Identification and Localization of Telocyte-Like Cells in Human Umbilical Cord Stroma

Identification and Localization of Telocyte-Like Cells in Human Umbilical Cord Stroma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification and Localization of Telocyte-Like Cells in Human Umbilical Cord Stroma Ezel Erkan, Ibrahim Alptekin, Bilge Serdaroglu, Serife Esra Cetinkaya, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7203915/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 20 You are reading this latest preprint version Abstract Human umbilical cord (hUC) connective tissue, known as Wharton's jelly, contains multipotent stromal cells (MSCs). Despite being considered the primary source of MSCs for regenerative purposes, hUC is one of the least studied fetal tissues. We conducted a detailed examination of normal hUCs (n = 16) and identified telocyte-like cells (TLCs) exhibiting both structural and phenotypical features similar to telocytes previously described in various tissues, including the placenta. TLCs were found to be concentrated around intervascular stromal clefts in the UC. These cells had thin, elongated bipolar cell bodies (9-fold higher in length/width ratio compared to MSCs), distinguishing them from the well-defined MSCs, which display abundant ER-Golgi systems and high collagen production. We confirmed the presence of TLCs with marker expression patterns including F-actin, vimentin, α-SMA, α-actinin, caveolin-1 + , c-Kit + , PDGFR-α + , CD34 – , CD73 + , CD90 + , and CD105 + , reflecting a distinct stromal identity, either adjacent to MSCs or possibly originating from them. Isolation, culture, and immunocytochemical labeling further confirmed the presence of TLCs, highlighting the diverse nature of hUC cell cultures. These two cell types (TLCs and MSCs) were observed in contact with each other or within their respective populations. Each of the methods used in this study contributed to the identification of these cells, but none alone was enough to definitively characterize them. The findings conclusively demonstrate the existence of TLCs in the hUC. This provides significant new evidence regarding the cellular heterogeneity of the stem cell niche and suggests a potential role for TLCs in the stromal network of this tissue. Biological sciences/Cell biology Biological sciences/Stem cells interstitial cell multipotent stromal cell telocyte umbilical cord stroma Wharton’s Jelly Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Telocytes are a recently identified type of stromal cell, first described by Popescu and Faussone-Pellegrini in 2010 as a novel interstitial cell population distinct from fibroblasts, interstitial Cajal cells, and mesenchymal stem cells 1 . Initially referred to as Interstitial Cajal-like Cells, they were later renamed “telocytes” to reflect their most distinguishing feature: extremely long, slender prolongations called telopodes 2 . Structurally, telocytes are defined by their small, often spindle-shaped or triangular cell bodies, from which emerge one to five telopodes. These telopodes can extend up to several hundred micrometers and exhibit a moniliform appearance due to alternating thin segments (podomers) and dilated segments (podoms), which contain few mitochondria, endoplasmic reticulum, and caveolae 2 , 3 . Telocytes are typically identified as their unique ultrastructure, and immunophenotype, where they may express markers such as CD34, c-Kit (CD117), PDGFRα/β, and vimentin, though these markers are not entirely specific 4 , 5 . Telocytes have a widespread tissue distribution and have been identified in numerous organs and systems. In the cardiovascular system, they are abundant in the myocardium, where they form complex networks around cardiomyocytes, blood vessels, and stem cell niches 6 . In the lungs, they are present in both bronchial and alveolar walls, suggesting a role in tissue organization and regeneration 2 . Similarly, telocytes are found in the gastrointestinal tract, liver, pancreas, skin, skeletal muscle, uterus, fallopian tubes as reviewed by Sanches et al. 7 , and placenta 8 , 9 , underscoring their diverse functional potential across organ systems. Functionally, telocytes are believed to play several critical roles in tissue homeostasis and repair. One of their primary proposed functions is intercellular signaling. They establish direct physical contacts with other cells, including stem cells, immune cells, nerve fibers, and endothelial cells, through homo- and heterocellular junctions such as gap junctions and nanocontacts 6 . Their telopodes form extensive 3D networks that are thought to coordinate local microenvironmental cues, facilitating mechanical support and chemical signaling. Another proposed function of telocytes is the regulation of stem cell niches. In various tissues, telocytes are located near resident stem/progenitor cells, with whom they appear to interact via paracrine signaling or direct contact, potentially guiding their proliferation and differentiation 10 . Additionally, telocytes may be involved in immune surveillance, extracellular matrix (ECM) remodeling, and electrophysiological modulation, particularly in excitable tissues such as the heart and gastrointestinal tract 10 – 12 . The human umbilical cord (hUC) is a fetus-derived tissue composed of two arteries and one vein embedded in a gelatinous ECM known as Wharton’s jelly. This matrix constitutes the stromal compartment, which is considered to contain a relatively homogeneous population of stromal cells with significant regenerative potential. Those cells, also called “multipotent/mesenchymal stromal cells (MSCs)” are the most extensively studied cell type in recent years due to their multilineage differentiation capacity, immunomodulatory properties, and paracrine signaling functions 13 . hUC-MSCs are considered advantageous compared to adult tissue-derived MSCs, as they are more primitive, exhibit higher proliferative capacity, and are collected via a non-invasive procedure as reviewed 14 . The ECM of Wharton’s jelly, rich in hyaluronic acid and collagen, provides a supportive scaffold and biochemical cues for cellular behavior and survival 15 . Due to these properties, the hUC stroma is increasingly recognized as a valuable source for regenerative medicine and cell-based therapies 16 . After extensive histological examinations including transmission electron microscopy (TEM) and three-dimensional high-resolution confocal image stacks, along with systematic analysis of hundreds of tissue sections, we characterized a group of cells resembling telocytes found in other tissues in 16 normal hUC samples and identified their localization within the stromal compartment. These cells appear either as a distinct stromal population adjacent to known MSCs or possibly as derivatives of MSCs. Due to the uncertainty regarding their origin and developmental lineage, we have opted to refer to them as “telocyte-like cells (TLCs)” rather than definitively classify them as “telocytes”. However, it remains unclear whether these cells differentiate from resident MSCs during pregnancy. Materials and Methods Collection of Umbilical Cords The study was approved by the Ankara University Ethical Review Board for Human Research (approval #I09-598-23). Written informed consent was obtained from full-term pregnant women (n = 16) prior to the procedure. UCs (n = 16) were collected during Caesarean deliveries from healthy singleton pregnancies (38 ± 2 weeks of gestation) in women aged 20–35 years from the Department of Obstetrics and Gynecology, Ankara University School of Medicine. Samples were collected approximately 20 cm from the umbilicus, specifically from the proximal one-third segment of the cord, as previously described 17 . The 12-cm cord segments were delivered to the laboratory within 4 hours post-delivery. Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Upon arrival, the cords were dissected in Leibovitz’s L-15 medium supplemented with 1% penicillin-streptomycin, 1% amphotericin B, and 1% L-glutamine. Transverse sections of the cords were prepared for various analyses as follows: (i) a 2-cm fresh tissue segment for stereomicroscopic examination; (ii) 1-cm segments fixed in paraformaldehyde (PFA) for frozen sectioning, immunostaining, and super-resolution confocal microscopy (SR-CM); (iii) 1-cm segments fixed in glutaraldehyde (GA) for transmission electron microscopy (TEM); (iv) 1-cm segments stored at − 80°C for qRT-PCR analysis; (v) a 5-cm segment used for explant cultures intended for flow cytometry and scanning electron microscopy (SEM). All methods were carried out in accordance with relevant guidelines and regulations. Stereo and Light Microscopy Tissue blocks were initially rinsed with sterile phosphate-buffered saline (PBS) and examined under a stereomicroscope equipped with episcopic illumination (Nikon, Japan) to assess macroscopic features, including surface topography and the integrity of the umbilical vessels. One-centimeter-thick tissue samples from each umbilical cord were fixed in 3.5% (w/v) paraformaldehyde (PFA) for 48 hours at 4°C. Following fixation, the samples were washed twice with PBS and incubated for three days in a 1.2 M sucrose solution containing 0.01% PFA. The tissue blocks were then embedded in Cryomatrix (Fisher Scientific, USA), frozen at − 25°C, and sectioned into 10–14 µm-thick slices using a cryostat (Shandon, UK). Cryosections were stained with standard hematoxylin–eosin (H&E). Imaging was performed using a bright-field AxioImager microscope (Zeiss, Germany) equipped with ×2.5, ×20, and ×40 objectives. Both individual and tiled image stacks were captured using an Axiocam 503 color camera and Zen Blue software (v2.3 Pro). Transmission Electron Microscopy (TEM) For ultrastructural analysis, three small tissue blocks (each a few mm³) representing distinct stromal regions – subepithelial stroma (SES), intervascular stroma (IVS), and perivascular stroma (PVS) – were collected from each umbilical cord (UC) sample. A rat intestinal tissue sample was also included as a positive control for telocytes. Samples were processed as previously described 18 . High-resolution images were obtained using a transmission electron microscope (Hitachi HT7800, Japan) equipped with a 19 MB digital camera. Image analysis and reconstruction were performed using Adobe Photoshop (version 2022). Additionally, semi-thin sections were examined using a super-resolution confocal microscope (Zeiss LSM880, Germany) with a photomultiplier tube detector at 40× magnification and 1.5–2.6× digital zoom. Immunostaining and Super‑Resolution Confocal Microscopy (SR-CM) Immunofluorescent double-labeling was performed on PFA-fixed frozen sections and cultured cells using a panel of primary antibodies specific to telocytes and UC stromal cells, along with appropriate secondary antibodies ( Supp. Table 1 ). Prior to antibody application, sections and cells were incubated in a blocking solution. Nuclear labeling was achieved using Hoechst 33258 (1 mg/mL) diluted in a 1:1 PBS/glycerol-based mounting medium. Antibody specificity was verified through both positive and negative controls, including omission of the primary antibody and staining of known positive tissues or structures. After PBS washing and blocking (1-hour incubation at + 4°C), sections were incubated with primary antibodies at 37°C for 3 hours. Following three PBS washes, secondary antibodies were applied for 2 hours at 37°C. The same protocol was followed for double immunofluorescence, using sequential incubation with two sets of primary and secondary antibodies, each followed by PBS washes. For F-actin visualization, 633-Phalloidin or FITC-Phalloidin was applied to cryosections for 40 minutes at room temperature in the dark. The phalloidin solution (1 mg/mL) was prepared by diluting a methanol-based, DMSO-containing stock 1:10 in PBS and incubating for 1 hour in the dark. For immunostaining of cultured cells grown on coverslips, cells were fixed in 3.5% PFA for 20–25 minutes, washed with PBS-azide, and stored at + 4°C in a humidified chamber. After blocking, cells were incubated with primary antibody for 90 minutes at 37°C, followed by PBS washing and secondary antibody incubation under the same conditions. For double labeling, a second round of primary and secondary antibody incubation was performed sequentially. F-actin labeling was carried out using either FITC- or 633-Phalloidin, followed by gentle PBS washing. Coverslips were mounted in a 1:1 PBS solution containing Hoechst 33258 (1 mg/mL), sealed with nail polish, and stored at + 4°C. Fluorescently labeled sections and cells were imaged using a Zeiss LSM-880 confocal microscope (Zeiss, Germany) equipped with an AiryScan® detector to obtain super-resolution images (70–100 nm) through pixel reassignment and deconvolution. Laser lines (405, 488, 543 and 633 nm) were selected based on the fluorophores used; phalloidin images acquired with the 633 nm laser were pseudo-colored red. Laser power and scanning settings were standardized across all experiments using reference histograms. Imaging was conducted with 20× (dry) and 40× (water immersion) objectives, using a zoom factor of 1.8–2.2. Z-stack volumes were automatically calculated and consistently aligned. Image data were analyzed using the 3D module of Zen Desk software (version 2.3). For each section, three to six representative regions were selected, and their coordinates were recorded. From each region, 10–60 optical slices were acquired to reconstruct 3D dual-channel composite images with a thickness of 12–15 µm. Quantitative Reverse Transcriptase PCR Analysis Following removal of blood vessels, two stromal-rich segments (~ 1 cm thick) were excised from each UC (n = 15), snap-frozen in liquid nitrogen, and stored at − 80°C until further processing. Samples were divided into three experimental groups (n = 5 per group) and pooled within each group to generate biological replicates for transcriptomic analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). For complementary DNA (cDNA) synthesis, 1 µg of total RNA per sample was reverse transcribed using a commercial reverse transcription kit (Medchem, USA). Quantitative real-time PCR was performed using EvaGreen Master Mix (Bio-Rad, UAE) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Primer sequences for target and reference gene (GAPDH) are listed ( Supp. Table 2 ). Amplification specificity was confirmed via melt curve analysis, and amplicon size was verified by electrophoresis on 2% agarose gels stained with RedSafe® (Intron Biotechnology, USA). Electrophoresis was performed at 150 V for 30–45 min, and bands were visualized using the ChemiDoc MP Imaging System (Bio-Rad, UAE). Expression profiles of eight target genes—vimentin, CD34, c-Kit, PDGFR-α, caveolin-1, CD73, CD90, and CD105—were evaluated. GAPDH served as the endogenous reference gene. Each target was assessed in technical triplicates across three biological replicates. Threshold cycle (Ct) values were recorded, and relative gene expression levels were calculated using the 2 −ΔCt method. Results are reported as mean ± standard deviation, based on descriptive statistical analysis. Cell Culture The primary aim of the hUC culture was to identify and isolate TLCs from the stromal cell populations using telocyte-specific markers via flow cytometry (see below) and fluorescence-activated cell sorting. Initial live-cell observations and morphometric measurements were carried out by Hoffman Modulation Contrast (HMC) imaging using 10× objective (Olympus, Japan). Additionally, immunofluorescence staining was performed to evaluate the expression of both telocyte- and hUC-MSC-specific markers in fixed cultures. Cell morphometry was applied to captured images to characterize cells at different time points. For explant cultures, 5 cm segments of UC were dissected in sterile Leibovitz’s L-15 medium, processed, and cryopreserved as previously described 19 . Scanning Electron Microscopy (SEM) Cultured cells grown on round glass coverslips (~ 90% confluency) were rinsed twice with Dulbecco’s PBS, then fixed with 200 µL of 2.5% glutaraldehyde (GA) for 30 minutes at room temperature. This was followed by two 15-minute washes with 0.1 M Sorenson’s buffer. Post-fixation was carried out in a 1:1 mixture of 1% osmium tetroxide (OsO₄) and 0.2 M Sorenson’s buffer for 30 minutes, followed by another two buffer washes. Dehydration was performed through a graded ethanol series (30%, 50%, 75%, 95%, and 100%), with each step lasting 15 minutes. Four coverslips were treated with a 1:1 ethanol–hexamethyldisilazane (HMDS) solution for 5 minutes and then transferred to pure HMDS until complete evaporation. Samples were mounted on aluminum stubs using carbon adhesive tabs, sputter-coated with a 15 nm layer of gold–palladium, and examined using a LEO 438 VP scanning electron microscope (UK) operated at 15 kEV in high-vacuum mode with a secondary electron detector. Cell morphology and dimensional measurements were assessed from the acquired SEM images. Flow Cytometry To quantify TLC populations in hUC-MSC cultures based on specific molecular markers, flow cytometry was performed using a BD FACS Canto II system (USA) on three sets of triplicate passage 2 (P 2 ) cell cultures. In the first and second experiment, primary antibodies were applied in pairs. P 2 cells detached with Tryple (Gibco, USA) and resuspended