**3. Pre-clinical data supporting HBO role in modulating EPO/EPOR signaling in HSCs**

Previously published work implicating erythropoietin (EPO) in HSC homing led investigators to examine the role of EPO/EPOR signaling in HSC homing and engraftment in vitro and in vivo pre-clinical models. Gonzalez et al. demonstrated that circulating HSCs rapidly decline after birth [27]. Interestingly, the decline in HSCs correlated with low EPO blood concentration. Additionally, the decline in HSCs being attributed to HSC BM homing, these observations suggested a possible role for EPO in BM homing and clearance of HSCs from the infant's circulation following birth. Investigators have pursued HBO as a potentially safe approach to effectively lower EPO as previously published [28]. The hypothesis was that lowering EPO at the time of hematopoietic stem/progenitor cell (HSPC) infusion will result in improved bone marrow homing and subsequent engraftment. Studies examining HBOT effects on hematopoietic stem cells are limited. On the other hand, HBOT has been shown to have minimal, if any, effects on blood counts during steady-state conditions [29]. The previously published and accumulated pre-clinical data that supports EPO's role in UCB engraftment are summarized in the next section [30].

To understand EPO effects on UCB CD34+ , the expression of EPOR was assessed by flow cytometry. Analyses of 5 UCB units revealed that on average 6.5% of CD34+ UCB cells express EPOR [30]. A significantly higher percentage of EPOR positive cells (45.7 ± 1.4%, **Figure 2**) was observed within the HSC (Lin<sup>−</sup> CD34+ CD38<sup>−</sup> CD45RA<sup>−</sup> CD90+ CD49f+ cells) population. EPOR positive cells were less among multipotent progenitor (MPP) (Lin<sup>−</sup> CD34+ CD38<sup>−</sup> CD45RA<sup>−</sup> CD90<sup>−</sup> CD49f<sup>−</sup> cells, 22.2 ± 0.3%) or the broader progenitor pool (Lin<sup>−</sup>CD34+ CD38+ cells, 25.1 ± 0.7%). To test whether a functional EPO-EPOR signaling cascade was activated in EPORexpressing UCB CD34+ cells, EPOR expression was depleted via RNA interference (RNAi), and the erythroid differentiation potential after culture in methylcellulose culture medium was compared to UCB CD34+ cells without EPOR depletion. Depletion of EPOR expression by RNAi greatly reduced the size of erythroid colonies and UCB CD34+ differentiation potential toward the erythroid lineage, indicating that EPO promotes functional EPO-EPOR signaling response in these cells [30].

#### **Figure 2.**

*Erythropoietin receptor expression on umbilical cord blood CD34+ cells and subsets (unpublished data).*

As earlier studies potentially implicated EPO signaling in hematopoietic stem/ progenitor cell (HSPC) homing [27], investigators tested if there were EPO-EPOR signaling effects on SDF-1-induced migration of UCB CD34+ HSPC, by examining UCB CD34+ CD38<sup>−</sup> cell transmigration toward an SDF-1 gradient after a preexposure of the cells to different concentrations of EPO. Exposure of UCB CD34+ CD38<sup>−</sup> to EPO significantly reduced their SDF-1-induced directional migration. Blocking EPO signaling by anti-EPOR or anti-EPO antibodies rescued SDF-1-induced migration of UCB CD34<sup>+</sup> cells for both CD34<sup>+</sup> CD38<sup>−</sup> and CD34+ CD38+ populations [30].

HBO treatment has been shown to reduce systemic EPO levels in healthy volunteers [28]. As previous in vitro studies indicated that EPO-EPOR signaling inhibits SDF-1-induced migration of UCB CD34+ cells, investigators examined whether HBO pre-treatment of mice prior to cell infusion enhances BM homing. First, investigators measured serum EPO levels in their murine transplant model 7 hours after HBO exposure (or 3 hours post UCB CD34+ infusion). HBO exposure significantly reduced serum EPO levels compared to controls (*p* < 0.0001). In addition, a higher percentage of the UCB CD34+ cells was seen in the BM of HBO-treated mice 3 hours posttransplant [30].

