|HIF-only Stem Cells Could Help Tissue Regeneration!|
Original article from STEM CELLS
Tissue stem cells are understood to contribute to the repair and regeneration of the inflamed and damaged tissues through the secretion of growth factors and antioxidants (Prockop 2009) which they express at high levels (Kim et al). Similarly, human embryonic stem cells (hESCs) have been reported to exert cytoprotective activity following tissue injury. However, as sites of injury and inflammation are characterized by hypoxia and production of reactive oxygen species (ROS) which induce p53 activation (Momcilovic et al), it has been suggested that stem cells may have specific mechanisms to maintain an undifferentiated state at the area of injury in order to serve their cytoprotective activity, such as the suppression of p53 during hypoxia and oxidative stress. Das et al now report that their studies using hESCs as a model system have uncovered a mechanism by which some hESCs, when exposed to hypoxia or oxidative stress, induce HIF-2a (Maxwell 2005) leading to a reduction in p53 levels and enhanced levels of Oct4 and Nanog. This elicits a highly cytoprotective and pluripotent state in hESCs which may benefit the surrounding tissues during the process of tissue regeneration.
The response of p53 to oxidative stress was first analysed over short time periods by exposure of hESCs (BGO1 and confirmed in H9) to extreme hypoxia for 4 hours followed by re-oxygenation (between 4 and 24 days exposure). This resulted in cell death and differentiation associated with increases in ROS and HIF-2a protein levels after hypoxia lasting until day 4 and an increase in p53 protein levels at day 4. Surviving cells were also shown to be positive for the pluripotency associated marker SSEA3 and also ABCG2, a HIF-2α target (Martin et al). Around 12% of SSEA3+ cells survived at day 4 and the fraction of cells that were dual positive for ABCG2 and SSEA3 increased at day 4 to around 8.5% from an initial 2%. SSEA3+/ABCG2+ cells also expressed more HIF-2α but less p53 than SSEA3+/ABCG2- cells, confirmed at the protein, mRNA and transcriptional activity level. Trypan blue viability assays also found that SSEA3+/ABCG2+ cells survived better (3.5 fold higher) than SSEA3+/ABCG2- cells and also that DNA damage was reduced in the SSEA3+/ABCG2+ fraction. These findings were then studied over longer time periods, which found that the high HIF-2α / low p53 / ABCG2+ phenotype (ABCG2+Hox) was stably sustained for around 2 weeks. Over a two week period Oct4 and Nanog protein expression was maintained at a high level, with their transcriptional activity 3 to 4 fold higher in ABCG2+Hox cells compared with ABCG2+ cells maintained in a normoxic state (ABCG2+Nox), whereafter levels returned to those observed for ABCG2+Nox cells. This suggests that ABCG2+Hox cells are in a state of increased pluripotency relative to normally cultured ESCs. ABCG2 levels themselves also followed this pattern, and additionally, Glutathione, a potent cellular antioxidant known to be secreted into the extracellular space by ABCG2 (Brechbuhl et al), was also higher in the ABCG2+Hox cells before returning to levels observed in ABCG2+Nox cells at 2 weeks.
Cytoprotective capabilities of these cells were then assayed by treating cardiomyocytes and neural cells (SH-SY5Y cells) exposed to oxidative stress with conditioned medium (CM) from day 8 post-hypoxia cultures; leading to a 10- to 12-fold increase in survival as compared to cells treated with CM from ABCG2+Nox cells. In vivo cytoprotection was assayed using a murine model of carboplatin-induced bone marrow stem cell toxicity (Das et al 2008) and CM from ABCG2+Hox cells led to a 17-fold and 10-fold increase in protection of HSCs and MSCs respectively, similar to levels observed when using the potent antioxidant N-acetyl-cysteine (NAC).
High levels of Nanog, as observed in ABCG2+Hox cells, have been linked to cells taking on characteristics of embryonic carcinoma (EC) cells, gaining a state of extreme self-sufficiency (Andrews 2002, Baker et al and Draper et al). Levels of FGF-2 and Bcl-2, involved in growth and regulation of apoptosis and associated with extreme self-sufficiency of ESCs and EC cells (Ardehali et al), were upregulated in ABCG2+Hox cells and single cell growth was also boosted to similar levels observed for Tera-2 EC cells. Teratoma forming assays also found an increase in efficiency in ABCG2+Hox cells over ABCG2+Nox cells to a level comparable to Tera-2 cells. Interestingly, some of the ABCG2+Nox cells contained in the Matrigel plug used in the transplantation assays to transplant the cells continued to express Nanog, and the authors suggest that some cells may have undergone reprogramming to acquire an ABCG2+Hox phenotype due to hypoxic conditions within the Matrigel plug. Additionally, it was noted that ABCG2+Hox xenograft-derived cells did not form secondary teratomas, suggesting a transient nature of this self-sufficiency phenotype. This is advantageous, as cells with increased pluripotency can exert their cytoprotective state without potentially leading to tumourigenesis.
The mechanism behind Nanog upregulation in ABCG2+Hox cells was next analysed, under the hypothesis that high HIF-2α might suppress p53 activity to allow the high Nanog state. HIF-2α inhibition was first shown to lead to an eight- to nine-fold reduction of the ABCG2+Hox fraction following the 1 day hypoxia treatment before re-oxygenation, and was further accompanied by a fourfold increase in apoptosis and an increase in p53 expression accompanied by a downregulation in Nanog and ABCG2. However, HIF-2α inhibition combined with p53 inhibition enhanced survival of the ABCG2+Hox fraction. The regulation of p53 by MDM2 (a negative regulator of p53 through ubiquitination and subsequent p53 degradation) was next analysed through the treatment of ABCG2+Hox cells with an MDMD2 inhibitor (Nutlin-3), which led to 9-fold reduction in the ABCG2+Hox cell number and decreases in Nanog, Oct-4, and ABCG2 levels, all presumably in response to an increase in p53 protein levels. High MDM2 and low p53 protein levels were found during the 2 weeks of re-oxygenation following hypoxia, with HIF-2α inhibition leading to a low MDM2 and high p53 state.
Taken together, this study supports the hypothesis that hypoxia/re-oxygenation leads to a subfraction of hESCs (ABCG2+) to increase HIF-2α which decreases p53 activity and leads to the upregulation of pluripotency-associated factors leading to an enhanced stemness state. This endows hESCs with enhanced survival and cytoprotective ability, through the increased secretion of glutathione and represents a novel mechanism of enhanced pluripotency whereby hESCs might contribute to wound repair when exposed to oxidative stress. The authors also put forward an interesting suggestion; that the enhanced stemness reprogramming may also be a form of “cell altruism (Das 2000)” that benefits the organism during early development.
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Brechbuhl HM et al.
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Das B et al.
Draper JS et al.
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STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.