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.

Nano-Hydroxyapatite vs. Xenografts: Synthesis, Characterization, and In Vitro Behavior

“…3− groups absorption bands identified at 472 cm −1 are characteristic of the ν 2 inclination of the O-P-O bond, the high-amplitude bands from 572 cm −1 , 601 cm −1 , and 963 cm −1 refer to the symmetric and asymmetric deformation modes of ν 4 O-OP, while the intense absorption bands in the range of 1040-1090 cm −1 correspond to the ν 3 P-O [69][70][71].…”

“…The strong absorption band at 874 cm −1 was assigned to the presence of CO 3 2− involved in B-type PO 4 3− substitution (ν 2[B] ), and the absence of a lower intensity for the absorption band at 880 cm −1 attributed to A-type carbonate substitution (ν 2[A] in OH − position) together with the missing band at around 3570 cm −1 for the stretching mode of OH − could be another piece of evidence. There are papers that presented methods to appreciate the content of CO 3 2− that substitutes PO 4 3− in bio-apatite (C/P), by measuring the amplitude of the absorption band at 1415-1420 cm −1 (as CO 3 2− quantity) and at 1011-1042 cm −1 corresponding to PO 4 3− quantity [69][70][71]. According to this method, the C/P ratio for HA1 was 0.028, that for BioOss was 0.125, and the highest content of carbonate for GenOs was 0.574, the latter containing a certain quantity of denatured collagen but without any confusion, and the characteristic absorption band for carbonyl (CO) group is placed at 1455 cm −1 [69][70][71].…”

“…There are papers that presented methods to appreciate the content of CO 3 2− that substitutes PO 4 3− in bio-apatite (C/P), by measuring the amplitude of the absorption band at 1415-1420 cm −1 (as CO 3 2− quantity) and at 1011-1042 cm −1 corresponding to PO 4 3− quantity [69][70][71]. According to this method, the C/P ratio for HA1 was 0.028, that for BioOss was 0.125, and the highest content of carbonate for GenOs was 0.574, the latter containing a certain quantity of denatured collagen but without any confusion, and the characteristic absorption band for carbonyl (CO) group is placed at 1455 cm −1 [69][70][71].…”

“…Each spectrum was collected at room temperature at a resolution of 4 cm −1 , and 32 samples were scanned between 4000 and 440 cm −1 . The obtained results were presented as the average of the 32 individual scanned samples for each Bio-Oss, GenOs, and HA1 powder and were compared with the theoretical available data [ 68 , 69 , 70 , 71 ].…”

Role of Monoamine Oxidase A (MAO-A) in Cardiac Aging

“…In an in-vitro model of cellular replicative senescence, higher levels of TRMP7 inward current have been recorded in human amniocytes, possibly as a result of activation of increased levels of endogenous ROS. Transient receptor potential (TRP)M7 like current density at -120mV was significantly increased in senescent amniocyte 76 .…”