in serum-free medium. The cell suspension was divided into three tubes: two for dual antibody staining [CD34 (1:500) + PDGFR-α (1:200), CD34 (1:500) + c-Kit (1:500)], and one for isotype control. After incubation with primary antibodies at room temperature for 30 minutes, secondary antibodies were added [FITC-conjugated goat anti-mouse or Cy3-conjugated goat anti-rabbit antibodies ( Supp. Table 1 )] and incubated in the dark. In the second experiment, the primary antibody incubation time was extended to 50 minutes, and the secondary antibodies were diluted to 1:2000. An unstained cell group was also included. Cells were divided into four tubes (two for staining, one isotype, one unstained), and processed similarly. After incubation with primary and secondary antibodies; samples were prepared and transported under cold, dark conditions for analysis. In the third experiment, primary antibodies were applied individually to evaluate their expression separately. Cells were divided into six tubes, with three assigned for single antibody staining (CD34, PDGFR-α, c-Kit) and the remaining for isotypes and unstained controls. Secondary antibodies were matched appropriately to their corresponding primary antibodies. In all three experiments, following incubation and washing steps, the samples were transferred under cold, dark conditions for analysis using FlowJo software. Marker positivity rates were determined relative to isotype controls and baseline autofluorescence. To validate and visually compare protein distributions observed by flow cytometry, smears were prepared from the analyzed cell suspensions and examined using SR-CM. For this, 10 µL of labeled or unlabeled cell suspensions were fixed in 90 µL of 3.5% PFA for 5–10 minutes, then centrifuged at 200 g for 10 minutes. The resulting pellets were resuspended in a 1:1 PBS solution containing Hoechst 33258 (1 mg/mL) for nuclear labeling. A 20 µL aliquot of the fixed suspension was placed on glass slides, covered with coverslips, and imaged using SR-CM. Results We began by examining UC blocks under a stereomicroscope to confirm their normal macroscopic features, including surface topography and the integrity of the umbilical vessels. Subsequently, HE-stained sections from 16 healthy deliveries were analyzed to identify stromal compartments in each section (Fig. 1 A–C). Slender cells with long, thin cytoplasmic processes were particularly prominent in the superficial (Fig. 1 D) and deep intervascular stroma (IVS) regions (Fig. 1 E), frequently observed encircling clefts – homogeneous ground substance-filled, mesh-like systems – in the superficial IVS. Semithin sections provided definitive confirmation of these cells and allowed for more precise morphological characterization. These cells exhibited multimicrometer-long, thin cytoplasmic extensions and a centrally located, ovoid, euchromatic nucleus. The extensions were often in close association with collagen bundles (Fig. 1 F and G ). These cells were embedded within a HE- and toluidine blue-stained semithin sections of human umbilical cords, enclosed by the umbilical cord epithelium (UCE). ( A ) Three main stromal regions with indistinct borders, identified from the outermost to the innermost, are: subepithelial stroma (SES), intervascular stroma (IVS), and perivascular stroma (PVS), the second classically known as Wharton’s jelly. ( B ) The IVS is further subdivided into superficial IVS (underlying to SES) and ( C ) deep IVS (overlying to PVS). ( D, E ) Telocyte-like cells (TLCs, arrowheads ) are clearly identified in both superficial and deep IVS regions, particularly encircling clefts ( asterisks ). ( F–H ) Higher magnification images obtained from semithin sections reveal that TLCs possess extremely long and thin cytoplasmic extensions called telopodes, which show irregularities along their length. These are embedded in a highly edematous and loose ECM, interspersed among MSCs ( arrows ). Scale bars: 1000 µm (A), 100 µm (B), 200 µm (C), 50 µm (D, E), 10 µm (F–H). highly edematous and loose ECM and were frequently found in proximity to wide-cuboid stromal cells, commonly identified as MSCs of Wharton’s jelly (Fig. 1 H). Then, we compared these cells with rat intestinal telocytes and found striking phenotypic similarities ( Supp. Figure 1 A). Morphometric analysis revealed that the mean length of the long-slender cells in hUCs was 92.46 µm (range: 48.93–120.08 µm; SD: 27.54), compared to 124 µm (range: 110.72–140.25 µm; SD: 14.85) in rat intestinal telocytes. Based on these comparisons and morphological features consistent with telocytes described in various tissues, we conclude that these long-slender cells are telocytes or telocyte-like cells (TLCs) in the hUC. In low magnification TEM observations, TLCs were distinguished by their extremely thin cytoplasmic extensions mostly neighboring to the clefts and embedded in a collagen-rich ECM (Fig. 2 ). TLCs exhibited small, euchromatic nuclei generally located centrally within their elongated cytoplasm, which contained poor protein-synthesizing organelles (Fig. 2 A–C). At higher magnifications, the plasma membrane was closely associated with extracellular vesicles (mean: 323.4 nm; range: 261–357 nm; SD: 40.16) (Fig. 2 C and D ), and the cell surface displayed interruptions with several caveolae (Fig. 2 D). Measurements of cytoplasmic width revealed a mean value of 143.72 nm (range: 62.8–220.82 nm; SD: 54.5) (Fig. 2 E and F ). Adjacent to TLCs, MSCs were characterized by their relatively wider and shorter cytoplasm, embedded within an abundant collagen matrix rich in protein-synthesizing organelles, and containing accumulations of widened ER cisternae and lipid-laden inclusions with periodic lamellae (Fig. 2 G and H ). To further distinguish these two cell types, we performed a morphometric analysis by calculating the length-to-width ratio (L/W) (Fig. 2 I). TLCs exhibited a mean ratio of 32.38 (range: 21.68–51.86; SD: 10.83), whereas MSCs showed a mean ratio of 3.60 (range: 1.96–5.43; SD: 0.94). As we had previously compared TLCs with rat intestinal telocytes in semithin sections, we also examined these cells in 50 nm ultrathin sections using TEM and observed a close similarity in both the fine structure of the cytoplasm and the length-to-width ratio ( Supp. Figure 1 B). For molecular characterization of TLCs, we collected a series of super-resolution 3D confocal images using specific fluorescent markers targeting cytoskeletal components, receptors, and cell surface proteins, along with well-established MSC markers. Initial validation was conducted using positive and negative controls, which involved either omitting the primary antibody or using appropriate tissues and/or tissue components. These controls are presented in Supp. Figure 1 . TEM observations of TLCs. ( A–C ) In low magnifications, they are distinguished by their extremely thin (as also shown in E and F ) cytoplasmic extensions embedded in a relatively scarse collagen fibers as opposed to abundant collagen bundles around MSCs (shown in G and H ). TLCs are also characterized by poor protein synthesizing organalles. However, they display several extracellular vesicles with close proximity to plasma membrane shown by white arrowheads in C and D and the cell surface displayed interruptions with several caveolae ( black arrows in D ). ( G–H ) MSCs are evident by their wider and shorter cytoplasms with rich in protein sythnesizing organelles, elaborate ER cisternae ( black arrowheads ) and lipid-laden inclusions ( white asterisks ). ( I ) The length-to-width ratio (L/W) was calculated as 32.38 for TLCs and 3.60 for MSCs. Black asterisks : Clefts; N : Nucleus. Scale bars: 3 µm (A–B), 1 µm (C–H). First, we labeled the entire UC sections with phalloidin to visualize F-actin filaments. As expected, all cell types from outer to inner regions including umbilical cord epithelium (UCE), vascular smooth muscle cells, and stromal cells were positive for phalloidin staining (Fig. 3 A). Therefore, this marker was not specific to TLCs. However, the general cell morphology, characterized by slender and thin cytoplasmic extensions, remained distinctive for TLCs. Additionally, F-actin showed the density and volume of cells in each region. Thus, TLCs were predominantly located in superficial IVS regions (Fig. 3 B). Secondly, we performed vimentin labeling which is a well-known positive marker for MSCs. Interestingly, long and slender shaped TLCs displayed stronger positivity compared to the one in relatively round-ovoid shaped MSCs with broader extensions (Fig. 3 C and D ). Thus, this finding has proven that TLCs are positive for vimentin. Another cytoskeletal protein tested was α-SMA, a smooth muscle cell marker that was found to be positive in both TLCs and MSCs (Fig. 3 E). The specificity of the antibody was confirmed by strong staining in the arterial wall of the UC ( Supp. Figure 1 I) and the absence of staining in the UCE. The actin-binding protein α-actinin, used as a marker for stromal cell labeling, was found to be positive in all stromal cells; however, a strong and punctate staining pattern was specifically observed in TLCs (Fig. 3 F). Finally, we tested three MSC markers –CD73, CD90, and CD105– to determine whether they are also expressed in TLCs, in parallel with neighboring MSCs. TLCs exhibited strong and diffuse positivity for CD73 and CD105, whereas CD90 staining was sparse and punctate (Fig. 3 G–I). Interestingly, a similar staining pattern was also observed in MSCs ( Supp. Figure 1 O–R). Another group of markers analyzed included cell surface proteins and receptors. Among these, CD34, PDGFR-α, c-Kit, and caveolin-1 were selected for their high specificity to telocytes. CD34 expression was completely absent in both TLCs and MSCs (Fig. 4 A and B ), even after testing four different antibodies derived from two species and three different commercial sources. This confirms previous reports indicating that CD34 negativity is a general phenotype of UC stroma. As a positive internal control, strong CD34 labeling was observed in the vein endothelium ( Supp. Figure 1 N). PDGFR-α showed strong plasmalemmal and weak cytoplasmic positivity in TLCs, particularly those surrounding clefts in the superficial IVS (Fig. 4 C and D ), often appearing embedded within dense collagen fibers. c-Kit, a tyrosine kinase receptor, was also expressed in TLCs, especially in those encircling clefts within the superficial IVS (Fig. 4 E and F ). At higher magnification (not shown), the cytoplasmic staining appeared strong and punctate. c-Kit positivity was also observed in arterial wall smooth muscle cells (not shown). Caveolin-1, a membrane-associated scaffolding protein, was detected in all TLCs within the superficial IVS as punctate cytoplasmic foci (Fig. 4 G and H ). A few positive cells were also identified in the SES, deep IVS, and PVS regions (not shown). Arterial wall smooth muscle cells again served as a positive internal control and were strongly caveolin-1 positive ( Supp. Figure 1 U). Markers specific to selected cell surface proteins and receptors in hUC sections. All images were deconvoluted and reconstructed from 16 consecutive optical sections along the z-axis to generate 3D image stacks. ( A , B ) CD34 showed minimal to no staining across all stromal regions. ( C , D ) PDGFR-α exhibited strong membranous and weak cytoplasmic positivity in TLCs, particularly those surrounding clefts ( asterisks in corresponding DIC images), which were enclosed by bundles of collagen fibers ( black arrows ) in the superficial IVS. ( E , F ) c-Kit expression was also strong in TLCs, especially those around clefts ( asterisks in corresponding DIC images) in the superficial IVS. ( G , H ) Caveolin-1 was localized in TLCs within the superficial IVS, appearing as cytoplasmic punctate staining. ★: UCE. Scale bars: 50 µm (A, B, E, F); 20 µm (C, D, G, H). Agarose gel electrophoresis confirmed the amplification of all eight target genes, with band intensities corresponding to expression levels (Fig. 5 A). Notably, CD34 exhibited the weakest bands, indicating lower transcript abundance compared to the others. To validate these findings, qRT-PCR data were further analyzed using descriptive statistical approaches. Ct values were normalized to the GAPDH reference gene, and relative expression levels were calculated using the 2 −ΔCt method ( Supp. Table 3 ). To facilitate interpretation of the broad dynamic range in expression, relative transcript levels were visualized using a logarithmic y-axis (Fig. 5 B). Among all targets, vimentin displayed the highest expression, followed by PDGFR-α and c-Kit. Moderate expression was observed for CD73, CD90, CD105, and caveolin-1, while CD34 consistently showed the lowest expression level. (Fig. 5 B). To further investigate the phenotype and marker expression of TLCs, we isolated the entire stromal cell population – including MSCs and TLCs – via explant culture. Cells harvested from the explants (P 0 ) were cryopreserved for up to two passages (P 2 ) and subsequently used for flow cytometric analysis, live-cell imaging with HMC, and post-fixation imaging by SEM and SR-CM. At passage 2 (P 2 ), initial cell attachment (T 0 ) was followed by the appearance of both broad-bodied cells, characteristic of MSCs, and extremely thin, elongated TLCs (Fig. 6 A and B ). These fusiform cells remained observable at T 36 and T 48 (Fig. 6 C–F). Throughout the culture period, TLCs were sparsely distributed and exhibited minimal proliferation, whereas adjacent MSCs expanded robustly, reaching confluency by approximately T 120 . To characterize the TLC phenotype, we conducted morphometric analysis on approximately 200 cells using composite HMC images. The average cell length was 604.69 µm (range: 272.27–1380.02 µm; SD: 339.7). In contrast, MSCs exhibited markedly different morphology, attaching to the substrate via flattened lamellipodia and displaying wider cytoplasmic domains. As the fine structural assessment, SEM further revealed cellular phenotype, cell surface and morphometric features of those elongated cells. The mean width of their cellular extensions was measured as 1.49 µm (range: 1.04–2.09 µm; SD: 0.43) (Fig. 6 G, I and J ). They occasionaly made contacts with the adjacent MSCs, which have extremely flat and broad cytoplasm extending nano tube-like spikes between each other (Fig. 6 G, H and I ). Elongated TLCs seemed attached to the substrate at two ends having a relatively flattaned cytoplasmic lamellipodia. ( A ) Agarose gel electrophoresis of the qRT-PCR products revealed detectable bands for all eight target genes. ( B ) Expression levels of eight target genes normalized to GAPDH are shown as mean ΔCt ± SD from three biological replicates (n = 3). Y-axis is presented on a logarithmic scale for better visualization of gene expression differences. Next, we aimed to isolate specific cell populations based on known markers such as CD34, PDGFR-α, and c-Kit using flow cytometry. However, immunophenotypic characterization of the entire live stromal cell population failed to distinguish any specific subpopulation using these markers ( Supp. Figure 2 ). This finding was confirmed through three sets of experiments involving single and double antibody labeling with varying antibody concentrations and incubation times. To further investigate, we examined the same live, unfixed cell populations using SR-CM to determine whether any marker expression undetected by flow cytometry could be visualized. SR-CM analysis also failed to detect any specific signal, supporting the initial flow cytometry findings ( Supp. Figure 2 ). In contrast, when the same cell populations were fixed and stained with the corresponding antibodies, clear and distinguishable labeling patterns were observed (Fig. 7 ), suggesting that these markers may not be reliably detectable in live, unfixed stromal cells under the tested conditions. Cytoskeletal proteins, MSC markers, cell surface proteins, and receptors in cultured mixed stromal cell populations. ( A ) F-actin and ( B ) vimentin exhibited strong filamentous staining in both MSCs and TLCs. ( C ) α-SMA were evident in stress-fibers rich MSCs while very little in TLCs. ( D ) In contrast, α-actinin were strongly positive in both cell types. ( E–G ) MSC markers (CD73, CD90, and CD105) were expressed in both populations, although CD90 showed slightly weaker staining. ( H ) CD34 was completely absent in all cells. ( I ) Caveolin-1 showed strong positivity in both cell types and showed a strong staining in ER-Golgi region in MSCs (see, inset). ( J ) PDGFR-α was expressed in both cell types, with prominent staining in the protrusions of TLCs. White arrowheads indicate elongating TLC telopods and white arrows indicates MSCs. Scale bars: 20 µm. Finally, we examined the cultured stromal cell populations grown on glass coverslips using the same panel of markers previously found to be positive in UC sections. These markers included those specific to cell surface proteins, receptors, cytoskeletal components, and MSC markers. F-actin was uniformly distributed in all cells, displaying strong filamentous staining characteristic of the typical stromal phenotype (Fig. 7 A). Vimentin expression was observed in both MSCs and elongated TLCs, with prominent filamentous localization (Fig. 7 B). Interestingly, α-SMA positivity was stronger due to the richness of stress-fibers in MSCs, while very little in TLC