reporter gene controlled by a Foxll promoter.

[0020] In some embodiments, the telocytes are capable of contracting extracellular matrix (ECM) by at least about 10%.

[0021] In some embodiments, the telocytes are capable of supporting growth of organoids in the absence of exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0022] In some embodiments, the telocytes are capable of supporting growth of organoids in culture in the absence of exogenous niche factors.

[0023] In some embodiments, there is provided a composition including organoids and the immortalized telocyte cell line disclosed herein in a culture medium.

[0024] In some embodiments, the telocytes express Foxll.

[0025] In some embodiments, the organoids are human organoids or mouse organoids.

[0026] In some embodiments, the organoids are derived from an organ or a tissue selected from small intestine, large intestine, colon, and hair follicles.

[0027] In some embodiments, there is provided the immortalized telocyte cell line disclosed herein and the composition disclosed herein, for use in a method of culturing organoids.

[0028] In some embodiments, the method does not include adding exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0029] In some embodiments, there is provided the immortalized telocyte cell line disclosed herein and the composition disclosed herein, for use in a method of screening drugs for treating a disease selected from inflammatory bowel disease (IBD), intestinal or colorectal cancer, and infectious disease.

[0030] In some embodiments, there is provided a method of culturing organoids, the method including contacting the organoids with the immortalized telocyte cell line disclosed herein in a culture medium, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0031] In some embodiments, there is provided a method of preparing a tissue culture model for an intestinal disease, the method including contacting intestinal organoids in a culture medium with the immortalized telocyte cell line disclosed herein, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0032] In some embodiments, the intestinal disease is selected from inflammatory bowel disease (IBD), intestinal or colorectal cancer, and infectious disease.

[0033] In some embodiments, there is provided a method of preparing intestinal tissue grafts for regenerative medicine, the method including contacting intestinal organoids in a culture medium with the immortalized telocyte cell line disclosed herein, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0034] In some embodiments, there is provided a method of preparing a mouse telocyte cell line, the method including: a. generating a mouse carrying a reporter gene controlled by a Foxl 1 promoter for labeling telocytes; b. isolating from the mouse intestinal mesenchymal cells including telocytes, including: dissecting mouse jejunum, isolating mesenchyme from the jejunum by removing epithelium, and digesting the mesenchyme with collagenase to produce single mesenchymal cells; c. culturing the mesenchymal cells in a culture medium; d. isolating telocytes by sorting the mesenchymal cells for cells expressing the reporter gene; and e. immortalizing the isolated telocytes.

[0035] In some embodiments, the immortalizing is performed by infecting the telocytes with viral particles expressing SV40 Large T antigen.

[0036] In some embodiments, the sorting is performed by fluorescence activated cell sorting (FACS).

[0037] In addition to the exemplary embodiments described above, further embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0038] BRIEF DESCRIPTION OF DRAWINGS

[0039] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0040] Figs. 1A-1M show that immortalized telocytes retain structural characteristics. Fig. 1A. Schematic illustration of the experimental strategy for creating the immortalized telocyte cell line. The jejunum from Foxll-Cre; Rosa26-mTmG mouse model was dissected, and the mesenchyme was isolated and sorted into green fluorescent protein (GFP)+telocytes. Sorted GFP+ telocytes were cultured for two weeks and were then infected with SV40 T-antigen viral particles and selected with hygromycin to obtain the infected cells. Fig. IB. Quantification of DAPI+nuclei at time 0 (to) and after 24 hours (t24h) revealed a significant 94% increase in DAPI+nuclei per region (n=30 regions per time point, p < 0.0001, Student's t-test), suggesting a cell cycle duration of approximately 25 hours for immortalized telocyte cell line. Fig. 1C. Live imaging of immortalized telocyte cell culture revealed key cellular characteristics of these cells. The boxed regions highlight the length of telopodes (194 pm, upper left panel), their branching pattern (upper right panel), and the presence of dilated regions, known as podoms (indicated by asterisk). Additionally, immortalized telocytes form a network by interconnecting through several telopodes and cellular protrusions (all panels). The boxed regions are enlarged below the main images. Scale bar 10 pm. Fig. ID. Quantification of nuclear content in GFP+immortalized telocytes based on DAPI staining. Pie chart shows that 91.2% of GFP+telocytes are mononucleated, while 9.8% are polynucleated (defined as having more than two nuclei per cell). P: polynucleated; M: mononucleated. Figs. 1E- 1G. Immunofluorescence of immortalized telocytes stained for phalloidin (red) revealed asymmetrical distribution of actin filaments beneath the cell membrane, along cellular extensions and in bulbous tips containing multiple fine projections. Fig. IE shows stress fibers (arrow) and a 226 pm long projection. Scale bar 10 pm. Fig. IF shows cytoneme-like structures (boxed region), enlarged in the right panel. Fig. 1G shows an actin-based projection measuring 293 pm in length that connects two telocytes, the boxed region highlights GFP membrane labeling along the extension. Green: GFP, red: phalloidin, blue: DAPI. Fig. 1H. Immunofluorescence of immortalized telocytes stained for a -tubulin (red) revealed asymmetrical distribution at the base of cellular extensions. Green: GFP, blue: DAPI. Scale bar 100 pm. Fig. II. Single-molecule RNA fluorescence in situ hybridization (smFISH) for Foxll mRNA (white) shows expression in GFP+immortalized telocytes. Scale bar 10 pm. The boxed regopm is enlarged in the right panel. Green: GFP; blue: DAPI. Fig. 1J. Quantification of Foxll mRNA expression in GFP+immortalized telocytes, measured by smFISH. Results show an average of 0.0054 Foxll mRNA molecules per pm3. Error bar represents standard deviation. Fig. IK. Immunofluorescence of GFP+immortalized telocytes for PDGFRa (red). All telocytes express PDGFRa, exhibiting two distinct expression levels: high and low. Green: GFP; blue: DAPI. Fig. IL. FACS analysis of GFP+immortalized telocytes stained for PDGFRa identifies two populations: PDGFRaHlghand PDGFRaLow, consistent with immunofluorescence data. Fig. IM Transmission electron microscopy images of GFP+immortalized telocytes reveal characteristic ultrastructural features, including long cellular extensions known as telopodes. These telopodes contain multivesicular bodies (Mv), rough endoplasmic reticulum (rER), caveolae (Cv), and mitochondria (M). Frequent vesicle budding (V) at the plasma membrane was also observed. Panel I (top right) shows an enlarged view of the boxed region in the left panel, while panels II and III (bottom left and right, respectively) provide further enlargement of the boxed area highlighted in panel I as II and III.

[0041] Figs. 2A-2H show that immortalized telocytes are heterogenous and retain the molecular expression profile characteristic of native telocytes. Fig. 2A. Uniform Manifold Approximation and Projection (UMAP) visualization of single-cell RNA-sequencing (scRNA-seq) data from immortalized telocytes. Left panel: four transcriptionally distinct clusters (clusters 0-3) were identified. Right panel: In vivo gene signatures of four telocyte subtypes (Crypt, Villus base, Villus mid, and Villus tip) were projected onto the immortalized telocyte dataset. Custer 0 showed enrichment for villus tip telocyte markers, while clusters 1 and 3 expressed a mixture of crypt and villus mid gene signature. Cluster 2 more closely resembles villus mid telocytes, indicating partial recapitulation of in vivo telocyte heterogeneity in the immortalized line. Fig. 2B. Ridge plot displaying the distribution of normalized signature scores for the four native intestinal telocyte populations (Crypt, Villus base, Villus mid and Villus tip) across four immortalized telocyte clusters (0-3). Each colored ridge shows the density of signature scores for one cluster; higher peaks indicate greater cell frequency at specific score values, while horizontal peak position represents predominant expression of the corresponding population’s gene signature. Cluster 0 is enriched for telocytes with high villus tip signature scores, cluster 2 for villus base and mid signatures, and clusters 1 and 3 show mixed expression patterns spanning multiple telocyte populations. Fig. 2C. Violin plots showing heterogeneous expression of Sox2, Wnt5a, Dclkl and CD34 across clusters of immortalized telocyte in scRNA-seq data. Each violin plot is accompanied (below it) by immunofluorescence staining showing cultured GFP+immortalized telocytes (green) further stained for Sox2, Dclkl, and CD34 (red), or with smFISH detection of Wnt5a mRNA within the telocyte extensions (white). Scale bars 50pm, boxed region 2 pm (enlarged on the right). Each plot depicts (left to right) clusters 0, 1, 2, and 3. Green: GFP, blue: DAPI. Fig. 2D presents dot plots showing the expression of unique genes in immortalized telocyte subtypes. Figs. 2E-2H present Gene Ontology pathway enrichment analyses of differentially expressed genes in immortalized subtypes (Biological Process). Fig. 2E. Cluster 0; Fig. 2F. Cluster 1; Fig. 2G. Cluster 2; Fig. 2H. Cluster 3. Scale bars are -Logio (adjusted p-value).

