The morphological analysis of the intestinal stroma revealed the presence of slender, elongated cells. They were in the subepithelial region around the folds’ base and along their length in both the proximal and distal intestine. Hematoxylin–eosin staining highlighted a typical distribution generating a supportive network within the interstitial space adjacent to the enterocytes’ basement membrane (Figure 1).

The observation using the light microscope revealed that this cell population was characterized by a peculiar moniliform shape due to the presence of a thin and elongated nucleus and stretched, slender cytoplasmic projections (Figure 2).

Moreover, Periodic Acid Schiff–Alcian Blue at pH 2.5 (PAS–AB 2.5), Masson’s trichrome, Mallory’s triple stain, Crossman’s trichrome histochemical staining (Figure 3 and Figure 4), and toluidine-blue-stained semi-thin sections (Figure 5) further emphasized the generation of a continuous elaborate network underneath the intestinal epithelium.

The examination with TEM confirmed the presence of stromal cells underlying the epithelium which were clearly distinguishable from the common fibroblasts found in the lamina propria. They presented an elongated nucleus containing heterochromatin clusters with limited cytoplasm and very long, thin projections. Moreover, they possessed an irregular shape with a variable number of branches, generally 2–3 per cell (Figure 6). These branches, called telopods (TPs), developed in a non-linear fashion, presented peripheral dilations, or podoms (Pm), that hosted cellular organelles and vacuoles (Figure 7), and were surrounded by collagen fibers (Figure 8).

Overall, histological and ultrastructural features confirmed the identity of these cells as telocytes.

In situ hybridization showed that pdgfrα was expressed in many cells located exclusively in the stroma. Here, two clearly distinct pdgfrα⁺ cell populations were visible. The first displayed high pdgfrα expression, had slender, elongated nuclei and was located just below the basement membrane of the intestinal epithelium. These cells created a continuous plexus that extended from the basal to the apical compartment (Figure 9A,B; Scale bar 100 µm). They showed more intense pdgfrα expression at the fold apex and along its length, while it tended to become lower around the folds’ base (Figure 9C,D; Scale bar 50 µm). The other pdgfrα ⁺ population was characterized by a low signal and was located in the innermost region of the lamina propria, away from the epithelium (Figure 8E; Scale bar 50 µm).

Foxl1+ cells were observed along the folds’ connective axis in both the proximal and distal intestine. Its expression was rare and limited to a few cells located in the peri-epithelial space at the folds’ base. In the distal intestine, foxl1⁺ cells were found not only around the folds’ base but also around the base of the secondary folds protruding from the complex plicae, which are characteristic of this region. As opposed to pdgfrα, scattered mRNA dots of foxl1 were also found in the epithelium lining the base of the folds (Figure 10).

Furthermore, pdgfrα and foxl1 transcripts were co-expressed in a few elongated cells localized just beneath the basal membrane, at the base and at the apex of the intestinal folds, indicating the presence of a small telocyte subpopulation along the whole intestinal length (Figure 11).