Endothelial Progenitor Cell Transplantation for Hematopoietic Regeneration.
Authors: Salter Alice B. , Muramoto Garrett G. , Himburg Heather , Meadows Sarah , Chao Nelson J. , Chute John P. 
Published: April 2020

Abstract
Hematopoietic stem cells (HSCs) reside in close association with bone marrow (BM) osteoblasts which help maintain the quiescent HSC pool over time. Hematopoietic stem and progenitor cells also reside in association with BM sinusoidal endothelial cells in vivo but the function of the BM vascular niche in regulating hematopoiesis is less well defined. We hypothesized that the BM endothelial cells function in vivo to support the proliferation and regeneration of hematopoietic stem/progenitor cells and that damage incurred by the BM vascular niche following myelotoxic therapies contributes to delayed hematopoietic recovery in vivo. To test this hypothesis, we generated primary endothelial progenitor cells (EPCs) from the peripheral blood of C57Bl6 mice and examined whether transplantation of allogeneic EPCs alone could facilitate earlier hematopoietic recovery in irradiated recipient BALB/c mice as compared to controls. Ten week old BALB/c mice were irradiated with 550 cGy total body irradiation at day 0 and then received either 1 x 10e6 EPCs intravenously at +4 hours and 2.5 x 10e6 EPCs daily intraperitoneally on days +1 to +4 or saline infusions at the same intervals. Microscopic analysis of femurs from BALB/c mice demonstrated significantly reduced BM cellularity at day +10 and +15, with recovery evident at day +20. EPC-transplanted mice also showed marked BM hypocellularity at day +10 but earlier recovery of BM cellularity at day +15. Total viable BM cell counts confirmed a 2-fold increase in viable BM cells in the EPC-transplanted group at day +15 compared to controls. Flow cytometric analysis revealed that EPC-transplanted mice had significantly earlier recovery of c-kit+sca-1-lin− progenitor cells at day +10, +12 and +15 compared to saline treated controls (means 0.9%, 0.2%, 1.3% vs. 0.2%, 0.06%, 0.5%; p=0.08, p=0.02, p=0.04, respectively). EPC-transplanted mice also demonstrated accelerated regeneration of c-kit+sca-1+lin− (“KSL”) stem/progenitor cells at day +15 compared to saline-treated controls (mean 0.09% vs. 0.03% KSL cells, p=0.04). Importantly, this accelerated recovery of hematopoiesis corresponded with more rapid recovery of hematologic parameters in EPC-transplanted mice compared to controls. EPC-transplanted mice demonstrated significantly earlier recovery of total white blood cells compared to controls by day +16 (mean 1.4 vs. 0.5, p&lt;0.01) and achieved recovery of absolute neutrophil count (ANC) &gt; 500 at day +18; conversely, controls did not achieve ANC &gt; 500 until day +30 (p&lt;0.01 for all timepoints, day +10 to +21). Platelet count recovery was also accelerated in EPC-transplanted mice with a platelet count of &gt;400,000 achieved at day +14 versus day +25 in the saline-treated controls (p&lt;0.01 for all timepoints, day +10 to +18). Preliminary studies using GFP+ endothelial cells have shown no evidence of engraftment of transplanted endothelial cells in the BM or spleen of recipient mice, suggesting that an EPC-elaborated soluble activity may account for the results observed. In ongoing studies, we are examining whether transplanted EPCs mediate these effects indirectly via accelerated recovery of MECA+ endothelial cells in the BM vascular niche or directly upon hematopoietic stem/progenitor cells. Taken together, these studies demonstrate that transplantation of primary EPCs alone mediates accelerated hematopoietic recovery following myelotoxic therapy and these effects are not impeded by immunologic barriers. These results also confirm the contribution of endothelial cells in supporting hematopoietic regeneration in vivo.