Pre-Administration of PLX-R18 Cells Protects Mice from Radiation-Induced Hematopoietic Failure and Lethality

Abstract

Acute Radiation Syndrome (ARS) is a syndrome involving damage to multiple organs caused by exposure to a high dose of ionizing radiation over a short period of time; even low doses of radiation damage the radiosensitive hematopoietic system and causes H-ARS. PLacenta eXpanded (PLX)-R18 is a 3D-expanded placenta-derived stromal cell product designated for the treatment of hematological disorders. These cells have been shown in vitro to secrete hematopoietic proteins, to stimulate colony formation, and to induce bone marrow migration. Previous studies in mice showed that PLX-R18 cells responded to radiation-induced hematopoietic failure by transiently secreting hematopoiesis related proteins to enhance reconstitution of the hematopoietic system. We assessed the potential effect of prophylactic PLX-R18 treatment on H-ARS. PLX-R18 cells were administered intramuscularly to C57BL/6 mice, −1 and 3 days after (LD70/30) total body irradiation. PLX R18 treatment significantly increased survival after irradiation (p < 0.0005). In addition, peripheral blood and bone marrow (BM) cellularity were monitored at several time points up to 30 days. PLX-R18 treatment significantly increased the number of colony-forming hematopoietic progenitors in the femoral BM and significantly raised peripheral blood cellularity. PLX-R18 administration attenuated biomarkers of bone marrow aplasia (EPO, FLT3L), sepsis (SAA), and systemic inflammation (sP-selectin and E-selectin) and attenuated radiation-induced inflammatory cytokines/chemokines and growth factors, including G-CSF, MIP-1a, MIP-1b, IL-2, IL-6 and MCP-1, In addition, PLX-R18 also ameliorated radiation-induced upregulation of pAKT. Taken together, prophylactic PLX-R18 administration may serve as a protection measure, mitigating bone marrow failure symptoms and systemic inflammation in the H-ARS model.

Keywords: PLX-R18; acute radiation syndrome; hematopoietic radiation injury; placenta-derived stromal cells; prophylactic countermeasure.

Figure 1

Survival of C57BL/6 male mice following total-body irradiation with 8 Gy at an estimated rate of 0.6 Gy/min and IM administration of two doses of PLX-R18 (2 million cells/dose) on 1 day prior to and 3 days post-TBI (■) or PlasmaLyte as vehicle (□).

Figure 2

Recovery of peripheral blood cells [white blood cells (WBC), neutrophils (NEU), platelets (PLT), and lymphocytes (LYM)] of unirradiated mice treated with vehicle PlasmaLyte (○) and PLX-R18 (●) and irradiated (8 Gy) mice treated with PlasmaLyte (∆) and PLX-R18 (▲). Either PlasmaLyte or PLX-R18 (2 million cells/dose) was administered (IM) 24 h prior to and 3 days post-TBI (indicated by arrows). Day 0 represents day of irradiation. Data represented are mean ± standard error of the mean (SEM) for n = 8 mice. Significant difference (p < 0.005–0.0125) between PLX-R18-treated and vehicle-treated irradiated group by ANOVA is indicated with an asterisk (*). Some data points in the figure do not have error bars that are visible because they are smaller than symbols. Bar graphs show the most significant data (day 21 post-TBI) for each of the cell type.

Figure 3

Effect of 8 Gy TBI on femoral bone marrow. Clonogenic potential of bone marrow cells was assessed by a CFU assay. Colony forming units (CFU) were assayed on days 1, 14, 30 and 45 after exposure. Cells from three femurs were pooled, counted, and each sample plated in duplicate to be scored 14 days after plating. Data are expressed as mean ± Standard error of mean (SEM). No statistically significant difference was determined between irradiated and naïve groups. Non-irradiated but administered with either PlasmaLyte or PLX-R18 were used as controls. *** indicates p = 0.00016.

Figure 4

PLX-R18 treatment promoted sternal bone marrow hematopoietic cell recovery after lethal exposure of 8 Gy TBI when administered first dose at 24 h prior to and 3 days post-TBI. Bone marrow megakaryocyte numbers were quantitated from histological sections from days 0 (2 h post-TBI), 2, 3, 14 and 30 post-TBI. Significant increase (indicated as *** p = 0.0011) in bone marrow cellularity and megakaryocytes were observed on day 30 post-TBI in the PLX-R18 treatment group. Data represented are mean ± standard error of the mean (SEM) for n = 8 mice. Representative H&E stained sternal bone marrow sections are shown for PlasmaLyte and PLX-R18 on day 30 post-TBI.

Figure 5

PLX-R18 restores levels of endothelial and radiation injury biomarkers. PLX-R18 administration inhibited radiation-induced elevated synthesis of various biomarkers in mouse serum when compared to vehicle-treated animals. The levels of EPO, SAA, PCT, Flt3L, E-selectin and sP-selectin were evaluated from serum from samples collected on days 3, 9, 14, 21 and 30 post-TBI by ELISA. Data represented are mean ± standard error of the mean (SEM) for n = 8 mice per group; *** p < 0.0001; ** p = 0.002.

Figure 6

Differential levels of circulatory cytokines & recovery by PLX-R18 by Luminex assay. Serum samples were subjected to an array of 23 cytokines. The four murine proteins were significantly elevated (n = 5/time point). * p < 0.05, ** p < 0.01, ***/**** p < 0.0001. That yellow symbol means the day of radiation. The arrow means the days we administered the PLX-R18.

Figure 7

Differential levels of cytokines & receptors from the autoimmune pathway as early and late markers of radiation injury and modulation by PLX-R18 treatment by PCR. Spleen samples were subjected to an array of 42 genes.

Figure 8

Differential levels of proteins and their phosphorylated forms as markers of inflammation due to radiation injury and effective amelioration by PLX-R18 treatment. Levels of AKT and pAKT were evaluated by Western blot analyses (A). The band density was quantified using image analyses software from BioRad (B) * indicates p < 0.05.