telopodes. α-actinin labeled both cell types (Fig. 7 C and D ). All three MSC markers tested were positive, although CD90 staining appeared slightly weaker compared to CD73 and CD105 (Fig. 7 E–G). CD34 expression was entirely absent in all cell types (Fig. 7 H). No c-Kit staining was recorded in any cultured cells (not shown). In contrast, caveolin-1 showed strong positivity, particularly in the ER-Golgi regions (Fig. 7 I). PDGFR-α was localized to the cell body and protrusions (Fig. 7 J). Discussion Telocytes are a recently characterized and multifunctional component of the interstitial space. With their distinctive morphology, strategic anatomical localization, and broad range of cellular interactions, they are increasingly recognized as key contributors to tissue homeostasis, regeneration, and intercellular communication, as reviewed by Sanches et al. 7 . The hUC, a unique form of loose connective tissue, is particularly rich in certain types of collagen fibers, interstitial space, ECM components, and stromal cells. Recent studies have emphasized the potential of hUC not only as a source of MSCs but also as a niche housing other specialized interstitial cells with distinct morphological and functional properties 20 . Karahuseyinoglu et al. 21 demonstrated that only a portion of stromal cells is responsive to neuronal induction and suggested that this may be due to the presence of heterogeneous cells populations derived from the UC stroma. Supporting evidence for this came from the study of Sarugaser et al. 22 , who showed that a portion of cells could not be differentiated into neurons. In this study, detailed histological analyses revealed a phenotypically distinct cell population within the hUC stroma that closely resembles telocytes. These TLCs were predominantly localized encircling the clefts of the “superficial” IVS as opposed to the “deep” IVS. We propose these novel terms – “superficial” for cleft-enriched regions and “deep” for cleft-devoid regions – to refine UC stromal nomenclature. The close spatial association of TLCs with known MSCs suggests a potential lineage relationship, possibly indicating a transdifferentiated state of MSCs. However, given the current lack of definitive evidence regarding their origin and developmental trajectory, we refer to these cells as “telocyte-like cells” rather than classifying them as true telocytes. Our initial light microscopic examinations, utilizing both thin and semi-thin stained sections, identified a variety of cells in the hUC stroma. Alongside the typical multiform, round, or ovoid MSCs, we observed slender cells with bipolar projections, consistent with previously described telocytes 23 . Our comparative analysis in rat intestinal submucosa further confirmed morphological similarities to telocytes described in that context 24 , 25 . TEM-based stitched panoramic micrographs revealed thin, elongated TLCs, phenotypically distinct from MSCs with broader cytoplasm. Morphometric analysis showed that the L/W ratio of TLCs was approximately nine times that of wider MSCs, confirming a heterogeneous stromal cell population. The average thickness of their cytoplasmic extensions measured 0.143 µm, consistent with previously reported values (~ 0.2 µm) 26 . TEM examination further revealed lipid inclusions and widened ER cisternae specifically within MSCs, whereas TLCs lacked these structures. Importantly, there was no direct evidence of collagen synthesis by TLCs in this fibrous stromal environment. These findings support our hypothesis that MSCs are the primary contributors to collagen production within the hUC stroma. As proposed by Bei et al. 12 , unlike fibroblasts, which are primarily responsible for ECM production, telocytes may instead facilitate intercellular communication by forming three-dimensional networks around collagen fibers. Furthermore, their close spatial association with collagen found around the clefts has led to the suggestion that telocytes may regulate collagen organization 27 . Specifically, they may modulate the turgor regulation of the umbilical cord. This concept aligns with Nanaev et al.'s earlier suggestion that myofibroblastic MSCs and their less differentiated precursors mark the jelly-filled stromal clefts of Wharton’s jelly. These cells, along with the surrounding meshwork of contractile cells, function as a mechanism to maintain cord turgor and prevent compression 28 . Based on these insights, it is plausible that TLCs contribute to ECM dynamics – particularly in collagen-rich environments such as Wharton’s jelly – by regulating matrix architecture rather than synthesizing its components directly. Frozen sections of UCs showed fine cytoplasmic staining of F-actin filaments across all stromal cell populations, with pronounced labeling using phalloidin dyes. TLCs were predominantly observed in both superficial and deep IVS, but were only rarely detected in the PVS. Vimentin, an intermediate filament protein characteristic of mesoderm–derived cells, was strongly expressed in both TLCs – particularly within their thin, elongated telopods – and in the broader cytoplasmic regions of MSCs. As expected, neither F-actin nor vimentin served as discriminatory markers, as both are also expressed in fibroblasts, endothelial cells, and tissue macrophages. However, consistent with previous studies 29 , our results support the mesenchymal stromal origin of TLCs based on their strong vimentin expression. Interestingly, these two cytoskeletal markers helped phenotypically distinguish between stromal subtypes: TLCs, characterized by flattened or small nuclei, exhibited more intense vimentin staining, whereas MSCs with round/ovoid nuclei displayed weaker staining. This differential expression pattern may aid in distinguishing stromal cell subsets based on nuclear morphology and cytoskeletal profiles. The next marker we tested was α-actinin, an actin-binding protein, which is involved in F-actin-based cytoskeletal organization. Our TLC and MSC findings align with our F-actin results and previous reports about telocytes suggesting that it is not a specific marker for telocytes, as we have noted its expression in both TLCs and MSCs. hUC-MSCs are often classified as myofibroblasts due to their expression of contractile proteins, including α-smooth muscle actin (α-SMA) 21 , 30 , 31 . Based on this, we hypothesized that α-SMA expression could serve as a distinguishing marker between MSCs and TLCs. However, immunostaining of tissue sections revealed that α-SMA did not clearly discriminate between the two cell types, as TLCs also exhibited weak α-SMA positivity. Interestingly, in culture conditions, a substantial portion of the MSC population showed strong α-SMA expression, primarily localized to their broad cytoplasmic regions, where fine arrays of stress fibers were clearly evident. In contrast, telopodes of TLCs lacked such organization. Liu et al. reported that although most telocytes are α-SMA negative, a subset associated with smooth muscle regions has been reported to express this protein 32 . Similarly, in the fetal placenta, cultured telocytes were found to express α-SMA, though their staining pattern was diffuse and lacked the characteristic stress fibers observed in smooth muscle cells 9 . These findings suggest that α-SMA expression alone is insufficient to definitively distinguish MSCs from TLCs and that functional diversity may exist among stromal cell subsets. Further studies are warranted to clarify the role and identity of α-SMA-positive cells within the hUC stroma. Furthermore, as discussed by Can & Karahuseyinoglu 33 , IVS cells exhibit longer and more numerous cytoplasmic processes compared to PVS cells. Notably, hUC myofibroblasts, more prevalent in PVS, likely originate from adjacent vascular smooth muscle cells due to morphological similarities and contractile properties. This suggests that stromal cells in superficial and deeper cord regions may have distinct embryological origins. To explore this further, evaluating cleft formation, vessel development, and their perivascular regions throughout the cord's developmental weeks during pregnancy would be crucial. hUC-MSCs are recognized to express CD73, CD90, and CD105, as documented 34 – 36 . TLCs displayed robust and diffuse positivity for CD73 and CD105, while CD90 staining was sporadic and punctate. There is variability in the literature regarding these marker profiles in telocytes. Studies identifying telocytes in placental tissues and those examining telocyte cultures have reported absence of CD90 expression in cultured telocytes 9 , 37 , while others noted higher CD90 levels compared to fibroblasts 7 . In our cultures, CD90 was weakly expressed across morphologically diverse cell types. Additionally, TLCs exhibited focal CD105 positivity, consistent with studies reporting telocytes to be CD105-positive 38 – 40 . CD73 expression was also detected in our cells, representing a novel finding in telocyte field. CD34 is a commonly used immunohistochemical marker for identifying telocytes and distinguishing them from other interstitial cells 41 , 42 . In contrast, UC stromal cells are typically CD34-negative 33 . In line with our findings and previously published studies 22 , 33 , 43 , 44 , we conclude that CD34 is not a reliable marker for identifying TLCs in UC tissue. This discrepancy may reflect tissue-specific expression patterns, as telocytes from different organs do not always exhibit the same marker profiles 26 . PDGFR-α has been widely used to distinguish telocytes from other interstitial cell types and is associated with tissue homeostasis and injury response 45 . Its expression is believed to play a key role in telocyte-mediated stromal signaling 25 . However, PDGFR-α is not specific to telocytes, as it can also be expressed by fibroblasts 7 . In our study, PDGFR-α expression was detected in TLCs, particularly in those lining the superficial IVS clefts and distributed among collagen bundles. PDGFR-α positivity was also maintained in thin, elongated TLCs under culture conditions. Notably, strong PDGFR-α expression was observed in the cytoplasmic extensions of these cells, supporting their potential role in intercellular communication and stromal network formation. c-Kit–positive telocytes have also been identified in fetal tissues such as the placenta, particularly in trophoblast-rich areas and around blood vessels. Reports on c-Kit expression in hUC-MSCs are inconsistent, with both positive and negative findings reported. In our observations, TLCs exhibited strong c-Kit both in the membrane and cytoplasmic loci in thin, elongated telopodes in both superficial and deep IVS regions. Besides TLCs, few positivities were also noted that corresponded to MSCs in the same tissue compartments. No c-Kit staining was recorded in any cultured cells. These findings do not support previous reports suggesting c-Kit as a distinguishing marker for telocytes, reinforcing its diagnostic value in identifying these cells within the UC stroma. c-Kit–positive telocytes have been identified in fetal tissues such as the placenta, particularly in trophoblast-rich regions and around blood vessels 9 . However, reports on c-Kit expression in hUC-MSCs remain inconsistent, with studies presenting both positive and negative findings 22 , 33 , 46 – 48 . In our observations, TLCs exhibited strong c-Kit expression in both membrane and cytoplasmic regions, particularly along their thin, elongated telopodes, located in both superficial and deep areas of the intervillous space (IVS). In addition to TLCs, a limited number of c-Kit–positive cells, likely corresponding to MSCs, were also observed within the same compartments. Notably, no c-Kit staining was detected in any of the cultured cell populations. These results do not support the previously proposed role of c-Kit as a distinguishing marker for telocytes in culture. Instead, they reinforce its diagnostic value for identifying telocytes specifically within the umbilical cord stroma. Caveolin-1 has been reported to localize prominently in the telopodes of telocytes, supporting its role in intercellular communication 49 , and its expression has also been confirmed in hUC-MSCs 50 . In our immunofluorescence analysis of UC sections, strong caveolin-1 positivity was observed in the thin, elongated TLCs surrounding the superficial IVS compartments, whereas broader MSCs in the deep IVS and PVS showed either membrane-localized or absent expression. In cultured cells, caveolin-1 exhibited perinuclear staining consistent with localization to the ER-Golgi apparatus and in nearly all cells. TEM further confirmed the presence of telocyte-specific caveolae, consistent with previous reports 26 . Notably, numerous extracellular vesicles were detected adjacent to these cell surface pits, supporting the hypothesis that telocytes participate in paracrine and/or juxtacrine signaling by transferring regulatory molecules to neighboring cells 51 , 52 . Collectively, our findings indicate that while caveolin-1 is expressed in both MSCs and TLCs, its subcellular distribution differs markedly, and these distinct localization patterns may aid in the identification of TLCs. We also attempted to isolate marker-specific cell populations using both dual and single surface markers, including CD34, PDGFR-α, and c-Kit—commonly used to distinguish telocytes from other interstitial cells 45 . However, flow cytometry analysis of live, unfixed cells did not yield a sufficient number of positively labeled cells under either dual-marker (CD34 + PDGFR-α and CD34 + c-Kit) or single-marker conditions. As a result, TLCs could not be reliably separated from other stromal cell populations in the UC, and establishing isolated subcultures was not feasible. These findings suggest that TLCs in Wharton’s jelly may express these markers at low levels, may not present them in unfixed conditions, or may require fixation for consistent antigen detection. To validate our immunofluorescence findings, qRT-PCR analysis was performed, revealing that vimentin expression was markedly elevated—ranging from 32- to 5200-fold higher—compared to the other seven target genes. This finding supports the mesodermal origin of all stromal cells in Wharton’s jelly and aligns with the strong vimentin immunoreactivity observed in both tissue sections and cultured cells. Notably, PDGFR-α and c-Kit showed higher transcript levels than CD73, CD90, CD105, and caveolin-1. Although previous studies have reported that telocytes express vimentin, CD34, and c-Kit 53 , our inability to isolate TLC populations precluded direct comparisons between MSCs and TLCs. Thus, our results are best interpreted as representative of the broader gene expression profile of UC stromal cells. Consistent with prior qRT-PCR studies 54 , we also observed CD34 negativity and CD73, CD90, and CD105 positivity in stromal cells. In conclusion, the qRT-PCR data corroborated our immunofluorescence results and confirmed that gene expression levels were consistent with corresponding protein expression patterns. In this study, cell cultures derived from hUCs revealed two morphologically distinct stromal populations. The lack of a standardized method for isolating a homogeneous hUC-MSC population has been previously noted 55 , along with reports of rapid proliferation and morphological variation in early passages 56 . The morphological heterogeneity of UC-derived stromal cells has also been documented by Coskun and Can 17 . They described two subtypes: type-1 cells, with broad cytoplasm and prominent filopodia, showing myofibroblastic characteristics, and type-2 cells, with a thinner, elongated, fibroblast-like appearance. Guenther et al. 56 reported five initial morphologies—triangular, star-shaped, flattened, elongated, and round—which gradually transitioned into a more homogeneous population dominated by flattened and elongated forms over time in culture. Our observations align with and contribute to this literature by confirming the morphological diversity of Wharton’s jelly stromal cells and highlighting two distinct cell types consistently observed both in tissue sections and in vitro cultures. These findings underscore the dynamic and heterogeneous nature of the UC stromal compartment. Furthermore, the average length of TLCs in our study was 604.69 µm, which falls within the reported 200–1000 µm range 57 . SEM, widely used for identifying telocytes due to its ability to visualize their fine projections and interactions 58,59 , revealed TLCs with long, thin processes in close contact with each other and with broader MSCs. These observations suggest that TLCs contribute to intercellular communication, supporting previous reports of telocyte networks playing a role in maintaining three-dimensional tissue architecture 10 , 52 . Conclusively, here we present the first evidence for the existence of TLCs in the hUC, shedding light on the cellular heterogeneity of the stem cell niche in this tissue. Our findings contribute to the understanding of how TLCs may support the structural and functional diversity within the Wharton's jelly microenvironment. Although each of the methods employed in this study contributed to the identification of these cells, none proved sufficient on their own to definitively characterize them. This limitation underscores the need for future advanced molecular studies – focusing on telocyte-specific markers and investigating gene, microRNA, and secretome profiles – to achieve a more precise definition of telocytes, particularly in fibrous stromal tissues that share overlapping markers. Moreover, exploring the physiological roles of telocytes during embryonic development may reveal novel insights with potential implications for regenerative medicine and tissue engineering. Importantly, considering the UC as a postnatal biological source of cells, the presence of TLC populations may open new avenues for innovative cell-based therapeutic strategies. Abbreviations ECM Extracellular matrix ER Endoplasmic reticulum F-actin Filamentous actin GA Glutaraldehyde H&E Hematoxylin–eosin HMC Hoffman Modulation Contrast hUC Human umbilical cord IVS Intervascular stroma LSM Laser scanning microscope MSC Multipotent stromal cell PFA Paraformaldehyde PVS Perivascular stroma qRT-PCR Quantitative reverse transcriptase PCR RT Room temperature SEM Scanning electron microscope SES Subepithelial stroma SR-CM Super resolution confocal microscopy TEM Transmission electron microscope TLC Telocyte-like cell UC Umbilical cord UCE Umbilical cord epithelium Declarations Declaration of Competing Interest The authors declare no conflicting of interest. Funding This study received partial funding from Ankara University Scientific Research Fund TYL-2024-3343 and TSG-2022-2545 to AC. Author Contribution E.E. contributed to the data collection and writing the manuscript. I.A. and B.S. contributed to the data collection. S.E.C. collected UC samples and provided clinical data. M.D. contributed to the execution and interpretation of the qRT-PCR experiments. F.T.C. contributed to the administration. A.C .contributed to the design of the study, writing the manuscript, data collection and final approval of manuscript. All authors read and approved the final manuscript. Acknowledgement The authors express their gratitude to Remzi Ata and Seyfettin Ülger for their technical assistance in TEM sample preparation and image collection. