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

Abstract The aim of the study was to evidence replicative senescence‐induced changes in human amniocytes via flow cytometry, quantitative reverse‐transcription‐polymerase chain reaction (qRT‐PCR) and automated/manual patch‐clamp. Both cryopreserved and senescent amniocytes cultured in BIO‐AMF‐2 medium featured high percentages of pluripotency cell surface antigens SSEA‐1, SSEA‐4, TRA1‐60, TRA1‐81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT‐PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence‐associated β‐galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)‐1α, IL‐6, IL‐8, transforming growth factor β, nuclear factor κB p65 expression), increases for cell cycle‐regulating genes (p16INK4A), cytoskeletal elements (β‐actin); HMGB1, c‐Myc, Bcl‐2 showed reduced changes and p21, MDM2 decreased. Via patch‐clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+‐dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 μmol/L exerted use‐dependent Na+ current block. Transient receptor potential (TRP)M7‐like current density at −120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.


| INTRODUC TI ON
Senescence represents an adaptive cellular phenomenon occurring in diploid cells upon repeated cell division cycles. Although to date there is no unique marker to distinguish senescent cells from differentiated non-proliferating cells, a combination of markers characterize distinct elements of the complex chain of molecular phenomena and events leading to this phenotype. 1,2 Senescence markers include: increased expression of cell cycle regulatory genes such as p16 INK4A 3,4 or p15 INK4B 5 that impede progression from G1 to S phase and trigger senescence-associated growth arrest (SAGA); a senescence-associated secretory phenotype (SASP) 6,7 with increased production and secretion of proinflammatory factors such as interleukin 6 (IL-6), IL-8, IL-1α, and subsequent increase in expression of a lysosomal splice variant of β-galactosidase, senescence-associated β-galactosidase (SA-β-Gal) 8,9 ; the presence of senescence-associated heterochromatin foci (SAHF) 10,11 and other epigenetic changes 12,13 ; activation of the DNA-damage-response (DDR) 14 reflected in increased expression of p53-binding protein 1 (53BP1) and the presence of γH2AX DNA lesion foci, initiated by phosphorylated variants of histone 2A such as γ-H2A.X. 15 There is a clear distinction between physiological replicative senescence (RS), involving telomere uncapping by repeated cell division, and stress-induced premature senescence (SIPS), 1 also called 'stasis', 10 triggered by a variety of external or internal stress factors such as oncogene activation (oncogene-induced senescence), chromatin disruption (genotoxic stress), endo/exogenous oxidative stress, X-ray exposure or aggressive chemotherapy, endo/exogenous mitogenic signals like growth-related oncogene α (GRO α) or circulating angiotensin II. 7 Although short-term senescence is beneficial by preventing cells to transform and thus propagate DNA damages, or promote transformation into tumour cells, 16 enhancing tissue remodelling and repair, including wound healing, 17 long-term effects of senescent cell accumulation are detrimental, by maintenance of tissue inflammation, 18 occupancy of stem-cell niches, 19 tumour promotion 20 and a number of age-related diseases, including atherosclerosis. 21 Senescent cells undergo a number of metabolic changes related to molecules involved in nutrient sensing: insulin-like growth factor 1 (IGF-1), involved in glucose sensing, and its associated signalling pathway, mammalian (or mechanistic) target of rapamycin (mTOR), acting as sensor of high aminoacid concentrations, 5′-adenosine monophosphate-activated protein kinase (AMPK) and sirtuin proteins, detecting nutrient scarcity, accumulation of advanced glycation endproducts (AGE) and phospho/sphingolipids. 22 The SASP involves activation of several signalling pathways, including mTOR, MAPK (mitogen-activated protein kinase), 23 phosphoinositide 3-kinase (PI3K), and GATA4/p62-mediated autophagy, 24 all targeting nuclear factor κB (NF-κB) and the CCAAT/enhancer binding protein β (C/ EBPβ), which in turn transactivate transcription of numerous proinflammatory genes, such as IL-6, IL-8, and their receptors IL-6R/GP80 and IL-8RB/CXCR2. [25][26][27] In addition, mTOR specifically activates translation of IL-1α, an early SASP element, which upon juxtacrine binding to its receptor activates via IL-1 receptor associated kinase 1 (IRAK1) a positive feedback loop with further upstream NF-κB activation. 28 Interleukin-6 also features an autocrine/paracrine positive feedback loop via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway that targets C/EBPβ, further increasing IL-6 and IL-8 expression. 29 This pathway is also activated by other SASP components, like monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), interferon I and II. 30 Another SASP factor, transforming growth factor β (TGF-β), in turn activates paracrine and autocrine positive feedback loops, triggering production of reactive oxygen species (ROS), DNA damage and DDR, and stable cell-cycle arrest by induction of p16 INK4A or p15 INK4B . 31 Because of deleterious effects of prolonged or excessive senescence, a number of therapies have been proposed as cures to extend lifespan and alleviate chronic age-related diseases, focused on apoptosis induction in senescent cells, their enhanced removal by activation of immune system, SASP modulation or prevention of SAGA. 32 Mesenchymal stem cells (MSC) of various origin (bone marrow, adipose tissue, dermis, placenta, amniotic fluid, deciduous teeth, synovial fluid or membrane, etc) 33 represent attractive therapeutic alternatives in diseases like ischaemic stroke, 34 brain or spine injuries, 35 autoimmune and other diseases, 36 graft-vs-host disease, 37 because of their capacity to limit local inflammation and apoptosis and to promote neovascularisation and healing. 38 In vitro culture and expansion are required to produce large quantities of MSC for clinical applications, but such protocols incur the risk of induction of senescence that limits or reverses their anti-inflammatory properties. [39][40][41] Several studies explored different molecular changes associated with in vitro or in vivo senescence of MSC, including proteasome activation and autophagy, 42-44 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  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.

