Telocytes in the Luminal GI Tract

Abstract

Telocytes are unique mesenchymal cells characterized by multiple remarkably long cytoplasmic extensions that extend hundreds of micron away from the cell body. Through these extensions, telocytes establish a 3-dimensional network by connecting with other telocytes and various cell types within the tissue. In the intestine, telocytes have emerged as an essential component of the stem cell niche, providing Wnt proteins that are critical for the proliferation of stem and progenitor cells. However, the analysis of single-cell RNA sequencing has revealed other stromal populations and mechanisms for niche organization, raising questions about the role of telocytes as a component of the stem cell niche. This review explores the current state-of-the-art, existing controversies, and potential future directions related to telocytes in the luminal gastrointestinal tract.

Summary.

Telocytes, distinctive stromal cells, have recently emerged as a crucial component of the intestinal stem cell niche. This review explores the present advancements, ongoing debates, and possible future paths concerning telocytes in the luminal gastrointestinal tract.

To maintain homeostasis, cells, extracellular matrix, and signaling pathways work together to ensure the proper function, structure, and overall stability of the tissue. Tissues consist of diverse cell types, each with specific functions and characteristics. The complexity of tissue homeostasis stems from dynamic processes and intricate interactions at the cellular and molecular levels. Consequently, functioning cell networks are critical for supporting homeostasis in multicellular organisms. These cell networks, also known as cellular communication systems, empower cells to coordinate their activities, respond to environmental changes, and maintain a balanced internal environment.

The single-cell layer of the intestinal epithelium can be described as a complex network where epithelial cells collaborate to carry out various functions in a spatially compartmentalized manner. This tissue organization is determined by gradients of morphogens produced by niche cells, which modulate the response of epithelial cells at different tissue coordinates. Intestinal epithelial cells are organized within repetitive crypt-villus units. The crypt acts as the proliferating compartment, housing stem and Paneth cells at the base, and both receive canonical Wnt signals. The mid-crypt region consists of actively dividing transit amplifying cells, whereas postmitotic, differentiated cells accumulate along the villi.1, 2, 3, 4, 5, 6, 7

Cells at the tip of each villus are shed off into the lumen but are promptly replaced by cells generated at the crypt, facilitating a highly regenerative process.⁸ To maintain homeostasis, the rate of shedding must be balanced with the rate of cell proliferation in the crypt, preventing gaps in the epithelial barrier. Therefore, the regulation of intestinal renewal requires a holistic, integrated perspective that views epithelial cells as both a continuous network and individual entities.

Telocytes, identified more recently as a distinct type of interstitial cell, were initially classified as interstitial Cajal-like cells. However, in 2010, it was shown by electron microscopy that telocytes are entirely separate from Cajal cells, which function as pacemaker cells in the control of smooth muscle contraction.^9,10 Telocytes are present in the interstitial space of many tissues and are distinguished by elongated cellular extensions known as telopodes, which can extend to lengths ranging from tens to hundreds of microns. Telocytes exhibit variable numbers of telopodes, and telopodes contain thin segments referred to as podomeres, each having a diameter of less than 200 nm. Additionally, telopodes feature dilated regions known as podoms, which hold caveolae (Ca²⁺ uptake/release units), mitochondria, and endoplasmic reticulum. Telopodes create a 3-dimensional network by overlapping cytoplasmic processes with other telocytes, establishing contact with the epithelium, immune cells, smooth muscle cells, fibroblasts, blood vessels, and nerve bundles.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Although this observation has led to the suggestion that telocytes may play a significant role in regulating homeostasis, this was experimentally tested only recently.

The initial experimental evidence indicating that telocytes are a crucial component in tissue homeostasis comes from the intestine, with 4 independent groups using 4 distinct markers to label telocytes. The Kaestner laboratory used the transcription factor FoxL1, the Peduto laboratory implicated CD34, the Virshup laboratory used the PDGF Receptor alpha (PDGFRα), and the Basler laboratory used the nuclear mediator of the Hedgehog pathway, Gli1.21, 22, 23, 24, 25, 26 The 4 groups demonstrated the existence of both a physical overlap and coexpression of the markers among FoxL1+, CD34+, PDGFRα+, and Gli1+ stromal cells. They used either an organoid-stroma coculture system, demonstrating enhanced organoid growth with the stromal compartment, or in vivo genetic engineering to delete the genes Porcn or Wls, which encode proteins essential for Wnt secretion, within the target cells. This led to crypt collapse, a reduction in epithelial proliferation, and a decrease in the number of intestinal stem cells, demonstrating that telocytes are the Wnt-producing niche cells.

