Hypoxia modulation on pd-JAO-dc. (A) Heat map showing the top 50 upregulated and downregulated genes in differential gene expression profile analysis of pd-JAO-dc maintained in hypoxic vs normoxic conditions in 5 pairs of samples. (B) Enrichment map shows the positive enriched biological process (derived from c5 GSEA-MSigDB) in pd-JAO-dc in normoxic vs hypoxic conditions. Nodes represent the functional gene sets, and the size is proportional to the size of the gene set. Edge thickness is proportional to the overlap between gene sets. Only gene sets that are enriched with an FDR < 0.05 are shown. Nodes with common biological functions were clustered by Auto Annotate Cytoscape plug-in using the MCL cluster. (C) Absolute expression of AK4, GLUT1, MYC, PROM1, and CA9 genes in pd-JAO-dc by droplet digital polymerase chain reaction analysis in both conditions. Heat map columns show the mean value of absolute copies per μL for all samples for each condition as log2. (D) Bars showing the enriched clusters of GSEA of top gene sets positively enriched in normoxic conditions compared with hypoxia. X-axis normalized enriched score (NES) value. (E) TEM images of fixed tBM pd-JAO systems after 5 days from the generation for 2 patients. Degraded mitochondria showed characteristic swellings, large portions of empty matrix, inside it are recognizable residues of cristae, and the double membrane is clearly visible. An autophagic body can be larger than a mitochondrion and inside of it, we can appreciate reticulum residues membranes and vesicles. Red arrow indicates an example of AVs in the cells. Scale bar, 2 μm and 500 nm, left and right respectively for both hypoxia and normoxia. (F) Number of AVs counted in the pd-JAO TEM section (hypoxia, N = 12 for patient #6 and N = 12 for patient #8, normoxia N = 11 for patient #6 and N = 7 for patient #8). Mean and SEM are shown. ∗∗P ≤ .01, ∗∗∗∗P ≤ .0001 by 2-tailed unpaired t test. AV: autophagic vesicles; M: mitochondria; N: nuclei.