Inflammatory Bowel Disease (IBD) is a multifactorial gastrointestinal condition encompassing two major forms of intestinal inflammation: Crohn’s disease (CD) and ulcerative colitis (UC). Both conditions are linked to auto-inflammatory reactions and genetic predispositions. Various drug therapies and biological treatments proposed to reduce IBD-associated inflammation. We induced IBD in a mouse model by stimulating bowel inflammation with an oral dextran sodium sulfate (DSS) beverage. Our novel cell therapy approach for IBD involves intramuscular (IM) and intraperitoneal (IP) delivery of non-matched, expanded, potent xenogeneic fetal human mesenchymal stromal cells (f-hPSCs) in 2 × 106 cell injections. This cell therapy has already been shown previously to induce pro-regenerative and anti-inflammatory effects in different systemic and local disorders, where the injected f-hPSCs were shown to respond to the stress of the host and secrete the adequate secretome in response to this stress. In the current study, the IP-injected f-hPSCs treatment of the DSS-induced IBD enhanced the regenerative processes of the damaged bowel and reduced the inflammatory process. This was associated with rapid regain of the mice’s weight and a decrease in inflammation-associated parameters, such as colon edema, bowel shortening, and a threefold increase in bowel mass, as estimated by increased colon weight and reduced length. This ratio best emphasized the induced inflammatory response associated with the decrease in the inflamed colon length with an increase in its mass. Although IM f-hPSCs delivery was somehow effective by a few parameters, the IP delivery produced a superior response. The IP f-hPSCs treated mice lost only ~15% of their weight at the peak of the IBD effect, compared to ~25% in untreated mice. A reduction in the inflammatory response of the gut was also indicated by a decrease in neutrophil infiltration, as assayed by a myeloperoxidase (MPO) assay. Additionally, a significant improvement in the histological score of the gut and faster recovery to 90% of its original size was observed. These findings suggest that f-hPSC treatments could serve as an effective and safe anti-inflammatory and pro-regenerative treatment for IBD.

Stem cell therapies for degenerative cartilage disease are limited by an incomplete understanding of hyaline cartilage formation and maintenance. Human bone marrow stromal cells/skeletal stem cells (hBMSCs/SSCs) produce stable hyaline cartilage when attached to hyaluronic acid-coated fibrin microbe- ads (HyA-FMBs), yet the mechanism remains unclear. In vitro, , hBMSC/SSC/HyA-FMB organoids exhibited reduced BMP signaling early in chondrogenic differentiation, followed by restoration of BMP signaling in chondrogenic IGFBP5+/MGP+ + / MGP + cells. Subsequently, human-induced pluripotent stem cell (hiPSC)-derived sclerotome cells were established (BMP inhibition) and then treated with transforming growth factor b (TGF-b) b )- /+ BMP2 and growth differentiation factor 5 (GDF5) (BMP signaling activation). TGF-b b alone elicited a weak chondrogenic response, but TGF-b/BMP2/GDF5 b /BMP2/GDF5 led to delamination of SOX9+ + aggregates (chondrospheroids) with high expression of COL2A1, , ACAN, , and PRG4 and minimal expression of COL10A1 and ALP in vitro. . While transplanted hBMSCs/SSCs/HyA-FMBs did not heal articular cartilage defects in immunocompromised rodents, chondrospheroid-derived cells/HyA-FMBs formed non-hypertrophic cartilage that persisted until at least 5 months in vivo. .

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Skin exposure to high-dose irradiation, as commonly practiced in radiotherapy, affects the different skin layers, causing dry and wet desquamation, hyperkeratosis fibrosis, hard to heal wounds and alopecia and damaged hair follicles. Fetal tissue mesenchymal stromal cells (f-hPSC) were isolated from excised human fetal placental tissue, based on their direct migration from the tissue samples to the tissue dish. The current study follows earlier reports on for the mitigation of acute radiation syndrome following whole body high-dose exposure with remotely injected f-hPSC. Both the head only and a back skin flap of mice were irradiated with 16 &18 Gy, respectively, by 6MeV clinical linear accelerator electron beam. In both locations, the irradiated skin areas developed early and late radiation induced skin damages, including cutaneous fibrosis, lesions, scaring and severe hair follicle loss and reduced hair pigmentation. Injection of 2 × 106 f-hPSC, 3 and 8 weeks following 16 Gy head irradiation, and 1 and 4 weeks following the 18 Gy back skin only irradiation, resulted in significantly faster healing of radiation induced damages, with reduction of wet desquamation as measured by surface moisture level and minor recovery of the skin viscoelasticity. Detailed histological morphometry showed a clear alleviation of radiation induced hyperkeratosis in f-hPSC treated mice, with significant regain of hair follicles density. Following 16 Gy head irradiation, the hair follicles density in the scalp skin was reduced significantly by almost a half relative to the controls. A nearly full recovery of hair density was found in the f-hPSC treated mice. In the 18 Gy irradiated back skin, the hair follicles density dropped in a late stage by ~70% relative to naïve controls. In irradiated f-hPSC treated mice, it was reduced by only ~30% and was significantly higher than the non-treated group. Our results suggest that local injections of xenogeneic f-hPSC could serve as a simple, safe and highly effective non-autologous pro-regenerative treatment for high-dose radiation induced skin insults. We expect that such treatment could also be applied for other irradiated organs.

