HIF-2α suppresses p53 to enhance the stemness and regenerative potential of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) have been reported to exert cytoprotective activity in the area of tissue injury. However, hypoxia/oxidative stress prevailing in the area of injury could activate p53, leading to death and differentiation of hESCs. Here we report that when exposed to hypoxia/oxidative stress, a small fraction of hESCs, namely the SSEA3+/ABCG2+ fraction undergoes a transient state of reprogramming to a low p53 and high hypoxia inducible factor (HIF)-2α state of transcriptional activity. This state can be sustained for a period of 2 weeks and is associated with enhanced transcriptional activity of Oct-4 and Nanog, concomitant with high teratomagenic potential. Conditioned medium obtained from the post-hypoxia SSEA3+/ABCG2+ hESCs showed cytoprotection both in vitro and in vivo. We termed this phenotype as the "enhanced stemness" state. We then demonstrated that the underlying molecular mechanism of this transient phenotype of enhanced stemness involved high Bcl-2, fibroblast growth factor (FGF)-2, and MDM2 expression and an altered state of the p53/MDM2 oscillation system. Specific silencing of HIF-2α and p53 resisted the reprogramming of SSEA3+/ABCG2+ to the enhanced stemness phenotype. Thus, our studies have uncovered a unique transient reprogramming activity in hESCs, the enhanced stemness reprogramming where a highly cytoprotective and undifferentiated state is achieved by transiently suppressing p53 activity. We suggest that this transient reprogramming is a form of stem cell altruism that benefits the surrounding tissues during the process of tissue regeneration.

Figure 1

SSEA3+/ABCG2+ fraction survives and exhibits high HIF-2α and low p53 activity during exposure to hypoxia/reoxygenation. (A): Morphological evidence of BGO1 human embryonic stem cell differentiation and death (image to the right) following exposure to extreme hypoxia followed by 4 days of reoxygenation. The hypoxia- and reoxygenation-treated cells showed elongated neurite processes including fibroblastic-like features as well as evidence of cell fragmentation. (B): The fold change in reactive oxygen species (DCFH-DA), p53, and HIF-2α in the BGO1 cells is shown in (A), following day-4 post-hypoxia/reoxygenation. (C): Flow cytometry showing expansion (a vs. b compartments) of the SSEA3+/ABCG2+ fraction on the day-4 post-hypoxia/reoxygenation. Inset shows the profile of isotype control. (D): Immunofluorescence labeling of SSEA3+/ABCG2− and SSEA3+/ABCG2+ fractions on day-4 post-hypoxia/reoxygenation showing a high HIF-2α and low p53 signal in the SSEA3+/ABCG2+ fraction (DAPI, nuclear stain). (E): The corresponding fold change in DCFH-DA, p53, and HIFs in the SSEA3+/ABCG2+ versus SSEA3+/ABCG2− fractions on the day-4 post-hypoxia/reoxygenation. (F): Protein levels of HIF-2α and p53 in the SSEA3+/ABCG2+ fraction were measured by ELISA. Note high HIF-2α versus low p53 until day 20. *, p < .05; n = 4. Scale bar = 40 μm (A); 10 μm (D). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DCFH-DA, 2′,7′-dichlorfluorescein-diacetate; HIF, hypoxia inducible factor.

Figure 2

A representative flow cytometry profile depicting the sustained expression of high Nanog and Oct-4 in the BGO1 ABCG2+hox cells following exposure to hypoxia/reoxygenation. The ABCG2+hox cells were flow cytometry sorted as shown in Figure 1D and maintained in the human embryonic stem cells (hESCs) culture media for next 3 weeks. In specific interval of time, the ESCs were fixed and subjected to flow cytometry expression of Nanog and Oct-4 as described [23]. The inset panel at the top of the prehypoxia panel indicates the isotype control. The BGO1 hESCs were fixed and permeabilized before subjecting to incubation with primary and secondary (Alexa 488 for Oct-4 and phycoerythrin for Nanog) antibodies. Results shown are representative of a typical experiment (n = 4; data from 5,000 single-cell events).

Figure 3

ABCG2+hox fraction exhibits a highly undifferentiated and cytoprotective state. (A): The fold change in the transcriptional activity of Oct-4 and Nanog in ABCG2+hox relative to ABCG2+nox cells. (B): The fold change of the GSH and ABCG2 levels in the ABCG2+hox relative to ABCG2+nox cells. The GSH level was measured in the CM obtained from the cells. The ABCG2 level was measured by ELISA. (C): Fold change in cell survival, measured by trypan blue exclusion, of human primary cardiomyocytes and neuronal-like cells (SHSY5Y N-type neuroblastoma cell line) [31] following exposure to conditioned medium obtained from human embryonic stem cells versus nonexposed medium. The antioxidant NAC was used as a positive control. (D): Fold change in the survival of the mouse HSCs and MSCs following treatment of carboplatin-treated mice with CM obtained from ABCG2+hox versus ABCG2+nox cells. *, p < .05; **, p < .001; A, B, C n = 4, and D, n = 3. Abbreviations: CM, conditioned media; GSH, glutathione; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; NAC, N-acetyl cysteine; TF, transcription factor.

Figure 4

ABCG2+hox fraction exhibits a phenotype of self-sufficiency. (A): The fold change in FGF-2 and Bcl-2 in ABCG2+hox cells relative to ABCG2+nox cells. (B): The single-cell clonogenic assay showing the alkaline phosphatase staining of abundant colony formation in the ABCG2+hox cell fraction. The histogram shows the significant fold increase in colonies derived from the ABCG2+hox fraction. Tera-2-derived colonies were used as a control. (C): The in vivo limiting dilution assay showing higher teratomagenic formation efficiency of ABCG2+hox versus ABCG2+nox. Tera-2 teratoma formation was used as a control. (D): Immunofluorescence staining on a frozen section of ABCG2+ cells containing Matrigel plug showing expression of Nanog positive cells. The quantification of the Nanog expressing cells is given in the Supporting Information Figure 6. *, p < .05; **, p < .001; ***, p < .00001, n = 4. Scale bar = 40 μm (B) and (D). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FGF-2, fibroblast growth factor-2.

5

HIF-2α is involved in the maintenance of the enhanced stemness state. (A): The fold change in the apoptosis (caspase-3 activity) of the ABCG2+hox (day-8 post-hypoxia) cells versus ABCG2+nox cells following treatment with various inhibitors of HIF-2α and p53. (B): The fold change of HIF-2α and p53 protein levels in the surviving cells of (A). (C): The fold change in Oct-4 and ABCG2 protein levels in the surviving cells of (A).*, p < .05; **, p < .001; n = 3. Abbreviations: HIF, hypoxia inducible factor; siRNA, small interfering RNA.

Figure 6

HIF-2α is involved in the generation of an altered p53/MDM2 oscillation in the ABCG2+hox. (A): The kinetic changes of p53 and MDM2 proteins in ABCG2+ hox cells immediately, (B) days after, and (C) and (D) following treatment with hypoxia inducible factor (HIF) inhibitor FM19G11 [21]. The p53 and MDM2 levels were checked by InCell western and the induction fold of p53 and MDM2 after reoxygenation was calculated at each time point. Data presented represent the mean of four experiments. Similar results of fold induction of p53 and MDM2 was obtained by ELISA measurement (described in text).