Fig 2: Generation and characterization of ACL-iPSC-SW163A (from a second donor using protocol#2). ACL-iPSC-SW163 derived using the scoring technique were grown to passage 20 (six colonies with pluripotent morphology were observed from 50,000 somatic cells giving an estimated efficiency of 0.012%). (A) ACL-iPSC-SW163A stained positive for antibodies to pluripotency markers NANOG, OCT-4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, with lack of early differentiation marker SSEA-1. (B) ACL-iPSC-SW163 displayed a normal karyotype. (C) ACL-iPSC can form embryoid bodies (EBs), (Ci) morphology of EBs at day 10, (Cii-Civ) phase images displaying a range of morphologies of following EB outgrowth for 5 days. (D) Following outgrowth, EBs produced cells positive for antibodies to markers of mesoderm (a-SMA), endoderm (GATA6), and ectoderm (Neurofilament). [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: Tumorigenic potential of dedifferentiated tumor cells in vivo(A) In vivo tumorigenic analysis strategies. The 14 d hypoxia (1%) or normoxia (21%) exposed CD133-CD15-NESTIN- Gl261-luc cells (104) were injected into the brains of female C57 mice. These mice were raised under normoxia (21% O2). Alternatively, the mice were intracerebrally injected with normoxia-treated CD133-CD15-NESTIN- Gl261-luc cells (3 × 104) and subsequently raised under hypoxia (10% O2) or normoxia (21% O2). (B) GL261 cells following 14 d of in vitro hypoxia (1% O2) had a stronger tumorigenic ability compared with the normoxia cells. The mice with in vivo injections of differentiated CD133-CD15-NESTIN- cells that were raised under hypoxia (10% O2) exhibited a stronger tumorigenic ability compared with the mice raised under normoxia. (C) The quantitative value of bioluminescence was significantly increased in hypoxia compared with normoxia (*P < 0.001, Paired-samples T Test). (D) Lethality analysis indicated there were significant higher death rates in hypoxia-exposed cell injection than normoxia-exposed cell injection, the same trend was found between the mice with normoxia-exposed cells raised under normoxia or hypoxia (Log-rank Test).

Fig 4: Pontin deficiency causes the defects in mouse ES cell maintenance.(a) WT and Pontin-deficient E5.0 embryos were stained using the TUNEL assay system. Apoptotic cells were shown by TUNEL (green) and nuclei were stained with DAPI (blue). The epiblast cells were immunostained with an anti-Oct4 antibody (red). Representative images are shown. Right side histogram represents the numbers of TUNEL-positive cells per embryo (mean±s.d.). P value was calculated by t-test (n=10 for each group; ***P=2.03 × 10-8). Magnification × 40. (b) Representative images show decreased SSEA-1 expression (red) in inner cell mass of Pontin-deficient embryos (n=4 for each group). (c,d) Reduction of Pontin expression during mouse embryo development stages (c) and EB differentiation (d). (e) Diagram of the strategy for depletion of Pontin using Pontinf/f; CreER ES cells. (f) Depletion of Pontin in Pontinf/f; CreER ES cells at indicated days after OHT treatment. (g) Growth curves of Pontinf/f; CreER ES cells in the absence or presence of OHT. These experiments were independently repeated three times. (h) Cell cycle-phase analysis of Pontinf/f; CreER ES cells in the absence or presence of OHT. Cells were harvested at 3 days after treatment with vehicle (95% EtOH) or OHT. Similar results were obtained from three independent experiments. (i) BrdU incorporation was used to determine the proportion of the cells in S phase. WT or Pontin-depleted ES cells were harvested at 3 days after vehicle or OHT treatment. The percentages of BrdU positive cells are graphed. Values are expressed as mean±s.d. of three independent experiments. ***P<0.001. (j) Representative images show the reduction of AP activity of Pontin-depleted ES cells compared with WT. Cells were stained at 3 days after vehicle or OHT treatment. Similar results were obtained from three independent experiments. Magnification × 10. (k) SSEA-1 expression in Pontin-depleted ES cells. Colonies of undifferentiated WT and Pontin-depleted ES cells were stained as indicated. Representative images are shown. Similar results were obtained from three independent experiments. Magnification × 20. (l) Knockdown of Pontin reduces reprogramming efficiency. The number of GFP-positive colonies was presented.

Fig 5: HIF1a promoted dedifferentiation under hypoxia(A) Immunofluorescence demonstrated GL261 neurospheres or primary GBM cultured at 1% hypoxia highly expressed HIF1a. However, the expression of HIF1a located in cell cytoplasm not nuclear as usual. (B) HIF1a mRNA level increased in a time-dependent manner, and the highest point was identified following hypoxia for 12 h (*P < 0.05, One-sample T Test). (C) Compared with control normoxia, HIF1a protein expressed only under 1% hypoxia conditions (*P < 0.05, One-sample T Test). (D) HIF1a-ShRNA3 and digoxin reduced HIF1a expression in U87 CD133-CD15-NESTIN- cells, and vector 3 had no influence on HIF1a levels. (E) The rate of GSCs marker CD133, CD15 and NESTIN showed no differences between U87 CD133-CD15-NESTIN- cells and HIF1a-Vector CD133-CD15-NESTIN- cells after hypoxia exposure 12 d; but there existed a significant lower expression of stem cell markers in HIF1a-ShRNA CD133-CD15-NESTIN- cells than control; and the digoxin group also showed a decrease of stem cell markers compared with control group (*P>0.05, *P < 0.05, ***P < 0.05, Paired-samples T Test). (F) The neurosphere formation rates of U87 HIF1a-ShRNA CD133-CD15-NESTIN- cells and digoxin treated cells were significantly lower than control without HIF1a interference (*P < 0.05, #P < 0.05, Paired-samples T Test). (G) There was no difference in the d3 or d7 cell survival rate (d3 surviving cells/d0 seeded cells; d7 surviving cells/d3 surviving cells) of the U87 HIF1a-ShRNA CD133-CD15-NESTIN- cells compared with cells without HIF1a interference. However, the survival rate of cells treated with digoxin slightly decreased (*P < 0.05, #P > 0.05, Paired-samples T Test). (H) VEGF mRNA expression in the U87 HIF1a-ShRNA CD133-CD15-NESTIN- cells and digoxin treated cells was lower than control without HIF1a interference (Paired-samples T Test). (I) VEGF protein expression significantly decreased in the U87 HIF1a-ShRNA cells and U87 cells treated with digoxin (Paired-samples T Test).