Figure 1.

Establishment of the transcriptional landscape of human stem cells and their derivatives under 39 °C heat stress. (A) A schematic of the workflow. The febrile temperature experiments were carried out in hESCs at early passages (between 50–60 passages), in hVECs and hVSMCs at passage 2 or 3, in hMSCs at passage 4 or 5, in hNSCs at passage 6 or 7, in hCMs and hNeurons at approximate day 21 from differentiation. (B) Rose diagram showing the numbers of 39 °C hyperthermia-associated differentially expressed genes (hereafter referred as hyperthermia DEGs) in the indicated cell types. (C and D) GO term and pathway enrichment analysis based on upregulated (C) or downregulated (D) hyperthermia DEGs across seven cell types. The color key from red to gray (C), or from blue to gray (D) indicates P values from low to high, respectively. (E) Western blot analysis of HSP90AA1 expression across seven cell types under control (37 °C) and febrile temperature (39 °C) culture conditions. (F–H) Flow cytometric analysis of apoptosis in hESCs (F), hVECs (G) and hMSCs (H) under control (37 °C) and febrile temperature (39 °C) culture conditions. Data are shown as the mean ± SEM, n = 3; *P < 0.05. (I and J) Immunofluorescence analysis of Ki67 expression in hVECs (I) and hMSCs (J) under control (37 °C) and febrile temperature (39 °C) culture conditions. Data are shown as the mean ± SEM, n = 3, **P < 0.01, ***P < 0.001. Scale bar, 25 µm. (K and L) Flow cytometric analysis of cell cycle in hVECs (K) and hMSCs (L) under control (37 °C) and febrile temperature (39 °C) culture conditions. Data are shown as the mean ± SEM, n = 3, ***P < 0.001. (M and N) Immunofluorescence analysis of 53BP1 and γH2AX expression in hVECs (M) and hMSCs (N) under control (37 °C) and febrile temperature (39 °C) culture conditions. Relative 53BP1 and γH2AX double-positive cells were calculated and are shown as the mean ± SEM, n = 3, *P < 0.05. Scale bar, 25 µm