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

Abstract Purpose Selected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis. Methods and materials f-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors. Results and conclusions The survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

The injection of placental stromal cells isolated from fetal human tissues (f-hPSC) was reported to indirectly induce tissue regeneration in different animal models. A procedure of f-hPSC isolation from fragments of both selected fresh or cryopreserved bulk placental neonate tissues is proposed, based on their high migratory potential,. The fragments of the desired fetal placental tissues are adhered to a culture dish by traces of diluted fibrin and covered with culture medium. Spontaneous migration of pure f-hPSC from the tissue fragments to the cell culture dishes is followed by their rapid expansion by numerous passages. The isolated f-hPSC express typical mesenchymal surface antigens, including CD29, CD105, CD166 and CD146, with negative expression of white blood cell lineage and endothelial cells markers. Optimal yields of f-hPSC cultures can also be obtained from tissue samples cryopreserved in medium composed of 10% dimethyl sulfoxide (M2SO) and 50% fetal calf serum. Slightly better yields are obtained with media supplemented with 1% human albumin. Medium with 5% M2SO and/or 0.25 mg/ml PEG yielded inferior results. The f-hPSC from fresh or cryopreserved tissues express similar cell markers and growth kinetics. The proposed isolation protocol may also be applied for high yield isolation of stromal cells from fresh and cryopreserved tissue of other organs.

Purpose: Osteoarthritic cartilage defects are unable to heal on their own and surgical approaches are often ineffective. Tissue engineering using transplantation of autologous chondrocytes is also limited. Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells), have been suggested as a source for cartilage reconstruction. However, studies using hBMSCs pre-differentiated into cartilage prior to transplantation failed to demonstrate formation of stable, hyaline-like cartilage that is resistant to hypertrophy in vivo. We previously determined that naive (untreated) hBMSCs were able to form stable, non-hypertrophic cartilage when transplanted subcutaneously in conjunction with fibrin microbeads covalently coated with hyaluronic acid (HyA-FMBs). In our current study, we are investigating the ability of hBMSC/HyA-FMB constructs to generate cartilage in an articular defect in immunocompromised rodents. Methods: FMB were manufactured by dehydrothermal crosslinking of concentrated thrombin activated fibrinogen emulsified in heated oil. The FMB were collected, rinsed in organic solvents and ethanol, and sieved for sizing. FMB were then coated by crosslinking HyA to their surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. hBMSCs were cultured from orthopedic surgery bony waste and cryopreserved in liquid nitrogen. Prior to implantation in rodents, hBMSCs were thawed and incubated for 12 hours with protamine and iron oxide nanoparticles for cell tracking. Cells were then incubated in suspension with HyA-FMBs for attachment for 90 minutes. hBMSC/HyA-FMB constructs were transplanted subcutaneously into the backs of NSG immunocompromised mice using standard procedures. To create intra-articular damage, the knee capsule was accessed from a medial parapatellar incision. The patella was laterally dislocated, and the distal femur immobilized using a miniature positioning clamp. A microdrill with a 0.5mm burr was used to drill a 1.5mm deep osteochondral defect. The defect was filled with hBMSC/HyA-FMB using a 0.5mm microcurrette. The patella was relocated and the incision closed using skin adhesive. A similar defect is being created in immunocompromised SRG rats (Hera Biolabs). Mice were euthanized on day 3 (to evaluate construct integrity), at 8 weeks (to examine early cartilage formation), at 16 weeks (to assess formation of mature cartilage), and at 28 weeks (to evaluate cartilage stability). Cell integration was determined using non-decalcified cryohistology and Prussian blue staining of iron nanoparticles. Cartilage formation was assessed using H&E, toluidine blue, and immunohistochemistry of COL1, COL2, COL10. The human origin of the cells was determined by human mitochondria immunohistochemistry. Results: Subcutaneous transplants of hBMSC/HyA-FMB formed stable hyaline-like cartilage at 28 weeks. Post osteochondral defect surgery, mice had uniform defect sizing, and no visually appreciable alterations in activity. Repair construct integrity, as assessed by cryohistology at 3 days, showed intact FMB with adherent hBMSCs visualized by Prussian Blue staining; growth of fibrous tissue was also detected. Intra-articular defect studies using larger cohorts of rodents and longer-term time points are currently underway. Conclusions: hBMSC/HyA-FMB constructs formed stable hyaline cartilage in a subcutaneous model in vivo. These data suggest that these constructs may be a viable method for repairing osteochondral defects. Further intra-articular experimentation is underway for clinical translation of this methodology.

