Faculty of Physics - University of Bucharest

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FACULTY OF PHYSICS

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2024-11-22 1:56

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Conference: Bucharest University Faculty of Physics 2018 Meeting

Section: Biophysics; Medical Physics

Title:

Senescence changes in mesenchymal stem cells cultures

Authors:

Răzvan AIRINI (1), Florin IORDACHE (2), Dorin ALEXANDRU (2), Lorand SAVU (3), Florin EPUREANU (1), Dan Florin MIHĂILESCU (1), Bogdan AMUZESCU (1),Horia MANIU (2)

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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

E-mail

airinirazvan@gmail.com

Keywords:

mesenchymal stem cell, amniotic fluid stem cell, senescence-associated secretory phenotype, automated patch-clamp, flow cytometry, qRT-PCR

Abstract:

Mesenchymal stem cells (MSC) could have medical applicability in health conditions such as heart diseases or brain injuries. However, their ability to reduce inflammation is at risk because in vitro culture protocols utilized to provide sufficient amounts of MSC induce in most cases senescence of these cells. In this study we compared cryopreserved and senescent human amniotic fluid stem cells (AFSC) via flow cytometry, qRT-PCR, and automated/manual patch-clamp. The expression of surface markers (CD29,CD31,CD45,CD44,CD49e,CD54,CD56,CD73,CD90, CD105,CD117,CD133,CD146) decreased in senescent AFSC by up to 82.3 %. The gene expression profile revealed a secretory phenotype of senescent cells, with mRNA level increases for IL-1alfa, IL-6, IL-8, TGF-beta, NF-kappaB p65, increases in cell cycle regulating genes like p16INK4A, beta-actin. HMGB1, c-Myc, Bcl-2 showed reduced changes and p21, MDM2 decreased. We identified through whole-cell patch-clamp experiments five components of ion currents: a prominent inactivatable (A-type) K+ current (IA), a delayed outward-rectifying K+ current (Ior), current fluctuations of BK channels (which were larger in cryopreserved cells), a voltage-dependent Na+ current (INa), and an inward rectifying K+ current (IKir), measured between -80 and -120 mV. We also studied effects of specific blockers on some of these current components. We noticed inhibitory effects of iberiotoxin 100 nM on BK channels current fluctuations (macroscopic time constant of onset 13.3 s) and the progressive decay in peak INa amplitude upon repeated channel opening when applying lidocaine 200 microM. We recorded TRPM7-like currents in AFSC using a special combination of Cs+-based external and internal solutions. Removal of divalent cations in the external solution unmasked large outward and inward currents carried by monovalent cations, and the phenomenon was reversible upon readmission of divalent cations. The average current density of inward TRPM7-like current at -120 mV was significantly higher in senescent AFSC, probably due to increased activation by reactive oxygen species asociated with senescence.

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(6), 463-476 https://doi.org/10.1007/s12576-016-0441-8

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.