• Poulsen Espersen posted an update 4 days, 10 hours ago

    Furthermore, neuronal APOE can also be expressed at high levels under stress conditions such as brain injury although APOE expression is normally low in healthy neurons. Although the existing literature suggest that APOE plays an important role in stem cell maintenance, one should note that the majority of these findings were generated from rodent models.However, such evidence has not been reported to this date.To reduce this knowledge gap, we conducted a short study examining the expression pattern of APOE gene and protein in human induced pluripotent stem cells undergoing neural induction in vitro.We found that gene expression is the highest in cells at the earliest stage of neural induction, whereas protein expression becomes more localised intracellularly, indicating that APOE expression pattern changes according to the differentiation state of cells.Cell line CTRMS human induced pluripotent stem cell line was reprogrammed from keratinocytes obtained from a neurotypical male.Centrifugation was done at revolutions per minute for min during neural passaging and, and at RPM for min during neural passaging. Passaging ratios were: for neural passaging and, and: for neural passaging. To ensure cell survival M Y inhibitor, was mixed with the plating medium at each neural passaging and then removed after hrs.Briefly, the random hexamers were annealed to total RNA at C for mins, then the synthesis was performed at C for mins, and then the reaction was terminated at C for mins.For gene expression analysis, realtime quantitative polymerase chain reaction according to the manufacturers instructions.Primary antibodies were incubated at C overnight followed by washings with TBS.Secondary antibodies conjugated with fluorescent dyes were incubated at room temperature for hours followed by washings with TBS.Imaging was done with IX inverted epifluorescence microscope. The mean, standard error of measurement, and number of biological replicates are reported.Our findings demonstrate that in cells at the earliest stage of neurodevelopment, human APOE gene expression is high, and APOE protein is not clearly localised at the intracellular region.Various combinations of small molecule inhibitors did not alter these patterns of expression.CTRM human iPSC line that was known to be homozygous for APOE was used as control. Neural passaging, and were carried out on D, D, and D, respectively.Total RNA extraction was made on cells that were not used for neural passaging on D, D, D, and D.Neural induction medium composition for each differentiation lineage and N:B medium composition are also shown.B APOE gene expression is reduced along neural induction regardless of lineage.In this study, qualitative analysis was performed on APOE immunocytochemistry results.To validate this observation, however, additional experiments with a more direct quantitative approach should be conducted.Since APOE has been shown to exist in both secreted and intracellular forms, it will be interesting to see which form of APOE is produced at each differentiation stage.It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells.Therefore, further investigations on secreted and intracellular APOE using quantitative buy Talazoparib approaches will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state.