Electrophysiology, immunophenotype, and gene expression characterization of senescent and cryopreserved human amniotic fluid stem cells

Abstract

We characterized human amniotic fluid stem cells (AFSC) in senescent cultures (6 weeks) versus cryopreserved cells using whole-cell patch-clamp, immunophenotyping, and differential gene expression profiling for senescence genes. We evidenced five ion current components (outward rectifier, A-type, inward rectifier, and big conductance Ca-dependent K currents, fast voltage-dependent Na currents). Senescent AFSC showed reduced expression of CD90, CD44, CD133, over 500-fold increase of interferon gamma and telomerase reverse transcriptase genes, increased cycle-dependent kinase 4 inhibitors, p53-binding protein 1, and decreased calreticulin and CD44. HLA-ABC immune expression was similar, and HLA-DR expression very low in both cell types. A subset of cryopreserved AFSC featured large inward rectifier K currents, voltage-dependent Na currents, and neural progenitor markers evidenced by immunophenotyping and RT-PCR. In all AFSC, in both culture conditions, at patch rupture the outward currents were very low, and they increased progressively over several minutes upon cytoplasm dialysis with pipette solution.

Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles

“…However, the effective clinical translation of AFCs based approaches requires in vitro culture expansion to achieve a sufficient number of cells and strict characterization of the cultures, two factors that may severely hamper their clinical use [5].…”

Senescence‐induced immunophenotype, gene expression and electrophysiology changes in human amniocytes

“…The electrical phenotype of amniocytes in the present study, obtained via automated/manual whole-cell patch-clamp, is similar to that found in our previous studies on human amniocytes. 47 Using the same voltage protocols and combination of solutions, we found similar percentages of cryopreserved and senescent cells expressing certain ion current components, and similar current densities, with some exceptions. Thus, BK fluctuations were present in all cells included in the present study, and in only ~70% of both cryopreserved and senescent amniocytes in the 2016 study, and the percentage of senescent amniocytes featuring I Kir was higher in the present study.…”

“…[39][40][41] Several studies explored different molecular changes associated with in vitro or in vivo senescence of MSC, including proteasome activation and autophagy, [42][43][44] effects of oxidative stress, 40,41 DDR and SAGA 45,46 in either control conditions or by induction of senescence via oxidative stress, doxorubicin, X-ray exposure or replicative exhaustion. 42 In a previous study 47 we demonstrated multiple differences between cryopreserved and senescent amniocytes including senescence-induced reduction in cell surface markers CD44, CD90, CD105, CD133 with preserved expression of CD29 and HLA-ABC, highly increased expression in senescent cells of in- Therefore, the aim of the present study is to further explore activation of specific signalling pathways associated with SASP and SAGA in our in vitro senescence model, and to correlate them with changes in ion currents assessed via automated or manual patch-clamp.…”

“…Cells were detached with accutase, Devices, Sunnyvalle, CA). The electrophysiology assay included several voltage-clamp protocols identical to those applied in our previous study by manual patch-clamp 47 for comparison. A general protocol for K + currents consisted in 300-ms voltage steps from −60 to +60 mV in 10-mV increments from a holding potential of −80 mV.…”

“…For identifying different ion current components in cryopreserved and senescent amniocytes we used the same solutions as in our previous study. 47 The extracellular solution contained (in mmol/L): NaCl 135, KCl 5.4, CaCl 2 1.8, MgCl 2 0.9, NaH 2 PO 4 0.33, HEPES 10, D-glucose 10, pH 7.40 at 25°C with NaOH. The internal (pipette) solution was composed of (in mmol/L): KCl 140, EGTA 5, HEPES 10, pH 7.21 at 25°C with KOH.…”