Klotho inhibits neuronal senescence in human brain organoids

摘要

a Representative images of sections of human cortical brain organoids derived from H9-ESCs cultured over 13 weeks in vitro and stained with SA-β-gal. Scale bar = 1000 µm. Dotted box indicates a magnified image. W is week. b Quantification of the percentage of SA-β-gal area normalized to the total area of each individual organoid derived from H9-ESCs. Data are presented as mean ± standard deviation. ****P < 0.0001 via one-way ANOVA. Number of independent experiments = 3. Total number of analyzed organoids = 72. W is week. c Representative images of sections of different ages of human organoids derived from H9-ESCs. Sections were stained with p21 antibody (red) and counterstained with Hoechst 33342 (blue). Scale bar = 65 µm. W is week. White dotted box indicates the magnified image. d Quantification of the percentage of total p21+ cells relative to the total number of cells per organoids derived from H9-ESCs. Data are presented as mean ± standard deviation. **P < 0.001, *P < 0.01 via one-way ANOVA. Number of independent experiments = 3. Total number of analyzed organoids = 63. W is week. e qRT-PCR of p16 and KL at different stages of in vitro culture of human cortical organoids derived from H9-ESCs. All values were normalized to GAPDH levels of their respective samples, and expressed relative to W1 values to obtain the fold change. Data are shown as mean ± standard error mean; Number of independent experiments = 4. Total number of analyzed organoids = 72; ***P < 0.001, ****P < 0.0001 via one-way ANOVA. W is week. f Western blots showing the protein levels of full-length KL and secreted KL in human cortical organoids derived from H9-ESCs cultured over 13 weeks. Actin was used for normalization. Star indicates the specific band of secreted KL, sKL indicates secreted KL. All blots derive from the same experiment and processed in parallel. g Bar graphs show the quantification of KL, as well as secreted KL levels obtained from f. Data are shown as mean ± standard error mean. The number of independent experiments = 4, total number of examined organoids is 72. sKL indicates secreted KL. h Normalized relative expression values of p16 and KL mRNA obtained from published RNA-seq studies. The fold change was calculated by dividing the normalized mRNA value of different weeks over W1. Data are shown as mean ± standard error mean. W is week. i Heatmap represents the expression of RNA-seq data of senescence increased and decreased markers in cortical organoids derived from iPSCs cultured over different time points. Relative expression data were further normalized between 0 (lowest) to 1 (highest) expression value of individual genes across different time points of organoids culture. Normalized expression values were color-coded with red values indicating upregulation and white values indicating downregulation. W is week.