| Amniotic fluid cell culture
For amniocytes isolation and culture we used methods similar to those previously described. 47

| Senescence-associated βgalactosidase staining
We used the SA-β-Gal staining method described by Dimri et al. 8 Amniocytes cultured in 60-mm diameter Petri dishes were washed gently twice with standard PBS, fixed in 2% formaldehyde plus 0.2% glutaraldehyde in PBS at pH 7.4 for 6 minutes, washed three times in PBS, then exposed for 12-16 hours at 37°C to X-Gal working solution, containing 1 mg X-Gal substrate per ml (5-bromo-4-chloro-3indolyl-β-D-galactoside from 20 mg/mL stock solution, BG-3-G; Sigma-Aldrich), a redox system composed of potassium ferro-and ferricyanide (5 mmol/L each), 40 mmol/L pH 6.0 citrate buffer (17.9 mL citric acid 0.1 mol/L plus 32.1 mL dibasic sodium phosphate 0.2 mol/L per 100 mL), NaCl 150 mmol/L and MgCl 2 2 mmol/L. Stained cells were placed in 70% glycerol in pure water, examined and photographed with a standard inverted microscope (Olympus CKX41), and stored at 4°C in a refrigerator.

| Surface markers assessment by flow cytometry
The expression of cell surface markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 10 5 Table S1.

