A.D. Vrapciu

Subsets of telocytes: Myocardial telocytes

Telocytes (TCs) are morphologically defined as small-sized cells with long, thin, moniliform processes called telopodes (Tps). Numerous papers imply that TCs are a distinctive cell type, and that transmission electron microscopy (TEM) is the gold standard tool for their identification. We aimed to reproduce previous studies on myocardial TCs to check their validity. For this purpose we performed an immunohistochemical study on human cardiac samples from six autopsied donor cadavers, using antibodies against CD10, CD31, CD34, CD146, Ki67, alpha-smooth muscle actin (α-SMA), Platelet-Derived Growth Factor Receptor-alpha (PDGFRα) and laminin. Additionally we performed a TEM study on cardiac samples from three human autopsied donor cadavers and five adult Sprague-Dawley rats. We found endothelial cells (ECs), cords, and filopodia-projecting endothelial tip cells (ETCs) that expressed CD10, CD31, CD34, CD146, and PDGFR-α. Often, endothelial cells closely neighbored the sarcolemmal basal laminae. Endothelial progenitor cells, as well as nascent capillaries, were CD31+/CD34+. Proliferative endothelial cells expressed Ki67. In larger vessels we found pericytes that expressed CD146 and α-SMA; scarce α-SMA-expressing spindle-shaped cells lining cardiomyocytes were suggestive of a pericytic role in angiogenic sprout guidance. The TEM study showed that endothelial tubes are almost exclusively found in the narrow myocardial interstitia. ECs that built them up appeared identical to the cells that previous TEM studies have suggested to be myocardial telocytes. A subset of stromal cells with TC-like phenotype and telopodes-like processes actually seem to configure blood vessels, and therefore belong to the endothelial lineage. This study shows that data presented in previous studies on myocardial telocytes is not enough to allow the reproducibility of the results. At least a subset of cells considered to be TCs might belong to the endothelial lineage.

Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.

CD146- and CD105-positive phenotypes of retinal ganglion cells. Are these in situ proofs of neuronal regeneration?

The in vivo identity of stem cells is not yet clear. Numerous studies involve the perivascular niches as providers of stem cells during regenerative processes. CD146, in humans, as well as gicerin, at chicken, play roles in neuronal development and neurites extension. CD146 is a marker of stemness but also a pericytary marker. Stem cells in vascular niches can differentiate in neural cells. By applying CD146 and CD105 antibodies on human retinas from glaucomatous eyes, CD146-positive retinal ganglion cells (RGCs) were found, some being placed in perivascular positions; ongoing processes of neurites extension were related to these neurons. On other hand, RGCs were positively labeled by CD105 antibodies. These results support the hypothesis that in glaucoma eyes the CD146-positive RGCs result from regenerative processes driven by stem cells in the retinal perivascular niches. Further experiments are needed to evaluate whether CR146-positive neurons indicate also a physiological process of maintenance of retina.

The molecular phenotypes of ureteral telocytes are layer-specific

Telocytes (TC) are the delicate interstitial (stromal) cells defined by their long, thin and moniliform processes termed telopodes. Numerous studies determined that different subsets of telocytes populate almost all tissues and attempted to relate these subsets to various functions, from cell signaling to tissue repair and regeneration. Extremely few studies addressed the urinary tract though few data on the molecular pattern of the urinary TCs actually exist. We therefore hypothesized that subsets of urinary TCs co-localize within the human ureter and we aimed at performing an immunohistochemical study to evaluate the tissue-specific molecular pattern of TCs. On sample tissues of proximal ureter drawn from ten human adult patients during surgery were applied primary antibodies against CD34, CD105, von Willebrand Factor, the heavy chain of smooth muscle myosin (SMM) and c-erbB-2. The molecular pattern indicated three different subsets of ureteral TCs which are neither endothelial nor epithelial in nature: (a) type I: the CD34-/CD105+ TCs of the superficial layer of lamina propria; (b) type II: the CD34+/CD105± myoid TCs of the deep layer of lamina propria and (c) type III: the CD34+/CD105+ perivascular TCs. Although apparently different, all these subsets of TCs could belong to the stem/progenitor niche of the ureter.

Stem potentialities of the human iris - An in situ immunohistochemical study.