[0042] Figs. 3A-3D show that immortalized telocytes exhibit matrix remodeling and contraction skills. Figs. 3A-3B. Immunofluorescence of GFP+immortalized telocytes for matrix metalloproteinase 10 (red, Fig. 3A) and alpha smooth muscle actin (aSMA) (red, Fig. 3B) reveals expression in a subset of GFP+immortalized telocytes (green). The boxed area on the left panel is enlarged in the middle panel. Green: GFP, blue: DAPI. Fig. 3C. Confocal images of Matrigel® domes with and without 10,000 GFP+immortalized telocytes. Images were acquired 0, 18, 42 and 92 hours post-seeding. Scale bar 1 pm. Upper panel: control (Matrigel® only), lower panel: Matrigel® with telocytes. Fig. 3D. Quantification of Matrigel® area over time, with and without GFP+immortalized telocytes, reveals significant matrix contraction in the presence of telocytes. Control Matrigel® (no cells) maintained a stable area throughout the time course, whereas Matrigel® seeded with telocytes exhibited progressive area reduction. A significant difference between conditions emerged at 92 hours (p = 0.0152). Overall group comparison confirmed a significant effect of telocytes on Matrigel® contraction (Mann-Whitney U = 47.0, p = 0.022). Error bars represent standard deviation.

[0043] Figs. 4A-4F show that immortalized telocytes support organoid growth in the absence of exogenous growth factors. Figs. 4A-4D. Photographs of mouse jejunum organoids cultured under three conditions: organoids alone, organoids supplemented with niche factors: EGF, Noggin, Rspol, bFGF (ENRF), and organoids cocultured with immortalized telocytes (no supplements). As shown in Fig. 4D, by day 4, immortalized telocytes establish close physical contact with the organoids (including a fluorescent signal from the GFP+ telocytes). Fig. 4E shows quantifications of organoid size, taken on days 2, 3, and 4. Both the photographs and the quantification show that organoids alone die within 48 hours unless supplemented with niche factors or co-cultured with immortalized telocytes. By day 2, organoids co-cultured with telocytes were significantly larger than those cultured with growth factors alone (n=4 replicates, at least 50 organoids per group, p < 0.05, Student’s t-test). At days 3 and 4, no significant differences in organoid size were observed between co-cultures with telocytes and those supplemented with niche factors. This is a solid indication that the immortalized telocyte cell line can be used as a substitute for niche factors, when maintaining organoids culture. By day 4, immortalized telocytes establish close physical contact with the organoids. Fig. 4F. Whole-mount immunofluorescence of organoids cultured for four days, either supplemented with niche factors or co-cultured with immortalized telocytes (right panel), stained for Ki67 (red, a marker of proliferation). In co-culture conditions, Ki67 expression is restricted to the base of the budding region, indicating spatial confinement of proliferating cells. In contrast, organoids grown with exogenous niche factors (left panel) show widespread Ki67 expression throughout the budding region. Asterisks indicate Ki67 staining in immortalized telocytes. Scale bar 100 pm. Blue: DAPI.

[0044] Figs. 5A-5G show that immortalized telocytes support human organoid growth. Fig. 5A. Human colon organoids were cultured under three conditions: DMEM basal medium, commercial growth medium (IntestiCult™ Stem Cell technologies#06010), and organoids co-cultured with GFP+immortalized telocytes in basal medium (no supplements). Images of human colon organoids show that organoids cultured in basal DMEM medium die, whereas organoids co- cultured with GFP+immortalized telocytes grow gradually similar to colon organoids cultured in growth medium. Note the close contact between organoids and telocytes. Fig. 5B. Representative images of human colon organoids on day 7, cultured under three different conditions: commercial growth medium (IntestiCult™ StemCell technologies#06010), commercial differentiation medium (IntestiCult™ StemCell technologies# 100-0214) and co-culture with GFP+immortalized telocytes in basal medium (no supplements). Organoids in commercial growth medium display a predominantly spherical morphology and larger size, whereas those in differentiation medium exhibit a more complex budded morphology. Organoids co-cultured with telocytes show a mixture of both spherical and budded morphologies. Scale bars 100 pm. Fig. 5C. Quantification of organoid surface area over a 6-day time course reveals distinct growth dynamics across culture conditions. Organoids in growth medium exhibited significantly larger surface areas compared to those in differentiation medium or co-cultured with telocytes, with statistical difference observed relative to differentiation medium (p < 0.001) and telocyte co-culture (p = 0.008). No significant difference in size was found between differentiation medium and telocyte groups after Bonferroni correction (p=0.018; not significant at a=0.017). Growth medium-cultured organoids displayed accelerated growth from day 3 onward, reaching peak surface area between days 4 and 6. In contrast, differentiation medium-supported organoids showed modest, steady growth, while those co-cultured with telocytes remained relatively stable throughout the time course. Error bars represent standard error of the mean. Statistical analysis was performed using Kruskal-Wallis test followed by Mann-Whitney U post hoc comparisons with Bonferroni correction. Fig. 5D. Immunofluorescence staining of human colon organoid sections on day 7, cultured under three conditions: commercial growth medium (IntestiCult™ StemCell Technologies#060I0, left panel), commercial differentiation medium (IntestiCult™ StemCell Technologies# 100-0214, middle panel) and co-culture with immortalized telocytes in basal medium (no supplements, right panel). Sections were stained for Sox9 (red), a marker of progenitor cells and E-cadherin (white), a marker of epithelial cells. Sox9 expression was broadly detected in organoids cultured in growth medium and in those co-cultured with telocytes. In contrast, organoids cultured in differentiation medium showed Sox9 expression restricted to the budded region. Blue: DAPI. Fig. 5E. Quantification of the percentage of Sox9+nuclei per organoid section under three culture conditions: commercial growth medium (IntestiCult™ StemCell Technologies #06010, left), commercial differentiation medium (IntestiCult™ StemCell Technologies #100-0214, middle) and co-culture with immortalized telocytes in basal medium (no supplements, right). Box plots represent the median, interquartile range, and full data distribution (whiskers), p < 0.01; ns: not significant. Fig. 5F. Immunohistochemistry for p-catenin, a marker of canonical Wnt signaling activity, in human colon organoid sections on day 7. Nuclei were counterstained with hematoxylin (purple). Arrows indicate representative nuclei with positive (nuclear) -catenin staining; asterisk mark nuclei lacking p -catenin signal. Organoids cultured in growth medium (left panel) exhibit widespread nuclear p-catenin staining, consistent with active Wnt signaling. In contrast, nuclear -catenin staining is restricted to budded regions in organoids cultured in differentiation medium (middle panel) and those co-cultured withtelocytes (right panel). Fig. 5G. Quantification of the percentage of nuclear p-catenin+nuclei per organoid section under three culture conditions: commercial growth medium (IntcstiCult™ Stem Cell Technologies #06010, left), commercial differentiation medium (IntcstiCult™ StemCell Technologies #100-0214, middle) and co-culture with immortalized telocytes in basal medium (no supplements, right). Box plots represent the median, interquartile range, and full data distribution (whiskers), p < 0.01; ns: not significant. Scale bars 50 pm.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0047] Telocytes establish an extensive 3D network along the basal side of the crypt-villus epithelium, playing a critical role as a stem cell niche by supplying Wnt proteins essential for stem cell function. Additionally, telocytes facilitate enterocyte differentiation. They serve as signaling hubs, expressing a wide array of signaling molecules from key pathways, including R-spondin, bone morphogenic protein (BMP), Transforming Growth Factor-P (TGF ), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), Notch and Sonic hedgehog (Shh). Notably, four spatially distinct telocyte subtypes were recently identified along the jejunum crypt-villus axis: crypt, villus base, villus mid and villus tip, each with a unique gene expression profile, correlating with active signaling pathways in the epithelium.

[0048] The crucial role of telocytes in maintaining intestinal homeostasis highlights the need for effective strategies to establish in vitro sources of these cells while preserving their in vivo features and functions. However, several challenges complicate the maintenance of primary telocyte cultures. Telocytes are present in very low numbers, constituting less than 1% of the intestinal stromal population, as determined by flow cytometry. Their unique characteristics and long cellular extensions render them particularly vulnerable to damage during dissociation and sorting procedures. Although current protocols for isolating mesenchymal cells from mouse intestines can yield a relatively high proportion of viable telocytes, their low abundance and non-proliferative nature present significant hurdles.

[0049] The present invention is directed to the preparation and use of an immortalized intestinal telocyte cell line that retains the key cellular and molecular characteristics of native telocytes. These immortalized telocytes continue to express key niche factors and support the growth of both mouse and human organoids in co-culture without the need for external factor supplementation.

[0050] Although telocytes have very important and useful functions in maintaining homeostasis, and supporting stem cell function and differentiation, isolating these cells is challenging due to their low abundance, slow proliferation rate, and delicate structure which makes them susceptible to damage during dissociation and sorting procedures.

[0051] In the present invention, the inventors have developed methods for isolating and immortalizing telocytes, to generate telocyte cell lines and facilitate using these cells in various applications. These methods include obtaining mice expressing a reporter gene (e.g., green fluorescent protein, GFP) driven by a forkhead box LI (Foxll) promoter, which effectively labels telocytes. After isolating mesenchymal cells from the mice jejunum, the telocytes were further isolated from the mesenchymal cells based on expression the reporter gene, and immortalized by Simian Virus 40 (SV40) Large T-antigen transformation to generate an immortalized cell line.

