Endothelial Cells Mediate the Repair of Human Bone Marrow Stem and Progenitor Cells Following Radiation Injury.

Abstract

The purposeful misuse of ionizing radiation has been recognized as a major bioterrorism threat in the United States. Although the myeloablative effects of ionizing radiation exposure have been well established, definitive treatments for potential victims of radiation injury are lacking. We have recently demonstrated that BM stem cells can be harvested from mice following exposure to lethal dose total body irradiation and can subsequently recover multilineage repopulating potential via ex vivo culture with microvascular endothelial cells (EC). However, it remains to be determined whether human hematopoietic stem cells (HSC) can be recovered in a similar manner following high dose radiation injury. In this study, we examined the capacity for ex vivo culture with and without primary human endothelial cells to support the cellular repair and recovery of primary human BM CD34+ cells following exposure to high dose irradiation. Exposure of primary BM CD34+ cells to 400 cGy resulted in a 40% decline in total viable cells and an 89% decline in CD34+CD38− cells compared to input despite 10 day culture with optimal concentrations of thrombopoietin, stem cell factor, and flt-3 ligand alone. Conversely, co-culture of 400 cGy irradiated BM CD34+ cells with primary human brain endothelial cells (LS-01) resulted in a 6-fold and 22-fold increase in total cells and CD34+CD38− cells compared to input, respectively. Non-contact cultures with LS-01 resulted in comparable recovery of total cells and the CD34+CD38− subset by 10 days. Concordantly, TSF-cultured progeny contained 5-fold greater numbers of early apoptotic and necrotic cells within the total and CD34+CD38− fractions as compared to the progeny of contact and non-contact endothelial cultures. Colony forming cell (CFC) assays demonstrated that 400 cGy exposure caused an 8-fold reduction in CFU-total compared to normal BM CD34+ cells and no recovery of CFU-GM, BFU-E, or CFU-Mix was observed following TSF culture. Co-culture with LS-01 resulted in the recovery of 50% of CFU-total compared to normal BM CD34+ cells. In order to assess HSC content post-irradiation, NOD/SCID mice were transplanted with normal BM CD34+ cells, 400 cGy irradiated BM CD34+ cells, and the progeny of 400 cGy irradiated cells following ex vivo culture. 100% of mice transplanted with normal BM CD34+ cells (7.5 x 105) demonstrated human hematopoietic engraftment at 6 weeks, whereas 0% of mice transplanted with 400 cGy irradiated BM CD34+ cells showed detectable human repopulation. Twenty-five percent of mice transplanted with 400 cGy irradiated/LS-01 cultured cells demonstrated human repopulation, whereas 0% of mice transplanted with 400 cGy irradiated/TSF-cultured cells showed human engraftment. These data demonstrate that ionizing radiation has a profoundly toxic effect on both human HSC and committed progenitor cells. Endothelial cells and endothelial cell-derived soluble factors appear to provide anti-apoptotic signals to BM progenitor cells following radiation damage, allowing cellular repair of committed progenitors as well as cells with in vivo repopulating capacity. Cytokine combinations alone, such as TSF, appear ineffective toward rescuing human BM stem/progenitor cells from radiation damage. Studies are ongoing to optimize the application of endothelial cells and endothelial cell-derived growth factors to stimulate HSC repair following radiation injury.