Hypoxia and Its Contributions to Regenerative Medicine

Stem cell technology is developing rapidly to bring tissue and organ regeneration from the foreground of current research to the hands of physicians for therapeutic interventions of injuries. Though this field is rapidly progressing, several limiting factors have reduced the efficacy and survival of many transplanted cells. To understand the limitations, a deeper understanding of the chemo–mechanical environment of an injury is needed. Tissue and organ development from specific progenitor cells is tightly controlled by the surrounding biochemical environment. Specifically, oxygen tension, otherwise known as the partial pressure of oxygen, is one of many critical factors playing into the differentiation process of cells into specific tissues. There is a delicate balance between hypoxia (a result of low oxygen tension) and normoxia through the cell life cycle, and this balance varies depending on the biological micro niche in which it resides. Tissue injuries are often accompanied by regions of ischaemia which have proved to negatively influence the survival of transplanted stem cells. This has brought about important adaptations in ex vivo tissue expansion protocol as well as in vivo injury therapies like transplantation of cardiac cells into the hypoxic environment of a recent myocardial infarction or other regions of ischaemic attacks. This review will present the progress of current knowledge on the role of oxygen tension in organogenesis and the significant clinical applications within stem cell therapies. Previously, it has been reported that stem cell fate differs with various oxygen tensions depending on lineage. Here, we first look into the initial uncertainty of the effects of hypoxia and stem cell fate as reported by Cicione et. al. (1) and the metabolic reasoning of the different cellular responses to hypoxia as reported by researcher Munoz et. al. from the Institute of Molecular Biophysics (2). We then look into the investigation by Tanya L. et al. on the effects of oxygen tension in iPS cells which reveals potential significance of hypoxia in cardiac development (3). Finally, we discuss the development and advances of hypoxic preconditioning of cells performed by Uksha S. et. al. (4) and the use of this approach for the ultimate therapy for in vivo application by Xiaofang Y. et. al. (5). Improving stem cell transplant efficiency is of great importance for the advancement of regenerative medicine and current research discussed in this review paves the way for this improvement.
Effects of Severe Hypoxia on Bone Marrow Mesenchymal Stem Cells Differentiation
The natural fate of stem cells can be influenced by the environmental oxygen tension. The goal of this study was to examine effects of hypoxia on differentiation for adipogenic, osteogenic, and chondrogenic lineages from mesenchymal stem cells (MSCs) derived from bone marrow and to see how each lineage differs. Bone marrow is one of the few organs maintained in the hypoxic state, which plays an important role during development and cell differentiation. This unique micro condition prompted researchers to probe into the effects of varying oxygen tension. It has already been demonstrated that in vivo hypoxic conditions for cellular expansion improved mesenchymal stem cell yield and time of expansion to through the chondrogenic lineage. In this particular study by Cicione et. al. (September 2013) (1), bone marrow mesenchymal cells were obtained and isolated from three hip replacement patients. These cells were cultured under standard conditions until the beginning of the differentiation process and characterized using flow cytometry via fluorescent antibody staining. Shortly after characterization, the cells were differentiated down three lineages: adipogenic, osteogenic, and chondrogenic under two oxygen tensions for 14-21 days. Normoxic conditions means that of normal atmospheric pressure with 21% oxygen, whereas hypoxic conditions refer to 1%. The differentiation for each lineage was done with standardized mediums. The results of the study were not uniform for all lineages. The adipogenic lineage under Normoxic condition resulted in a positive stain test for lipid formation whereas the hypoxic condition revealed no lipid formation. This histological outcome was confirmed with qPCR by investigating unique marker genes. The Osteogenic differentiation was initially histologically assessed with calcium deposit staining. Again, the normoxic lineage successfully differentiated as indicated by red staining whereas the hypoxic lineage experienced substantial reduction in differentiation. These results were also confirmed via qPCR for osteogenic markers. The same results followed for chondrogenic lineages. The major findings of this study reinforced the concept that oxygen is a stem cell regulator. Standard in vitro cultures are performed under standard 21% oxygen concentration but normal tissue experiences oxygen pressures between 1% and 13%. This experiment is in agreement with the niche model, which states that stem cells are characterized by a unique microenvironment of low oxygen tension that contributes to maintaining proliferation of stem cells without differentiation. The authors do address the contradicting results with past studies and identify many variable confounding factors between each study. For example, it is generally accepted that chondrogenesis is favored in hypoxia supported by the fact that devascularized nature and subsequent low oxygen tension within cartilage. Although this study clearly demonstrates an inhibitory effect of hypoxia on cell differentiation by observation of a reduction of down the three lineages studied, the fact that previous reports differ warrants further investigation into the mechanisms.
