Hyperbaric oxygen improves engraftment of ex-vivo expanded and gene transduced human CD34⁺ cells in a murine model of umbilical cord blood transplantation

Abstract

Delayed engraftment and graft failure represent major obstacles to successful umbilical cord blood (UCB) transplantation. Herein, we evaluated the use of hyperbaric oxygen (HBO) therapy as an intervention to improve human UCB stem/progenitor cell engraftment in an immune deficient mouse model. Six- to eight-week old NSG mice were sublethally irradiated 24 hours prior to CD34⁺ UCB cell transplant. Irradiated mice were separated into a non-HBO group (where mice remained under normoxic conditions) and the HBO group (where mice received 2 hours of HBO therapy; 100% oxygen at 2.5 atmospheres absolute). Four hours after completing HBO therapy, both groups intravenously received CD34⁺ UCB cells that were transduced with a lentivirus carrying luciferase gene and expanded for in vivo imaging. Mice were imaged and then sacrificed at one of 10 times up to 4.5 months post-transplant. HBO treated mice demonstrated significantly improved bone marrow, peripheral blood, and spleen retention and subsequent engraftment. In addition, HBO significantly improved peripheral, spleen and bone marrow engraftment of human myeloid and B-cell subsets. In vivo imaging demonstrated that HBO mice had significantly higher ventral and dorsal bioluminescence values. These studies suggest that HBO treatment of NSG mice prior to UCB CD34⁺ cell infusion significantly improves engraftment.

Keywords: CD34(+); Engraftment; Hyperbaric oxygen; Umbilical cord blood.

Figure-1. Impact of hyperbaric oxygen on umbilical cord blood CD34⁺ cell tissue retention/engraftment measured by flow cytometry

Peripheral blood, bone marrow, and spleen homogenates were processed for human CD45 staining and flow cytometry to determine percentage of human CD45⁺ cell retention (up to 72 hours) or engraftment (data after 72 hours). The percentage of human CD45⁺ cells was measured for each mouse and time point for each tissue. The data was log-transformed and plotted on the scatter plot graph. Mean human CD45⁺ cells were connected for each group at each time point (Blue: HBO, red: non-HBO, and green: control). In these experiments, HBO mice demonstrated a statistically significant increase in the percentage of human CD45⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. The difference in the percentage of human CD45⁺ cells between the two groups widened overtime.

Figure-2. Impact of hyperbaric oxygen (HBO) on umbilical cord blood CD34⁺ cell engraftment determined by bioluminescence

Transplanted mice were imaged in vivo using IVIS spectrum imager. Prior to imaging, mice were injected with D-Luciferin. Subsequently, mice were imaged ventrally then dorsally. Images were quantified using living image software version 4.0 and) measurements were expressed as flux i.e. photons/second (p/s). A: a representative in vivo image of the dorsal aspect of HBO and non-HBO mice obtained at month 3 time point. A measureable difference in bioluminescence signal between the 2 groups is demonstrated. Individual scales are shown to correspond to bioluminescence. B: photon flux log transformed data depicted in a scatter-plot. Mean photon flux values were connected for each group at each time point (Blue: HBO and red: non-HBO). In these experiments, HBO mice demonstrated a statistically significant increase in photon flux over non-HBO mice. The difference between the two groups of mice was more significant at the later time points of 3 and 4 months. C: In vivo imaging of the ventral aspect of HBO and non-HBO mice at baseline and 3 hours post-transplant. At baseline, (10 minutes after UCB CD34⁺ cell injection) both groups of mice demonstrated bioluminescence in the thoracic area. On the other hand, only HBO mice demonstrated central thoracic bioluminescence 3-hours post-transplant. For the 3 hour time point, 5 mice from the non-HBO pool and 5 mice from HBO pool were grouped. Abbreviation: HBO stands for hyperbaric oxygen.

Figure-2. Impact of hyperbaric oxygen (HBO) on umbilical cord blood CD34⁺ cell engraftment determined by bioluminescence

Transplanted mice were imaged in vivo using IVIS spectrum imager. Prior to imaging, mice were injected with D-Luciferin. Subsequently, mice were imaged ventrally then dorsally. Images were quantified using living image software version 4.0 and) measurements were expressed as flux i.e. photons/second (p/s). A: a representative in vivo image of the dorsal aspect of HBO and non-HBO mice obtained at month 3 time point. A measureable difference in bioluminescence signal between the 2 groups is demonstrated. Individual scales are shown to correspond to bioluminescence. B: photon flux log transformed data depicted in a scatter-plot. Mean photon flux values were connected for each group at each time point (Blue: HBO and red: non-HBO). In these experiments, HBO mice demonstrated a statistically significant increase in photon flux over non-HBO mice. The difference between the two groups of mice was more significant at the later time points of 3 and 4 months. C: In vivo imaging of the ventral aspect of HBO and non-HBO mice at baseline and 3 hours post-transplant. At baseline, (10 minutes after UCB CD34⁺ cell injection) both groups of mice demonstrated bioluminescence in the thoracic area. On the other hand, only HBO mice demonstrated central thoracic bioluminescence 3-hours post-transplant. For the 3 hour time point, 5 mice from the non-HBO pool and 5 mice from HBO pool were grouped. Abbreviation: HBO stands for hyperbaric oxygen.