| Solutions and chemicals
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. For the study of TRPM7-like currents we prepared solutions similar to those used in our previous studies 49

| Proof of RS of amniocyte cultures via lack of EdU incorporation and SA-β-Gal staining
We tested for the presence of cell proliferation in amniocyte cultures via EdU incorporation (10 μmol/L for 24 hours), followed by Click-iT ® reaction with AlexaFluor ® 488 and flow cytometry ( Figure 1A-C).
Cell cultures loaded with EdU were tested in two technical replicates for each condition. While cryopreserved amniocytes featured high percentages of EdU incorporation during S phase of cell cycle, this phenomenon was absent for senescent amniocytes. We also proved senescence via the widely used SA-β-Gal staining method applied to amniocytes cultured on 60-mm Petri dishes ( Figure 1D,E). After 24 hours of incubation at 37°C with the X-Gal substrate-containing staining solution, senescent amniocytes, which have been replated at low density, showed specific blue staining, while this phenomenon was absent for cryopreserved amniocytes. The percentages of cells featuring characteristic SA-β-Gal staining (obtained by counting >200 cells for each condition) were 7% for cryopreserved vs 93% for senescent amniocytes.   Table S2 of Supporting Information).

| Identification of multiple ion currents by patch-clamp and pharmacology assays
We performed automated whole-cell patch-clamp experiments on n = 17 cryopreserved amniocytes, using three voltage-clamp protocols and a combination of physiological K + -based external and internal solutions, as described in Section 2. Unfortunately, senescent amniocytes were far more difficult to assess via cytocentering patch-clamp, presumably because of a stiffer cytoskeleton, therefore we tested only two cells by automated patch-clamp and 15 cells by  Figure 4C). Figure 4D shows voltage-dependent Na + currents (I Na ), while Figure 4E Figure 5A. The current densities, numbers and percentages of cells where these five current components were identified for cryopreserved and senescent amniocytes are exposed in Table 1. We also studied effects of specific blockers on some of these current components, as shown in Figure 5. Thus, Figure 5A Table S3 and Figures S1 and S2 of Supporting Information applied at a frequency of 30 Hz. Figure 5D shows progressive decay in peak I Na amplitude produced by lidocaine upon repeated channel opening, and Figure 5C summarizes average relative peak I Na values for the first five stimuli of the protocol.

| Levels of TRPM7 current assessed by patchclamp
We studied TRPM7-like currents in amniocytes using the same double-ramp voltage protocol and a special combination of Cs + -based external and internal solutions, as shown in Section 2. The internal solution was Mg 2+ -free and contained ATP, two conditions required for TRPM7 channels activation. As shown in Figure 6A (for a cryopreserved amniocyte) and Figure 6C (for a senescent amniocyte), removal of divalent cations in the external solution unmasked large outward and inward currents carried by monovalent cations, with an I-V curve similar to that recorded in cardiomyocytes in previous experiments. 49 Figures 6B and 6D show time plots of current levels at −120 and +80 mV measured on consecutive recordings at 10-second intervals in these two cells. In both cases perfusion of divalent cation-free (DVF) external solution produced large currents, and the phenomenon was reversible upon readmission of divalent cations. Table 1 summarizes average TRPM7-like current densities at −120 and +80 mV (computed as differences between current levels during and before perfusion with divalent-free solution) and percentages of reversibility for n = 8 cryopreserved and n = 8 senescent amniocytes. Average current densities were higher in senescent amniocytes (for values at −120 mV the difference was statistically significant, P = 0.0185, two-tailed Student's t test for independent samples), and the reversibility was generally very good.