Analysis of Human Mesenchymal Stem Cell Metabolism During Proliferation and Osteogenic Differentiation Under Different Oxygen Tensions
Mesenchymal stem cells (MSC’s) are an important source in bone tissue engineering for grafting. Grafts from these differentiated cells are, at this time, inferior to those obtained from satellite bone within the same patient. This is due to failed survivability of the differentiated and transplanted cells. Munoz et. al. (November 2013), investigated the changes in metabolic phenotype that occur after differentiation from bone marrow mesenchymal stem cells to osteoblasts. The metabolism is significant as the cell metabolic requirements must be met by the resources of its environment. Because these cells are transplanted from a nutrient rich in vitro environment to a site of injury with a reduction nutrients and blood flow there is potential for a disruption in the cell needs. hMSC’s reside in a 2% hypoxic environment which as demonstrated by the previous research, maintains stem cell identity and proliferation in vitro (2). These cells exhibit metabolic production of lactate and are sensitive to to electron transport inhibitors indicating Glycolysis and oxidative phosphorylation as their primary metabolic phenotype. On the other hand, osteoblasts reside in an environment with 5-9% oxygen tension as they are highly vascularized. Osteoblasts respond to oxygen differently with high mitochondrial activity and moderate oxygen supply required. These researchers hypothesize that “osteogenic differentiation is associated with changes in metabolic phenotype, leading to differential responses to changes in oxygen tension”. They utilized gas chromatography–mass spectrometry to profile cells metabolism of 13C-labeled glucose in both hMSC and the hMSC-derived osteoblasts (hMSC-OS) at 2% and 20% oxygen tensions. Metabolites were profiled and allowed for a detailed schematic of cellular metabolism. The metabolic profile for hMSC at 2% reveals an abundance of glycolytic enzymes, increased glucose consumption, and associated lactate efflux. It was confirmed that the cellular metabolism does change after differentiation from MSC to osteoblasts. Hypoxia appears to inhibit glycolytic carbons into the TCA cycle in the osteoblast. The strong connection between glycolysis and TCA cycle in derived osteoblasts relative to hMSC suggests a greater reliance on oxygen availability for osteoblast survival. The results of this study show mesenchymal stem cells metabolism that allow for their own proliferation in a hypoxic environment. Probing into the reasoning behind MSC’s behavior in hypoxic environments aids in the development of and understanding their behavior in ischaemic sites of injury.
Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells
The ultimate application of stem cell therapies may undoubtedly be that of ex vivo organogenesis. However, just as previously discussed studies have shown, oxygen levels differing from that of a cells natural niche have inhibitory effects on differentiation. At this point, it is known that hypoxia can enhance pluripotency while the effect of hypoxia on differentiation of various cell types can be enhanced or repressed. (The most current literature has mostly focused on the repressive effects). Cardiac organogenesis is one of the areas of study, hindered by this factor. In this particular study, the researchers aimed to understand the effect of Hypoxia Inducible Factor alpha 1 modulated wnt signaling on the differentiation of iPS cells into beating cardiac cells. In the case of cardiac cells, hypoxia is known to cause a myriad of cardiac abnormalities. Hypoxia induced genes like HIF 1 are important in development of cardiac structure and vessel formation. HIF is active under hypoxic conditions and activates genes that respond to a hypoxic environment. Previous studies demonstrated the involvement of HIF in the Wnt pathway. Here, Tanya L et al. (November 2013) hypothesize that “Wnt path may be involved in modulating a cardiogenic response to hypoxia during differentiation”. They investigated role of hypoxia on the differentiation of cardiomyocytes from mouse iPS cells and the expression of genes within the Wnt pathway. iPS cells that were differentiated down cardiac lineages for 14 days of culture under normoxic conditions presented with spontaneous contractions. These embryoid bodies were further characterized to be functional cardiomyocytes. iPS cells were also sent down the same lineage but with short 24 hour bouts of hypoxic conditioning. After 13 days, no cells presented with spontaneous contractions. Hypoxia increased HIF 1 alpha and wnt target genes suggesting involvement of the wnt/beta-catenin pathway, which has a role in embryonic development of axis. The results indicated that hypoxia impairs cardiomyocyte differentiation and activates wnt signaling in undifferentiated iPS cells. This study further expresses the importance of oxygen tension in stem cell differentiation and suggests that hypoxia may play a role in early cardiogenesis.
Preconditioning Stem Cells With Caspase Inhibition and Hyperoxia Prior to Hypoxia Exposure Increases Cell Proliferation
Myocardial infarction is a result of arterial occlusion which results in downstream ischemia, necrosis, dysfunction, and failure. Stem cell therapy on infarct hearts can reduce the magnitude of these adverse outcomes and possible reverse the process of heart attack. With ischemia as the result of reduced delivery of oxygen (hypoxia) in the heart, survival of transplanted stem cells to the injured area is severely reduced. Specifically, the transplanted cells undergo apoptosis rather than self renewal as one may expect based on the previous studies discussed. This is likel;ey due to the fact that the ischaemic environment does not meet the metabolic needs of the differentiated cell. The previous studies described the effects of hypoxic environment on Mesenchymal stem cells resulting in restriction of differentiation. After transplantation however, the major observation is that of cellular apoptosis. Regardless, transplanted stem cell survival is still the main focus. With the ultimate goal of increasing transplant survival, Uksha S. et. al. (November 2013) investigated preconditioning of MSC cells. Here, preconditioning of hyperoxia was used as previous studies indicated positive outcomes by hyperoxygenating the organism during ischaemic attack. The effects of preconditioning of rat MSC’s with hyperoxia or Z-VAD-FMK pan caspase inhibitor or both were studied in a hypoxic environment to mimic the infarct heart. Both methods of preconditioning reduced MSC apoptosis and improved overall cell survival. These cells were not transplanted, rather exposed to an environment similar to that of where they would be transplanted. The following study provides application of preconditioning and real transplantation of cells to an ischaemic injury.