According to recent findings multiple human tissues harbor stem cells which, in turn, have different levels of stemness. We performed an immunohistochemical study on paraffin-embedded samples to test if the in situ stromal cells of the iris of the human eye (EI) have immune stem/progenitor phenotypes. Eviscerated post-traumatic eyes from eight patients were studied. These irises were found to contain fibroblastoid stromal cells with a CD34+/CD45+/CD105+/CD117+/DOG1+/PDGFR-α+/vimentin+/nestin-/collagen III- phenotype. These were assumed to be possible stem/progenitor cells involved in physiological processes of iridial stromal maintenance. All the vascular endothelia were CD34+/CD105+/vimentin+. Newly formed nestin+ endothelia were also found; this finding was supported by evidence of filopodia-projecting CD34+ endothelial tip cells, which demonstrated active processes of sprouting angiogenesis. The phenotype of the stromal cells also suggests a role of the circulating fibrocytes in iridial regenerative processes.

Variable relations of the trochlear nerve with the pontomesencephalic segment of the superior cerebellar artery

The superior cerebellar artery (SCA) is, perhaps, the most anatomically constant cerebellar artery which, in its lateral pontomesencephalic course, is crossed above by the trochlear nerve (CNIV). The SCA may determine, as an offending vessel, CNIV compression and superior oblique myokymia and thus surgical decompression may be indicated. In this regard an accurate knowledge of the variational possibilities of the SCA–CNIV is needed. Such rare neurovascular variants are reported here. The variables are determined by the length of the SCA, and the course of the CNIV as referred to the rostral (RT) and caudal (CT) trunks of the SCA. The CNIV may be pinched between the origins of the RT and CT, may pass above the RT or the SCA main trunk, and even between the primary branches of the RT. The CNIV was found compressed between the RT and the brainstem. Perhaps the most spectacular variation was a CNIV coursing through an arterial ring formed by the RT and CT which were anastomosed distally to the CNIV. The possibilities of neurovascular relations between the CNIV and the SCA should be considered when CNIV palsy, or surgical decompression, are estimated.

Anoctamin 1 Positive Esophageal Interstitial Cajal Cells in Late Stage Human Embryos

Interstitial cells of Cajal (ICCs) are located in various smooth muscle organs and act as pacemaker cells, or ensure neuromodulation or mechanosensory roles. The study aims to investigate functional states of human ICCs in morphogenesis, focusing on the anoctamin 1 phenotype. The investigation was performed in five late stage human embryos with lengths varying between 23 and 29 mm. Immunohistochemistry on paraffin embedded specimens was performed for a series of antibodies: α‐smooth muscle actin (α‐SMA), desmin, CD31, CD34, CD117/c‐kit, DOG1, and nestin. Longitudinal and circular muscle layers were α‐SMA+/desmin+/nestin+. An immature microvascular layer located in the inner submucosa was CD34+/CD31+/α‐SMA+/nestin+; endothelial tip cells were supporting active processes of sprouting angiogenesis. A CD34+/CD31‐ mesenchymal network was found in the circular muscle layer. CD117/c‐kit+ multipolar ICCs with dichotomizing processes were found mostly in the myenteric plexus layer; processes were configuring a network within the circular muscle layer where intramuscular ICCs were scarcely found. A strong DOG1+ reaction was found for the ICCs of the myenteric plexus layer apposed on the outer surface of the circular muscle layer, and for the intramuscular ICCs. The evidence of a sublayer of DOG1+ myenteric ICCs is suggestive for a subpopulation of ICCs being qualified for pacemaking at this early developmental stage. Anat Rec, 297:301–307, 2014.

Regenerative Potential of Human Schneiderian Membrane: Progenitor Cells and Epithelial-Mesenchymal Transition

