Human Placenta-Derived Stromal Cells Rescue Mice from Radiation-Induced Bone Marrow Failure: A Cytof-Based Mechanistic Analysis
Authors: Allen Hoshea , Sher Noa , Rekhes Shiran , Pinzur Lena , Prezma Tal , Gorodetsky Raphael , Volk Hans Dieter , Aberman Zami , Ofir Racheli 
Published: October 2019

Abstract
Acute Radiation Syndrome (ARS) is a set of health effects involving damage to multiple organs caused by exposure to high dose ionizing radiation over a short period of time. Even low doses damage the radio-sensitive hematopoietic system (causing H-ARS). We probed the mechanism of action by which a 3D-expanded placenta-derived stromal cell product designated for the treatment of hematological disorders alleviates symptoms in the H-ARS mouse model. These cells have been shown in vitro to secrete hematopoietic proteins, stimulate colony formation, and induce bone marrow (BM) migration.
Previous studies show that cells administered intramuscularly to C3H/HeN and C57BL/6 mice, 1 and 5 days after (LD70/30) total body irradiation, rescue radio-induced weight loss, survival, and peripheral blood (PB) and BM cellularity. The injected cells transiently secrete haematopoiesis related proteins to enhance reconstitution of the hematopoietic system and further induce endogenous cells to secrete a panel of cytokines.
Analysis of PB and BM taken from the experimental endpoint (day 23) of cell-treated irradiated mice indicated rescue of blood lineages to levels near those of naïve mice.
CyTOF analysis of BM cells indicated that in the myeloid lineage, the percentages of neutrophils and monocytes were consistently higher in cell-treated irradiated mice than in naïve mice (2.6±0.08 fold for neutrophils and 1.4±0.1 fold for monocytes). There was also a 1.5±0.5 fold increase in the percentage of the lymphoid lineage pDCs and a 2.8±0.9 fold reduction in the percentage of T cells in the BM of the treated compared to naïve mice. On the other hand, the percentage of B cells and NK cells were more similar when compared to naïve mice (1.2±0.4 fold for B cells and 1.1±0.2 fold for NK cells).
Neutrophils and monocytes were also elevated in the PB of cell-treated irradiated mice (1.3±0.09 fold for neutrophils and 1.7±0.2 fold for monocytes), and within the monocyte population, percentages of classical and non-classical monocytes were essentially identical to naïve mice (0.97±0.05 fold for classical and 1.1±0.1 fold for non-classical). In cell-treated mice, B cells showed evidence of active recovery from pro-B to the immature stage of development when compared to naïve mice (4.4±0.7 fold increase for pro-B cells in BM and 5.5±0.8 fold increase for immature B cells in blood). In addition, CD8+ effector memory T cells showed a 1.9±0.3 fold increase in treated compared to naïve mice.
The effect of placenta-derived stromal cells on human BM migration in vitro was also analyzed by CyTOF. Results indicated that the stromal cell product may induce significant maturation of early-stage neutrophils to late-stage neutrophils and significantly enhance the migration of neutrophils 17.9±1.4 fold compared to control. Active induction of migration of monocytes (2.4±0.0004 fold), eosinophils (2.6±0.1 fold), and endothelial cells (2.1±0.2 fold) was also observed.
A number of these beneficial effects may be ascribed to cytokines known to be secreted by the cell product in high concentration. IL-8 and osteopontin are highly chemotactic toward neutrophils and MCP1 is highly chemotactic toward monocytes. These cytokines may contribute to the observed migration of human BM neutrophils and monocytes in vitro as well as to the increased percentage of neutrophils and monocytes in the blood of the cell-treated irradiated mice. In addition, cytokines known to be involved in neutrophil granulopoiesis, i.e. SCF, G-CSF, GM-CSF, IL-3 and IL-6, are present in the secretome of the cell product and may contribute to the observed maturation of early-stage neutrophils in human BM.
Taken together, placenta-derived stromal cells have the capacity to alleviate symptoms of BM failure in the H-ARS model and rescue multiple blood lineages via a complex mechanism of action based on the secretion of multiple proteins acting on multiple hematopoietic lineages. Due to this combined mechanism of action involving the induction of cell migration, proliferation and differentiation, as well as an adaptive secretion profile based on the changing environment within the animal, placenta-derived cells are able to rescue from BM failure multiple cell lineages of the hematopoietic system to near-normal levels.

Disclosures
Allen: Pluristem: Employment. Sher:Pluristem: Employment. Rekhes:Pluristem: Employment. Pinzur:Pluristem: Employment. Prezma:Pluristem: Employment. Gorodetsky:Pluristem: Consultancy. Volk:Pluristem: Consultancy. Aberman:Pluristem: Employment. Ofir:Pluristem: Employment.