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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-23 18:01 |
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Conference: Bucharest University Faculty of Physics 2019 Meeting
Section: Biophysics; Medical Physics
Title:
Expression of pluripotency, surface and senescence markers in human amniocytes and in vitro induction of neural progenitor formation
Authors:
Bogdan AMUZESCU (1), Bogdan Florin EPUREANU (1), Florin IORDACHE (2), Antonia Teona DEFTU (1), Răzvan AIRINI (1), Dorin ALEXANDRU (2), Lorand SAVU (3,4), Dan Florin MIHAILESCU (1), Horia MANIU (2)
*
Affiliation:
1 Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest, Bucharest, Romania
2 Dept. Regenerative Medicine, “N. Simionescu” Institute of Cell Biology and Pathology, Bucharest, Romania
3 Genetic Lab S.R.L., Bucharest, Romania
4 Fundeni Clinical Institute, Bucharest, Romania
E-mail
bogdan@biologie.kappa.ro
Keywords:
Amniocyte, mesenchymal stem cell, neural progenitor, replicative senescence, patch-clamp, indirect immunofluorescence, flow cytometry, qRT-PCR
Abstract:
We assessed functional and molecular properties of human amniocytes isolated from amniotic fluid samples taken for routine diagnosis by amniocentesis at Genetic Lab upon written informed consent of patients. The cells were cultured in BIO-AMF-2 medium and cryopreserved in 10 % dimethylsulfoxide or maintained in vitro for 6 consecutive weeks without passages to achieve replicative senescence. Senescent amniocytes featured large percentages of staining with senescence-associated beta-galactosidase (93 % vs. 7 % in cryopreserved cells) and lack of 5-ethynyl-2’-deoxyuridine (EdU) incorporation (by flow cytometry upon AlexaFluor®488 binding to EdU) after 24 h of exposure to the compound. We evidenced by flow cytometry the presence of mesenchymal stem cell markers CD29, CD44, CD146, CD49e, CD90, CD73, CD56, CD54, CD105; absence of hematopoietic stem cell markers CD45, CD117, CD133; presence of pluripotency cell surface antigens SSEA-1, SSEA-4, TRA1-60, TRA1-81 (by flow cytometry) and pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT-PCR) in both populations, indicating an expansion of the stem cell population during cell culturing. We also evidenced increased expression in senescent amniocytes of inflammatory genes (IL-1alpha, IL-6, IL-8, TGF-beta, NF-kappaB p65), cell cycle regulatory genes (p16INK4A), and cytoskeletal elements (beta-actin). Further, we attempted neural differentiation of cryopreserved amniocytes by culturing them in 6-well plates in a special medium (ENStemA-Neural Expansion Medium, Millipore) supplemented with all trans-retinoic acid (R2625, Sigma) 20 μM, beta-FGF (GF003, EMD Millipore) 20 ng/mL, and EGF (Life technologies) 20 ng/mL. After a few days the cell shape changed from typical mesenchymal to rounded with multiple dendrite-like prolongations, and cell divisions decreased in frequency. After two weeks the neural-progenitor like cells featured positive indirect immunofluorescent staining for betaIII-tubulin, intermediate weight neurofilaments (NF-200), and glial fibrillary acidic protein (GFAP). However, by patch-clamp we identified only delayed rectifier outward K+ currents, seldom BK or inward rectifier K+ currents, and no inward Na+ or Ca2+ current.
Acknowledgement:
This study was funded from Competitiveness Operational Programme 2014–2020 project P_37_675 (contract no. 146/2016), Priority Axis 1, Action 1.1.4, co-financed by the European Funds for Regional Development and Romanian Government funds, from grant PNII-III-P1-1.1-PD-2016-1660 (No.19/2018) BIOPRINT, and from grant PN-III-P1-1.2-PCCDI-2017-0820 / 67PCCDI/2018 (MIMOSA).
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