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SC_11-1098_sm_supplFigure1.tif274KSupplementary Figure 1: (a) A schematic representation of the experimental design of hypoxia/re-oxygenation. (b) A representative (n = 4) flow cytometry profile of post-hypoxia BGO1 cells depicting sustained expression of HIF-2α mainly confined to the ABCG2+ fraction on day-4 of re-oxygenation. The BGO1 hESCs cells were fixed, and stained with both ABCG2 and HIF-2α antibodies, and then counterstained with FITC and PE secondary antibodies respectively. The insert on the top of the far-left flow cytometry profile represents the isotype control. (n = 4; data from 5,000 single-cell events). * hESCs cultures were allowed to grow on average 5-7 days resulting in colonies as depicted in Figure 1A (main text).
SC_11-1098_sm_supplFigure2.tif92KSupplementary Figure 2: (a) Real time PCR (QPCR) data showing the expression of p53, HIF- 2α, and HIF-1α expression in the SSEA3+/ABCG2+ and SSEA3+/ABCG2- fractions of BGO1 hESCs between pre-hypoxia and day-4 post hypoxia. QPCR data was first normalized to GAPDH value and then analyzed using ΔΔ CT method as described22,23 (b) The SSEA3+/ABCG2+ fraction shows high HIF-2α and low p53 activity as measured by the transcriptional activity assay. (c) The ABCG2- versus ABCG2+ fraction of SSEA3+ hESCs showed higher ROS generation, Oxidative DNA damage, and Caspase-3 activity. The BGO1 hESCs cells were exposed to hypoxia, harvested on day-4 re-oxygenation, and the values were expressed as the fold change between day-4 and pre-hypoxia. The **p<0.001; n = 3
SC_11-1098_sm_supplFigure3.tif211KSupplementary Figure 3: H9 derived ABCG2+ hox fraction exhibits highly undifferentiated and cytoprotective state a&b. The fold change in the transcriptional activity of HIF-2α, p53, Oct-4 and Nanog in ABCG2+hox relative to ABCG2+nox cells. c. Fold change in the survival of the mouse HSCs (CD34+ fraction) and MSCs (CD271+ fraction) following treatment of carboplatin-treated mice with conditioned media (CM) derived from the ABCG2+hox versus ABCG2+nox. *p<0.05, **p<0.001, ***<0.0001, n = 3
SC_11-1098_sm_supplFigure4.tif1896KSupplementary Figure 4: In vivo pluripotency of ABCG2+hox cells. The right panel shows an enlarged area of the left panel (Square box) depicting immature cartilage (Red arrow), and gastrointestinal glands (Yellow arrow). ABCG2+hox cells (1x104 cells) were injected to NOD/SCID mice subcutaneously as described23, and a representative 1.5 cm size teratoma was subjected to histological examination by performing H&E staining.
SC_11-1098_sm_supplFigure5.tif227KSupplementary Figure 5: Representative flow cytometry images that quantify results presented in Figure 4d, main text. The 10 day old subcutaneous matrigel plugs (n = 5) containing BGO1 hESCs derived SSEA3+/ABCG2+ cells [obtained either before hypoxia (ABCG2+nox) or after day-4 post-hypoxia (ABCG2+hox)] were subjected to enzymatic dissociation 22 and staining with antibody against Nanog28 followed by flow cytometry (5,000 single-cell events were collected). Whereas the ABCG2+nox matrigel plugs contained 3.1 ± 0.5 percentage of Nanog positive cells, the ABCG2+hox matrigel plugs contained 23.5 ± 1.2 percentage of Nanog positive cells (n = 5, *p<0.0001).
SC_11-1098_sm_supplFigure6.tif96KSupplementary Figure 6: (a) QPCR and ELISA data showing the silencing of HIF-2α and p53 by Accell siRNA in ABCG2+hox. The viability tested by Trypan blue showed no significant toxicity following either with targeted (T) or non-targeted (NT) siRNA treatment. The HIF-2α and p53 silencing were assessed by QPCR and ELISA respectively 72-hours after the addition of Accell siRNA 1μM to ABCG2+hox cells (day-4 post hypoxia). The data was compared to the untreated cells. QPCR data was first normalized to GAPDH value and then analyzed using ΔΔ CT method as described22,23 (b) Fold reduction of post-hypoxia (day 4) ABCG2+ hox cells following siRNA HIF-2α or FM19G11 (0.5μM/ 48 hours) or Nutlin-3 (10μM/48 hours) treatment compared to untreated BGO1 cells. The BGO1 cells treated with above mentioned siRNA or inhibitors were exposed to hypoxia, and the percentage of ABCG2+hox was evaluated by flow cytometry on day-4 post hypoxia. Note that siRNA HIF-1α silencing showed no detectable changes in the percentage of ABCG2+hox. n=3. **p<0.001. We achieved HIF-1α silencing of >80% protein level by ELISA (not shown).
SC_11-1098_sm_supplFigure7.tif150KSupplementary Figure 7: (a) The kinetic changes of the levels of p53 and MDM2 proteins (as measured by InCell western) depicted as induction fold in the SSEA3+/ABCG2- fraction immediately and (b) days after hypoxia/reoxygenation. (c) The kinetic changes of the levels of p53 and MDM2 proteins (as measured by ELISA) depicted as induction fold in the hESC line H9 derived SSEA3+/ABCG2+ fraction days after exposure to hypoxia, and (d) represent the state of p53/MDM2 kinetic changes of (c) when the siRNA HIF-2α treated H9 cells were exposed to hypoxia. Data presented represent the mean of four experiments (a&b) and three experiments (c & d).
SC_11-1098_sm_supplTable1.tif283KSupplementary Table 1

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