Nevertheless, single-cell analyses have revealed distinct subpopulations within these critical stromal cells. Specifically, the Shivdasani group categorized PDGFRα+ cells into 3 groups based on their PDGFRα expression levels. Those expressing high levels of PDGFRα, concentrated at the crypt-villus junction and coexpressing FoxL1 with abundant BMPs, were labeled as telocytes. Conversely, cells expressing lower levels of PDGFRα were further classified based on CD81 expression. Two subpopulations emerged: one expressing CD81, Ackr4, and the BMP inhibitor Gremlin1 (Grem1), termed trophocytes, located beneath the muscularis mucosae; and another subpopulation that was CD81 negative. Both subpopulations were shown to possess supportive activity in the intestine and colon.^27,28 Additional analysis confirmed this classification and further refined it by subdividing the PDGFRα low CD81-negative class into 2 subgroups: Fgfr2+ and Igfbp5+/CD90+ cells. Remarkably, in vivo transplantation and lineage tracing experiments have revealed that the subsets found in the adult intestine originate from Gli1-expressing precursors present in the embryonic day 12.5 intestine.²⁹ Grem1-driven lineage tracing revealed labeling of telocytes located at the crypt-villus junction.³⁰ However, PDGFRα low Grem1+ cells were excluded from telocyte classification because of the absence of FoxL1 expression in single-cell RNA sequencing. Consequently, this exclusion led to the inference that Grem1+ cells, rather than telocytes, serve as the niche providing canonical Wnt signals. The suggested mechanism for organizing Wnt-secreting stem cell niches within the crypt architecture involves self-organization based on the distance from the BMP signaling center, facilitated by subepithelial telocytes along the crypt top.²⁸ Further analysis to characterize the subpopulations of FoxL1+ cells is essential for a more comprehensive understanding of the relationship between Grem1+ and FoxL1+ cells. Of note, given the low sensitivity of single-cell RNA sequencing, transcripts of lowly expressed genes, such as FoxL1, are often undercounted.

Regarding telocytes, their population was further stratified based on their location along the crypt-villus axis. Telocytes positioned along the villus tip were found to express Lgr5, a hallmark of the epithelial stem cell marker, and noncanonical Wnt proteins. This observation suggests a spatial shift from canonical to noncanonical Wnt signaling in the villus epithelium.³¹ Furthermore, it has been demonstrated that villus tip telocytes not only govern epithelial differentiation but also regulate villus tip endothelial cell polarization and fenestration into the epithelial-facing side. Such organization optimizes the nutrient absorption process (Figure 1 and Figure 2).³² A simplified schematic summarizing the current understanding of telocytes in intestinal homeostasis (Figure 2).

Figure 1.

Villus tip telocyte (VTT) ablation perturbs villus tip vessel polarization and epithelial enterocyte expression. (A) Endothelial cell nuclei are polarized toward the villus core (white arrowheads, perinuclear VEGFR2 staining) or epithelial side (yellow arrowheads) of villus tip vessels. VTT depletion (Lgr5 DTA) disturbs villus tip endothelial cell polarization. Scale bar 50μm. (B) smFISH validation (white dots) of enterocyte villus tip gene Ada that is changed 48 hours following VTT ablation. Scale bar 20μm.

Reprinted with permission from Bernier-Latmani et al³² and Bahar Halpern et al.³¹

Figure 2.

Telocytes are closely appositioned to the intestinal epithelium and blood vessels, alining along the crypt-villus axis. Their structure extends around a cluster of approximately a dozen epithelial cells, creating a continous network along the basal side of the entire crypt-villus epithelium.

The association between telocytes to blood vessels has been further clarified by tracing the lineage of the established pericyte marker NG2 (Cspg4) in both the intestine and stomach. Interestingly, in addition to marking Rgs4 high pericytes, NG2 showed significant overlap in expression with pericryptal cells coexpressing FoxL1, termed pericyte-like, which includes telocytes. To evaluate the importance of these cells as a stem cell niche during regeneration, Wls was deleted in NG2+ Cre-derived cells following irradiation. The survival and proliferation of both stomach and intestinal progenitor and stem cells were affected, highlighting the crucial role of NG2+ FoxL1+ pericyte-like/telocytes as the stem cell niche.³³

In contrast to the highly regenerative abilities of the intestinal epithelium, telocytes naturally remain in a quiescent state and do not feasibly divide. This property enables telocytes to preserve a memory of homeostasis and offers an advantage in supporting tissue health. The slow turnover of telocytes was demonstrated through genetic pulse labeling using Gremlin1-CreERT2 reporter mice. It took approximately a year after a tamoxifen pulse for the entire telocyte network to express the GFP lineage label.³⁰ However, it has been reported that, following perturbation, the telocyte population can expand significantly. After inducing colitis through DSS treatment, a condition requiring increased regeneration, the number of RBP1+ cells, a subpopulation of Gli1+ cells in the colon, notably increased during the recovery period.²⁴ The observed increase in the telocyte population after injury may be attributed to proliferation, as has been proposed for the pericryptal NG2+ FoxL1+ pericyte-like/telocyte population after irradiation.³³ However, it remains unclear whether the ability of telocytes to proliferate is limited to a specific subpopulation under certain conditions, what triggers this, and how it affects the telocyte network.