[0052] As disclosed herein, the immortalized telocyte cell line of the invention was shown to maintain structural characteristics typical to intestinal telocytes, including long cytoplasmic projections (telopodes) including podom regions (see Fig. 1). These projections were found to be enriched in hallmark features typical of native telocytes, including multivesicular bodies, electron- dense vesicles, caveolae, abundant rough endoplasmic reticulum (rER), mitochondria and extracellular vesicles budding from the plasma membrane (50-200nm diameter), and clear membrane invaginations (Fig. IM).

[0053] Additionally, the immortalized telocytes of the invention were further shown to express the telocyte transcription factor Foxll (Fig. II) and the surface telocyte markers PDGFRa (Figs. 1K- 1L).

[0054] As also shown herein, the immortalized telocytes of the invention were found to include four telocyte clusters defined by clustering analysis based on single-cell RNA-sequencing (scRNA- seq) data. These subtypes were found by comparing signatures of differentially expressed genes (DEGs) to closely mirror the spatially defined telocyte subtypes found in the murine jejunum in vivo: crypt, villus base, villus mid and villus tip, each defined by unique gene expression profiles (Figs. 2A-2B). The four clusters were further found to exhibit differential expression of SRY-box 2 protein (Sox2), Wnt5a, doublecortin-like kinase 1 (Dclkl), and CD34 (Fig. 2C).

[0055] The immortalized telocytes of the invention were also shown to possess extracellular matrix (ECM) remodeling capabilities, as shown by expression of metalloproteinase 10 (MMP10) and a- smooth muscle actin (aSMA), relevant for ECM modulation and contraction (Figs. 3A-3B), and more directly in a Matrigel®-based contraction assay (Figs. 3C-3D).

[0056] Finally, the present invention shows that the immortalized telocytes of the invention are capable of supporting organoid growth in the absence of exogenous niche factors (EGF, Noggin, R-spondin, and basic fibroblast growth factor (bFGF), Figs. 4A-4E). Importantly, in contrast with addition of exogenous growth factors, in co-culture with immortalized telocytes Ki67 was restricted to the base of budding structures, suggesting a role in spatial regulation (Fig. 4F). Furthermore, the present invention shows support of organoid growth by mouse telocytes also for human colon organoids (Fig. 5A). Interestingly, organoids co-cultured with telocytes displayed both a spheric morphology (similar to culture in a growth medium) and a budded morphology (similar to culture in a differentiation medium, Fig. 5B), suggesting that the immortalized telocyte cell line can simultaneously promote features associated with both stem cell expansion and differentiation.

[0057] An immortalized telocyte cell line

[0058] In some embodiments, the present invention provides an immortalized telocyte cell line.

[0059] The term “immortalized telocyte cell line”, as used herein, refers to a population of telocytes that have been genetically modified or otherwise treated to overcome the normal limits on cell division, thereby acquiring the ability to proliferate indefinitely in vitro, while retaining key morphological, molecular, and functional characteristics of native telocytes.

[0060] In some embodiments, the immortalized telocyte cell line has a shorter cell cycle duration than native telocytes, which have a rather low proliferation rate. In some embodiments, the immortalized telocyte cell line has a cell cycle duration of less than 30 hours. In some embodiments, the immortalized telocyte cell line has a cell cycle duration of about 24-30 hours. In some embodiments, the immortalized telocyte cell line has a cell cycle duration of about 25 hours.

[0061] In some embodiments, the immortalized telocyte cell line is capable of undergoing at least 10 cell cycles. In some embodiments, the immortalized telocyte cell line is capable of undergoing at least 15 cell cycles. In some embodiments, the immortalized telocyte cell line is capable of undergoing at least 20 cell cycles.

[0062] In some embodiments, the immortalized telocyte cell line is viable in culture for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 24 months, or 36 months. In some embodiments, the immortalized telocyte cell line is viable in culture for at least one month. In some embodiments, the immortalized telocyte cell line is viable in culture for at least 2 months. In some embodiments, the immortalized telocyte cell line is viable in culture for at least a year.

[0063] Telocytes have been identified in a wide range of mammalian tissues and organs. They are broadly distributed as interstitial (stromal) cells involved in structural support, intercellular signaling, and maintenance of local stem-cell niches. Nonlimiting examples fortissues and organs including telocytes include: gastrointestinal system (small intestine, large intestine, stomach, esophagus), respiratory system (lung, pleura, trachea), cardiovascular system (myocardium, pericardium, blood vessels), reproductive organs (uterus, fallopian tubes, placenta, testis, epididymis), urinary system (kidney, ureter, urinary bladder), digestive glands and accessory organs (liver, pancreas, salivary glands), musculoskeletal system (skeletal muscle, tendons, fascia, bone marrow), nervous system (peripheral nerves, meninges, choroid plexus), skin and integument (dermis, subcutaneous connective tissue, hair follicles, sweat glands), sensory organs (eye, cornea, sclera, choroid). Accordingly, the telocytes used for establishing the telocyte cell line may be derived from any of the organs, systems, and tissues in the above list.

[0064] In some embodiments, the telocytes are derived from an organ or tissue selected from small intestine, large intestine, colon, and hair follicles.

[0065] In some embodiments, the telocytes are derived from small intestine or colon.

[0066] The term “derived from”, as used herein, means that the source of the cells from which the cell line was established is the indicated tissue or organ.

[0067] In some embodiments, the telocytes are derived from a mammal, such as a human, a mouse, a rat, a pig, a dog, a sheep, a goat, a cow, or a monkey. In some embodiments, the telocytes are derived from a human. In some embodiments, the telocytes are derived from a mouse.

[0068] Intestinal telocytes are characterized by the expression of the transcription factor Foxll. Accordingly in some embodiments, the telocytes express Foxl 1.

[0069] In addition to Foxll, telocytes typically express certain genes, which may be further used to define this cell line. Such gene include platelet-derived growth factor receptor alpha (PDGFRa) and CD34. Additional genes also expressed in telocytes include matrix metalloproteinase 10 (MMP10), alpha-smooth muscle actin (aS MA), Glioma-associated oncogene homolog 1 (Glil), Gremlin 1, R-spondin 3 (Rspo3), Noggin, Wnt2b, Wnt5a, bone morphogenic proteins 1 (BMP1) and 4 (BMP4), bone morphogenetic protein receptor type-lB (BMPR1B), Chordin, Bambi, Wnt4, chondroitin sulfate proteoglycan 4 (CSPG4), tenascin-C (TNC), and Dclkl.

[0070] In some embodiments, at least a portion of the telocytes express PDGFRa. In some embodiments, at least about 50% of the telocytes express PDGFRa. In some embodiments, about 100% of the telocytes express PDGFRa. In some embodiments, at least a portion of the telocytes express CD34.

[0071] In some embodiments, at least a portion of the telocytes express a gene selected from matrix metalloproteinase 10 (MMP10), alpha-smooth muscle actin (aS MA), Glioma-associated oncogene homolog 1 (Glil), Gremlin 1, R-spondin 3 (Rspo3), Noggin, Wnt2b, Wnt5a, bone morphogenic proteins 1 (BMP1) and 4 (BMP4), bone morphogenetic protein receptor type-lB (BMPR1B), Chordin, Bambi, Wnt4, chondroitin sulfate proteoglycan 4 (CSPG4), tenascin-C (TNC), and Dclkl.

[0072] In some embodiments, at least a portion of the telocytes express MMP10. In some embodiments, at least a portion of the telocytes express aSMA.

[0073] In some embodiments, the portion of telocytes is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In some embodiments, the portion of telocytes is at least about 10%. In some embodiments, the portion of telocytes is at least about 30%. In some embodiments, the portion of telocytes is at least about 50%. In some embodiments, the portion of telocytes is at least about 70%.

[0074] In some embodiments, the telocytes include at least two distinct populations, one population expressing PDGFRa at a high level (PDGFRahlgh) and another population expressing PDGFRa at a low level (PDGFRalow). In some embodiments, the PDGFRahlghpopulation has a mean fluorescence intensity that is at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher compared to that of the PDGFRalowpopulation. In some embodiments, the PDGFRahlghpopulation has a mean fluorescence intensity that is at least about 2 times higher compared to that of the PDGFRalowpopulation. In some embodiments, the PDGFRahlghpopulation has a mean fluorescence intensity that is at least about 5 times higher compared to that of the PDGFRalowpopulation. In some embodiments, the PDGFRahlghpopulation has a mean fluorescence intensity that is at least about 10 times higher compared to that of the PDGFRalowpopulation. In some embodiments, the number of cells in the telocyte cell line that are PDGFRahlghis at least twice the numbers of cells that are PDGFRa10w.

[0075] As describe herein, the telocyte cell line was found to mirror native telocytes in that it includes four populations which diverge in gene expression, each having different characteristics (see Fig. 2). The complexity of this cell type and the presence of the various populations appear to enable the various functions of these cells.

[0076] In some embodiments, the telocyte cell line includes subpopulations which differ in expression levels of at least one gene selected from Sox2, Wnt5a, Dclkl, and CD34. Native telocytes have many morphological features which characterize them and are important for their function. As disclosed herein, such features include long cytoplasmic projections (telopodes) including podom regions. Telopodes are elongated, thin cytoplasmic projections extending from the telocyte cell body, typically ranging from several tens to several hundreds of micrometers in length. Each telopode exhibits a characteristic alternating ultrastructural pattern consisting of podoms, which are short, dilated segments that contain organelles such as mitochondria, caveolae, elements of rough and smooth endoplasmic reticulum, and occasionally lysosomes or vesicles; and podomers, which are thin, filamentous segments (often <0.2 pm in diameter) connecting the podoms and giving the telopode a beaded or moniliform appearance under electron microscopy.

