First Documentation of Cell-to-Cell Peroxisome Transfer in Hematopoietic Stem and Progenitor Cells: A Method to "Relieve Stress"?

Cell to cell communication is key to survival. Recently it has been demonstrated that intercellular transfer of mitochondria occurs and can aid in marrow recovery after irradiation. Peroxisomes are a single membrane bound organelle responsible for various functions including lipid metabolism and protecting the cell from oxidative damage by reactive oxygen species (ROS). We have created the first mouse model in which peroxisomes can be tracked in living tissues using GFP tagged with a peroxisome targeting signal sequence (PTS), under the control of the Rosa promoter. Such GFP-PTS animals showed widespread peroxisomal GFP expression in all organs including hematopoietic stem and progenitor cells (HSPC) such as LSK-SLAM.

Marrow stroma cell lines were established from GFP-PTS animals. In vitro peroxisome transfer from stroma to HSPC showed a transfer rate of up to 0.5 %, which could be achieved only when cell-to-cell contact took place; the use of transwells inhibited transfer. When HSPCs were simulated to proliferate, peroxisome transfer dropped by 50.1% (p<0.0001). The ability of HSPC to take up peroxisomes in vivo was tested in a model of hematopoietic cell transplant (HCT). Using GFP-PTS mice as recipients with wild-type (WT) marrow donors, we found that after busulfan or radiation (9 Gy) conditioning, peroxisome transfer could occur into WT adoptively HSPC within 48 hours after HCT at a rate of 1%. At 3 months post HCT, bone marrow mononuclear cells (BMMC) and Lin^-SCA1⁺CKIT⁺ (LSK) were 100% donor and maintained GFP⁺peroxisomes at a frequency of 0.9% which were transferred from surrounding niche/stroma cells.

To test HSPC to HSPC peroxisome transfer, sublethal conditioning (4.5 Gy) of WT was used to generate mixed chimerism models with marrow from GFP-PTS mice used as donor. Total peripheral or marrow engraftment was 45-55% donor as expected at 3 months post-HCT. WT BMMC could take up peroxisomes at a 0.9% frequency, while LSK had a significantly higher peroxisome uptake of 3.9% (p=0.02) indicating greater efficiency. Results were similar if GFP-PTS⁺ animals were recipients in a sublethal transplant setting. Remarkably, we could also demonstrate that peroxisome transfer can occur between GFP-PTS donor hematopoietic cells and WT cells in the liver, spleen, thymus, and brain after HCT.

The genetic deletion of peroxisomes (via pex1 knockout) in animals resulted in ROS levels 10.3-fold over that of wild-type in PBMC, BMMC, and LSK (p<0.0001) as assessed by CellROX assay. Such animals had a severe defect in growth, had normal hematopoiesis, and increased radiation sensitivity indicating the importance of peroxisomes in reducing oxidative stress. To test the hypothesis that peroxisome transfer is utilized in situations of increased oxidative stress, pex1 KO stroma lines were exposed to increasing doses of radiation followed by incubation with GFP-PTS⁺ BMMC. Radiation treatment enhanced peroxisome transfer 5.1-fold over no-irradiated control (p<0.01), and cells receiving donor peroxisomes had greater viability (84% vs 68% viable, p<0.01) and reduced levels oxidative stress 24.8% compared to cell not receiving peroxisomes (p=0.03).

The entire system was reproduced in a zebrafish model expressing GFP-PTS. Sublethally irradiated WT zebrafish engrafted donor GFP-PTS+ marrow at 50% and showed a significantly high rate of peroxisome uptake of into WT HSPS of 30% at 3 months post-HCT, suggesting some species may perform peroxisome transfer at much greater rates than others.

In conclusion, we have demonstrated, for the first time, that peroxisome transfer can occur between a wide variety of cell types in vitro and in vivo, and HSPC-to-HSPC peroxisome transfer seems to be the most robust. The ability of peroxisome transfer to rescue cells from radiation-induced ROS and death was demonstrated in vitro while in vivo experiments are proceeding. There may be other far-reaching effects in terms of altering cellular metabolism occurring as a result of peroxisome transfer remaining to be discovered.

Gupta:Blue Rock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vertex Pharmaceuticals: Consultancy.