The Role of Endoglin in the Immunomodulatory Capacities of Mesenchymal Stem Cells and the Relationship to Hyperbaric Oxygen Therapy

Abstract

Mesenchymal stem cells (MSCs), a type of “adult” stem cell found in most organs, represent an emerging tool in the field of regenerative medicine. In the setting of myocardial injury, for example, MSCs have been shown to promote repair and recovery. Studies have been hampered, however, by variation in MSC phenotypes, as defined by cell surface marker expression, and the absence of a clear consensus of what MSC phenotypes are best able to support regeneration. Providing insight into the regenerative capabilities of varying MSCs phenotypes is crucial to their continued success in therapies. One cell surface marker that has been shown to have functional relevance to regenerative medicine is endoglin (CD105). Endoglin is a type I transmembrane protein that functions as an auxiliary receptor for the TGFβ receptor complex. The absence of endoglin on MSCs reportedly defines a population with a greater propensity for cardiovascular differentiation and greater capacity to support myocardial repair. However, despite their ability to improve post-infarct cardiac function and reduce the size of resultant scars, the retention of these cells in myocardial tissue varies from only 3%-6%. Thus, transdifferentiation seems an unlikely explanation for the regenerative effects. In addition to their capacity for multipotential differentiation, MSCs also have a reported ability to suppress various immune responses, including suppression of stimulated T-cell proliferation. I hypothesized that the absence of CD105 may identify a population of MSCs with an altered capacity to modulate the immunologic milieu at the time of myocardial injury, and this difference in immunomodulatory function accounts for the improved outcomes. To investigate this idea, MSCs that either express endoglin (CD105+) or not (CD105-) were co-cultured with T-cells and effects on stimulated T-cell proliferation examined. Surprisingly, neither CD105+ nor CD105- MSCs were able to suppress proliferation of either CD4+ or CD8+ T-cells. In light of this observation, effects of MSCs on T-cell differentiation were assessed. Notably, co-culture of both CD105+ and CD105- MSCs with CD4+ T-cells showed a striking effect on T-cell differentiation. Syngeneic MSCs induced Th2 skewing, with increased expression of IL-4 and IL-10 and a marked decrease in IL-17 expression. The presence of CD105 in the MSCs influenced this outcome, with a more pronounced decrease in IL-17 expression and increased IL-4 secretion. Effects of allogeneic MSCs on T-cell differentiation were also examined. Due to the reported “immune privilege” of MSCs, it has been proposed that allogeneic MSCs may be utilized to expand the pool of MSCs available for clinical use. In an allogeneic co-culture system, both CD105+ and CD105- MSCs significantly affected T-cell differentiation. Compared to syngeneic MSCs, allogeneic MSCs stimulated higher expression of IL-4, IL-5, and IL-10, as well as increased secretion of IL-4 and IL-10. There were fewer differences between CD105+ and CD105- MSCs. However, CD105- MSCs induced much less IL-2 and IFNγ compared to CD105+ MSCs. These results indicate that MSCs influence T-cell differentiation, resulting in a Th2 skewing with increased production of the immunosuppressive cytokine IL-10 and, in the case of syngeneic cells, diminished Th17 differentiation. This effect could be essential for the previously described MSC-induced cardiac functional preservation, and could explain differences between CD105+ and CD105- phenotypes. In addition to these mechanistic studies, I also examined potential clinical relevance of CD105 expression in MSCs in umbilical cord blood transplantation. This question arose from a clinical study was underway at the University of Kansas Cancer Center that focused on using hyperbaric oxygen (HBO) for improving clinical outcomes post umbilical cord blood (UCB) transplant. In the observation of post-transplant transfusion requirements, HBO-treated patients required less supportive blood products than historic UCB-recipients. Furthermore, they experienced decreased time to transfusion independence. However, upon examining the correlation between levels of erythropoietin in these HBO-patients to their transfusion requirements, the data did not match with animal studies, which showed a reduction in erythropoietin was the causative method by which HBO improved engraftment. Therefore, we examined the supportive role of MSCs in response to HBO in the hematopoietic niche. This included observing changes in CD105 on MSCs after exposure to HBO, as endoglin is a known hypoxia response gene. While the observation of immunomodulatory factors produced by MSCs as a result of HBO is still underway, a downregulation of CD105 12 hours after exposure to HBO was observed. This correlates to the homing window of hematopoietic stem cells in the context of transplant and may be indicative of changes in MSCs providing a more supportive bone marrow microenvironment after exposure to HBO-therapy.