One of the key features of telocytes is their ability to establish connections with other telocytes and various cell types by forming homocellular and/or heterocellular junctions through their telopodes. This process creates a continuous 3-dimensional network.^10,12,15,34, 35, 36, 37, 38, 39, 40, 41 Electron microscopy often reveals that telopodes are linked by point contacts and electron-dense nanostructures. The 2 cell membranes are separated by a narrow gap (10–30 nm), suggesting a molecular interaction between different telocytes and other cell types and a potential flow of information along the network. This is crucial for understanding how telocytes may contribute to the control of homeostasis. In the intestine, the telocyte network accompanies stem cells and their derivatives along the crypt-villus axis during their path of differentiation. On one hand, this allows telocytes to view the epithelium from a broad perspective and integrate data. However, on the other hand, through the telopodes, telocytes establish individual cell-cell specific communication.

It is possible that telocyte networks exist and support stem cells and their progeny in multiple species and tissues. Nevertheless, their identification may have been challenging because of the difficulty in observing delicate membranous cellular structures within tissues.

Future studies aiming to characterize telocyte-telocyte cell-cell interactions and the mechanisms through which they communicate with each other and other cells will enhance the understanding of how telocytes control homeostasis. Interfering with the telocyte network without affecting the function of individual telocytes will reveal the operative role of this network.

At each level of organization (cells, tissues, organs) structure is closely related to function. Because intestinal enterocytes look very different from skeletal muscle cells, telocyte unique cell structure adapts. The polarized and unique structure of telocytes, with multiple processes reaching a hundred microns’ distance from the cell body, allows an individual telocyte to contact and potentially signal several epithelial cells, including other types of cells. Villus tip telocytes regulate epithelial and endothelial villus tip cells.^31,32 A fundamental question is how do telocytes achieve directed cell-specific communication without influencing a broad domain of cells?

Cell-cell communication in response to diverse stimuli and cues requires a highly dynamic response of the interacting cells to external stimuli. These cells sense environmental cues, by expressing surface receptors to external ligands, and adapt their molecular identity to fulfill a biologic purpose. At the cellular level, exposure to a variety of signals simultaneously, sometimes from multiple sources, demands a quick, coordinated, local, precise, and very specific reaction. The polarized cell structure of telocytes necessitates remote subcellular compartments to precisely remodel their protein profile in response to specific stimuli.

Subcellular RNA localization has been identified as a prominent mechanism ensuring local protein production in various polarized cells, such as oocytes,^42,43 migrating fibroblasts,44, 45, 46 and neurons.47, 48, 49, 50, 51 Previous studies have demonstrated that mRNA molecules of different sets of signaling ligands are localized within telopodes, juxtaposed to the epithelium exactly at the site expected to be enriched for the corresponding ligand proteins.²² Furthermore, telopodes contain a substantial amount of endoplasmic reticulum, indicating the presence of machinery necessary for local protein synthesis. Exploring the cell biology of telopodes as a potential independent compartment from the cell soma and the machinery for subcellular compartmentalization of mRNAs and proteins within telopodes will uncover the mechanisms that enable telocytes’ potential integrative yet individual intensive cross-talk.

Revealing the role of telocytes in intestinal stem cell function has presented exciting opportunities to address fundamental questions across biology and explore mechanisms related to tissue repair and disease. However, it also brings forth significant technical challenges in the field. What conditions are necessary to effectively detect and visualize a delicate membranous network within tissues? Additionally, how can protocols be developed to dissociate telocytes within tissues and characterize them in vitro, especially considering the presence of extremely long cellular protrusions?

The development of methods for more accurate characterization of individual telocytes and their 3-dimensional network in vivo, and the investigation of the cell biology of both individual telocytes and their network, holds the potential to significantly inform and transform the understanding of epithelium and its regulation. Regardless, the identification of telocytes that are in closest contact to and potentially sense and signal all intestinal epithelial cells is a paradigm shift in understanding the mechanisms by which epithelial cell fate decisions are made and morphogenetic fields are established.