[0077] As shown herein (especially Fig. 1), the immortalized telocyte cell line maintains these important morphological features.

[0078] Accordingly, in some embodiments, the telocytes display morphological features including at least one cellular projection having a length of at least about 10 microns. In some embodiments, the telocytes display morphological features including cellular projections having a length of at least about 20, 50, or 100 microns.

[0079] In some embodiments, the at least one cellular projection includes multivesicular bodies, caveolae, rough endoplasmic reticulum (rER), mitochondria, and / or extracellular vesicles.

[0080] As described herein, the preparation of the immortalized telocyte cell line includes isolating mesenchymal cells from mouse jejunum, isolating telocytes from the mesenchymal cells, and immortalizing the isolated telocytes.

[0081] The strategy for isolating the telocytes from other mesenchymal cells is based on using a reporter gene that is expressed mainly in telocytes. To achieve this, the reporter gene expression is driven by a Foxl 1 promoter. In the present application, a mouse carrying a Cre recombinase expressed from a Foxll promoter was crossed with a mouse carrying a construct which would express green fluorescent protein (GFP) following Cre recombination. The result is that only the telocytes, which express Foxll, would activate Cre recombination, leading to expression of the GFP. However, other reporter genes may be used, including using a reporter gene (such as GFP or a similar reporter) directly transcribed from the Foxl 1 promoter.

[0082] However, although expression of Foxll is characteristic of telocytes, there are additional genes which characterize telocytes and may be also used, such as PDGFRa, Glil, and CD34.

[0083] In some embodiments, the telocytes express a reporter gene controlled by a promoter for a telocyte-specific gene. In some embodiments, the telocyte-specific gene is selected from Foxll PDGFRa, Glil, and CD34.

[0084] The immortalization of the telocytes may be done by any method known in the art for cell line preparation. Some nonlimiting examples include viral oncogene-mediated immortalization (SV40 Large T antigen, adenovirus E1A / E1B, human papillomavirus E6 / E7), telomerase-based immortalization (human telomerase reverse transcriptase, hTERT combined with viral oncogene), conditional immortalization systems (temperature-sensitive SV40 Large T antigen, inducible promoter systems, Cre-removable cassettes), and spontaneous or chemical immortalization (spontaneous mutation, chemical mutagen exposure).

[0085] In the present application, the method used was Simian Virus 40 (SV40) transformation. Briefly, SV40-mediated immortalization establishes a permanent cell line by viral expression of the SV40 Large T antigen, which disables the Rb and p53 tumor-suppressor pathways, thereby preventing growth arrest and allowing continuous cell division while preserving cell-specific traits.

[0086] In some embodiments, the immortalized telocytes include DNA encoding a viral antigen. In some embodiments, the immortalized telocytes include DNA encoding a viral antigen selected from SV40 large T antigen, adenovirus E1A / E1B, and human papillomavirus E6 / E7. In some embodiments, the immortalized telocytes include DNA encoding Simian virus 40 (SV40) large T antigen.

[0087] As explained above, the immortalized telocytes of the invention possess both ECM remodeling and contractile capabilities, as shown by expression of metalloproteinase 10 (MmplO) and a-smooth muscle actin (aSMA), relevant for ECM modulation and contraction (Figs. 3A- 3B), and more directly in a Matrigel®-based contraction assay (Figs. 3C-3D).

[0088] In some embodiments, the telocytes are capable of contracting ECM. In some embodiments, the contracting of the ECM is by at least 10%. In some embodiments, the contracting of the ECM is by at least 20%. In some embodiments, the contracting of the ECM is by at least 30%. In some embodiments, the contracting of the ECM is by at least 30% after 48 hours. In some embodiments, the contracting of the ECM is by at least 50%. In some embodiments, the contracting of the ECM is by at least 50% after 92 hours.

[0089] One of the main applications of the immortalized telocyte cell line of the invention is the ability of the telocytes to support growth of organoids in culture.

[0090] Organoids are three-dimensional (3D) multicellular structures grown in vitro from stem cells or progenitor cells that self-organize through cell differentiation and spatial patterning to recapitulate, at least in part, the cellular composition, architecture, and functional characteristics of a corresponding organ or tissue. Organoids can serve as a powerful tool in research, as well as be used for the preparation of tissue and organ substitutes. Crucially, the establishment and maintenance of organoids require supplementation with niche factors that support the formation of organized and functional tissues.

[0091] As shown above, the telocytes of the invention were capable of supporting both human and mouse organoids in a cell culture without the need for adding certain niche factors.

[0092] The term “supporting growth”, as used herein with reference to the telocyte cell line supporting growth of organoids, means maintaining the viability, structural integrity, and expansion ability of organoids over a defined culture period under conditions in which organoids cultured without the supporting cells would otherwise fail to survive or expand.

[0093] In some embodiments, organoid growth is considered to be supported when the organoids remain viable and their morphologically remains intact for at least 24, 48, or 72 hours, or for at least 4, 5, 6, 7, 8, 9, or 10 days in culture. In some embodiments, organoid growth is considered to be supported when the organoids exhibit increase in size, budding, or proliferation and / or differentiation after 24, 48, or 72 hours, or for at least 4, 5, 6, 7, 8, 9, or 10 days in culture.

[0094] In some embodiments, the immortalized telocyte cell line of the invention supports in the organoids culture at least one feature selected from: viability, growth, sternness, and differentiation.

[0095] The term “niche factors”, as used herein, relates to signaling molecules, extracellular matrix components, or cell-surface ligands that collectively maintain the self-renewal, proliferation, and differentiation balance of stem cells within their native microenvironment (the stem cell niche) or in vitro culture systems replicating that environment. These factors act by activating or modulating canonical developmental signaling pathways such as Wnt, BMP, EGF, FGF, Notch, and TGF-p, thereby preserving stem cell identity or directing lineage commitment. Nonlimiting examples for niche factors include Wnt3a, Wnt2b, R-spondin 1, R-spondin 2, R-spondin 3, Noggin, Gremlin 1, Gremlin 2, Chordin, Epidermal growth factor (EGF), Fibroblast growth factor 10 (FGF 10), basic fibroblast growth factor (FGF2), TGF-P inhibitors (e.g., A83-01), Delta-like ligand 1 (Dill), Jagged 1, laminin, collagen IV.

[0096] The term “exogenous”, as used herein, means that the exogenous niche factors are not secreted by the telocytes or by the organoids and are provided from an external source. The advantage of the telocytes is that the telocytes naturally secrete the niche factors which do not need to be provided from an external source.

[0097] In some embodiments, the telocytes are capable of supporting growth of organoids in the absence of exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0098] In some embodiments, the telocytes are capable of supporting growth of organoids in the absence of exogenous niche factors.

[0099] Compositions including the telocyte cell line and organoids

[0100] In some embodiments, there is provided a composition including organoids and an immortalized telocyte cell line in a culture medium.

[0101] Definitions and embodiments mentioned above and which may be relevant to the embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0102] In some embodiments, the telocytes express Foxll.

[0103] In some embodiments, the telocytes include DNA encoding SV40 large T antigen.

[0104] In some embodiments, the telocytes are derived from intestinal tissue, such as small intestine or colon.

[0105] The organoids may be derived from various mammals, including but not limited to human, mouse, rat, pig, cow, sheep, goat, dog, cat, and monkey.

[0106] In some embodiments, the organoids are mammalian organoids. In some embodiments, the organoids are human organoids. In some embodiments, the organoids are mouse organoids.

[0107] The organoids may be derived from various tissues, including intestine (small intestine, colon), stomach, esophagus, pancreas, liver, gallbladder, lung, trachea, uterus, fallopian tube, ovary, testis, kidney, bladder, heart, vascular tissue, skeletal muscle, and skin.

[0108] In some embodiments, the organoids are intestinal organoids.

[0109] In some embodiments, the organoids are derived from stem cells. In some embodiments, the organoids are derived from differentiated cells.

[0110] In some embodiments, the composition is essentially devoid of exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF). In some embodiments, the composition is essentially devoid of exogenous niche factors.

[0111] The term “essentially devoid of”, as used herein, means that the composition lacks the indicated niche factors, but may still include residual levels of niche factors that do not have functional significance. In some embodiments, the residual level is less than 10%, 5%, or 1% of the level of a niche factor that is required for it to have an effect on the organoids.

[0112] The culture medium may be any culture medium suitable for growing organoids, but not including niche factors as disclose above. In some embodiments, the culture medium is a standard organoid culture medium not including niche factor. Nonlimiting examples for a tissue culture media include media based on DMEM / F12, and as further described in the examples.

[0113] Methods of culturing organoids by using the telocyte cell line

[0114] Stem cells offer vast potential in regenerative medicine, disease modeling, toxicology screening, and cultivated food production. Yet, precisely controlling stem cell behavior, such as maintaining a naive state or directing differentiation, remains a significant challenge. Establishing heterogenous, in vitro-maintained telocyte networks promise to more faithfully recapitulate the physiological processes governing stem cell differentiation, by providing a more native microenvironment than exogenous factor cocktails alone.