Figure-2. Impact of hyperbaric oxygen (HBO) on umbilical cord blood CD34⁺ cell engraftment determined by bioluminescence

Transplanted mice were imaged in vivo using IVIS spectrum imager. Prior to imaging, mice were injected with D-Luciferin. Subsequently, mice were imaged ventrally then dorsally. Images were quantified using living image software version 4.0 and) measurements were expressed as flux i.e. photons/second (p/s). A: a representative in vivo image of the dorsal aspect of HBO and non-HBO mice obtained at month 3 time point. A measureable difference in bioluminescence signal between the 2 groups is demonstrated. Individual scales are shown to correspond to bioluminescence. B: photon flux log transformed data depicted in a scatter-plot. Mean photon flux values were connected for each group at each time point (Blue: HBO and red: non-HBO). In these experiments, HBO mice demonstrated a statistically significant increase in photon flux over non-HBO mice. The difference between the two groups of mice was more significant at the later time points of 3 and 4 months. C: In vivo imaging of the ventral aspect of HBO and non-HBO mice at baseline and 3 hours post-transplant. At baseline, (10 minutes after UCB CD34⁺ cell injection) both groups of mice demonstrated bioluminescence in the thoracic area. On the other hand, only HBO mice demonstrated central thoracic bioluminescence 3-hours post-transplant. For the 3 hour time point, 5 mice from the non-HBO pool and 5 mice from HBO pool were grouped. Abbreviation: HBO stands for hyperbaric oxygen.

Figure-3. Impact of hyperbaric oxygen on umbilical cord blood CD34⁺ cell subsets engraftment

Peripheral blood, bone marrow, and spleen were harvested from HBO and non-HBO mice. Subset engraftment was assessed by flow cytometry staining for human CD33 (myeloid subset-Fig.3A), human CD 19 (B-cell subset-Fig.3B), and human CD3 (T cell subset-Fig.3C) antibodies. The percentage of human CD19, CD33, or CD3 positive cells was measured for each mouse and time point for each tissue. The data was log-transformed and plotted on the scatterplot graph. Mean human CD19, CD33, and CD3 positive cells were connected for each group for each time point (Blue: HBO, red: non-HBO, and green: control). A: HBO mice demonstrated a statistically significant increase in the percentage of human CD33⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. B:HBO mice demonstrated a statistically significant increase in the percentage of human CD19⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. The difference in the percentage of human CD33⁺ and CD19⁺ cells between the two groups widened overtime. C: HBO mice demonstrated a statistically significant increase in the percentage of human CD3⁺ cells across time only in peripheral blood compared to non-HBO mice.

Figure-3. Impact of hyperbaric oxygen on umbilical cord blood CD34⁺ cell subsets engraftment

Peripheral blood, bone marrow, and spleen were harvested from HBO and non-HBO mice. Subset engraftment was assessed by flow cytometry staining for human CD33 (myeloid subset-Fig.3A), human CD 19 (B-cell subset-Fig.3B), and human CD3 (T cell subset-Fig.3C) antibodies. The percentage of human CD19, CD33, or CD3 positive cells was measured for each mouse and time point for each tissue. The data was log-transformed and plotted on the scatterplot graph. Mean human CD19, CD33, and CD3 positive cells were connected for each group for each time point (Blue: HBO, red: non-HBO, and green: control). A: HBO mice demonstrated a statistically significant increase in the percentage of human CD33⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. B:HBO mice demonstrated a statistically significant increase in the percentage of human CD19⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. The difference in the percentage of human CD33⁺ and CD19⁺ cells between the two groups widened overtime. C: HBO mice demonstrated a statistically significant increase in the percentage of human CD3⁺ cells across time only in peripheral blood compared to non-HBO mice.

Figure-3. Impact of hyperbaric oxygen on umbilical cord blood CD34⁺ cell subsets engraftment

Peripheral blood, bone marrow, and spleen were harvested from HBO and non-HBO mice. Subset engraftment was assessed by flow cytometry staining for human CD33 (myeloid subset-Fig.3A), human CD 19 (B-cell subset-Fig.3B), and human CD3 (T cell subset-Fig.3C) antibodies. The percentage of human CD19, CD33, or CD3 positive cells was measured for each mouse and time point for each tissue. The data was log-transformed and plotted on the scatterplot graph. Mean human CD19, CD33, and CD3 positive cells were connected for each group for each time point (Blue: HBO, red: non-HBO, and green: control). A: HBO mice demonstrated a statistically significant increase in the percentage of human CD33⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. B:HBO mice demonstrated a statistically significant increase in the percentage of human CD19⁺ cells in peripheral blood, bone marrow, and spleen over non-HBO mice. The difference in the percentage of human CD33⁺ and CD19⁺ cells between the two groups widened overtime. C: HBO mice demonstrated a statistically significant increase in the percentage of human CD3⁺ cells across time only in peripheral blood compared to non-HBO mice.