Intden imagej5/31/2023 Prior to antibody incubation, the cells were permeabilized and blocked with 0.1% Triton X-100 in PBS with 5% normal goat serum (NGS) (Bionordika, S-1000) for 30 min at RT. Cells were passaged using trypsin 0.05% EDTA 0.2 g/l (Life Technologies) and were used for experiments directly after differentiation.Ĭells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, P6148) in 1× PBS for 15 min at RT and washed 3 times with 1× PBS. Additionally, 20 ng/ml ciliary neurotrophic factor (CNTF ThermoFisher, PHC7015) was added to the medium for the last 2 weeks of culture. The following factors were added to the medium just before use: basic fibroblast growth factor (bFGF) 10 ng/ml (ThermoFisher, 13256029), heregulin β-1 10 ng/ml (Sigma-Aldrich, SRP3055), activin A 10 ng/ml (Peprotech, 120-14E) and insulin-like growth factor 1 (IGF-1) 200 ng/ml (Sigma-Aldrich, SRP3069). The cells were cultured in Advanced DMEM/F12 (ThermoFisher, 12634–010) supplemented with 1% penicillin streptomycin (ThermoFisher, 15140–122), 1% B27 supplement (ThermoFisher, 17504–044), 1% non-essential amino acids (Merc Millipore) and 1% L-glutamine (ThermoFisher, 25030–024). Human astrocytes were generated from neuroepithelial-like stem (NES) cells, produced from human induced pluripotent stem cells (iPSCs, Cntrl9 cell line). The aim of the present study was to investigate the impact of Aβ pathology on mitochondria functionality and energy metabolism in human induced pluripotent cell (hiPSC)-derived astrocytes using a battery of different methods, with the intention to fill this knowledge gap. However, in which way Aβ storage affects the astrocytes energy production remains unclear. Interestingly, astrocytes also forward major histocompatibility complex class II (MHCII) molecules using the same mechanism, indicating spreading of inflammation in addition to toxic protein aggregates. Moreover, we have shown that astrocytes transfer protein aggregates to neighboring cells via thin protrusions called tunneling nanotubes (TNTs). The astrocytic accumulation of Aβ results in severe cellular stress and the release of extracellular vesicles (EVs) containing neurotoxic content, which could be of relevance for AD progression. We have previously reported that cultured astrocytes engulf large amounts of aggregated Aβ, but then store, rather than degrade the ingested material.
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