[0115] Co-culturing telocytes with organoids, rather than relying solely on niche factors, offers a valuable and less costly approach for studying the molecular and cellular mechanisms involved in stem cell growth and differentiation within their niche. This method enables the exploration of reciprocal communication and bidirectional signaling between stem cells and their niche-dynamic that cannot fully captured through external factors alone.

[0116] In some embodiments, there is provided a method of culturing organoids, the method including contacting the organoids with the immortalized telocyte cell line disclosed herein in an culture medium, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0117] Definitions and embodiments mentioned above and which may be relevant to the embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0118] In some embodiments, the organoids are intestinal organoids.

[0119] In some embodiments, the method includes the following steps:

[0120] - isolating epithelial stem cells or crypts from intestine and embedding the crypts in a laminin-rich hydrogel (such as Matrigel®) to obtain organoids;

[0121] - dissociating the organoids embedded in the laminin-rich hydrogel;

[0122] - adding the immortalized telocyte cell line disclosed herein to the dissociated organoids and embedding the combination of organoids and telocyte cell line in a laminin-rich hydrogel, wherein the method does not include a step of adding niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0123] In some embodiments, there is provided the immortalized telocyte cell line disclosed herein or the composition disclosed herein for use in a method of culturing organoids.

[0124] The disclosed method of culturing organoids, as well as the cultured organoids, may be used for various applications including applications relevant to research and diagnostics.

[0125] The term “crypt”, as used herein relates to invaginated epithelial structures located at the base of the intestinal mucosa in both the small intestine and colon. They house and protect the stem and progenitor cell compartment responsible for continuous epithelial renewal. They are also known as crypts of Lieberkuhn.

[0126] Some specific examples are provided below.

[0127] An organoid culture in which the organoids are prepared from cells of a subject having a disease may be used as a model for the disease, e.g., for testing the effects various treatments or agents.

[0128] In some embodiments, there is provided a method of preparing a tissue culture model for an intestinal disease, the method including contacting intestinal organoids with the immortalized telocyte cell line disclosed herein in a culture medium, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0129] In some embodiments, there is provided a method of screening of drugs for treating an intestinal disease, the method including contacting intestinal organoids with the immortalized telocyte cell line disclosed herein in a culture medium, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0130] In some embodiments, the intestinal disease is selected from inflammatory bowel disease (IBD), intestinal or colorectal cancer, and infectious disease.

[0131] In some embodiments, the organoids are derived from a subject afflicted with the intestinal disease.

[0132] A further use of the organoid cultures with telocyte cell lines of the invention is for preparing tissue grafts for replacing affected tissues in a subject.

[0133] In some embodiments, there is provided a method of preparing intestinal tissue grafts for regenerative medicine, the method including contacting intestinal organoids with the immortalized telocyte cell line disclosed herein in a culture medium, wherein the method does not include a step of adding to the culture medium exogenous niche factors including epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

[0134] For applications with require more sternness, the telocytes may be separated by their PDGFRa expression level into a PDGFRalowpopulation and a PDGFRahlghpopulation (as defined above), and only PDGFRalowmay be used. An example for such an application is culturing of organoids from colorectal cancer.

[0135] For applications with require more differentiation, the telocytes may be separated by their PDGFRa expression level into a PDGFRalowpopulation and a PDGFRahlghpopulation (as defined above), and only PDGFRahlghmay be used. An example for such an application is fortissue culture models which mimic villus biology and interaction with the microbiome.

[0136] Definitions and embodiments mentioned above and which may be relevant to the embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0137] Methods of preparing an intestinal telocyte cell line

[0138] In some embodiments, there is provided a method of preparing a mouse telocyte cell line, the method including: a. generating a mouse carrying a reporter gene controlled by a Foxl 1 promoter for labeling telocytes; b. isolating from the mouse intestinal mesenchymal cells comprising telocytes, including: dissecting mouse jejunum, isolating mesenchyme from the jejunum by removing epithelium, and digesting the mesenchyme with collagenase to produce single mesenchymal cells; c. culturing the mesenchymal cells in a culture medium; d. isolating telocytes by sorting the mesenchymal cells for cells expressing the reporter gene; and e. immortalizing the isolated telocytes.

[0139] Definitions and embodiments mentioned above and which may be relevant to the embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis). The tissue culture medium may be any suitable tissue culture medium such as, e.g., a DMEM-based culture medium, as disclosed in the example section.

[0140] In some embodiments, the immortalizing is performed by infecting the telocytes with viral particles expressing SV40 Large T antigen.

[0141] In some embodiments, the sorting is performed by fluorescence activated cell sorting (FACS).

[0142] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0143] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0144] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0145] The term “treating” or “treatment”, as used herein, refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease and / or symptoms attributed to the disease. The term encompasses both inhibiting the disease, i.e. arresting its development; and ameliorating the disease, i.e. causing regression of the disease, e.g., by eliminating or ameliorating its symptoms.

[0146] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0147] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

[0148] Materials and Methods

[0149] Mouse models

[0150] Foxll-Cre mice (Sackett et al., 2007, Genesis 45, 518-522) were crossed with Rosa- membrane -targeted dimer tomato protein (mT) or membrane targeted green fluorescent protein (mG) (Rosa-mTmG) (Jackson Laboratories, Bar Harbor, ME #007676).

[0151] All animal experiments were approved by the Animal Care and Use Committee of the Hebrew University of Jerusalem.

[0152] Intestinal mesenchyme isolation and culture

[0153] Foxll-Cre; Rosa-mTmG mouse jejunum was dissected and mesenchyme was isolated as described in Canella et al., 2023, J Vis Exp. 2023, Mar 24:( 193). Briefly, the intestine was cut into 0.5 cm pieces. The epithelium was removed using 1 mM DL-dithiothreitol (DTT) and 2 mM EDTA. The remaining mesenchyme was then digested with 100 U / ml collagenase type VIII and 75 mg / ml DNase I in CM1640. Both primary and immortalized mesenchymal cells were cultured in DMEM medium containing 15% Fetal Bovine Serum and 1% Penicillin-Streptomycin.

[0154] Immortalization by SV-40 Large T-antigen infection

[0155] FACS-sorted GFP+telocytes were infected with viral particles expressing the SV40 Large T antigen (Addgene #170255), two weeks after sorting. Virus production followed standard procedures, involving transfection of a lentiviral vector encoding the protein, pHR-AR8.2 packaging vector, and the pCMV-VSV-G envelope plasmid into HEK293T cells. After transfection, supernatants containing viral particles were collected and concentrated by centrifugation. Intestinal mesenchymal cells were infected with 50pl of concentrated viral particles in 5-cm dishes, and selection was performed with Hygromycin (Thermo Fisher Scientific).

[0156] Fluorescence activated cell sorting (FACS) analysis

[0157] For single-cell suspension preparation, both primary and immortalized mesenchymal cells were incubated for 7 minutes with 0.25% Trypsin / lmM EDTA, then centrifuged at 1800 rpm for 5 minutes at 4°C. Single cells were resuspended in FACS buffer (5% FBS / PBS without Ca2+and Mg2+). To optimize fluorescence compensation settings, anti-mouse (BD #552843) and anti-rat (BD #552845) beads were used. Cells were sorted based on GFP signals. For FACS analysis GFP+immortalized telocytes, were incubated for 30 minutes with the antibodies listed in Table 1. Table 1: Antibodies for Immunofluorescence and immunohistochemistry

[0158] Immunofluorescence and immunohistochemistry Cultured cells and organoids were fixed in 4% PFA for 20 minutes at room temperature.

[0159] After fixation, samples were incubated with primary and secondary antibodies diluted in CAS- Block (Invitrogen 008120) and imaged on a Nikon Eclipse Ti2 confocal microscope (Nikon, Japan).

[0160] For p-catenin immunohistochemistry, organoids embedded in Matrigel® were fixed, processed for paraffin embedding, and sectioned. Antigen retrieval was performed using citrate buffer (pH=6) in a pressure cooker (Electron Microscopy Science). Sections were incubated with a P-catenin primary antibody diluted in Starting block™ (ThermoFisher #37542). For signal amplification, a biotinylated anti-mouse secondary antibody followed by the VECTASTAIN® ABC-HRP kit (Vector #PK-4000) was used. The signal was developed using 3,3- diaminobenzidine tetrahydrochloride (DAB) as the chromogen. Counterstaining was performed by incubating slides in hematoxylin (Sigma #0506002) for 30 seconds. Slides were then briefly rinsed with tap water, dehydrated, and mounted.

[0161] For Phalloidin staining cultured telocytes were fixed in 4% PFA in PBS for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 in PBS for 5 minutes. After thorough washing with PBS, cells were incubated with TRITC-conjugated phalloidin (MP Biomedicals, 160069) diluted 1:200 in PBS containing 1% BSA for 30 minutes at room temperature in the dark. Excess stain was removed by additional PBS washes. Nuclei were counterstained with DAPI (Sigma-Aldrich, #D9564). Images were acquired using a Nikon Eclipse Ti2 confocal microscope (Nikon, Japan).

[0162] Image analysis and processing were carried out using Fiji or NIS Elements software.

[0163] Transmission Electron Microscopy

[0164] Approximately 750 GFP+telocytes were cultured on an 8-chamber Lab-Tek slide and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde prepared in 0.1M cacodylate buffer (pH 7.4) for 4 hours at room temperature, followed by overnight fixation at 4°C. The cells were rinsed four times for 10 minutes each in 0.1 M cacodylate buffer, then post-fixed and stained with 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer for 1 hour.