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets used and/or analyzed during the current study are also available from the corresponding author on reasonable request. References Popescu, L. M. & Faussone-Pellegrini, M. S. 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The Treasury of Wharton's Jelly. Stem Cell Rev Rep 18, 1627–1638, (2015). 10.1007/s12015-021-10217-8 (2022). 57 Kang, Y. et al. Skin telocytes versus fibroblasts: two distinct dermal cell populations. J Cell Mol Med 19, 2530–2539, doi:10.1111/jcmm.12671 (2015). 58 Li, H., Lu, S., Liu, H., Ge, J. & Zhang, H. Scanning electron microscope evidence of telocytes in vasculature. J Cell Mol Med 18, 1486–1489, doi:10.1111/jcmm.12333 (2014). 59 Hussein, M. M. & Mokhtar, D. M. The roles of telocytes in lung development and angiogenesis: An immunohistochemical, ultrastructural, scanning electron microscopy and morphometrical study. Dev Biol 443, 137–152, doi:10.1016/j.ydbio.2018.09.010 (2018). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7203915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503752098,"identity":"56d3ea5c-4fe5-4207-98b7-87e7ad363743","order_by":0,"name":"Ezel Erkan","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Ezel","middleName":"","lastName":"Erkan","suffix":""},{"id":503752099,"identity":"422e7ae3-1ca6-47ed-b421-b6a645dd3b83","order_by":1,"name":"Ibrahim Alptekin","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Alptekin","suffix":""},{"id":503752100,"identity":"822b16b7-f311-4020-a6d0-92f38d6cfd6f","order_by":2,"name":"Bilge Serdaroglu","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Bilge","middleName":"","lastName":"Serdaroglu","suffix":""},{"id":503752101,"identity":"a08e2467-ab93-4c1b-8cec-e8ddcf47b77f","order_by":3,"name":"Serife Esra Cetinkaya","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Serife","middleName":"Esra","lastName":"Cetinkaya","suffix":""},{"id":503752102,"identity":"890886c9-6ff0-4b32-9b38-fa4da67062f6","order_by":4,"name":"Mohammadreza Dastouri","email":"","orcid":"","institution":"Ankara Medipol University","correspondingAuthor":false,"prefix":"","firstName":"Mohammadreza","middleName":"","lastName":"Dastouri","suffix":""},{"id":503752103,"identity":"489843dc-45fa-4776-b9b7-358d5a309802","order_by":5,"name":"Ferda Topal Celikkan","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Ferda","middleName":"Topal","lastName":"Celikkan","suffix":""},{"id":503752104,"identity":"1bf01b50-242a-410f-a439-cbcbb2cef8e5","order_by":6,"name":"Alp Can","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACdjBpw8DAA+EzNhDUwgwm05C1MBOl5TAJWvibeQ9++LnjvLw5z+FnH34w2MhuOMB/7AM+LRKH+ZIle8/cNtzZ22Y8s4chzXjDAWbmGXitOcxjxsDbdptxw3kGY6DjDieCtODVIQ/Uwvi37Zz9hvPsnxn/MPwnrMUAqIWZt+1A4oazPcbMPAwHCGsxBPpFWrYtOXnDmTPFzDIGycYzDzMb49Uid7z34Me3bXa2G86kb2Z8U2En23e88TFeLfD4gLqTgYFgTKJpGQWjYBSMglGABQAAI4xGKEZvF1EAAAAASUVORK5CYII=","orcid":"","institution":"Ankara University","correspondingAuthor":true,"prefix":"","firstName":"Alp","middleName":"","lastName":"Can","suffix":""}],"badges":[],"createdAt":"2025-07-24 09:23:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7203915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7203915/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-26282-9","type":"published","date":"2025-11-07T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89677822,"identity":"228ce1dd-d985-4e60-ad4d-a3f0568ef24b","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2085533,"visible":true,"origin":"","legend":"\u003cp\u003eHE- and toluidine blue-stained semithin sections of \u0026nbsp;\u0026nbsp;human umbilical cords, enclosed by the umbilical cord epithelium (UCE). (\u003cstrong\u003eA\u003c/strong\u003e) \u0026nbsp;\u0026nbsp;Three main stromal regions with indistinct borders, identified from the \u0026nbsp;\u0026nbsp;outermost to the innermost, are: subepithelial stroma (SES), intervascular \u0026nbsp;\u0026nbsp;stroma (IVS), and perivascular stroma (PVS), the second classically known as \u0026nbsp;\u0026nbsp;Wharton’s jelly. (\u003cstrong\u003eB\u003c/strong\u003e) The IVS is further subdivided into superficial \u0026nbsp;\u0026nbsp;IVS (underlying to SES) and (\u003cstrong\u003eC\u003c/strong\u003e) deep IVS (overlying to PVS). (\u003cstrong\u003eD, E\u003c/strong\u003e) \u0026nbsp;\u0026nbsp;Telocyte-like cells (TLCs, \u003cstrong\u003earrowheads\u003c/strong\u003e) are clearly identified in both \u0026nbsp;\u0026nbsp;superficial and deep IVS regions, particularly encircling clefts (\u003cstrong\u003easterisks\u003c/strong\u003e). \u0026nbsp;\u0026nbsp;(\u003cstrong\u003eF–H\u003c/strong\u003e) Higher magnification images obtained from semithin sections \u0026nbsp;\u0026nbsp;reveal that TLCs possess extremely long and thin cytoplasmic extensions \u0026nbsp;\u0026nbsp;called telopodes, which show irregularities along their length. These are \u0026nbsp;\u0026nbsp;embedded in a highly edematous and loose ECM, interspersed among MSCs (\u003cstrong\u003earrows\u003c/strong\u003e). \u0026nbsp;\u0026nbsp;Scale bars: 1000 µm (A), 100 µm (B), 200 µm (C), 50 µm (D, E), 10 µm (F–H).\u003c/p\u003e","description":"","filename":"ErkanetalFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/bdfce28689ab0762b9984407.jpg"},{"id":89677821,"identity":"fe09d96a-b3e7-4018-b3c0-de1a9813deb6","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1494240,"visible":true,"origin":"","legend":"\u003cp\u003eTEM observations of TLCs. (\u003cstrong\u003eA–C\u003c/strong\u003e) In low magnifications, they are distinguished by their extremely thin (as also shown in \u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) cytoplasmic extensions embedded in a relatively scarse collagen fibers as opposed to abundant collagen bundles around MSCs (shown in \u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e). TLCs are also characterized by poor protein synthesizing organalles. However, they display several extracellular vesicles with close proximity to plasma membrane shown by \u003cstrong\u003ewhite arrowheads\u003c/strong\u003e in \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e and the cell surface displayed interruptions with several caveolae (\u003cstrong\u003eblack arrows\u003c/strong\u003e in \u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eG–H\u003c/strong\u003e) MSCs are evident by their wider and shorter cytoplasms with rich in protein sythnesizing organelles, elaborate ER cisternae (\u003cstrong\u003eblack arrowheads\u003c/strong\u003e) and lipid-laden inclusions (\u003cstrong\u003ewhite\u003c/strong\u003e \u003cstrong\u003easterisks\u003c/strong\u003e). (\u003cstrong\u003eI\u003c/strong\u003e) The length-to-width ratio (L/W) was calculated as 32.38 for TLCs and 3.60 for MSCs. \u003cstrong\u003eBlack asterisks\u003c/strong\u003e: Clefts; \u003cstrong\u003eN\u003c/strong\u003e: Nucleus. Scale bars: 3 µm (A–B), 1 µm (C–H).\u003c/p\u003e","description":"","filename":"ErkanetalFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/8ffa2869cf262a911f0e24c7.jpg"},{"id":89677823,"identity":"76b6be3e-7299-4e91-8185-9834800e8995","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":979831,"visible":true,"origin":"","legend":"\u003cp\u003eCommon cytoskeletal and MSC markers in hUC sections. All images were deconvoluted and reconstructed from 16 consecutive optical sections along the z-axis to generate 3D image stacks. (\u003cstrong\u003eA\u003c/strong\u003e) F-actin is widely expressed in all cell types across tissue compartments, from the UCE to vascular smooth muscle cells (\u003cstrong\u003easterisk\u003c/strong\u003e), and is therefore not specific to TLCs or MSCs. (\u003cstrong\u003eB\u003c/strong\u003e) However, F-actin staining reveals cytoplasmic morphology, allowing identification of TLCs (\u003cstrong\u003ewhite arrowheads\u003c/strong\u003e) by their long, slender shape and preferential localization to the superficial intervascular stroma (IVS). (\u003cstrong\u003eC, D\u003c/strong\u003e) Vimentin highlights long, slender TLCs (\u003cstrong\u003ewhite arrowheads\u003c/strong\u003e), with stronger signal than that observed in round-to-ovoid MSCs with broader extensions (\u003cstrong\u003ewhite arrows\u003c/strong\u003e). (\u003cstrong\u003eE\u003c/strong\u003e) α-SMA is expressed in both TLCs (\u003cstrong\u003ewhite arrowheads\u003c/strong\u003e) and MSCs. (\u003cstrong\u003eF\u003c/strong\u003e) α-actinin is positive in all stromal cells; however, strong and punctate staining is specifically observed in TLCs (\u003cstrong\u003ewhite arrowheads\u003c/strong\u003e). (\u003cstrong\u003eG–I\u003c/strong\u003e) TLCs exhibit strong and diffuse positivity for CD73 and CD105, while CD90 staining is sparse and punctate. Scale bars: 50 µm (A, B, D); 20 µm (C, E–I).\u003c/p\u003e","description":"","filename":"ErkanetalFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/0b830bf4ae97170f6c2684f4.jpg"},{"id":89679096,"identity":"1e3d2051-567e-43d5-91ff-d73a34429e79","added_by":"auto","created_at":"2025-08-22 14:15:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":760779,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers specific to selected cell surface proteins and receptors in hUC sections. All images were deconvoluted and reconstructed from 16 consecutive optical sections along the z-axis to generate 3D image stacks. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) CD34 showed minimal to no staining across all stromal regions. (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) PDGFR-α exhibited strong membranous and weak cytoplasmic positivity in TLCs, particularly those surrounding clefts (\u003cstrong\u003easterisks\u003c/strong\u003e in corresponding DIC images), which were enclosed by bundles of collagen fibers (\u003cstrong\u003eblack arrows\u003c/strong\u003e) in the superficial IVS. (\u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e) c-Kit expression was also strong in TLCs, especially those around clefts (\u003cstrong\u003easterisks\u003c/strong\u003e in corresponding DIC images) in the superficial IVS. (\u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e) Caveolin-1 was localized in TLCs within the superficial IVS, appearing as cytoplasmic punctate staining. ★: UCE. Scale bars: 50 µm (A, B, E, F); 20 µm (C, D, G, H).\u003c/p\u003e","description":"","filename":"ErkanetalFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/cf1f57b3df4e603b0302a3dc.jpg"},{"id":89677826,"identity":"06eb3df2-740c-417a-8456-977e43399969","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":286279,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Agarose gel electrophoresis of the qRT-PCR products revealed detectable bands for all eight target genes. (\u003cstrong\u003eB\u003c/strong\u003e) Expression levels of eight target genes normalized to GAPDH are shown as mean ΔCt ± SD from three biological replicates (n=3). Y-axis is presented on a logarithmic scale for better visualization of gene expression differences.\u003c/p\u003e","description":"","filename":"ErkanetalFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/881e596439badb410152a02a.jpg"},{"id":89678381,"identity":"12d1ea80-e0ab-493c-9030-f685c46e2b72","added_by":"auto","created_at":"2025-08-22 14:07:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1000420,"visible":true,"origin":"","legend":"\u003cp\u003eExplant cultures of UC stroma at P\u003csub\u003e2\u003c/sub\u003e imaged by HMC and SEM. (\u003cstrong\u003eA–F\u003c/strong\u003e) Following initial cell attachment (T\u003csub\u003e0\u003c/sub\u003e), both broad-bodied cells characteristic of MSCs (\u003cstrong\u003ewhite arrows\u003c/strong\u003e) and extremely thin, elongated TLCs (\u003cstrong\u003eblack arrowheads\u003c/strong\u003e) appeared. The average length of these fusiform TLCs was 604.69 µm, and they remained observable at T\u003csub\u003e36\u003c/sub\u003e and T\u003csub\u003e48\u003c/sub\u003e. (\u003cstrong\u003eG–J\u003c/strong\u003e) SEM analysis revealed that TLC extensions had a mean width of 1.49 µm (\u003cstrong\u003eblack arrowheads\u003c/strong\u003e), with ovoid nuclei (\u003cstrong\u003eblack arrows\u003c/strong\u003e). Occasional contacts between TLCs were noted (see \u003cstrong\u003eH\u003c/strong\u003e). TLCs appeared attached to the substrate at both ends with relatively small cytoplasmic lamellipodia. In contrast, MSCs exhibited distinct morphology, characterized by large, flattened lamellipodia, extremely broad and flat cytoplasmic domains, and nanotube-like projections (see \u003cstrong\u003eG\u003c/strong\u003e) extending between neighboring cells. Scale bars: 20 µm (A, G, I); 50 µm (B–F); 10 µm (H, J).\u003c/p\u003e","description":"","filename":"ErkanetalFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/22e55b4b7568748d1b11f673.jpg"},{"id":89679098,"identity":"580ddd45-cadb-4b4f-b1a0-850243e86155","added_by":"auto","created_at":"2025-08-22 14:15:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":616347,"visible":true,"origin":"","legend":"\u003cp\u003eCytoskeletal proteins, MSC markers, cell surface proteins, and receptors in cultured mixed stromal cell populations. (\u003cstrong\u003eA\u003c/strong\u003e) F-actin and (\u003cstrong\u003eB\u003c/strong\u003e) vimentin exhibited strong filamentous staining in both MSCs and TLCs. (\u003cstrong\u003eC\u003c/strong\u003e) α-SMA were evident in stress-fibers rich MSCs while very little in TLCs. (\u003cstrong\u003eD\u003c/strong\u003e) In contrast, α-actinin were strongly positive in both cell types. (\u003cstrong\u003eE–G\u003c/strong\u003e) MSC markers (CD73, CD90, and CD105) were expressed in both populations, although CD90 showed slightly weaker staining. (\u003cstrong\u003eH\u003c/strong\u003e) CD34 was completely absent in all cells. (\u003cstrong\u003eI\u003c/strong\u003e) Caveolin-1 showed strong positivity in both cell types and showed a strong staining in ER-Golgi region in MSCs (see, inset). (\u003cstrong\u003eJ\u003c/strong\u003e) PDGFR-α was expressed in both cell types, with prominent staining in the protrusions of TLCs. \u003cstrong\u003eWhite arrowheads\u003c/strong\u003e indicate elongating TLC telopods and \u003cstrong\u003ewhite arrows\u003c/strong\u003e indicates MSCs. Scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"ErkanetalFig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/21c4f9c2020544f1e38b2b22.jpg"},{"id":95564108,"identity":"7f5aed75-9717-48ba-b41a-65298643bd5a","added_by":"auto","created_at":"2025-11-10 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8292752,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/2f882319-8712-4d8d-a249-17a4e2694c8b.pdf"},{"id":89678375,"identity":"58ee5c3d-20eb-415d-bdc0-18c3ac015129","added_by":"auto","created_at":"2025-08-22 14:07:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17027,"visible":true,"origin":"","legend":"","description":"","filename":"ErkanetalSuppTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/4d7f6ce1f1c3cedc84623ba8.docx"},{"id":89677820,"identity":"902c815e-f5c9-495d-a737-96be516a974b","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17035,"visible":true,"origin":"","legend":"","description":"","filename":"ErkanetalSuppTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/ff6a899b6cae766ca49c97ee.docx"},{"id":89679095,"identity":"256f0360-5fd9-41a3-a708-dd5ceed3f50b","added_by":"auto","created_at":"2025-08-22 14:15:54","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15624,"visible":true,"origin":"","legend":"","description":"","filename":"ErkanetalSuppTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/97bbf6050888e4165bf7ff48.docx"},{"id":89677832,"identity":"d00398b8-aa1f-43be-a67c-23518c907c3b","added_by":"auto","created_at":"2025-08-22 13:59:54","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":604201,"visible":true,"origin":"","legend":"","description":"","filename":"ErkanetalSuppFig1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/8ffbc51aef1732b4bc57ed83.docx"},{"id":89678385,"identity":"902f04df-b350-4086-964f-641bd9855346","added_by":"auto","created_at":"2025-08-22 14:07:54","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":371294,"visible":true,"origin":"","legend":"","description":"","filename":"ErkanetalSuppFig2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203915/v1/dfde66b4fb4fb72008664c63.