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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-23 18:24 |
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Conference: Bucharest University Faculty of Physics 2017 Meeting
Section: Biophysics; Medical Physics
Title:
Characterization of cryopreserved and senescent amniotic fluid stem cells in culture
Authors:
Bogdan AMUZESCU (1), Florin IORDACHE (2), Andrei CONSTANTINESCU (2), Eugen ANDREI (2), Ferdinand HALITZCHI (1), Lorand SAVU (3), Horia MANIU (2)
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Affiliation:
1) Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest
2) Dept. Regenerative Medicine, “N. Simionescu” Institute of Cell Biology and Pathology, Bucharest
3) GeneticLab SRL, Bucharest
E-mail
bogdan@biologie.kappa.ro
Keywords:
Amniotic fluid stem cell, neural differentiation, patch-clamp, immunofluorescence, flow cytometry, RT-PCR
Abstract:
Mesenchymal stem cells (MSC) have been used recently in a variety of medical applications, including traumatic or ischemic injury of the central or peripheral nervous system, due to ease of preparation from different sources and lack of bioethical concerns. Regenerative medicine applications require steady-state marker expression and functional properties. Therefore we have tested two preparations of human amniotic fluid stem cells (AFSC) (a particular type of MSC): cryopreserved AFSC (frozen after 1-3 passages and tested in the first week after replating) and senescent AFSC (cultured for 6 weeks with medium exchange without passage). AFSC were obtained from amniocentesis samples (upon written informed consent), cultured in AmnioMax (Life Technologies) and cryopreserved in 10% DMSO. We performed whole-cell patch-clamp experiments, immunophenotyping by flow cytometry, gene expression profiling with the RT2 Profiler PCR Array Human Cellular Senescence kit (Qiagen), immunofluorescence and RT-PCR for neural progenitor markers. Both types of cells featured steady outward rectifying currents (Ior) with an inactivatable (A-type) component (IA), current fluctuations suggestive of big conductance Ca-dependent K+ channels (BK), fast voltage-dependent Na+ currents (INa). IA was less expressed in cryopreserved cells, and a subset of them featured an inward rectifying current component (IK ir) and larger INa, suggestive of neuronal differentiation. To further prove this differentiation we evidenced neuronal markers beta3-tubulin, NF-200 and CD56 (NCAM) via immunofluorescence and flow cytometry, and markers neuroD1 and GIRK2 via RT-PCR. The immunophenotype showed decreases in CD44, CD90 and CD133 levels in senescent vs. cryopreserved AFSC, the presence of HLA-ABC and absence of HLA-DR, while the senescence gene profiling showed marked increases for interferon-gamma and telomerase reverse transcriptase (TERT) transcription levels in senescent AFSC. In conclusion, senescent AFSC showed specific changes in immunophenotype and gene expression profile, while cryopreserved AFSC featured spontaneous differentiation into neural precursors.
References:
Iordache F., Constantinescu A., Andrei E., Amuzescu B., Halitzchi F., Savu L., Maniu H. (2016): Electrophysiology, immunophenotype, and gene expression characterization of senescent and cryopreserved human amniotic fluid stem cells, J. Physiol. Sci. 66:463-476
Acknowledgement:
This work was supported from grants 1877/2014 ‘‘SORTIS’’ to Horia Maniu and
PN2 80/2012 to Aurel Popa-Wagner.
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