[0165] After staining, the cells were washed four times in cacodylate buffer and dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, for 10 minutes each, followed by three washes in 100% anhydrous ethanol for 20 minutes each). Dehydrated cells were infiltrated with Agar 100 resin in ethanol at increasing concentrations (25%, 50%, 75%, and 100%) for 16 hours per step, then embedded in fresh resin and polymerized at 60°C for 48 hours.

[0166] The resin-embedded cells were sectioned into ultrathin slices (80 nm) using a diamond knife on Leica Reichert Ultracut S microtome. Sections were mounted onto 200-mesh-thin-bar copper grids and sequentially stained with uranyl acetate and lead citrate for 10 minutes each. The prepared sections were imaged using a Tecnai 12 TEM (120kV, Phillips, Eindhoven, the Netherlands) equipped with Phurona camera and RADIUS software (Emsis GmbH, Munster, Germany).

[0167] Single-cell RNA sequencing and analysis

[0168] Single-cell RNA-sequencing (scRNA-seq) was performed on immortalized GFP+telocytes using the 10X Genomics Chromium Single Cell 3' v3 platform. Raw sequencing data were processed with Cell Ranger (v6.1.2) and aligned to the mouse mmlO genome. Downstream analysis was performed using Seurat (v4.3.0).

[0169] Quality control filtering was applied to remove low-quality cells, excluding cells with fewer than 4,000 or more than 10,000 detected genes, fewer than 10,000 or more than 150,000 UMI counts, or more than 10% mitochondrial transcript content. Doublets were identified and excluded using DoubletFinder with parameters pN=0.25, pK=0.09, and an expected doublet rate of 20%. Data normalization was carried out using the Seurat NormalizeData function. Highly variable genes (8,000 features) were selected using the variance stabilizing transformation (VST) method. Cell cycle effects were regressed out by including the difference between S and G2M phase scores during data scaling. Principal component analysis (PCA) was then applied, and the first 30 principal components were used for neighbor detection and clustering.

[0170] Cell clustering was performed in Seurat using the shared nearest neighbor (SNN) graphbased Louvain algorithm (resolution=0.035), yielding four distinct clusters. To compare immortalized telocytes with in vivo telocyte populations, gene signatures for the four native telocyte clusters (crypt, villus base, villus mid, and villus tip) were defined by selecting the top 150 differentially expressed genes (DEGs) from each native cluster. For each cluster, DEGs were ranked by log2 fold change, and the top 150 genes were used to generate cluster-specific gene signatures. These signatures were then applied to the immortalized dataset by calculating module scores using Seurat’s AddModule Score function. Each immortalized telocyte was assigned to an in vivo cluster based on its highest moule score among the four cluster-specific signatures.

[0171] For two-dimensional visualization, Uniform Manifold Approximation and Projection (UMAP) was conducted using the first 30 principal components. Differential expression analysis between clusters was performed using the Wilcoxon rank-sum test with Bonferroni correction. Genes were considered differentially expressed if they had an adjusted p-value < 0.05, were expressed in more than 25% of the cells in the cluster, and had a log2 fold-change greater than 0.25.

[0172] Differentially expressed genes were identified by comparing each telocyte cluster to the other three clusters. The resulting gene lists were analyzed for functional enrichment analysis using the ggplot2 R package to identify unique Gene Ontology (GO) terms. The GO annotations displayed in Figs. 2E-2H were selected manually to ensure a diverse representation of biological processes and to minimize repetitive or redundant terms. This approach was taken to provide a clearer and more informative summary of the functional enrichment analysis results.

[0173] Single-molecule RNA Fluorescence in-situ hybridization (smFISH)

[0174] Custom probe libraries, designed using the Stellaris FISH Probe Designer software (Biosearch Technologies, Inc., Petaluma, CA) and labeled with Cy5 or TMR were used to hybridize with the target coding RNA. For hybridization preparation, cells were seeded on coverslips and fixed with 3.7% formaldehyde for 10 minutes, following established an protocol (Raj et al., 2008, Nat Methods 5, 877-879). Antibody staining was performed by diluting the GFP antibody in hybridization buffer, followed by applying the Alexa 488 secondary antibody diluted in GLOX buffer for 20 minutes. Quantification of Foxll mRNA concentration was conducted using the TransQuant custom MATLAB program.

[0175] Matrigel® contraction assay

[0176] GFP+Immortalized telocytes were seeded at a density of 10,000 cells in 30pl Matrigel® (BD Biosciences, 356230). Images of Matrigel® domes were captured at 1,18, 42 and 92 hours postseeding. The circumference of Matrigel® domes was measured using Fiji, and area quantification was analyzed and plotted in GraphPad Prism 9.5.1.

[0177] Mouse organoid-telocytes co-culture assay

[0178] For the generation of organoids, crypts were isolated from mouse jejunum of 10 to 15-week- old wild-type mice as described in Mahe et al., 2013, Curr Protoc Mouse Biol 3, 217-240. Briefly, the jejunum was flushed with ice-cold PBS, cut into pieces smaller than 5mm, and crypts were isolated by mechanical dissociation in PBS with 2mM EDTA on ice. Crypts were embedded onto Matrigel® (BD Biosciences, 356230), 30pl per well in 24-well-plate.

[0179] Crypt-derived mouse organoids were cultured in DMEM / F12 (Gibco) containing GlutaMax™ (1: 100, Gibco) and Penicillin-Streptomycin (1: 100 Biological Industries) supplemented with B-27 (1:50 Gibco), mouse Noggin (100 ng / ml Peprotech 250-38), mouse Rspol (500 ng / ml Peprotech, 315-32), mouse EGF (20 ng / ml Peprotech, 315-09), and human basic FGF (bFGF, 10 ng / ml Peprotech, 450-33). The medium was changed every 2 days until co-culture.

[0180] For co-culture, GFP+immortalized telocytes were trypsinized (0.25% Trypsin-ImM EDTA, 7 minutes at 37°C), then pelleted by centrifugation (1,800 rpm, 5 minutes at 4°C). Organoids were dissociated mechanically, and 10,000 telocytes were embedded with organoid fragments in 30pl of Matrigel® per well. Co-cultures were maintained in standard organoid culture medium without added niche factors unless otherwise specified.

[0181] Three experimental conditions were established: organoids alone, organoids supplemented with niche factors (Noggin, Rspol, EGF, bFGF), organoids cocultured with immortalized telocytes (no supplements). Organoid growth was monitored over four consecutive days. Phasecontrast images were acquired daily, and organoid diameters were quantified using Fiji (ImageJ). Statistical analysis was performed using GraphPad Prism 9.5.1.

[0182] Human organoid-telocyte co-culture assay

[0183] Healthy human colon organoids were obtained from the Organoid Center at the Hadassah- Hebrew University Medical Organization (Jerusalem), under ethics approval (Helsinki protocol HMO-20-921). Co-culture with immortalized telocytes was performed following the same protocol used for mouse organoids. Human organoids were cultured in either Intesticult™ Organoid Growth Medium (Stem Cell Technologies ##06010) or Intesticult™ Organoid Differentiation Medium (StemCell Technologies #100-0214). For co-culture experiments, human colon organoids were maintained in DMEM / F12 (Gibco#l 1320033), supplemented with GlutaMax (1: 100, Gibco #35050061) and Penicillin-Streptomycin (1: 100 Biological Industries), in the absence of commercially added niche factors.

[0184] Example 1: Immortalized telocytes retain structural characteristics

[0185] An immortalized telocyte cell line was developed by crossing Foxll -promoter-driven Cre mice with Rosa-mTmG reporter mice (Muzumdar et al., 2007, Genesis 45. 593-605), which express a plasma membrane-bound green fluorescence protein (GFP), enabling clear visualization of the telocytes (Fig. 1A). The mouse jejunum was dissected and the mesenchyme isolated and cultured for two weeks to recover from potential dissociation damage. Following recovery, the mesenchymal cells were dissociated and GFP+telocytes were isolated by flow cytometry.

[0186] To achieve immortalization, the Simian Virus 40 (SV40) Large T-antigen protocol was employed, which is a well-established method known for its simplicity and reliability in immortalizing various cell types. GFP+telocytes were infected with viral particles expressing the SV40 Large T-antigen. Viral particles were produced by transfecting a lentiviral vector encoding the protein into HEK293T cells. The immortalized cells were expanded through passages (e.g., 7- 9 passages) before being cryopreserved for further analysis. In this context, “immortalization” refers to the acquisition of a markedly shorter cell cycle - approximately 25 hours - as measured by DAPI+nuclei over a 24-hour period (Fig. IB).

[0187] Importantly, the cells retained their defining phenotypic features through multiple passages.

[0188] Because immortalization can affect cellular identity and morphology, a comprehensive characterization of the cell line was performed. Immortalized telocytes showed variable GFP intensity and adopted a thin, flat morphology with extremely long cytoplasmic projections, often stretching several hundred microns from the cell body. These projections featured strong, localized GFP signals, consistent with podom regions of telopodes, which is the hallmark structures of native telocytes (Fig. 1C). A small fraction of cells exhibited multinucleation (Fig. ID), a known side effect of SV40 Large T-antigen-mediated immortalization.

