**4.1 Effect of hypoxia on gene transcription**

The survival and functioning of stem cells in hypoxic environment depends upon their metabolic switch controlled by hypoxia inducible factors (HIFs). HIFs are transcription factors that are present in eukaryotes. They have two subunits, that is, alpha(α) and beta(β). Their α subunit has three isoforms (HIF1-3). The post translational modification of α subunit depends on hydroxylation which is oxygen dependent. When intracellular oxygen falls, α subunit forms a stable α/β dimer because hydroxylation did not occur. This dimer is transcriptionally active; it enters into the nucleus, binds to hypoxia response elements and initiates transcription of hypoxia sensitive genes [52, 53]. HIF3-α is the negative regulator of HIF1 and HIF2 (**Figure 1**).

Effect of hypoxia on HIF- α is different for different types of stem cells

GRP78-Akt axis induced by HIF1α is important in augmenting functions like proliferation and survival of MSCs under hypoxia [54].

### **4.2 Reactive oxygen species (ROS) and hypoxia**

Reactive oxygen species are oxygen containing substances that are produced in cellular metabolism. They are detrimental to cellular functions. Hypoxic

#### **Figure 1.**

*Mechanism of HIF1 formation and transcription of hypoxia sensitive genes. HIF (hypoxia inducible factor), HRE (hypoxia response element).*

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result in loss of stemness [67].

*Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal…*

preconditioning results in up regulation of Nuclear factor erythroid 2 related factor 2 (NRF2), which is a redox sensitive transcription factor involved in regulation of antioxidant genes [55]. Hypoxic preconditioning results in increased glycolytic metabolism and decreased tricarboxylic acid (TCA) cycle and oxidative phosphorylation. This mechanism leads to decreased mitochondrial ROS production and increase in the levels of antioxidant enzymes [52, 56]. Unbalanced redox homeostasis can cause stem cells aging and decreased proliferation. Hypoxic preconditioning augments redox metabolism [54] ROS acts paradoxically, at higher levels its cause's damage, and at lower levels it plays a role of signaling molecule. ROS also controls the hydroxylation of HIF1a, it causes inactivation of prolyl-hydroxylase enzymes (PHD), as a result degradation of alpha subunit does not occur and HIF1 formation

**5. Hypoxic preconditioning improves regenerative potential of cells**

Hypoxic preconditioning improves survival and stemness of cells and has been investigated in a number studies. SOX2, OCT4, NANOG and c-Myc are the markers that show stemness of cells. It has been found that stem cells grown in hypoxia are more viable, have decreased apoptosis through effects on HIF1a and

When cultured under hypoxic (3 or 5% oxygen) condition for 5 days, stem cell

PI3K/Akt signaling pathway get activated in cells exposed to hypoxia which in turn regulates many genes of cell cycle and CDK2 resulting in increased selfrenewal and decreased apoptosis. Under hypoxic conditions cells switch their metabolism more towards glycolytic pathways and less towards oxidative phosphorylation resulting in less reactive oxygen species production (ROS) and more

Hypoxia (1%) results in decreased senescence, increased lifespan of mesenchymal stem cells and were able to maintain proliferation rate, morphology and genetic stability [61]. Cryopreserved adipose derived stem cells cultured at 2 and 5% oxygen tension resulted in increased number and viability as compared to counterparts grown at 21% oxygen. In addition, all stemness related gene expression NANOG, SOX-2, REX-1, and OCT-4 were much higher in hypoxia group than in normoxia group. Another group demonstrated the upregulation of stemness related genes OCT4, SOX2 and NANOG in MSC grown in 3% oxygen culture conditions [62, 63]. Stemness of MSCs remains preserved in hypoxic cultures. Hypoxia results in decreased expression of apoptotic BCL-2 and CASP3 and increased expression of anti-apoptotic genes [64]. Stem cells derived from apical papilla of wisdom teeth also showed increased proliferation and upregulation of SSEA4 which is an embryonic stem cell marker surface antigen. Human umbilical cord derived MSC grown in a culture under 5% oxygen showed better proliferation and maintenance of stemness [65]. MSC derived from different sources showed increased expression of pluripotency markers Oct4, C-Myc and Nanog when cultured at 5% oxygen concentration levels [66]. Low oxygen tension also helped iPSCs from liver cells to preserve their stemness and decrease the time to switch from G1 phase to S phase, increase proliferation but the physiological oxygen level of tissue of origin should be kept in mind because these cells were grown in 10% oxygen level. The same group showed that culturing these cells at very low level

**5.1 Effect of hypoxia on stemness and survival of stem cells**

markers were found to be statistically higher in dental pulp MSC [59].

production of antioxidant enzymes [49, 60].

*DOI: http://dx.doi.org/10.5772/intechopen.93217*

occurs [57].

p53 pathways [58].

*Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal… DOI: http://dx.doi.org/10.5772/intechopen.93217*

preconditioning results in up regulation of Nuclear factor erythroid 2 related factor 2 (NRF2), which is a redox sensitive transcription factor involved in regulation of antioxidant genes [55]. Hypoxic preconditioning results in increased glycolytic metabolism and decreased tricarboxylic acid (TCA) cycle and oxidative phosphorylation. This mechanism leads to decreased mitochondrial ROS production and increase in the levels of antioxidant enzymes [52, 56]. Unbalanced redox homeostasis can cause stem cells aging and decreased proliferation. Hypoxic preconditioning augments redox metabolism [54] ROS acts paradoxically, at higher levels its cause's damage, and at lower levels it plays a role of signaling molecule. ROS also controls the hydroxylation of HIF1a, it causes inactivation of prolyl-hydroxylase enzymes (PHD), as a result degradation of alpha subunit does not occur and HIF1 formation occurs [57].
