title: Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia.
abstract: BACKGROUND
  Endothelial dysfunction is found in different pathologies such as diabetes and renal and heart diseases, representing one of the major health problems. The reduced vasodilation of impaired endothelium starts a prothrombotic state associated with irregular blood flow. We aimed to explore the potential of amniotic fluid stem (AFS) cells as a source for regenerative medicine in this field; for the first time, we focused on third-trimester amniotic fluid AFS cells and compared them with the already-described AFS cells from the second trimester.
  
  
  METHODS
  Cells from the two trimesters were cultured, selected and expanded in normoxia (20 % oxygen) and hypoxia (5 % oxygen). Cells were analysed to compare markers, proliferation rate and differentiation abilities. Endothelial potential was assessed not only in vitro-Matrigel tube formation assay, acetylated human low-density lipoprotein (AcLDL) uptake-but also in vivo (Matrigel plug with cell injection and two animal models). Specifically, for the latter, we used established protocols to assess the involvement of AFS cells in two different mouse models of endothelial dysfunction: (1) a chronic ischemia model with local injection of cells and (2) an electric carotid damage where cells were systemically injected.
  
  
  RESULTS
  We isolated and expanded AFS cells from third-trimester amniotic fluid samples by using CD117 as a selection marker. Hypoxia enhanced the proliferation rate, the surface protein pattern was conserved between the trimesters and comparable differentiation was achieved after culture in both normoxia and hypoxia. Notably, the expression of early endothelial transcription factors and AngiomiRs was detected before and after induction. When in vivo, AFS cells from both trimesters expanded in hypoxia were able to rescue the surface blood flow when locally injected in mice after chronic ischemia damage, and importantly AFS cells at term of gestation possessed enhanced ability to fix carotid artery electric damage compared with AFS cells from the second trimester.
  
  
  CONCLUSIONS
  To the best of our knowledge, this is the first research work that fully characterizes AFS cells from the third trimester for regenerative medicine purposes. The results highlight how AFS cells, in particular at term of gestation and cultured in hypoxia, can be considered a promising source of stem cells possessing significant endothelial regenerative potential.
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. schiavo.andrea.alex@gmail.com.|Department of Woman and Children Health, University of Padova, via Giustinani 2, 35100, Padova, Italy. schiavo.andrea.alex@gmail.com.
authors.forename: Andrea Alex
authors.initials: AA
authors.lastname: Schiavo
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. c.franzin@irpcds.org.
authors.forename: Chiara
authors.initials: C
authors.lastname: Franzin
authors.affiliation: Venetian Institute of Molecular Medicine, via Orus 2, 35129, Padova, Italy. mattia.albiero@gmail.com.|Medicine Department (DIMED), University of Padova, via Giustiniani 2, 35100, Padova, Italy. mattia.albiero@gmail.com.
authors.forename: Mattia
authors.initials: M
authors.lastname: Albiero
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. m.piccoli@irpcds.org.
authors.forename: Martina
authors.initials: M
authors.lastname: Piccoli
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. giovanna.spiro@gmail.com.|Medicine Department (DIMED), University of Padova, via Giustiniani 2, 35100, Padova, Italy. giovanna.spiro@gmail.com.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Spiro
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. e.bertin@irpcds.org.
authors.forename: Enrica
authors.initials: E
authors.lastname: Bertin
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. l.urbani@ucl.ac.uk.|Stem Cells and Regenerative Medicine Section, Developmental biology and Cancer Program, Institute of Child Health, University College London, 30 Guilford Street, WC1N 1EH, London, UK. l.urbani@ucl.ac.uk.
authors.forename: Luca
authors.initials: L
authors.lastname: Urbani
authors.affiliation: Department of Woman and Children Health, University of Padova, via Giustinani 2, 35100, Padova, Italy. visentin78@gmail.com.
authors.forename: Silvia
authors.initials: S
authors.lastname: Visentin
authors.affiliation: Department of Woman and Children Health, University of Padova, via Giustinani 2, 35100, Padova, Italy. erich.cosmi@unipd.it.
authors.forename: Erich
authors.initials: E
authors.lastname: Cosmi
authors.affiliation: Venetian Institute of Molecular Medicine, via Orus 2, 35129, Padova, Italy. gianpaolo.fadini@unipd.it.|Medicine Department (DIMED), University of Padova, via Giustiniani 2, 35100, Padova, Italy. gianpaolo.fadini@unipd.it.
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Fadini
authors.affiliation: Stem Cells and Regenerative Medicine Section, Developmental biology and Cancer Program, Institute of Child Health, University College London, 30 Guilford Street, WC1N 1EH, London, UK. paolo.decoppi@gosh.nhs.uk.
authors.forename: Paolo
authors.initials: P
authors.lastname: De Coppi
authors.affiliation: Stem Cells and Regenerative Medicine Laboratory, Foundation Institute of Pediatric Research Città della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy. m.pozzobon@irpcds.org.
authors.forename: Michela
authors.initials: M
authors.lastname: Pozzobon
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology; D000818:Animals; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D066026:Endothelial Progenitor Cells / Q000502:physiology* / Q000637:transplantation; D005260:Female; D006614:Hindlimb / Q000098:blood supply; D006801:Humans; D007511:Ischemia / Q000628:therapy; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic; D011247:Pregnancy; D011263:Pregnancy Trimester, Third