Mechanism of Action of PLX-R18, a Placental-Derived Cellular Therapy for the Treatment of Radiation-Induced Bone Marrow Failure

Abstract Bone marrow (BM) failure occurs in individuals who fail to produce sufficient red blood cells, white blood cells, or platelets. This may be a result of damage to hematopoietic stem cells by a congenital defect or by exposure to a noxious substance or factor. PLX-R18 are 3D-expanded placenta-derived cells, with biological properties including a profound capacity to protect and regenerate bone marrow. The cells secrete a broad array of cytokines including G-CSF, IL-6, MCP-1, MCP-3 and GRO that contribute to the reconstitution of the hematopoietic and immune systems. To assess their therapeutic potential, PLX-RAD cells were administered to C3H/HeN male mice intramuscularly, one and five days following 7.7 Gy total body irradiation. Body weight and animal survival were monitored for 3 weeks, then the animals were euthanized for BM and blood analysis. In the PLX-R18-treated group, 10 out of 11 (91%) animals survived, compared to only 4 out of 9 in the vehicle treated group (44%), (P &lt; 0.05). Weight reduction after irradiation was also improved in the PLX-RAD treated mice where the average weight loss was less than 10% compared to almost 20% for vehicle-treated controls. Consistent with the improved survival, cell counts of all the three hematopoietic lineages were significantly increased in the BM and blood of PLX-R18 treated mice as compared to the control animals, and attained close to normal levels. Analysis of plasma in the irradiated treated animals detected the presence of critical, PLX-R18-derived (human) cytokines as well as alterations in the equivalent murine cytokines, suggesting a direct role of PLX-R18 secreted cytokines in animal survival. Interestingly, human cytokines were detected only after irradiation and were not present in the plasma of sham non-irradiated animals treated with PLX-R18. This may imply that the active in vivo cytokine secretion by PLX-R18 is a response to signals from the environment in the irradiated animals. Already by day 4-6 after irradiation, the number of colony forming progenitors in the BM of PLX-R18 treated animals was significantly higher than in control animals suggesting that earlier regeneration of BM induced by PLX-R18 secreted cytokines results in improved blood counts and increased survival. Studies in vitro demonstrated that PLX-R18-derived conditioned medium induced the formation of all colony types in a methyl cellulose colony formation assay as well as a 3.1 fold-increase in the number of migrating cells in a BM migration assay compared to the SDF-1-supplemented positive control. These results, in vivo and in vitro, reveal key clues to the resolution of the underlying mechanism of action. Administration of the novel cell product PLX-R18 markedly improved survival and recovery of the three hematopoietic blood lineages after radiation induced BM failure indicating potential as a highly effective therapy for general radiation-induced BM damage, or in a nuclear disaster scenario. Further clinical studies are planned. Disclosures Ofir: Pluristem ltd: Employment. Pinzur:Pluristem ltd: Employment. Aberman:Pluristem ltd: Employment. Gorodetsky:Pluristem ltd: Consultancy. Volk:Pluristem ltd: Consultancy. </jats:sec

Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia

Abstract 545: PLacental eXpanded (PLX) Cell Treatment Ameliorates Preeclampsia Induced by TLR3 or TLR7 Activation in Mice

PLacental eXpanded (PLX) cells (Pluristem Therapeutics Inc.) are human placenta-derived, mesenchymal-like adherent stromal cells that release proteins in response to the environment of the host. PLX cells are non-immunogenic and have been shown to decrease inflammation and increase angiogenesis in inflammatory and ischemic conditions. Therefore, we tested whether PLX cell treatment could attenuate symptoms of preeclampsia (PE) in mice. We hypothesized that one-time PLX cell treatment would decrease the pregnancy-dependent hypertension, proteinuria, endothelial dysfunction, splenomegaly, inflammation, and placental injury induced by Toll-like receptor (TLR) activation during pregnancy. Pregnant C57BL/6 mice were given ip injections of saline vehicle (P), the TLR3 agonist poly I:C (PPIC), or the TLR7 agonist R837 (PR) on days 13, 15, and 17 of gestation. P, PPIC, and PR mice were also given either plasmalyte A (PLA, vehicle) or PLX cells (1 million) by im injection in the right leg on gestational day 14 (n=8 in each group). PLX cell treatment progressively decreased SBP over 3 days in PPIC and PR mice and had no effect in P control mice (day 17 SBP in mmHg: P+PLA = 100±4, P+PLX = 96±4, PPIC+PLA = 144±3, PPIC+PLX = 111±1, PR+PLA = 145±2, PR+PLX = 106±3; PPIC+PLA and PR+PLA p&lt;0.05 vs. P+PLA). PLX cell treatment also normalized the urinary protein/creatinine ratio and aortic endothelium-dependent relaxation responses in PPIC and PR mice to that of P mice while having no significant effects on the number of fetuses or incidence of fetal demise per litter. Inflammation plays a central role in the development of TLR-induced PE and PLX cell treatment reduced spleen weight/body weight ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL-6 levels, and restored plasma IL-4 levels in PPIC and PR mice. Additionally, PLX cell treatment reduced fibrin deposition in the placental vasculature and significantly reduced placental HIF-1alpha protein levels. These data demonstrate that one-time PLX cell treatment after PE is induced was able to decrease inflammation, proteinuric hypertension, endothelial dysfunction, and placental injury in mice and may be beneficial in women with PE.</jats:p

Human PLacental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF

AbstractMesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2×105 PLX cells, added in a trans-well system, conferred 30–60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240ng/ml Semaxanib (anti VEGF165) and/or 400ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells

Human Placenta-Derived Stromal Cells Rescue Mice from Radiation-Induced Bone Marrow Failure: A Cytof-Based Mechanistic Analysis

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. </jats:sec