In the same murine model, investigators evaluated the impact of HBO treatment on peripheral blood, BM, and spleen retention at early time points (24–72 hours), which correlates with BM homing, and up to 4.5 months, which correlates with long-term engraftment. Efficient support of human cell engraftment has been reported in 6–8-week-old female NSG mice NOD/SCID/ IL-2Rgcnull [31] model. Briefly, sublethally irradiated NSG mice, after 24 hours, were treated with HBO for 2 hours (HBO) or without HBO in the control group. Next, approximately 105 CD34-selected UCB cells were infused into each mouse 6 hours following the start of HBO. Mice were euthanized at different time points; peripheral blood, BM, and spleen tissue were harvested; and engraftment was analyzed by flow cytometry. The degree of engraftment was determined by measuring the percentage of human CD45-expressing cells. For HBO therapy, 100% oxygen was delivered at 2.5 atmospheres absolute (ATA) in a single-place chamber. In murine in vivo model, HBO-treated mice had significantly improved BM (*p* = 0.0067), peripheral blood (*p* = 0.0131), and spleen (*p* = 0.0293) engraftment [32], the impact of which was more pronounced toward later time points at 3 and 4 months.

**155**

**Figure 3.**

**Figure 4.**

*EPO treatment enriches CD71<sup>+</sup>*

*(unpublished data).*

*engraftment.*

*Effect of Hyperbaric Oxygen on Hematopoietic Stem Cell Transplantation*

EPO has been shown to impact hematopoietic progenitor cells differentiation [33].

 *early erythroid cells (A) and correlates with active STAT3 signaling (B)* 

cell differentiation was

cells to the BM, thus improv-

cell infu-

 *cells.* 

Because HBOT lowers EPO levels in posttransplant, the impact of a low EPO

*Gene expression data analysis evaluating erythropoietin (EPO) treatment effects on UCB CD34+*

*The mechanisms by which hyperbaric oxygen therapy (HBO) affects hematopoietic stem/progenitor cell* 

examined. HBO mice demonstrated significantly lower numbers of burst-forming unit-erythroid (BFU-E) (*p* = 0.043) and increasing numbers of colony-forming unit-granulocyte/macrophage (CFU-G/M) (*p* = 0.05) 1 week following transplant. Interestingly, despite reduced BFU-E in the in vivo experiments, investigators observed a favorable trend in red blood cell (RBC) time to transfusion indepen-

These findings suggest that lowering the recipient EPO levels favors UCB CD34<sup>+</sup> engraftment by affecting two important HSC functions: BM homing and HSPC differentiation (**Figure 3**). Lower recipient EPO at the time of UCB CD34+

sion results in less early erythroid differentiation of infused progenitor cells. This

ing long-term multi-lineage engraftment. In confirmatory experiments utilizing

environment induced by HBO on human UCB CD34+

leads to early homing of undifferentiated UCB CD34<sup>+</sup>

dence (TTI) in their pilot study.

*DOI: http://dx.doi.org/10.5772/intechopen.85223*

*Effect of Hyperbaric Oxygen on Hematopoietic Stem Cell Transplantation DOI: http://dx.doi.org/10.5772/intechopen.85223*

#### **Figure 3.**

*Advances in Hematologic Malignancies*

As earlier studies potentially implicated EPO signaling in hematopoietic stem/ progenitor cell (HSPC) homing [27], investigators tested if there were EPO-EPOR

sure of the cells to different concentrations of EPO. Exposure of UCB CD34+

to EPO significantly reduced their SDF-1-induced directional migration. Blocking EPO signaling by anti-EPOR or anti-EPO antibodies rescued SDF-1-induced migra-

HBO treatment has been shown to reduce systemic EPO levels in healthy volunteers [28]. As previous in vitro studies indicated that EPO-EPOR signaling inhibits

HBO pre-treatment of mice prior to cell infusion enhances BM homing. First, investigators measured serum EPO levels in their murine transplant model 7 hours after

reduced serum EPO levels compared to controls (*p* < 0.0001). In addition, a higher

In the same murine model, investigators evaluated the impact of HBO treatment on peripheral blood, BM, and spleen retention at early time points (24–72 hours), which correlates with BM homing, and up to 4.5 months, which correlates with long-term engraftment. Efficient support of human cell engraftment has been reported in 6–8-week-old female NSG mice NOD/SCID/ IL-2Rgcnull [31] model. Briefly, sublethally irradiated NSG mice, after 24 hours, were treated with HBO for 2 hours (HBO) or without HBO in the control group.