Hypoxic Preconditioning with Cobalt of Bone Marrow Mesenchymal Stem Cells Improves Cell Migration and Enhances Therapy for Treatment of Ischemic Acute Kidney Injury
Xiaofang Yu et. al 2013 demonstrated the improved efficacy of kidney MSC transplant therapy in vivo by means of hypoxic preconditioning of MSC. This type of preconditioning differs from the that of Ukshas’ therapy with hyperoxia and caspase inhibition in that it trains the cells for hypoxia prior to transplant. The particular therapy targeted acute kidney injury characterized by poor renal function. Currently, the only management of such injury is dialysis. The specific need now is renal tubular epithelial cell repair. Though stem cell therapy has shown progress, it has been met with reduced cell survival in ischaemic sites. They used cobalt as a hypoxic mimetic preconditioner by culturing MSC in cobalt for 24 hours. Relative to normoxic conditions the Cobalt hypoxic culture showed increased expression of Hypoxic Inducible Factor alpha and enhanced HMC migration and retention to site of ischemic injury. Additionally, the severity of acute injury was reduced with preconditioned MSC. Blood urea nitrogen and serum creatine, both indicators of acute kidney injury, were present in lower quantities in rats receiving the hypoxic preconditioned cells. This study supports hypoxic MSC conditioning prior to transplantation for increased cell survival in ischaemic regions whose oxygen tension is even lower that the standard tension of that tissue. This particular approach with cobalt induced hypoxia is not particularly useful as cobalt is toxic to mammals. There is need for further investigation into modes of hypoxic preconditioning that are not harmful to the organic system.
Discussion / Conclusion
The need for regenerative medicine is pushing the boundaries of current stem cell research. At the heart of the push is the realized benefits of regenerating tissues. Patients would no longer need to worry about donor match and availability, host-graft rejection, need for use of growth factors, or viral vectors. Physicians could reduce adverse outcomes of an anticipated myocardial infarction or other ischaemic attacks. Myocardial infarction is of particular interest due to such a high incidence worldwide. Though the research discussed has focus on in vivo stem cell therapy, the knowledge can be applied to ex vivo organ synthesis and other stem cell frontiers. The benefits are seemingly endless, but limitations exist that have reduced the survival of cells applied in transplant therapies. Specifically, Oxygen tension has been recognized as a major regulator of differentiation and its effects differ depending on the natural niche of the cell to be differentiated. Understanding how hypoxia plays a role in an stem cells natural environment as well as areas to where they migrate is of critical importance in understanding how such therapy works. In an effort to rationalize the behavior (self renewal) of stem cells in a naturally hypoxic environment, one may consider a reduced oxidative stress.The connection between hypoxia of the natural stem cell niche and the effects of an ischaemic injury on stem cells is not entirely clear. The apoptotic effects of ischaemic injuries on stem cells can be explained by the failure to provide cells with necessary oxygen and nutrient demands based on the changes in metabolic activity associated with differentiation (2). The development of preconditioning of stem cells prior to transplantation has since become a viable option for transplant survival. To date the best strategy promoting stem cell survival after transplant is hypoxic preconditioning. Specifically, intermittent bouts of hypoxic stress followed by normoxia as well as pharmacological induction of hypoxia have proved most effective (5). The mechanism behind such preconditioning brings Hypoxia Inducible Factor (HIF-alpha 1) into question (3). HIF leads to modulation of many genes involved in angiogenesis, mitochondrial respiration and biogenesis, glycolysis, cell proliferation, and cell apoptosis (6). Though hypoxic conditions have been shown to fairly effective at preventing apoptosis and ensuring cell survival after graft, the future of preconditioning lies in pharmaceutical approaches to mimic hypoxic conditions. though Cobalt has this effect, it is toxic to the organic body. This has prompted research into other pharmaceuticals to have a similar and safer effect. Perhaps genetic therapy to induce expression of factors relevant to hypoxic environment is a potential means of preconditioning. Investigation into every aspect which promotes stem cell transplant survival will contribute to the reality of regenerative medicine in clinical applications.

References

6. Tsai YP, Wu KJ. Hypoxia-regulated target genes implicated in tumor metastasis. J Biomed Sci. 2012;20:102.

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