[0189] During active proliferation, cells assumed a rounded morphology with multiple protrusions. After division, cells spread out into large, thin structures maintaining continuous intercellular contacts and extending long projections to neighboring cells, ultimately forming interconnected networks. This behavior mirrors the architecture of subepithelial telocytes along the intestinal crypt-villus axis.

[0190] Immortalized telocytes also displayed cellular polarization, with actin cytoskeleton components concentrated in specific regions. Phalloidin staining revealed asymmetric distribution of parallel actin filaments arranged in a regular, striped pattern beneath the plasma membrane (Fig. IE), indicative of contractile properties. The projections (telopodes) varied in thickness and were actin-rich (Figs. 1E-1G), with some appearing continuous with stress fibers (Fig. IE arrow). Many extensions ended in bulbous tips containing multiple fine projections, suggestive of cytoneme-like structures (Fig. IF, see boxed region). Immunofluore scent staining for a -tubulin showed localized microtubule enrichment at the base of finer projections (Fig. 1H), implicating coordinated microtubule-actin dynamics in projection extension.

[0191] To validate lineage fidelity, Foxll expression was confirmed using single-molecule RNA fluorescence in situ hybridization (smFISH) (Fig. II). Immortalized GFP+telocytes maintained detectable expression of Foxl 1 mRNA, confirming preservation of key aspects of their original identity. The expression of PDGFRa, a recognized marker of subepithelial telocytes in the intestine, was next evaluated using both immunofluorescence and flow cytometry analyses. Two distinct subpopulations were identified: PDGFRahlghand PDGFRalow(Figs. 1K-1L).

[0192] To assess the ultrastructural integrity of the immortalized cells, transmission electron microscopy (TEM) was performed. Immortalized telocytes exhibited exceptionally long projections, extending several dozen microns, that often extended beyond the field of the TEM grid (Fig. IM). These telopodes were enriched in hallmark features typical of native telocytes, including: multivesicular bodies, electron-dense vesicles, caveolae, abundant rough endoplasmic reticulum (rER), mitochondria and extracellular vesicles budding from the plasma membrane (50- 200nm diameter), as well as clear membrane invaginations. These organelles and structural features are consistent with previous descriptions of telopodes in native telocytes.

[0193] In sum, it was surprising that despite the genetic manipulation, the immortalized telocytes preserved key morphological and ultrastructural features of native telocytes.

[0194] Example 2: Immortalized telocytes preserve molecular expression profile

[0195] To evaluate whether immortalized telocytes preserved native subtype diversity, single-cell RNA-Sequencing (scRNA-Seq) was performed, identifying four distinct clusters closely mirroring the spatially defined telocyte subtypes found in the murine jejunum in vivo: crypt, villus base, villus mid and villus tip, each with unique gene expression profiles. One cluster was particularly enriched for villus tip telocyte markers (cluster 0), while the others showed overlapping gene signatures with multiple native subtypes (Figs. 2A-2B). To compare immortalized telocytes with in vivo telocyte populations, gene signatures for four native telocyte clusters (Crypt, Villus base, Villus mid, and Villus Tip) were used to calculate module scores in Seurat using the AddModule Score function. Each cell was assigned to the in vivo cluster with the highest module scores, enabling functional comparison between immortalized and native telocyte populations.

[0196] Key markers, including Sox2, Wnt5a, Dclkl, and CD34, were differentially expressed across clusters (Fig. 2C). Notably, Sox2 was highly enriched in several clusters of the immortalized telocyte line. However, this gene is typically absent from telocytes in the adult murine intestine and is only expressed during developmental stages (unpublished data). This finding suggests that the process of cellular immortalization and release from senescence may alter telocyte gene expression, leading to reactivation of Sox2, a gene more commonly associated with developmental or progenitor states. Although Sox2 is not expressed by adult intestinal telocytes in vivo, its enrichment in the immortalized clusters likely reflects a shift toward a more progenitor-like or proliferative state under in vitro conditions. Thus, immortalization appears to preserve telocyte heterogeneity, while also inducing distinct gene expression changes compared to primary telocytes in vivo.

[0197] In addition, Wnt5a mRNA molecules were localized along the extensions of telocytes, as revealed by single molecule RNA FISH (smFISH) (Fig. 2C). Correspondingly, rER was observed within these extensions by TEM (Fig. IM). This co-localization raises the intriguing possibility that local translation may occur within telocyte extensions.

[0198] Importantly, signaling molecules central to Wnt, Bmp, Egfr, Tgfb, Fgf and Hedgehog pathways were expressed, primarily in clusters 1 and 3, underscoring the maintenance of essential stem-cell niche functional properties in this model (Fig. 2D).

[0199] Next, gene ontology analysis was performed across the four identified cell clusters (Figs. 2E-2H). The data revealed distinct patterns of gene activation among clusters 0 and 2. Specifically, cluster 0 (Fig. 2E) exhibited a unique transcriptional landscape, with enrichment for genes involved in metabolic processes, ribosome and macromolecule biogenesis, and chromosome organization, functions reminiscent of those seen in villus tip telocytes. These properties suggest that the cell line can dynamically adapt to diverse conditions. In contrast, cluster 1 (Fig. 2F), which carries the signature of crypt telocytes, typically involved in stem cell maintenance and epithelial proliferation, is relatively underrepresented, possibly explaining the cell line’s tendency to promote organoid differentiation over proliferation and thus support a balanced organoid growth profile. Cluster 2 (Fig. 2G) was enriched for genes related to both actin filament-based processes and muscle structure development, as observed in native villus base telocytes, as well as genes involved in immune regulation and defense responses, which are characteristic of native villus mid telocytes. Cluster 3 (Fig. 2H) showed enrichment for broader biological processes, such as regulation of signaling, cell-cell communication, and multicellular organism development. Interestingly, gene ontology related to neuronal synapse organization and axon development was also enriched in the telocyte cell line. This suggests a close relationship between the regulation of neuronal extension and communication with telocytes.

[0200] Overall, this molecular analysis highlights the capacity of this cell line to sustain distinct gene expression profiles and produce stem cell niche molecules, even ex vivo and in the absence of their native tissue environment.

[0201] Example 3: Immortalized telocytes exhibit matrix remodeling and contraction skills

[0202] To further characterize key functional properties of the immortalized telocytes, and features that are challenging to dissect in vivo, the in vitro culture system was used. Given the polarized organization of cytoskeletal elements in immortalized telocytes, the focus was on examining their potential for contractility and extracellular matrix (ECM) remodeling.

[0203] First, the expression of matrix metalloproteinase 10 (MmplO), an enzyme crucial for ECM degradation and modulation of inflammatory bowel disease (IBD), and a-smooth muscle actin (aSMA), a marker of contractile cells were assessed by immunostaining. Both MmplO and aSMA were detected in a subset of immortalized telocytes (Figs. 3A-3B), indicating persistent cellular heterogeneity.

[0204] To directly evaluate their ability to remodel the ECM, a Matrigel®-based contraction assay was performed. Immortalized telocytes were embedded within Matrigel® at a density of 10,000 cells per 30pl dome. After 42 hours in culture, telocytes had extended their processes, spreading throughout the Matrigel® (Fig. 3C). Notably, significant reduction in the Matrigel® dome size was observed, reflecting robust cell-matrix interaction and active 3D contractility (Figs. 3C-3D).

[0205] These findings demonstrate that immortalized telocytes possess both ECM remodeling and contractile capabilities. The observed gel contraction and cellular morphological rearrangements highlight their potential to influence tissue architecture through mechanical interactions with the matrix, highlighting their functional relevance in the tissue microenvironment.

[0206] Example 4: Immortalized telocytes support organoid growth in the absence of exogenous growth factors

[0207] To determine whether immortalized telocytes may be used as a substitute for exogenous growth factors, their ability to maintain organoid growth without additional niche factors was assessed. Organoids, three-dimensional tissue structures derived from pluripotent or adult stem cells, have become invaluable tools in fundamental and biomedical research for modeling development, disease, drug responses, and personalized medicine. Successful organoid formation typically requires supplementation with exogenous niche factors, including EGF, Noggin, R- spondin, and bFGF (ENRF), to support tissue organization and functionality.

[0208] Immortalized telocytes were co-cultured with mouse small intestinal organoids embedded in Matrigel®, comparing three conditions: organoids alone with no external supplements, organoids with ENRF supplementation, and organoids co-cultured with immortalized telocytes (Figs. 4A-4D). After one day of culture, organoids co-cultured with telocytes displayed a larger and more robust spheroid morphology relative to both control groups (Fig. 4A).

[0209] Over a four-day period, organoids cultured without exogenous niche factors demonstrated progressive deterioration, with significant loss of structure apparent by day 2 (Fig. 4B). In stark contrast, organoids co-cultured with telocytes sustained growth, exhibited prominent budding, and developed crypt-like projections by day 3 (Fig. 4C) and were similarly effective in maintaining organoid growth as the ENRF supplement. By day 4, these organoids maintained a characteristic budded morphology, with telocytes closely opposed to the basal side of the epithelium (Fig. 4D), mimicking the positioning of telocytes in the intestine. Fig. 4E shows the organoid sizes during the experiment.