6 hours following the start of HBO. Mice were euthanized at different time points; peripheral blood, BM, and spleen tissue were harvested; and engraftment was analyzed by flow cytometry. The degree of engraftment was determined by measuring the percentage of human CD45-expressing cells. For HBO therapy, 100% oxygen was delivered at 2.5 atmospheres absolute (ATA) in a single-place chamber. In murine in vivo model, HBO-treated mice had significantly improved BM (*p* = 0.0067), peripheral blood (*p* = 0.0131), and spleen (*p* = 0.0293) engraftment [32], the impact of which was more pronounced toward later time points at

CD38<sup>−</sup> cell transmigration toward an SDF-1 gradient after a preexpo-

CD38<sup>−</sup> and CD34+

HSPC, by examining

populations [30].

CD38+

 *cells and subsets (unpublished data).*

infusion). HBO exposure significantly

cells, investigators examined whether

cells was seen in the BM of HBO-treated mice 3 hours

CD34-selected UCB cells were infused into each mouse

CD38<sup>−</sup>

signaling effects on SDF-1-induced migration of UCB CD34+

*Erythropoietin receptor expression on umbilical cord blood CD34+*

cells for both CD34<sup>+</sup>

SDF-1-induced migration of UCB CD34+

HBO exposure (or 3 hours post UCB CD34+

percentage of the UCB CD34+

posttransplant [30].

Next, approximately 105

**154**

3 and 4 months.

UCB CD34<sup>+</sup>

**Figure 2.**

tion of UCB CD34<sup>+</sup>

*The mechanisms by which hyperbaric oxygen therapy (HBO) affects hematopoietic stem/progenitor cell engraftment.*

#### **Figure 4.**

*Gene expression data analysis evaluating erythropoietin (EPO) treatment effects on UCB CD34+ cells. EPO treatment enriches CD71<sup>+</sup> early erythroid cells (A) and correlates with active STAT3 signaling (B) (unpublished data).*

EPO has been shown to impact hematopoietic progenitor cells differentiation [33]. Because HBOT lowers EPO levels in posttransplant, the impact of a low EPO environment induced by HBO on human UCB CD34+ cell differentiation was examined. HBO mice demonstrated significantly lower numbers of burst-forming unit-erythroid (BFU-E) (*p* = 0.043) and increasing numbers of colony-forming unit-granulocyte/macrophage (CFU-G/M) (*p* = 0.05) 1 week following transplant. Interestingly, despite reduced BFU-E in the in vivo experiments, investigators observed a favorable trend in red blood cell (RBC) time to transfusion independence (TTI) in their pilot study.

These findings suggest that lowering the recipient EPO levels favors UCB CD34<sup>+</sup> engraftment by affecting two important HSC functions: BM homing and HSPC differentiation (**Figure 3**). Lower recipient EPO at the time of UCB CD34+ cell infusion results in less early erythroid differentiation of infused progenitor cells. This leads to early homing of undifferentiated UCB CD34<sup>+</sup> cells to the BM, thus improving long-term multi-lineage engraftment. In confirmatory experiments utilizing

RNA-seq for transcriptional assessment, investigators found that EPO treatment of UCB CD34+ cells enriches CD71<sup>+</sup> early erythroid cells, consistent with early erythroid commitment (**Figure 4**). In the same data set, EPO treatment was associated with signal transducer and activator of transcription 3 (STAT3) pathway activation (**Figure 4**). Importantly, signal transducer and activator of transcription 3 (STAT3) is a known downstream effector of EPOR signal transduction [34–37].