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and Localization of Telocyte-Like Cells in Human Umbilical Cord Stroma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTelocytes are a recently identified type of stromal cell, first described by Popescu and Faussone-Pellegrini in 2010 as a novel interstitial cell population distinct from fibroblasts, interstitial Cajal cells, and mesenchymal stem cells \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Initially referred to as Interstitial Cajal-like Cells, they were later renamed \u0026ldquo;telocytes\u0026rdquo; to reflect their most distinguishing feature: extremely long, slender prolongations called telopodes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Structurally, telocytes are defined by their small, often spindle-shaped or triangular cell bodies, from which emerge one to five telopodes. These telopodes can extend up to several hundred micrometers and exhibit a moniliform appearance due to alternating thin segments (podomers) and dilated segments (podoms), which contain few mitochondria, endoplasmic reticulum, and caveolae \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Telocytes are typically identified as their unique ultrastructure, and immunophenotype, where they may express markers such as CD34, c-Kit (CD117), PDGFRα/β, and vimentin, though these markers are not entirely specific \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTelocytes have a widespread tissue distribution and have been identified in numerous organs and systems. In the cardiovascular system, they are abundant in the myocardium, where they form complex networks around cardiomyocytes, blood vessels, and stem cell niches \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In the lungs, they are present in both bronchial and alveolar walls, suggesting a role in tissue organization and regeneration \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Similarly, telocytes are found in the gastrointestinal tract, liver, pancreas, skin, skeletal muscle, uterus, fallopian tubes as reviewed by Sanches et al. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and placenta \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, underscoring their diverse functional potential across organ systems.\u003c/p\u003e\u003cp\u003eFunctionally, telocytes are believed to play several critical roles in tissue homeostasis and repair. One of their primary proposed functions is intercellular signaling. They establish direct physical contacts with other cells, including stem cells, immune cells, nerve fibers, and endothelial cells, through homo- and heterocellular junctions such as gap junctions and nanocontacts \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Their telopodes form extensive 3D networks that are thought to coordinate local microenvironmental cues, facilitating mechanical support and chemical signaling. Another proposed function of telocytes is the regulation of stem cell niches. In various tissues, telocytes are located near resident stem/progenitor cells, with whom they appear to interact via paracrine signaling or direct contact, potentially guiding their proliferation and differentiation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, telocytes may be involved in immune surveillance, extracellular matrix (ECM) remodeling, and electrophysiological modulation, particularly in excitable tissues such as the heart and gastrointestinal tract \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe human umbilical cord (hUC) is a fetus-derived tissue composed of two arteries and one vein embedded in a gelatinous ECM known as Wharton\u0026rsquo;s jelly. This matrix constitutes the stromal compartment, which is considered to contain a relatively homogeneous population of stromal cells with significant regenerative potential. Those cells, also called \u0026ldquo;multipotent/mesenchymal stromal cells (MSCs)\u0026rdquo; are the most extensively studied cell type in recent years due to their multilineage differentiation capacity, immunomodulatory properties, and paracrine signaling functions \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. hUC-MSCs are considered advantageous compared to adult tissue-derived MSCs, as they are more primitive, exhibit higher proliferative capacity, and are collected via a non-invasive procedure as reviewed \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The ECM of Wharton\u0026rsquo;s jelly, rich in hyaluronic acid and collagen, provides a supportive scaffold and biochemical cues for cellular behavior and survival \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Due to these properties, the hUC stroma is increasingly recognized as a valuable source for regenerative medicine and cell-based therapies \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAfter extensive histological examinations including transmission electron microscopy (TEM) and three-dimensional high-resolution confocal image stacks, along with systematic analysis of hundreds of tissue sections, we characterized a group of cells resembling telocytes found in other tissues in 16 normal hUC samples and identified their localization within the stromal compartment. These cells appear either as a distinct stromal population adjacent to known MSCs or possibly as derivatives of MSCs. Due to the uncertainty regarding their origin and developmental lineage, we have opted to refer to them as \u0026ldquo;telocyte-like cells (TLCs)\u0026rdquo; rather than definitively classify them as \u0026ldquo;telocytes\u0026rdquo;. However, it remains unclear whether these cells differentiate from resident MSCs during pregnancy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eCollection of Umbilical Cords\u003c/em\u003e\u003c/p\u003e\u003cp\u003e The study was approved by the Ankara University Ethical Review Board for Human Research (approval #I09-598-23). Written informed consent was obtained from full-term pregnant women (n\u0026thinsp;=\u0026thinsp;16) prior to the procedure. UCs (n\u0026thinsp;=\u0026thinsp;16) were collected during Caesarean deliveries from healthy singleton pregnancies (38\u0026thinsp;\u0026plusmn;\u0026thinsp;2 weeks of gestation) in women aged 20\u0026ndash;35 years from the Department of Obstetrics and Gynecology, Ankara University School of Medicine. Samples were collected approximately 20 cm from the umbilicus, specifically from the proximal one-third segment of the cord, as previously described \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The 12-cm cord segments were delivered to the laboratory within 4 hours post-delivery. Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Upon arrival, the cords were dissected in Leibovitz\u0026rsquo;s L-15 medium supplemented with 1% penicillin-streptomycin, 1% amphotericin B, and 1% L-glutamine. Transverse sections of the cords were prepared for various analyses as follows: (i) a 2-cm fresh tissue segment for stereomicroscopic examination; (ii) 1-cm segments fixed in paraformaldehyde (PFA) for frozen sectioning, immunostaining, and super-resolution confocal microscopy (SR-CM); (iii) 1-cm segments fixed in glutaraldehyde (GA) for transmission electron microscopy (TEM); (iv) 1-cm segments stored at \u0026minus;\u0026thinsp;80\u0026deg;C for qRT-PCR analysis; (v) a 5-cm segment used for explant cultures intended for flow cytometry and scanning electron microscopy (SEM). All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStereo and Light Microscopy\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTissue blocks were initially rinsed with sterile phosphate-buffered saline (PBS) and examined under a stereomicroscope equipped with episcopic illumination (Nikon, Japan) to assess macroscopic features, including surface topography and the integrity of the umbilical vessels. One-centimeter-thick tissue samples from each umbilical cord were fixed in 3.5% (w/v) paraformaldehyde (PFA) for 48 hours at 4\u0026deg;C. Following fixation, the samples were washed twice with PBS and incubated for three days in a 1.2 M sucrose solution containing 0.01% PFA. The tissue blocks were then embedded in Cryomatrix (Fisher Scientific, USA), frozen at \u0026minus;\u0026thinsp;25\u0026deg;C, and sectioned into 10\u0026ndash;14 \u0026micro;m-thick slices using a cryostat (Shandon, UK). Cryosections were stained with standard hematoxylin\u0026ndash;eosin (H\u0026amp;E). Imaging was performed using a bright-field AxioImager microscope (Zeiss, Germany) equipped with \u0026times;2.5, \u0026times;20, and \u0026times;40 objectives. Both individual and tiled image stacks were captured using an Axiocam 503 color camera and Zen Blue software (v2.3 Pro).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTransmission Electron Microscopy (TEM)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFor ultrastructural analysis, three small tissue blocks (each a few mm\u0026sup3;) representing distinct stromal regions \u0026ndash; subepithelial stroma (SES), intervascular stroma (IVS), and perivascular stroma (PVS) \u0026ndash; were collected from each umbilical cord (UC) sample. A rat intestinal tissue sample was also included as a positive control for telocytes. Samples were processed as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. High-resolution images were obtained using a transmission electron microscope (Hitachi HT7800, Japan) equipped with a 19 MB digital camera. Image analysis and reconstruction were performed using Adobe Photoshop (version 2022). Additionally, semi-thin sections were examined using a super-resolution confocal microscope (Zeiss LSM880, Germany) with a photomultiplier tube detector at 40\u0026times; magnification and 1.5\u0026ndash;2.6\u0026times; digital zoom.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunostaining and Super‑Resolution Confocal Microscopy (SR-CM)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescent double-labeling was performed on PFA-fixed frozen sections and cultured cells using a panel of primary antibodies specific to telocytes and UC stromal cells, along with appropriate secondary antibodies (\u003cb\u003eSupp. Table\u0026nbsp;1\u003c/b\u003e). Prior to antibody application, sections and cells were incubated in a blocking solution. Nuclear labeling was achieved using Hoechst 33258 (1 mg/mL) diluted in a 1:1 PBS/glycerol-based mounting medium. Antibody specificity was verified through both positive and negative controls, including omission of the primary antibody and staining of known positive tissues or structures. After PBS washing and blocking (1-hour incubation at +\u0026thinsp;4\u0026deg;C), sections were incubated with primary antibodies at 37\u0026deg;C for 3 hours. Following three PBS washes, secondary antibodies were applied for 2 hours at 37\u0026deg;C. The same protocol was followed for double immunofluorescence, using sequential incubation with two sets of primary and secondary antibodies, each followed by PBS washes. For F-actin visualization, 633-Phalloidin or FITC-Phalloidin was applied to cryosections for 40 minutes at room temperature in the dark. The phalloidin solution (1 mg/mL) was prepared by diluting a methanol-based, DMSO-containing stock 1:10 in PBS and incubating for 1 hour in the dark.\u003c/p\u003e\u003cp\u003eFor immunostaining of cultured cells grown on coverslips, cells were fixed in 3.5% PFA for 20\u0026ndash;25 minutes, washed with PBS-azide, and stored at +\u0026thinsp;4\u0026deg;C in a humidified chamber. After blocking, cells were incubated with primary antibody for 90 minutes at 37\u0026deg;C, followed by PBS washing and secondary antibody incubation under the same conditions. For double labeling, a second round of primary and secondary antibody incubation was performed sequentially. F-actin labeling was carried out using either FITC- or 633-Phalloidin, followed by gentle PBS washing. Coverslips were mounted in a 1:1 PBS solution containing Hoechst 33258 (1 mg/mL), sealed with nail polish, and stored at +\u0026thinsp;4\u0026deg;C.\u003c/p\u003e\u003cp\u003eFluorescently labeled sections and cells were imaged using a Zeiss LSM-880 confocal microscope (Zeiss, Germany) equipped with an AiryScan\u0026reg; detector to obtain super-resolution images (70\u0026ndash;100 nm) through pixel reassignment and deconvolution. Laser lines (405, 488, 543 and 633 nm) were selected based on the fluorophores used; phalloidin images acquired with the 633 nm laser were pseudo-colored red. Laser power and scanning settings were standardized across all experiments using reference histograms. Imaging was conducted with 20\u0026times; (dry) and 40\u0026times; (water immersion) objectives, using a zoom factor of 1.8\u0026ndash;2.2. Z-stack volumes were automatically calculated and consistently aligned. Image data were analyzed using the 3D module of Zen Desk software (version 2.3). For each section, three to six representative regions were selected, and their coordinates were recorded. From each region, 10\u0026ndash;60 optical slices were acquired to reconstruct 3D dual-channel composite images with a thickness of 12\u0026ndash;15 \u0026micro;m.\u003c/p\u003e\u003cp\u003e\u003cem\u003eQuantitative Reverse Transcriptase PCR Analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFollowing removal of blood vessels, two stromal-rich segments (~\u0026thinsp;1 cm thick) were excised from each UC (n\u0026thinsp;=\u0026thinsp;15), snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing. Samples were divided into three experimental groups (n\u0026thinsp;=\u0026thinsp;5 per group) and pooled within each group to generate biological replicates for transcriptomic analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). For complementary DNA (cDNA) synthesis, 1 \u0026micro;g of total RNA per sample was reverse transcribed using a commercial reverse transcription kit (Medchem, USA). Quantitative real-time PCR was performed using EvaGreen Master Mix (Bio-Rad, UAE) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Primer sequences for target and reference gene (GAPDH) are listed (\u003cb\u003eSupp. Table\u0026nbsp;2\u003c/b\u003e). Amplification specificity was confirmed via melt curve analysis, and amplicon size was verified by electrophoresis on 2% agarose gels stained with RedSafe\u0026reg; (Intron Biotechnology, USA). Electrophoresis was performed at 150 V for 30\u0026ndash;45 min, and bands were visualized using the ChemiDoc MP Imaging System (Bio-Rad, UAE). Expression profiles of eight target genes\u0026mdash;vimentin, CD34, c-Kit, PDGFR-α, caveolin-1, CD73, CD90, and CD105\u0026mdash;were evaluated. GAPDH served as the endogenous reference gene. Each target was assessed in technical triplicates across three biological replicates. Threshold cycle (Ct) values were recorded, and relative gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e method. Results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, based on descriptive statistical analysis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell Culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe primary aim of the hUC culture was to identify and isolate TLCs from the stromal cell populations using telocyte-specific markers via flow cytometry (see below) and fluorescence-activated cell sorting. Initial live-cell observations and morphometric measurements were carried out by Hoffman Modulation Contrast (HMC) imaging using 10\u0026times; objective (Olympus, Japan). Additionally, immunofluorescence staining was performed to evaluate the expression of both telocyte- and hUC-MSC-specific markers in fixed cultures. Cell morphometry was applied to captured images to characterize cells at different time points. For explant cultures, 5 cm segments of UC were dissected in sterile Leibovitz\u0026rsquo;s L-15 medium, processed, and cryopreserved as previously described \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eScanning Electron Microscopy (SEM)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCultured cells grown on round glass coverslips (~\u0026thinsp;90% confluency) were rinsed twice with Dulbecco\u0026rsquo;s PBS, then fixed with 200 \u0026micro;L of 2.5% glutaraldehyde (GA) for 30 minutes at room temperature. This was followed by two 15-minute washes with 0.1 M Sorenson\u0026rsquo;s buffer. Post-fixation was carried out in a 1:1 mixture of 1% osmium tetroxide (OsO₄) and 0.2 M Sorenson\u0026rsquo;s buffer for 30 minutes, followed by another two buffer washes. Dehydration was performed through a graded ethanol series (30%, 50%, 75%, 95%, and 100%), with each step lasting 15 minutes. Four coverslips were treated with a 1:1 ethanol\u0026ndash;hexamethyldisilazane (HMDS) solution for 5 minutes and then transferred to pure HMDS until complete evaporation. Samples were mounted on aluminum stubs using carbon adhesive tabs, sputter-coated with a 15 nm layer of gold\u0026ndash;palladium, and examined using a LEO 438 VP scanning electron microscope (UK) operated at 15 kEV in high-vacuum mode with a secondary electron detector. Cell morphology and dimensional measurements were assessed from the acquired SEM images.