[0210] To further assess stem cell activity, the expression of Ki67 (a proliferation marker) was compared between the co-cultures on day 4. In co-culture with immortalized telocytes, Ki67 was restricted to the base of budding structures, whereas organoids maintained with ENRF factors showed broader marker expression throughout the budding compartments (Fig. 4F). These findings suggest that immortalized telocytes not only support organoid growth in the absence of exogenous factors but may also promote spatially regulated stem cell activity and differentiation.

[0211] Collectively, these data demonstrate that immortalized telocytes are a potent source of niche signals and can sustain organoid self-renewal and organization without additional growth factor supplements. As such, this cell line provides a valuable platform for in vitro studies of stem cellniche interactions under both physiological and pathological conditions.

[0212] Example 5: Immortalized telocytes support human organoid growth

[0213] Surprisingly, the immortalized mouse telocyte cell line can also support human colon organoids in the absence of externally supplied niche factors. Notably, while organoids cultured without external support began deteriorating as early as day 2, those co-cultured with immortalized telocytes survived, established close physical associations with the telocytes, and gradually developed cystic structures. This morphological development was similar to organoids grown in commercially enriched growth medium (Fig. 5A).

[0214] Remarkably, this supportive effect persisted despite species differences: the immortalized telocytes originated from mouse small intestine, whereas the organoids were derived from human colon tissue. Nevertheless, the telocytes facilitated organoid growth and preserved key colonspecific features. These findings suggest that telocytes exhibit a flexible secretory profile and are capable of providing niche-supporting factors, potentially modulated by dynamic interactions with the epithelial cells.

[0215] Next, the effects of telocyte co-culture on organoid sternness states were compared relative to standard growth and differentiation conditions over a seven-day period. Organoids cultured in differentiation medium exhibited a multi-budded morphology, while those in growth medium maintained a spheroid morphology. Interestingly, organoids co-cultured with telocytes displayed both spheroid and budded morphologies (Fig. 5B). During the first two days, their size was comparable to organoids cultured in growth medium. However, over time, organoids co-cultured with telocytes reached a growth plateau, resembling the size profile of organoids in differentiation medium, whereas those in growth medium continued to expand.

[0216] Furthermore, when organoid surface was evaluated (Fig. 5C), it was demonstrated that organoids cultured in growth medium undergo rapid expansion, reaching maximal surface area between days 4 and 6, whereas organoids maintained in differentiation medium or co-cultured with telocytes show modest or stable surface area over the same period. While organoids in growth medium were significantly larger by day 6 than those in differentiation medium or telocyte co-culture, there was no statistically significant difference between the differentiation and telocyte groups after a Bonferroni correction. Notably, telocyte co-culture supports a stable, intermediate organoid size, suggesting that telocytes promote a more balanced growth-to- differentiation ratio compared to conditions favoring pure proliferation or differentiation.

[0217] Immunostaining for the progenitor cell marker Sox9 and active canonical Wnt signaling (as indicated by nuclear p-catenin) revealed that Sox9 levels in telocyte-co-cultured organoids were similar to those maintained in growth medium. However, a marked reduction in nuclear p-catenin was observed in the telocyte co-cultures compared to organoids maintained under standard growth conditions (Fig. 5D-5G). These results suggest that the cell line promotes the maintenance of a progenitor cell state, potentially at the expense of sustaining true sternness within the organoids.

[0218] In conclusion, these findings indicate that immortalized telocytes are able to support human colon organoid growth and help maintain aspects of cellular heterogeneity and the stem cell niche, even across species barriers. These results suggest that this intestinal telocyte cell line can serve as a promising and versatile tool for investigating stem cell-niche interactions in both mouse and human systems, and could have potential applications in preclinical research.

Claims

CLAIMSWhat is claimed is:

1. An immortalized telocyte cell line comprising telocytes comprising DNA encoding Simian virus 40 (SV40) large T antigen.

2. The immortalized telocyte cell line of claim 1, wherein the telocytes are derived from intestine.

3. The immortalized telocyte cell line of claim 1 or 2, wherein the telocytes are mouse telocytes or human telocytes.

4. The immortalized telocyte cell line of any one of claims 1-3, wherein the telocytes express Foxl 1.

5. The immortalized telocyte cell line of any one of claims 1-4, wherein at least a portion of the telocytes express platelet-derived growth factor receptor alpha (PDGFRa).

6. The immortalized telocyte cell line of claim 5, wherein the portion of telocytes expressing PDGFRa is at least about 50%.

7. The immortalized telocyte cell line of claim 5 or 6, including a population expressing PDGFRa at a high level and a population expressing PDGFRa at a low level, and wherein the high expression level is at least about 2 fold higher compared to the low expression level.

8. The immortalized telocyte cell line of any one of claims 1-7, wherein at least a portion of the telocytes expresses a gene selected from matrix metalloproteinase 10 (MMP10) and alphasmooth muscle actin (aSMA).

9. The immortalized telocyte cell line of claim 8, wherein the portion of telocytes expressing a gene selected from MMP10 and aSMA is at least about 10%.

10. The immortalized telocyte cell line of any one of claims 1-9, wherein at least a portion of the telocytes express a gene selected from Glioma-associated oncogene homolog 1 (Glil), Gremlin 1, R-spondin 3 (Rspo3), Noggin, Wnt2b, Wnt5a, bone morphogenic proteins 1 (BMP1) and 4 (BMP4), bone morphogenetic protein receptor type- IB (BMPR1B), Chordin, Bambi, Wnt4, chondroitin sulfate proteoglycan 4 (CSPG4), tenascin-C (TNC), and Dclkl.

11. The immortalized telocyte cell line of any one of claims 1-10, further comprising subpopulations which differ in expression levels of at least one gene selected from SRY -box 2 (Sox2), Wnt5a, doublecortin-like kinase 1 (Dclkl), and CD34.

12. The immortalized telocyte cell line of any one of claims 1-11, wherein the telocytes display morphological features comprising at least one cellular projection having a length of at least about 10 microns.

13. The immortalized telocyte cell line of claim 12, wherein the at least one cellular projection comprises multivesicular bodies, caveolae, rough endoplasmic reticulum (rER), mitochondria, and / or extracellular vesicles.

14. The immortalized telocyte cell line of any one of claims 1-13, wherein the telocytes express a reporter gene controlled by a Foxll promoter.

15. The immortalized telocyte cell line of any one of claims 1-14, wherein the telocytes are capable of contracting extracellular matrix (ECM) by at least about 10%.

16. The immortalized telocyte cell line of any one of claims 1-15, wherein the telocytes are capable of supporting growth of organoids in the absence of exogenous niche factors comprising epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

17. The immortalized telocyte cell line of any one of claims 1-16, wherein the telocytes are capable of supporting growth of organoids in culture in the absence of exogenous niche factors.

18. A composition comprising organoids and the immortalized telocyte cell line of any one of claims 1-17 in a culture medium.

19. The composition of claim 18, wherein the telocytes express Foxll.

20. The composition of claim 18 or 19, wherein the organoids are human organoids or mouse organoids.

21. The composition of any one of claims 17-19, wherein the organoids are derived from an organ or a tissue selected from small intestine, large intestine, colon, and hair follicles.

22. The immortalized cell line of any one of claims 1-17 or the composition of any one of claims 18-21, for use in a method of culturing organoids.

23. The immortalized cell line or the composition for use of claim 22, wherein the method does not comprise adding exogenous niche factors comprising epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

24. The immortalized cell line of any one of claims 1-17 or the composition of any one of claims 17-20, for use in a method of screening drugs for treating a disease selected from inflammatory bowel disease (IBD), intestinal or colorectal cancer, and infectious disease.

25. A method of culturing organoids, the method comprising contacting the organoids with the immortalized telocyte cell line of any one of claims 1-17 in a culture medium, wherein the method does not comprise a step of adding to the culture medium exogenous niche factors comprising epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

26. A method of preparing a tissue culture model for an intestinal disease, the method comprising contacting intestinal organoids in a culture medium with the immortalized telocyte cell line of any one of claims 1-17, wherein the method does not comprise a step of adding to the culture medium exogenous niche factors comprising epidermal growth factor (EGF), Noggin, R- spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

27. The method of claim 25, wherein the intestinal disease is selected from inflammatory bowel disease (IBD), intestinal or colorectal cancer, and infectious disease.

28. A method of preparing intestinal tissue grafts for regenerative medicine, the method comprising contacting intestinal organoids in a culture medium with the immortalized telocyte cell line of any one of claims 1-17, wherein the method does not comprise a step of adding to the culture medium exogenous niche factors comprising epidermal growth factor (EGF), Noggin, R-spondin 1 (Rspol), and basic fibroblast growth factor (bFGF).

29. A method of preparing a mouse telocyte cell line, the method comprising: a. generating a mouse carrying a reporter gene controlled by a Foxl 1 promoter for labeling telocyte s; b. isolating from the mouse intestinal mesenchymal cells comprising telocytes, comprising: dissecting mouse jejunum, isolating mesenchyme from the jejunum by removing epithelium, and digesting the mesenchyme with collagenase to produce single mesenchymal cells;c. culturing the mesenchymal cells in a culture medium; d. isolating telocytes by sorting the mesenchymal cells for cells expressing the reporter gene; and e. immortalizing the isolated telocytes.

30. The method of claim 29, wherein the immortalizing is performed by infecting the telocytes with viral particles expressing SV40 Large T antigen.

31. The method of claim 29 or 30, wherein the sorting is performed by fluorescence activated cell sorting (FACS).

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