| D ISCUSS I ON
The immunophenotype of cryopreserved amniocytes features expression of cellular surface markers CD29, CD44, CD49e, CD54, CD56, CD73, CD90, CD105 and CD146. Interestingly, in senescent amniocytes the expression of these markers is decreased. Putative explanations for this reduced surface marker expression in senescent amniocytes include excessive glycocalix proliferation, senescencedown-regulated gene expression, or reduced protein synthesis as an energy-sparing mechanism, but the real cause remains elusive. There are at least two major pathways that regulate senescence-the p53/p21 and p16 INK4A /pRb. Although senescent amniocytes underwent growth arrest, they continued to be metabolically active, with changes in gene expression, morphology, cytoskeleton reorganization, and in activity of SA-β-Gal. The molecular pathways triggering senescence involve retinoblastoma protein (pRb) or p53, which activate cyclin-dependent kinase inhibitors p16 and p21, respectively. Depending on the cellular type, these pathways can be different, but they can also influence each other and cooperate to induce senescence. Other inducers involved in senescence are c-Myc, p300, Bcl-2. Moreover, p16 INK4A became a biomarker of aging, 54 besides its tumour suppressor role. Our results demonstrate high levels of p16 INK4A in senescent cells. Furthermore, MDM2 is a negative regulator of p53 activity, being sequestered by p14arf (the alternate reading frame product of p16 INK4A ) in the nucleoli, where it prevents export of p53 from the nucleus to the cytoplasm for degradation by the 26S proteasome subunit. 55 Senescence is associated with loss of p53 activity that greatly enhances the SASP, 7 as confirmed by our study; small molecule drugs that can restore function of tumour-suppressing genes such as p53 may be used as treatment for senescence-associated conditions.
Recent data showed that c-Myc can activate senescence through stromal secretion of TGF-β. 56 The SASP is associated with the expression of factors such as IL-1, IL-1R, IL-6, IL-8, MCPs, MIPs (macrophage inflammatory proteins), TGF-β. All these molecules initiate a signalling cascade that activates NF-kB and/or F I G U R E 3 Differences in gene expression levels between senescent and cryopreserved amniocytes. The fold-change in expression in senescent amniocytes (taking expression levels in cryopreserved amniocytes as reference) was computed as 2 −ΔC T . Error bars represent SD of multiple replicates in senescent amniocytes computed vs average values in cryopreserved amniocytes. The equal expression level is marked with a horizontal dashed line. See also Tables S1 and S2 of Supporting Information. IL, interleukin; NF-κB, nuclear factor κB; TGF-β, transforming growth factor β C/EBPβ. 57 We showed that SASP of human amniocytes includes high levels of IL-1, IL-6, IL-8, TGF, eNOS and NF-κB (p65 component). Secretion of these inflammatory factors by senescent cells suggests local or systemic chronic inflammation, which leads to age-related diseases such as tissue degeneration and hyperplasia.
These cytokines also sustain SAGA. Furthermore, the SASP includes at least one alarmin, HMGB1, which can initiate an inflammatory response and interact with p53, 58 as shown by us via a 2.6-fold increase in HMGB1 expression in senescent vs cryopreserved amniocytes.
F I G U R E 4 Ion currents recorded in amniocytes. (A and B) Two voltage-clamp recordings in senescent amniocytes via automated patch-clamp, using a general protocol for K + currents (voltage steps shown in insert of A). I or and big conductance Ca 2+ -dependent K + (BK) fluctuations are visible in both recordings, while I A can be noticed only in (A); (C) same protocol applied to a cryopreserved amniocyte with very small current levels; single-channel BK openings can be noticed at larger depolarizations (+20 to +60 mV); (D) voltage-dependent Na + current (I Na ) in a senescent amniocyte (voltage protocol shown in insert); (E) double-ramp voltage-clamp protocol (from −120 to +80 mV and back) applied to a senescent amniocyte; a small T-type Ca 2+ channel-like current with a threshold of ~−50 mV is present on the ascending ramp. I A , inactivatable A-type K + current; I or , outward rectifier K + current 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   for this difference could be the use of a different cell culture medium (BIO-AMF-2) that better preserves pluripotency and avoids auto/ paracrine neural differentiation. The higher levels of TRPM7-like currents in senescent vs cryopreserved amniocytes may be explained by increased levels of oxidative stress associated with the SASP leading to activation of this current. 59 The importance of tetraethylammonium-sensitive delayed outward rectifier K + currents for cell proliferation properties of embryonic and induced pluripotent stem cells has been described. 60 Similarly, TRPM7 is an ion channel with astounding roles in physiology and physiopathology, including internal Mg 2+ ho-

ACK N OWLED G EM ENTS
The study was funded from Competitiveness Operational