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFlow Cytometry\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo quantify TLC populations in hUC-MSC cultures based on specific molecular markers, flow cytometry was performed using a BD FACS Canto II system (USA) on three sets of triplicate passage 2 (P\u003csub\u003e2\u003c/sub\u003e) cell cultures. In the first and second experiment, primary antibodies were applied in pairs. P\u003csub\u003e2\u003c/sub\u003e cells detached with Tryple (Gibco, USA) and resuspended in serum-free medium. The cell suspension was divided into three tubes: two for dual antibody staining [CD34 (1:500)\u0026thinsp;+\u0026thinsp;PDGFR-α (1:200), CD34 (1:500)\u0026thinsp;+\u0026thinsp;c-Kit (1:500)], and one for isotype control. After incubation with primary antibodies at room temperature for 30 minutes, secondary antibodies were added [FITC-conjugated goat anti-mouse or Cy3-conjugated goat anti-rabbit antibodies (\u003cb\u003eSupp. Table\u0026nbsp;1\u003c/b\u003e)] and incubated in the dark. In the second experiment, the primary antibody incubation time was extended to 50 minutes, and the secondary antibodies were diluted to 1:2000. An unstained cell group was also included. Cells were divided into four tubes (two for staining, one isotype, one unstained), and processed similarly. After incubation with primary and secondary antibodies; samples were prepared and transported under cold, dark conditions for analysis. In the third experiment, primary antibodies were applied individually to evaluate their expression separately. Cells were divided into six tubes, with three assigned for single antibody staining (CD34, PDGFR-α, c-Kit) and the remaining for isotypes and unstained controls. Secondary antibodies were matched appropriately to their corresponding primary antibodies. In all three experiments, following incubation and washing steps, the samples were transferred under cold, dark conditions for analysis using FlowJo software. Marker positivity rates were determined relative to isotype controls and baseline autofluorescence.\u003c/p\u003e\u003cp\u003eTo validate and visually compare protein distributions observed by flow cytometry, smears were prepared from the analyzed cell suspensions and examined using SR-CM. For this, 10 \u0026micro;L of labeled or unlabeled cell suspensions were fixed in 90 \u0026micro;L of 3.5% PFA for 5\u0026ndash;10 minutes, then centrifuged at 200\u003cem\u003eg\u003c/em\u003e for 10 minutes. The resulting pellets were resuspended in a 1:1 PBS solution containing Hoechst 33258 (1 mg/mL) for nuclear labeling. A 20 \u0026micro;L aliquot of the fixed suspension was placed on glass slides, covered with coverslips, and imaged using SR-CM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe began by examining UC blocks under a stereomicroscope to confirm their normal macroscopic features, including surface topography and the integrity of the umbilical vessels. Subsequently, HE-stained sections from 16 healthy deliveries were analyzed to identify stromal compartments in each section (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C). Slender cells with long, thin cytoplasmic processes were particularly prominent in the superficial (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and deep intervascular stroma (IVS) regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), frequently observed encircling clefts \u0026ndash; homogeneous ground substance-filled, mesh-like systems \u0026ndash; in the superficial IVS. Semithin sections provided definitive confirmation of these cells and allowed for more precise morphological characterization. These cells exhibited multimicrometer-long, thin cytoplasmic extensions and a centrally located, ovoid, euchromatic nucleus. The extensions were often in close association with collagen bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cb\u003eG\u003c/b\u003e). These cells were embedded within a\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHE- and toluidine blue-stained semithin sections of human umbilical cords, enclosed by the umbilical cord epithelium (UCE). (\u003cb\u003eA\u003c/b\u003e) Three main stromal regions with indistinct borders, identified from the outermost to the innermost, are: subepithelial stroma (SES), intervascular stroma (IVS), and perivascular stroma (PVS), the second classically known as Wharton\u0026rsquo;s jelly. (\u003cb\u003eB\u003c/b\u003e) The IVS is further subdivided into superficial IVS (underlying to SES) and (\u003cb\u003eC\u003c/b\u003e) deep IVS (overlying to PVS). (\u003cb\u003eD, E\u003c/b\u003e) Telocyte-like cells (TLCs, \u003cb\u003earrowheads\u003c/b\u003e) are clearly identified in both superficial and deep IVS regions, particularly encircling clefts (\u003cb\u003easterisks\u003c/b\u003e). (\u003cb\u003eF\u0026ndash;H\u003c/b\u003e) Higher magnification images obtained from semithin sections reveal that TLCs possess extremely long and thin cytoplasmic extensions called telopodes, which show irregularities along their length. These are embedded in a highly edematous and loose ECM, interspersed among MSCs (\u003cb\u003earrows\u003c/b\u003e). Scale bars: 1000 \u0026micro;m (A), 100 \u0026micro;m (B), 200 \u0026micro;m (C), 50 \u0026micro;m (D, E), 10 \u0026micro;m (F\u0026ndash;H).\u003c/p\u003e\u003cp\u003ehighly edematous and loose ECM and were frequently found in proximity to wide-cuboid stromal cells, commonly identified as MSCs of Wharton\u0026rsquo;s jelly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eThen, we compared these cells with rat intestinal telocytes and found striking phenotypic similarities (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Morphometric analysis revealed that the mean length of the long-slender cells in hUCs was 92.46 \u0026micro;m (range: 48.93\u0026ndash;120.08 \u0026micro;m; SD: 27.54), compared to 124 \u0026micro;m (range: 110.72\u0026ndash;140.25 \u0026micro;m; SD: 14.85) in rat intestinal telocytes. Based on these comparisons and morphological features consistent with telocytes described in various tissues, we conclude that these long-slender cells are telocytes or telocyte-like cells (TLCs) in the hUC.\u003c/p\u003e\u003cp\u003eIn low magnification TEM observations, TLCs were distinguished by their extremely thin cytoplasmic extensions mostly neighboring to the clefts and embedded in a collagen-rich ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). TLCs exhibited small, euchromatic nuclei generally located centrally within their elongated cytoplasm, which contained poor protein-synthesizing organelles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C). At higher magnifications, the plasma membrane was closely associated with extracellular vesicles (mean: 323.4 nm; range: 261\u0026ndash;357 nm; SD: 40.16) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cb\u003eD\u003c/b\u003e), and the cell surface displayed interruptions with several caveolae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Measurements of cytoplasmic width revealed a mean value of 143.72 nm (range: 62.8\u0026ndash;220.82 nm; SD: 54.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cb\u003eF\u003c/b\u003e). Adjacent to TLCs, MSCs were characterized by their relatively wider and shorter cytoplasm, embedded within an abundant collagen matrix rich in protein-synthesizing organelles, and containing accumulations of widened ER cisternae and lipid-laden inclusions with periodic lamellae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cb\u003eH\u003c/b\u003e). To further distinguish these two cell types, we performed a morphometric analysis by calculating the length-to-width ratio (L/W) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). TLCs exhibited a mean ratio of 32.38 (range: 21.68\u0026ndash;51.86; SD: 10.83), whereas MSCs showed a mean ratio of 3.60 (range: 1.96\u0026ndash;5.43; SD: 0.94). As we had previously compared TLCs with rat intestinal telocytes in semithin sections, we also examined these cells in 50 nm ultrathin sections using TEM and observed a close similarity in both the fine structure of the cytoplasm and the length-to-width ratio (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFor molecular characterization of TLCs, we collected a series of super-resolution 3D confocal images using specific fluorescent markers targeting cytoskeletal components, receptors, and cell surface proteins, along with well-established MSC markers. Initial validation was conducted using positive and negative controls, which involved either omitting the primary antibody or using appropriate tissues and/or tissue components. These controls are presented in \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTEM observations of TLCs. (\u003cb\u003eA\u0026ndash;C\u003c/b\u003e) In low magnifications, they are distinguished by their extremely thin (as also shown in \u003cb\u003eE\u003c/b\u003e and \u003cb\u003eF\u003c/b\u003e) cytoplasmic extensions embedded in a relatively scarse collagen fibers as opposed to abundant collagen bundles around MSCs (shown in \u003cb\u003eG\u003c/b\u003e and \u003cb\u003eH\u003c/b\u003e). TLCs are also characterized by poor protein synthesizing organalles. However, they display several extracellular vesicles with close proximity to plasma membrane shown by \u003cb\u003ewhite arrowheads\u003c/b\u003e in \u003cb\u003eC\u003c/b\u003e and \u003cb\u003eD\u003c/b\u003e and the cell surface displayed interruptions with several caveolae (\u003cb\u003eblack arrows\u003c/b\u003e in \u003cb\u003eD\u003c/b\u003e). (\u003cb\u003eG\u0026ndash;H\u003c/b\u003e) MSCs are evident by their wider and shorter cytoplasms with rich in protein sythnesizing organelles, elaborate ER cisternae (\u003cb\u003eblack arrowheads\u003c/b\u003e) and lipid-laden inclusions (\u003cb\u003ewhite asterisks\u003c/b\u003e). (\u003cb\u003eI\u003c/b\u003e) The length-to-width ratio (L/W) was calculated as 32.38 for TLCs and 3.60 for MSCs. \u003cb\u003eBlack asterisks\u003c/b\u003e: Clefts; \u003cb\u003eN\u003c/b\u003e: Nucleus. Scale bars: 3 \u0026micro;m (A\u0026ndash;B), 1 \u0026micro;m (C\u0026ndash;H).\u003c/p\u003e\u003cp\u003eFirst, we labeled the entire UC sections with phalloidin to visualize F-actin filaments. As expected, all cell types from outer to inner regions including umbilical cord epithelium (UCE), vascular smooth muscle cells, and stromal cells were positive for phalloidin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Therefore, this marker was not specific to TLCs. However, the general cell morphology, characterized by slender and thin cytoplasmic extensions, remained distinctive for TLCs. Additionally, F-actin showed the density and volume of cells in each region. Thus, TLCs were predominantly located in superficial IVS regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Secondly, we performed vimentin labeling which is a well-known positive marker for MSCs. Interestingly, long and slender shaped TLCs displayed stronger positivity compared to the one in relatively round-ovoid shaped MSCs with broader extensions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cb\u003eD\u003c/b\u003e). Thus, this finding has proven that TLCs are positive for vimentin. Another cytoskeletal protein tested was α-SMA, a smooth muscle cell marker that was found to be positive in both TLCs and MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The specificity of the antibody was confirmed by strong staining in the arterial wall of the UC (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) and the absence of staining in the UCE. The actin-binding protein α-actinin, used as a marker for stromal cell labeling, was found to be positive in all stromal cells; however, a strong and punctate staining pattern was specifically observed in TLCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Finally, we tested three MSC markers \u0026ndash;CD73, CD90, and CD105\u0026ndash; to determine whether they are also expressed in TLCs, in parallel with neighboring MSCs. TLCs exhibited strong and diffuse positivity for CD73 and CD105, whereas CD90 staining was sparse and punctate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;I). Interestingly, a similar staining pattern was also observed in MSCs (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO\u0026ndash;R).\u003c/p\u003e\u003cp\u003eAnother group of markers analyzed included cell surface proteins and receptors. Among these, CD34, PDGFR-α, c-Kit, and caveolin-1 were selected for their high specificity to telocytes. CD34 expression was completely absent in both TLCs and MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cb\u003eB\u003c/b\u003e), even after testing four different antibodies derived from two species and three different commercial sources. This confirms previous reports indicating that CD34 negativity is a general phenotype of UC stroma. As a positive internal control, strong CD34 labeling was observed in the vein endothelium (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). PDGFR-α showed strong plasmalemmal and weak cytoplasmic positivity in TLCs, particularly those surrounding clefts in the superficial IVS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cb\u003eD\u003c/b\u003e), often appearing embedded within dense collagen fibers. c-Kit, a tyrosine kinase receptor, was also expressed in TLCs, especially in those encircling clefts within the superficial IVS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cb\u003eF\u003c/b\u003e). At higher magnification (not shown), the cytoplasmic staining appeared strong and punctate. c-Kit positivity was also observed in arterial wall smooth muscle cells (not shown). Caveolin-1, a membrane-associated scaffolding protein, was detected in all TLCs within the superficial IVS as punctate cytoplasmic foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cb\u003eH\u003c/b\u003e). A few positive cells were also identified in the SES, deep IVS, and PVS regions (not shown). Arterial wall smooth muscle cells again served as a positive internal control and were strongly caveolin-1 positive (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eU).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMarkers specific to selected cell surface proteins and receptors in hUC sections. All images were deconvoluted and reconstructed from 16 consecutive optical sections along the z-axis to generate 3D image stacks. (\u003cb\u003eA\u003c/b\u003e, \u003cb\u003eB\u003c/b\u003e) CD34 showed minimal to no staining across all stromal regions. (\u003cb\u003eC\u003c/b\u003e, \u003cb\u003eD\u003c/b\u003e) PDGFR-α exhibited strong membranous and weak cytoplasmic positivity in TLCs, particularly those surrounding clefts (\u003cb\u003easterisks\u003c/b\u003e in corresponding DIC images), which were enclosed by bundles of collagen fibers (\u003cb\u003eblack arrows\u003c/b\u003e) in the superficial IVS. (\u003cb\u003eE\u003c/b\u003e, \u003cb\u003eF\u003c/b\u003e) c-Kit expression was also strong in TLCs, especially those around clefts (\u003cb\u003easterisks\u003c/b\u003e in corresponding DIC images) in the superficial IVS. (\u003cb\u003eG\u003c/b\u003e, \u003cb\u003eH\u003c/b\u003e) Caveolin-1 was localized in TLCs within the superficial IVS, appearing as cytoplasmic punctate staining. ★: UCE. Scale bars: 50 \u0026micro;m (A, B, E, F); 20 \u0026micro;m (C, D, G, H).\u003c/p\u003e\u003cp\u003eAgarose gel electrophoresis confirmed the amplification of all eight target genes, with band intensities corresponding to expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Notably, CD34 exhibited the weakest bands, indicating lower transcript abundance compared to the others. To validate these findings, qRT-PCR data were further analyzed using descriptive statistical approaches. Ct values were normalized to the GAPDH reference gene, and relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e method (\u003cb\u003eSupp. Table\u0026nbsp;3\u003c/b\u003e). To facilitate interpretation of the broad dynamic range in expression, relative transcript levels were visualized using a logarithmic y-axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among all targets, vimentin displayed the highest expression, followed by PDGFR-α and c-Kit. Moderate expression was observed for CD73, CD90, CD105, and caveolin-1, while CD34 consistently showed the lowest expression level. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo further investigate the phenotype and marker expression of TLCs, we isolated the entire stromal cell population \u0026ndash; including MSCs and TLCs \u0026ndash; via explant culture. Cells harvested from the explants (P\u003csub\u003e0\u003c/sub\u003e) were cryopreserved for up to two passages (P\u003csub\u003e2\u003c/sub\u003e) and subsequently used for flow cytometric analysis, live-cell imaging with HMC, and post-fixation imaging by SEM and SR-CM. At passage 2 (P\u003csub\u003e2\u003c/sub\u003e), initial cell attachment (T\u003csub\u003e0\u003c/sub\u003e) was followed by the appearance of both broad-bodied cells, characteristic of MSCs, and extremely thin, elongated TLCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cb\u003eB\u003c/b\u003e). These fusiform cells remained observable at T\u003csub\u003e36\u003c/sub\u003e and T\u003csub\u003e48\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;F). Throughout the culture period, TLCs were sparsely distributed and exhibited minimal proliferation, whereas adjacent MSCs expanded robustly, reaching confluency by approximately T\u003csub\u003e120\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eTo characterize the TLC phenotype, we conducted morphometric analysis on approximately 200 cells using composite HMC images. The average cell length was 604.69 \u0026micro;m (range: 272.27\u0026ndash;1380.02 \u0026micro;m; SD: 339.7). In contrast, MSCs exhibited markedly different morphology, attaching to the substrate via flattened lamellipodia and displaying wider cytoplasmic domains. As the fine structural assessment, SEM further revealed cellular phenotype, cell surface and morphometric features of those elongated cells. The mean width of their cellular extensions was measured as 1.49 \u0026micro;m (range: 1.04\u0026ndash;2.09 \u0026micro;m; SD: 0.43) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I and \u003cb\u003eJ\u003c/b\u003e). They occasionaly made contacts with the adjacent MSCs, which have extremely flat and broad cytoplasm extending nano tube-like spikes between each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H and \u003cb\u003eI\u003c/b\u003e). Elongated TLCs seemed attached to the substrate at two ends having a relatively flattaned cytoplasmic lamellipodia.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e(\u003cb\u003eA\u003c/b\u003e) Agarose gel electrophoresis of the qRT-PCR products revealed detectable bands for all eight target genes. (\u003cb\u003eB\u003c/b\u003e) Expression levels of eight target genes normalized to GAPDH are shown as mean ΔCt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three biological replicates (n\u0026thinsp;=\u0026thinsp;3). Y-axis is presented on a logarithmic scale for better visualization of gene expression differences.\u003c/p\u003e\u003cp\u003eNext, we aimed to isolate specific cell populations based on known markers such as CD34, PDGFR-α, and c-Kit using flow cytometry. However, immunophenotypic characterization of the entire live stromal cell population failed to distinguish any specific subpopulation using these markers (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding was confirmed through three sets of experiments involving single and double antibody labeling with varying antibody concentrations and incubation times. To further investigate, we examined the same live, unfixed cell populations using SR-CM to determine whether any marker expression undetected by flow cytometry could be visualized. SR-CM analysis also failed to detect any specific signal, supporting the initial flow cytometry findings (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, when the same cell populations were fixed and stained with the corresponding antibodies, clear and distinguishable labeling patterns were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting that these markers may not be reliably detectable in live, unfixed stromal cells under the tested conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCytoskeletal proteins, MSC markers, cell surface proteins, and receptors in cultured mixed stromal cell populations. (\u003cb\u003eA\u003c/b\u003e) F-actin and (\u003cb\u003eB\u003c/b\u003e) vimentin exhibited strong filamentous staining in both MSCs and TLCs. (\u003cb\u003eC\u003c/b\u003e) α-SMA were evident in stress-fibers rich MSCs while very little in TLCs. (\u003cb\u003eD\u003c/b\u003e) In contrast, α-actinin were strongly positive in both cell types. (\u003cb\u003eE\u0026ndash;G\u003c/b\u003e) MSC markers (CD73, CD90, and CD105) were expressed in both populations, although CD90 showed slightly weaker staining. (\u003cb\u003eH\u003c/b\u003e) CD34 was completely absent in all cells. (\u003cb\u003eI\u003c/b\u003e) Caveolin-1 showed strong positivity in both cell types and showed a strong staining in ER-Golgi region in MSCs (see, inset). (\u003cb\u003eJ\u003c/b\u003e) PDGFR-α was expressed in both cell types, with prominent staining in the protrusions of TLCs. \u003cb\u003eWhite arrowheads\u003c/b\u003e indicate elongating TLC telopods and \u003cb\u003ewhite arrows\u003c/b\u003e indicates MSCs. Scale bars: 20 \u0026micro;m.\u003c/p\u003e\u003cp\u003eFinally, we examined the cultured stromal cell populations grown on glass coverslips using the same panel of markers previously found to be positive in UC sections. These markers included those specific to cell surface proteins, receptors, cytoskeletal components, and MSC markers. F-actin was uniformly distributed in all cells, displaying strong filamentous staining characteristic of the typical stromal phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Vimentin expression was observed in both MSCs and elongated TLCs, with prominent filamentous localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Interestingly, α-SMA positivity was stronger due to the richness of stress-fibers in MSCs, while very little in TLC telopodes. α-actinin labeled both cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cb\u003eD\u003c/b\u003e). All three MSC markers tested were positive, although CD90 staining appeared slightly weaker compared to CD73 and CD105 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;G). CD34 expression was entirely absent in all cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). No c-Kit staining was recorded in any cultured cells (not shown). In contrast, caveolin-1 showed strong positivity, particularly in the ER-Golgi regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). PDGFR-α was localized to the cell body and protrusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTelocytes are a recently characterized and multifunctional component of the interstitial space. With their distinctive morphology, strategic anatomical localization, and broad range of cellular interactions, they are increasingly recognized as key contributors to tissue homeostasis, regeneration, and intercellular communication, as reviewed by Sanches et al. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe hUC, a unique form of loose connective tissue, is particularly rich in certain types of collagen fibers, interstitial space, ECM components, and stromal cells. Recent studies have emphasized the potential of hUC not only as a source of MSCs but also as a niche housing other specialized interstitial cells with distinct morphological and functional properties \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Karahuseyinoglu et al. \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e demonstrated that only a portion of stromal cells is responsive to neuronal induction and suggested that this may be due to the presence of heterogeneous cells populations derived from the UC stroma. Supporting evidence for this came from the study of Sarugaser et al. \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, who showed that a portion of cells could not be differentiated into neurons.\u003c/p\u003e\u003cp\u003eIn this study, detailed histological analyses revealed a phenotypically distinct cell population within the hUC stroma that closely resembles telocytes. These TLCs were predominantly localized encircling the clefts of the \u0026ldquo;superficial\u0026rdquo; IVS as opposed to the \u0026ldquo;deep\u0026rdquo; IVS. We propose these novel terms \u0026ndash; \u0026ldquo;superficial\u0026rdquo; for cleft-enriched regions and \u0026ldquo;deep\u0026rdquo; for cleft-devoid regions \u0026ndash; to refine UC stromal nomenclature. The close spatial association of TLCs with known MSCs suggests a potential lineage relationship, possibly indicating a transdifferentiated state of MSCs. However, given the current lack of definitive evidence regarding their origin and developmental trajectory, we refer to these cells as \u0026ldquo;telocyte-like cells\u0026rdquo; rather than classifying them as true telocytes.\u003c/p\u003e\u003cp\u003eOur initial light microscopic examinations, utilizing both thin and semi-thin stained sections, identified a variety of cells in the hUC stroma. Alongside the typical multiform, round, or ovoid MSCs, we observed slender cells with bipolar projections, consistent with previously described telocytes \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our comparative analysis in rat intestinal submucosa further confirmed morphological similarities to telocytes described in that context \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. TEM-based stitched panoramic micrographs revealed thin, elongated TLCs, phenotypically distinct from MSCs with broader cytoplasm. Morphometric analysis showed that the L/W ratio of TLCs was approximately nine times that of wider MSCs, confirming a heterogeneous stromal cell population. The average thickness of their cytoplasmic extensions measured 0.143 \u0026micro;m, consistent with previously reported values (~\u0026thinsp;0.2 \u0026micro;m) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTEM examination further revealed lipid inclusions and widened ER cisternae specifically within MSCs, whereas TLCs lacked these structures. Importantly, there was no direct evidence of collagen synthesis by TLCs in this fibrous stromal environment. These findings support our hypothesis that MSCs are the primary contributors to collagen production within the hUC stroma. As proposed by Bei et al. \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, unlike fibroblasts, which are primarily responsible for ECM production, telocytes may instead facilitate intercellular communication by forming three-dimensional networks around collagen fibers. Furthermore, their close spatial association with collagen found around the clefts has led to the suggestion that telocytes may regulate collagen organization \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Specifically, they may modulate the turgor regulation of the umbilical cord. This concept aligns with Nanaev et al.'s earlier suggestion that myofibroblastic MSCs and their less differentiated precursors mark the jelly-filled stromal clefts of Wharton\u0026rsquo;s jelly. These cells, along with the surrounding meshwork of contractile cells, function as a mechanism to maintain cord turgor and prevent compression \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Based on these insights, it is plausible that TLCs contribute to ECM dynamics \u0026ndash; particularly in collagen-rich environments such as Wharton\u0026rsquo;s jelly \u0026ndash; by regulating matrix architecture rather than synthesizing its components directly.\u003c/p\u003e\u003cp\u003eFrozen sections of UCs showed fine cytoplasmic staining of F-actin filaments across all stromal cell populations, with pronounced labeling using phalloidin dyes. TLCs were predominantly observed in both superficial and deep IVS, but were only rarely detected in the PVS. Vimentin, an intermediate filament protein characteristic of mesoderm\u0026ndash;derived cells, was strongly expressed in both TLCs \u0026ndash; particularly within their thin, elongated telopods \u0026ndash; and in the broader cytoplasmic regions of MSCs. As expected, neither F-actin nor vimentin served as discriminatory markers, as both are also expressed in fibroblasts, endothelial cells, and tissue macrophages. However, consistent with previous studies \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, our results support the mesenchymal stromal origin of TLCs based on their strong vimentin expression. Interestingly, these two cytoskeletal markers helped phenotypically distinguish between stromal subtypes: TLCs, characterized by flattened or small nuclei, exhibited more intense vimentin staining, whereas MSCs with round/ovoid nuclei displayed weaker staining. This differential expression pattern may aid in distinguishing stromal cell subsets based on nuclear morphology and cytoskeletal profiles.\u003c/p\u003e\u003cp\u003eThe next marker we tested was α-actinin, an actin-binding protein, which is involved in F-actin-based cytoskeletal organization. Our TLC and MSC findings align with our F-actin results and previous reports about telocytes suggesting that it is not a specific marker for telocytes, as we have noted its expression in both TLCs and MSCs. hUC-MSCs are often classified as myofibroblasts due to their expression of contractile proteins, including α-smooth muscle actin (α-SMA) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Based on this, we hypothesized that α-SMA expression could serve as a distinguishing marker between MSCs and TLCs. However, immunostaining of tissue sections revealed that α-SMA did not clearly discriminate between the two cell types, as TLCs also exhibited weak α-SMA positivity. Interestingly, in culture conditions, a substantial portion of the MSC population showed strong α-SMA expression, primarily localized to their broad cytoplasmic regions, where fine arrays of stress fibers were clearly evident. In contrast, telopodes of TLCs lacked such organization. Liu et al. reported that although most telocytes are α-SMA negative, a subset associated with smooth muscle regions has been reported to express this protein \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Similarly, in the fetal placenta, cultured telocytes were found to express α-SMA, though their staining pattern was diffuse and lacked the characteristic stress fibers observed in smooth muscle cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These findings suggest that α-SMA expression alone is insufficient to definitively distinguish MSCs from TLCs and that functional diversity may exist among stromal cell subsets. Further studies are warranted to clarify the role and identity of α-SMA-positive cells within the hUC stroma. Furthermore, as discussed by Can \u0026amp; Karahuseyinoglu \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, IVS cells exhibit longer and more numerous cytoplasmic processes compared to PVS cells. Notably, hUC myofibroblasts, more prevalent in PVS, likely originate from adjacent vascular smooth muscle cells due to morphological similarities and contractile properties. This suggests that stromal cells in superficial and deeper cord regions may have distinct embryological origins. To explore this further, evaluating cleft formation, vessel development, and their perivascular regions throughout the cord's developmental weeks during pregnancy would be crucial.\u003c/p\u003e\u003cp\u003ehUC-MSCs are recognized to express CD73, CD90, and CD105, as documented \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. TLCs displayed robust and diffuse positivity for CD73 and CD105, while CD90 staining was sporadic and punctate. There is variability in the literature regarding these marker profiles in telocytes. Studies identifying telocytes in placental tissues and those examining telocyte cultures have reported absence of CD90 expression in cultured telocytes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, while others noted higher CD90 levels compared to fibroblasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In our cultures, CD90 was weakly expressed across morphologically diverse cell types. Additionally, TLCs exhibited focal CD105 positivity, consistent with studies reporting telocytes to be CD105-positive \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. CD73 expression was also detected in our cells, representing a novel finding in telocyte field.\u003c/p\u003e\u003cp\u003eCD34 is a commonly used immunohistochemical marker for identifying telocytes and distinguishing them from other interstitial cells \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In contrast, UC stromal cells are typically CD34-negative \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In line with our findings and previously published studies \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we conclude that CD34 is not a reliable marker for identifying TLCs in UC tissue. This discrepancy may reflect tissue-specific expression patterns, as telocytes from different organs do not always exhibit the same marker profiles \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePDGFR-α has been widely used to distinguish telocytes from other interstitial cell types and is associated with tissue homeostasis and injury response \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Its expression is believed to play a key role in telocyte-mediated stromal signaling \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, PDGFR-α is not specific to telocytes, as it can also be expressed by fibroblasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In our study, PDGFR-α expression was detected in TLCs, particularly in those lining the superficial IVS clefts and distributed among collagen bundles. PDGFR-α positivity was also maintained in thin, elongated TLCs under culture conditions. Notably, strong PDGFR-α expression was observed in the cytoplasmic extensions of these cells, supporting their potential role in intercellular communication and stromal network formation.