An innate osteogenic potential of the Schneiderian membrane (SM) is progressively assessed in studies ranging from non‐human species to human subjects. It has relevance for endosteal placement and osseointegration. Nestin‐expressing osteogenic progenitor cells are allegedly involved in bone formation and remodelling. Nestin phenotype was not assessed previously in human SM. We therefore aimed to fill that particular gap in the literature. Bioptic samples of human adult SM were obtained during surgery from eight adult patients, operated for non‐malignant pathologies. Immunohistochemistry on paraffin‐embedded tissue samples used primary antibodies against nestin, CD45, CD146, cytokeratin 7 (CK7), and alpha‐smooth muscle actin (α‐SMA). Nestin expression was consistently found in endothelial cells, and was scarcely encountered in pericytes, putative stromal stem/progenitor cells, as well as in glandular epithelial cells. Moreover, woven bone formation in the periosteal layer of the SM can also be regarded as evidence of the osteogenic potential of this membrane. Nestin and CD45 expression in cells of the primary bone supports the osteogenic potential of SM nestin‐expressing cells and a possible involvement of hematopoietic stem cells in maxillary sinus floor remodeling. CD146, a known inducer of epithelial‐mesenchymal transition (EMT), was expressed in epithelia, as was CK7. Isolated stromal cells were found expressing CD146, CK7 and α‐SMA, suggesting that regenerative processes happening in the SM may also involve processes of EMT which generate stem/progenitor cells. This study provides additional evidence for the regenerative potential of the Schneiderian membrane and identifies potential roles for cells of its stem niche in osteogenesis. Anat Rec, 298:2132–2140, 2015.

The human retinal stem niches could overlap a vascular anatomical pattern.

Stem cells are undifferentiated cells which have the unique potential to self-renew and to supply, via intermediate stages of transit amplifying cells (TACs), differentiated cells. Within the stem cell niches not only heterologous cells, but also differentiated progeny of stem cells provide regulation to the stem cells parents. Stem cells can differentiate in various cell types during tissue maintenance or repair. Mesenchymal stem/stromal cells (MSCs) are the best candidates for regenerative medicine. However, the standard for identifying MSCs indicates their plastic adherence in standard culture conditions. Beyond a great diversity of biomarkers identifying MSCs in in vitro conditions (such as the positive expression of CD73, CD90 and CD105, and negative expression of CD14, CD34 and CD45), their exact identity in vivo is not yet clear, although it was suggested either a pericytic, or a fibroblastic origin (Lv et al., 2014). The most reliable molecules to sort MSCs are Stro-1, SSEA-4 and CD146 (Lv et al., 2014). Evidence suggests that neural progenitors may exist throughout life in the human retina (Mayer et al., 2005). Different sources for replacement of retinal neurons have been identified. Nevertheless, extrinsic providers of stem/progenitor cells, such as the bone marrow, could supply the adult retina during regenerative processes. It is intended here to bring arguments for a better focus in researches on the perivascular stem niches of human retina. This perspective results from observational studies in humans and experimental studies are further needed to assess the validity of the different perivascular stem niches. The retinal stem cells (RSCs) hypothesis resulted from observations in lower vertebrates (Kiyama et al., 2012) of multipotent cells of the ciliary marginal zone (CMZ) which constantly produce throughout life new retinal cells; the CMZ niche is anatomically related to the peripheral retina. All types of retinal neurons can arise from the CMZ (Kiyama et al., 2012). Human RSCs were tested in vitro and were found able to survive, migrate, integrate and to differentiate, especially in photoreceptors (Coles et al., 2004). However, are the CMZ-derived RSCs able, in vivo, to completely supply retina with progenitors, even the central retina? Retinal pericytes share in rodents some phenotypic features, such as the CD146 expression, with archetypal MSCs (Wittig et al., 2013). Rat aorta pericytes were induced to differentiate into neural-like phenotypes (Montiel-Eulefi et al., 2012). A differentiation scheme for pericytes was presented for human adult adipose tissue, according to which pericytes with stem cell properties, including the CD146 expression, differentiate from the inside out, give rise to adventitial CD146+ TACs, then to CD146− MSC-like supra-adventitial stromal cells (Zimmerlin et al., 2012). Expression of a stem/progenitor phenotype in vascular smooth muscle cells (VSMCs) is an age-related process associated with arterial remodelling (Ferlosio et al., 2012). Proliferating VSMCs undergo a process of dedifferentiation and this could be equally true for pericytes. We previously discussed that CD146-positive human retinal ganglion cells could result from regenerative processes driven by stem cells in the retinal perivascular niches (Vrapciu et al., 2014). This is consistent with studies of CD146+ pericytes, which determined and strongly support the concept of perivascular stem niche (Crisan et al., 2012; Chen et al., 2013). In order to identify neural progenitor cells for therapies of central nervous system disorders, such as posttraumatic injuries or neurodegenerative disorders, two pericytes subtypes were described in mouse experiments; in type-2, Nestin-GFP+/NG2-DsRed+/CD146+, pericyte-derived Tuj1+/ CD146+/PDGFRbeta+/NG2+ cells, the nerve growth factor receptor was exclusively expressed, thus it can identify pericytes with neurogenic potential (Birbrair et al., 2013). We found S100 protein+ cells in the optic nerve head (ONH) (Figure 1), which suggests the presence of neural cells. Neurons were not previously assessed in the ONH. They could be the result of a perivascular stem niche activation related to an ongoing process of regeneration. This is supported by CD146+ cells with neuronal morphologies which were found nearing vessels within the inner layers of central retina and the ONH (Figure 1). Thus, along vessels deriving from the central artery of retina a perivascular niche could supply with newly formed neurons the ganglion cell layer of retina during reparatory/regenerative processes. Tissue samples of a previously described lot (Vrapciu et al., 2014) were used for CD146 (clone N1238, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, U.K., 1:50) and S100 (polyclonal, Dako, Glostrup Denmark, 1:400) protein labeling. Figure 1 CD146 (A–C) and S100 protein (D) labeling of human retina. Vascular/progenitor stem cells able to differentiate into endothelial and smooth muscle cells can be equally native vascular wall residents and bone marrow-derived cells. Pericytes are able to repair endothelia, in a centripetal/luminal direction but are also able to replenish, in a centrifugal manner, the perivascular tissue-specific differentiated cells. The vascular-related stem/progenitor niche could be regarded anatomically, being superposed on the vascular architecture of an organ/tissue. In this regard, the eye, which is supplied from two vascular systems (i.e., the ciliary arteries and the central retinal artery), possesses two different vascular/perivascular stem niches, which may not be exclusive. The ciliary arteries niche can be viewed as a stem/progenitor anatomic supplier of the CMZ. The central artery of retina which courses through the optic disc, or the ONH, further branches within the nerve fibers retinal layer being thus anatomically able to support retinal ganglion cells replenishment from the vascular/perivascular stem niche. However, regenerative processes in the outer layers of the central retina could also rely on the ciliary arteries system. So, the stem/progenitor niche of the central retinal artery could relate to retinal ganglion cells regeneration and the arteries of the uvea could be regarded, but not exclusively, as stem/progenitor providers of the outer retinal layers. Further studies are mandatory to assess the stem potentiality of the choroid which is anatomically able to ensure regeneration of the central retina, in a similar manner to the CMZ which is able to supply stem/progenitor cells in the peripheral retina.

