title: Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles.
abstract: An increasing number of studies on stem cells suggests that the therapeutic effect they exert is primarily mediated by a paracrine regulation through extracellular vesicles (EVs) giving solid grounds for stem cell EVs to be exploited as agents for treating diseases or for restoring damaged tissues and organs. Due to their capacity to differentiate in all embryonic germ layers, amniotic fluid stem cells (AFCs), represent a highly promising cell type for tissue regeneration, which however is still poorly studied and in turn underutilized. In view of this, we conducted a first investigation on the expression of human hTERT gene - known to be among the key triggers of organ regeneration - in AFCs and in the EVs they secrete.
  
  
  
  Isolated AFCs were evaluated by RT-qPCR for hTERT expression. The clones expressing the highest levels of transcript, were analyzed by Immunofluorescence imaging and Nuclear/cytoplasmic fractionation in order to evaluate hTERT subcellular localization. We then separated EVs from FBS depleted culture medium by serial (ultra) centrifugations steps and characterized them using Western blotting, Atomic force Microscopy and Nanoplasmonic assay.
  
  
  
  We first demonstrated that primary cultures of AFCs express the gene hTERT at different levels. Then we evidenced that in AFCs with the higher transcript levels, the hTERT protein is present in the nuclear and cytoplasmic compartment. Finally, we found that cytosolic hTERT is embodied in the EVs that AFCs secrete in the extracellular milieu.
  
  
  
  Our study demonstrates for the first time the expression of the full protein hTERT by AFCs and its release outside the cell mediated by EVs, indicating a new extra telomeric role for this protein. This finding represents an initial but crucial evidence for considering AFCs derived EVs as new potential sources for tissue regeneration.
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy. Electronic address: annalisa.radeghieri@unibs.it.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Radeghieri
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Savio
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zendrini
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Di Noto
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Salvi
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bergese
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Italy, Viale Europa 11, 25123, Brescia, Italy. Electronic address: giovanna.piovani@unibs.it.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Piovani
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000201:enzymology*; D002469:Cell Separation; D003593:Cytoplasm / Q000201:enzymology*; D000067128:Extracellular Vesicles / Q000201:enzymology; D006801:Humans; D018625:Microscopy, Atomic Force; D012038:Regeneration; D013234:Stem Cells / Q000201:enzymology* / Q000502:physiology; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D019098:Telomerase
keywords: Amniotic fluid cells; Extracellular vesicles; Gene expression; Intracellular distribution; hTERT