\u003c/p\u003e\u003cp\u003ec-Kit\u0026ndash;positive telocytes have also been identified in fetal tissues such as the placenta, particularly in trophoblast-rich areas and around blood vessels. Reports on c-Kit expression in hUC-MSCs are inconsistent, with both positive and negative findings reported. In our observations, TLCs exhibited strong c-Kit both in the membrane and cytoplasmic loci in thin, elongated telopodes in both superficial and deep IVS regions. Besides TLCs, few positivities were also noted that corresponded to MSCs in the same tissue compartments. No c-Kit staining was recorded in any cultured cells. These findings do not support previous reports suggesting c-Kit as a distinguishing marker for telocytes, reinforcing its diagnostic value in identifying these cells within the UC stroma.\u003c/p\u003e\u003cp\u003ec-Kit\u0026ndash;positive telocytes have been identified in fetal tissues such as the placenta, particularly in trophoblast-rich regions and around blood vessels \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, reports on c-Kit expression in hUC-MSCs remain inconsistent, with studies presenting both positive and negative findings \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In our observations, TLCs exhibited strong c-Kit expression in both membrane and cytoplasmic regions, particularly along their thin, elongated telopodes, located in both superficial and deep areas of the intervillous space (IVS). In addition to TLCs, a limited number of c-Kit\u0026ndash;positive cells, likely corresponding to MSCs, were also observed within the same compartments. Notably, no c-Kit staining was detected in any of the cultured cell populations. These results do not support the previously proposed role of c-Kit as a distinguishing marker for telocytes in culture. Instead, they reinforce its diagnostic value for identifying telocytes specifically within the umbilical cord stroma.\u003c/p\u003e\u003cp\u003eCaveolin-1 has been reported to localize prominently in the telopodes of telocytes, supporting its role in intercellular communication \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and its expression has also been confirmed in hUC-MSCs \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In our immunofluorescence analysis of UC sections, strong caveolin-1 positivity was observed in the thin, elongated TLCs surrounding the superficial IVS compartments, whereas broader MSCs in the deep IVS and PVS showed either membrane-localized or absent expression. In cultured cells, caveolin-1 exhibited perinuclear staining consistent with localization to the ER-Golgi apparatus and in nearly all cells. TEM further confirmed the presence of telocyte-specific caveolae, consistent with previous reports \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, numerous extracellular vesicles were detected adjacent to these cell surface pits, supporting the hypothesis that telocytes participate in paracrine and/or juxtacrine signaling by transferring regulatory molecules to neighboring cells \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Collectively, our findings indicate that while caveolin-1 is expressed in both MSCs and TLCs, its subcellular distribution differs markedly, and these distinct localization patterns may aid in the identification of TLCs.\u003c/p\u003e\u003cp\u003eWe also attempted to isolate marker-specific cell populations using both dual and single surface markers, including CD34, PDGFR-α, and c-Kit\u0026mdash;commonly used to distinguish telocytes from other interstitial cells \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, flow cytometry analysis of live, unfixed cells did not yield a sufficient number of positively labeled cells under either dual-marker (CD34\u0026thinsp;+\u0026thinsp;PDGFR-α and CD34\u0026thinsp;+\u0026thinsp;c-Kit) or single-marker conditions. As a result, TLCs could not be reliably separated from other stromal cell populations in the UC, and establishing isolated subcultures was not feasible. These findings suggest that TLCs in Wharton\u0026rsquo;s jelly may express these markers at low levels, may not present them in unfixed conditions, or may require fixation for consistent antigen detection.\u003c/p\u003e\u003cp\u003eTo validate our immunofluorescence findings, qRT-PCR analysis was performed, revealing that vimentin expression was markedly elevated\u0026mdash;ranging from 32- to 5200-fold higher\u0026mdash;compared to the other seven target genes. This finding supports the mesodermal origin of all stromal cells in Wharton\u0026rsquo;s jelly and aligns with the strong vimentin immunoreactivity observed in both tissue sections and cultured cells. Notably, PDGFR-α and c-Kit showed higher transcript levels than CD73, CD90, CD105, and caveolin-1. Although previous studies have reported that telocytes express vimentin, CD34, and c-Kit \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, our inability to isolate TLC populations precluded direct comparisons between MSCs and TLCs. Thus, our results are best interpreted as representative of the broader gene expression profile of UC stromal cells. Consistent with prior qRT-PCR studies \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, we also observed CD34 negativity and CD73, CD90, and CD105 positivity in stromal cells. In conclusion, the qRT-PCR data corroborated our immunofluorescence results and confirmed that gene expression levels were consistent with corresponding protein expression patterns.\u003c/p\u003e\u003cp\u003eIn this study, cell cultures derived from hUCs revealed two morphologically distinct stromal populations. The lack of a standardized method for isolating a homogeneous hUC-MSC population has been previously noted \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, along with reports of rapid proliferation and morphological variation in early passages \u003csup\u003e56\u003c/sup\u003e. The morphological heterogeneity of UC-derived stromal cells has also been documented by Coskun and Can \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. They described two subtypes: type-1 cells, with broad cytoplasm and prominent filopodia, showing myofibroblastic characteristics, and type-2 cells, with a thinner, elongated, fibroblast-like appearance. Guenther et al. \u003csup\u003e56\u003c/sup\u003e reported five initial morphologies\u0026mdash;triangular, star-shaped, flattened, elongated, and round\u0026mdash;which gradually transitioned into a more homogeneous population dominated by flattened and elongated forms over time in culture. Our observations align with and contribute to this literature by confirming the morphological diversity of Wharton\u0026rsquo;s jelly stromal cells and highlighting two distinct cell types consistently observed both in tissue sections and in vitro cultures. These findings underscore the dynamic and heterogeneous nature of the UC stromal compartment. Furthermore, the average length of TLCs in our study was 604.69 \u0026micro;m, which falls within the reported 200\u0026ndash;1000 \u0026micro;m range \u003csup\u003e57\u003c/sup\u003e. SEM, widely used for identifying telocytes due to its ability to visualize their fine projections and interactions \u003csup\u003e58,59\u003c/sup\u003e, revealed TLCs with long, thin processes in close contact with each other and with broader MSCs. These observations suggest that TLCs contribute to intercellular communication, supporting previous reports of telocyte networks playing a role in maintaining three-dimensional tissue architecture \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eConclusively, here we present the first evidence for the existence of TLCs in the hUC, shedding light on the cellular heterogeneity of the stem cell niche in this tissue. Our findings contribute to the understanding of how TLCs may support the structural and functional diversity within the Wharton's jelly microenvironment. Although each of the methods employed in this study contributed to the identification of these cells, none proved sufficient on their own to definitively characterize them. This limitation underscores the need for future advanced molecular studies \u0026ndash; focusing on telocyte-specific markers and investigating gene, microRNA, and secretome profiles \u0026ndash; to achieve a more precise definition of telocytes, particularly in fibrous stromal tissues that share overlapping markers. Moreover, exploring the physiological roles of telocytes during embryonic development may reveal novel insights with potential implications for regenerative medicine and tissue engineering. Importantly, considering the UC as a postnatal biological source of cells, the presence of TLC populations may open new avenues for innovative cell-based therapeutic strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eExtracellular matrix\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndoplasmic reticulum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eF-actin\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFilamentous actin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutaraldehyde\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHematoxylin\u0026ndash;eosin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHoffman Modulation Contrast\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ehUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman umbilical cord\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntervascular stroma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLSM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLaser scanning microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultipotent stromal cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParaformaldehyde\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePVS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePerivascular stroma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eQuantitative reverse transcriptase PCR\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRoom temperature\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eScanning electron microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSES\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSubepithelial stroma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSR-CM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuper resolution confocal microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransmission electron microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTelocyte-like cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUmbilical cord\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUCE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUmbilical cord epithelium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicting of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study received partial funding from Ankara University Scientific Research Fund TYL-2024-3343 and TSG-2022-2545 to AC.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.E. contributed to the data collection and writing the manuscript. I.A. and B.S. contributed to the data collection. S.E.C. collected UC samples and provided clinical data. M.D. contributed to the execution and interpretation of the qRT-PCR experiments. F.T.C. contributed to the administration. A.C .contributed to the design of the study, writing the manuscript, data collection and final approval of manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to Remzi Ata and Seyfettin \u0026Uuml;lger for their technical assistance in TEM sample preparation and image collection.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets used and/or analyzed during the current study are also available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePopescu, L. M. \u0026amp; Faussone-Pellegrini, M. S. TELOCYTES - a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 729\u0026ndash;740. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1582-4934.2010.01059.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1582-4934.2010.01059.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePopescu, L. M., Gherghiceanu, M., Suciu, L. C., Manole, C. G. \u0026amp; Hinescu, M. E. 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The roles of telocytes in lung development and angiogenesis: An immunohistochemical, ultrastructural, scanning electron microscopy and morphometrical study. \u003cem\u003eDev Biol\u003c/em\u003e 443, 137\u0026ndash;152, doi:10.1016/j.ydbio.2018.09.010 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"interstitial cell, multipotent stromal cell, telocyte, umbilical cord stroma, Wharton’s Jelly","lastPublishedDoi":"10.21203/rs.3.rs-7203915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7203915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman umbilical cord (hUC) connective tissue, known as Wharton's jelly, contains multipotent stromal cells (MSCs). Despite being considered the primary source of MSCs for regenerative purposes, hUC is one of the least studied fetal tissues. We conducted a detailed examination of normal hUCs (n\u0026thinsp;=\u0026thinsp;16) and identified telocyte-like cells (TLCs) exhibiting both structural and phenotypical features similar to telocytes previously described in various tissues, including the placenta. TLCs were found to be concentrated around intervascular stromal clefts in the UC. These cells had thin, elongated bipolar cell bodies (9-fold higher in length/width ratio compared to MSCs), distinguishing them from the well-defined MSCs, which display abundant ER-Golgi systems and high collagen production. We confirmed the presence of TLCs with marker expression patterns including F-actin, vimentin, α-SMA, α-actinin, caveolin-1\u003csup\u003e+\u003c/sup\u003e, c-Kit\u003csup\u003e+\u003c/sup\u003e, PDGFR-α\u003csup\u003e+\u003c/sup\u003e, CD34\u003csup\u003e\u0026ndash;\u003c/sup\u003e, CD73\u003csup\u003e+\u003c/sup\u003e, CD90\u003csup\u003e+\u003c/sup\u003e, and CD105\u003csup\u003e+\u003c/sup\u003e, reflecting a distinct stromal identity, either adjacent to MSCs or possibly originating from them. Isolation, culture, and immunocytochemical labeling further confirmed the presence of TLCs, highlighting the diverse nature of hUC cell cultures. These two cell types (TLCs and MSCs) were observed in contact with each other or within their respective populations. Each of the methods used in this study contributed to the identification of these cells, but none alone was enough to definitively characterize them. The findings conclusively demonstrate the existence of TLCs in the hUC. This provides significant new evidence regarding the cellular heterogeneity of the stem cell niche and suggests a potential role for TLCs in the stromal network of this tissue.\u003c/p\u003e","manuscriptTitle":"Identification and Localization of Telocyte-Like Cells in Human Umbilical Cord Stroma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 13:59:49","doi":"10.21203/rs.3.rs-7203915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-26T13:13:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T15:09:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T13:01:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-21T15:01:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262443139098637855520104855644533582720","date":"2025-08-20T12:47:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T12:33:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T19:06:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63689135491384952135315396948485433585","date":"2025-08-19T02:37:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202432456991869623022330150419445331247","date":"2025-08-17T06:47:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120722698107437297390742948266640289404","date":"2025-08-15T14:33:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T11:51:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66300576726452967529495435851099827243","date":"2025-08-15T11:04:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44535214363741158604982008031547949758","date":"2025-08-15T08:23:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178781605822177668590789865358579167765","date":"2025-08-15T06:35:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242966493265799172345453995197213086066","date":"2025-08-15T05:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-15T05:29:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T05:20:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-31T08:45:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T07:32:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-29T07:28:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"192e5a35-259b-4f5d-bc97-d7c9e66c40c8","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53519404,"name":"Biological sciences/Cell biology"},{"id":53519405,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-11-10T16:03:33+00:00","versionOfRecord":{"articleIdentity":"rs-7203915","link":"https://doi.org/10.1038/s41598-025-26282-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-07 15:58:06","publishedOnDateReadable":"November 7th, 2025"},"versionCreatedAt":"2025-08-22 13:59:49","video":"","vorDoi":"10.1038/s41598-025-26282-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-26282-9","workflowStages":[]},"version":"v1","identity":"rs-7203915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7203915","identity":"rs-7203915","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}