Stromal cells/telocytes and endothelial progenitors in the perivascular niches of the trigeminal ganglion

Stromal cells/telocytes (SCs/TCs) were recently described in the human adult trigeminal ganglion (TG). As some markers are equally expressed in SCs/TCs and endothelial cells, we hypothesized that a subset of the TG SCs/TCs is in fact represented by endothelial progenitor cells of a myelomonocytic origin. This study aimed to evaluate whether the interstitial cells of the human adult TG correlate with the myelomonocytic lineage. We used primary antibodies for c-erbB2/HER-2, CD31, nestin, CD10, CD117/c-kit, von Willebrand factor (vWF), CD34, Stro-1, CD146, α-smooth muscle actin (α-SMA), CD68, VEGFR-2 and cytokeratin 7 (CK7). The TG pial mesothelium and subpial vascular microstroma expressed c-erbB2/HER-2, CK7 and VEGFR-2. SCs/TCs neighbouring the neuronoglial units (NGUs) also expressed HER-2, which suggests a pial origin. These cells were also positive for CD10, CD31, CD34, CD68 and nestin. Endothelial cells expressed CD10, CD31, CD34, CD146, nestin and vWF. We also found vasculogenic networks with spindle-shaped and stellate endothelial progenitors expressing CD10, CD31, CD34, CD68, CD146 and VEGFR-2. Isolated mesenchymal stromal cells expressed Stro-1, CD146, CK7, c-kit and nestin. Pericytes expressed α-SMA and CD146. Using transmission electron microscopy (TEM), we found endothelial-specific Weibel-Palade bodies in spindle-shaped stromal progenitors. Our study supports the hypothesis that an intrinsic vasculogenic niche potentially involved in microvascular maintenance and repair might be present in the human adult trigeminal ganglion and that it might be supplied by either the pial mesothelium or the bone marrow niche.