Abstract
Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31−/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα−/vimentin+) and fibroblasts (CD31−/CD34−/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.
Highlights
Telocytes (TCs) are a long-neglected peculiar type of stromal cells that were firstly described by Popescu and Faussone-Pellegrini in 2010 [1] and that have been subsequently identified, over the last decade, in both perivascular and interstitial compartments of a variety of organs in humans and other vertebrates [2,3,4,5,6,7,8,9,10,11,12]
Since the most widely used TC marker, namely CD34, is expressed by endothelial cells (ECs), the first step of the protocol consisted of a microbead-based cell selection for the pan-endothelial cell surface marker CD31, known as platelet/endothelial cell adhesion molecule-1, to separate CD31+ cells from CD31−
The ability of TCs to form complex interstitial networks and communicate with neighboring cells by establishing multiple intercellular contacts or releasing extracellular vesicles containing bioactive molecules, together with the evidence that such TC networks are severely impaired in a variety of pathologies, led to proposing that these cells may exert a wide spectrum of functions important for the maintenance of tissue homeostasis [2,4,5,10,28,29,57,58]
Summary
Telocytes (TCs) are a long-neglected peculiar type of stromal cells that were firstly described by Popescu and Faussone-Pellegrini in 2010 [1] and that have been subsequently identified, over the last decade, in both perivascular and interstitial compartments of a variety of organs in humans and other vertebrates [2,3,4,5,6,7,8,9,10,11,12]. Since CD34 is strongly expressed by vascular endothelial cells (ECs), CD34/CD31 double immunostaining has been proven to be extremely useful to unequivocally distinguish between CD34+/CD31− TCs and CD34+/CD31+ ECs of blood vessels, especially considering that the latter, when captured in tissue sections, may often exhibit an elongated profile resembling that of TCs [7,8,9,24,25] Another marker commonly used to identify TCs by immunohistochemistry is platelet-derived growth factor receptor (PDGFR)α that has been demonstrated to be coexpressed with CD34 in TCs from different organs [5,8,9,10,11,25,26,27]
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