Fig 2: PTMS transferring from htNSC to neurons in the hypothalamus.(A) Representative brain sections of 3-mo C57BL/6 mice were immunostained for PTMS and NSC marker Sox2, focusing on NSC-positive regions including the third ventricle (3V) region of the hypothalamus (HT), dentate gyrus of hippocampus (Hip), and sub-ventricle zone of forebrain (SVZ). DAPI nuclear staining was included. (B) Brain sections across the cortex or the hippocampus of 3-mo male C57BL/6 mouse were generated and co-immunostained for PTMS and neuronal marker NeuN. Representative regions of parietal cortex and hippocampal dentate gyrus are presented. High-magnification views (High-mag) of outlined regions in merge images were included to show more details. (C) 3-mo C57BL/6 mice received unilateral injection of Sox2 promoter-driven PTMS-HA lentivirus in the mediobasal hypothalamus (MBH). Brain sections were made at day 7 postinjection and immunostained with Sox2 and neuronal marker HuC/D. 3V region of MBH containing Sox2-positive cells and MBH parenchyma containing HuC/D-positive neurons are presented in left and right panels, respectively. Small arrows pointed to co-localization of HA with Sox2 (left panel) or HuC/D (right panel). Scale bar, 100 µm.

Fig 3: Hypoxia promoted an increase in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133. (a) The RT-qPCR analysis showed an upregulation in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 in a time-dependent manner in sorted A549 cells under hypoxic conditions (*P<0.05). For SOX-2, the peak expression was after 12 h of hypoxia, and the expression then decreased slightly but remained statistically higher than that of the control cells. The expression of OCT-4, KLF-4, Nanog Lin-28A and CD133 was highest after 9 h of hypoxia. (b, c) Western blot analysis showed that the transcription factors and stem cell markers were not expressed under normoxia. However, there was an increase in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 in a time-dependent manner after hypoxia treatment in sorted A549 cells (*P<0.05).

Fig 4: Brain organoid generated by vertical mixing showed inverted structure in comparison with brain organoid generated by orbital mixing.a Schematic diagram of conditions used to induce brain organoid by orbital mixing (upper schema) and by vertical mixing (lower schema). b, c Immunostaining for neural progenitor (SOX2, magenta) and neuron (MAP2, green) in brain organoid generated by orbital mixing (b) or vertical mixing (c) on Day 56. d Quantification of SOX2-positive area in the peripheral region of brain organoid on Day 56. The peripheral region was defined as 100 µm inside from the edge of brain organoid. Brain organoid from vertical mixing showed higher percentage of SOX2-positive area in peripheral region in comparison with brain organoid from orbital mixing. Data represent mean ± SD (n = 6 for brain organoids by orbital mixing, n = 7 for brain organoids by vertical mixing). Difference between the two conditions was analyzed by Student’s two-tailed t-test (*p < 0.0001). e, f Immunostaining for markers of neural progenitors (SOX2, blue), intermediate neural progenitors (TBR2, gray), and cortical neurons (CTIP2, red; MAP2, green) in brain organoids by orbital mixing (e) or vertical mixing (f) on Day 56. g, h Immunostaining for markers of ventricular neuroepithelial cells (N-CADHERIN: N-CAD, gray), neural progenitors (SOX2, magenta), and cortical neurons (MAP2, green) in brain organoids by orbital mixing (g) or vertical mixing (h) on Day 56. Note the apical side of organoids generated by orbital mixing is located inside organoids, while that of organoids generated by vertical mixing is located at the surface of organoids. i Schematic diagram shows the difference of brain organoids by orbital mixing and by vertical mixing. Scale bars = 500 µm (b, c), 100 µm (e, f), 50 µm (g, h).

Fig 5: Generation and in vitro efficacy of the SOX2 epigenetic silencer.(A) Constructs were generated on the basis of human SOX2 TF and the epigenetic domain KRAB, DNMT3A (3A), and DNMT3L (3L), and V5 was added as tag. A factor based on human NEUROD1 TF and the epigenetic domain KRAB, DNMT3A (3A), DNMT3L (3L), and V5 were added as additional control. (B) Growth curve of SNB19 cells infected with the indicated constructs indicates that SES is able to kill the cells after 12 days in culture. ***P = 0.001; statistically compared with two-way analysis of variance (ANOVA). n = 3. (C) Microphotographs of the cells at the indicated time points from the infection with either mock-expressing (GFP, green fluorescent protein) or SES-expressing lentiviruses. (D) Western blot (WB) for V5, SOX2, and calnexin (CNX) (as loading control) in SNB19 cells either not infected or infected with lentivirus carrying GFP or SES. Scale bar, 100 µm (C).