AbstractBackgroundMost current cell‐based regenerative therapies are based on the indirect induction of the affected tissues repair. Xenogeneic cell‐based treatment with expanded human placenta stromal cells, predominantly from fetal origin (PLX‐RAD cells), were shown to mitigate significantly acute radiation syndrome (ARS) following high dose irradiation in mice, with expedited regain of weight loss and haematopoietic function. The current mechanistic study explores the indirect effect of the secretome of PLX‐RAD cells in the rescue of the irradiated mice.MethodsThe mitigation of the ARS was investigated following two intramuscularly (IM) injected 2 × 106 PLX‐RAD cells, 1 and 5 days following 7.7 Gy irradiation. The mice survival rate and their blood or bone marrow (BM) cell counts were followed up and correlated with multiplex immunoassay of a panel of related human proteins of PLX‐RAD derived secretome, as well as endogenous secretion of related mouse proteins. PLX‐RAD secretome was also tested in vitro for its effect on the induction of the migration of BM progenitors.ResultsA 7.7 Gy whole body mice irradiation resulted in ~25% survival by 21 days. Treatment with two IM injections of 2 × 106 PLX‐RAD cells on days 1 and 5 after irradiation mitigated highly significantly the subsequent lethal ARS, with survival rate increase to nearly 100% and fast regain of the initial weight loss (P < 0,0001). This was associated with a significant faster haematopoiesis recovery from day 9 onwards (P < 0.01). Nine out of the 65 human proteins tested were highly significantly elevated in the mouse circulation, peaking on days 6–9 after irradiation, relative to negligible levels in non‐irradiated PLX‐RAD injected mice (P < 0.01). The highly elevated proteins included human G‐CSF, GRO, MCP‐1, IL‐6 and lL‐8, reaching >500 pg/mL, while MCP‐3, ENA, Eotaxin and fractalkine levels ranged between ~60–160pg/mL. The detected radiation‐induced PLX‐RAD secretome correlated well with the timing of the fast haematopoiesis regeneration. The radiation‐induced PLX‐RAD secretome seemed to reinforce the delayed high levels secretion of related mouse endogenous cytokines, including GCSF, KC, MCP‐1 and IL‐6. Additional supportive in vitro studies also confirmed the ability of cultured PLX‐RAD secretome to induce accelerated migration of BM progenitors.ConclusionsA well‐regulated and orchestrated secretion of major pro‐regenerative BM supporting secretome in high dose irradiated mice, treated with xenogeneic IM injected PLX‐RAD cells, can explain the observed mitigation of ARS. This seemed to coincide with faster haematopoiesis regeneration, regain of severe weight loss and the increased survival rate. The ARS‐related stress signals activating the IM injected PLX‐RAD cells for the remote secretion of the relevant human proteins deserve further investigation.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) with no effective treatment available for the chronic-progressive stage. Cell therapy is a promising therapeutic approach for attenuating the immune-mediated CNS process. Isolated and expanded human placental stromal cells (hPSCs) possess potent immunomodulatory and trophic properties, making them a good candidate for MS therapy. We examined the potential of hPSC therapy in preventing the onset or attenuating the course of established disease in a murine MS model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. We examined the feasibility of hPSC systemic delivery by intramuscular (i.m.) implantation rather than the commonly used intravenous injection, which is dose-limiting and carries the risk of pulmonary obstruction. Our findings showed significant attenuation of the disease only when hPSCs were injected directly to the central nervous system. Intramuscular implanted hPSCs survived at the site of injection for at least 2 months and elicited extensive local immune responses. Intramuscular hPSC implantation before disease onset caused a delay in the appearance of clinical signs and reduced the severity of a relapse induced by repeated challenge with the autoantigen. Intramuscular implantation after disease onset did not affect its course. Thus, pathological analysis of CNS tissue did not show inhibition of neuroinflammation in i.m. hPSC-implanted mice. Moreover, no apparent effect was seen on the proliferative response of peripheral lymph node cells in these animals. We conclude that to maximize their therapeutic potential in MS, hPSCs should be delivered directly to the affected CNS. Stem Cells Translational Medicine 2017;6:1286-1294.

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.

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.

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.

Targeted cancer immunotherapy is challenging due to the cellular diversity and imposed immune tolerance in the tumor microenvironment (TME). A promising route to overcome those drawbacks may be by activating innate immune cells (IIC) in the TME, toward tumor destruction. Studies have shown the ability to "re-educate" pro-tumor-activated IIC toward antitumor responses. The current research aims to stimulate such activation using a combination of innate activators loaded onto microparticles (MP). Four inducers of Toll-like receptors 4 and 7, complement C5a receptor (C5aR) and gamma Fc receptor and their combinations were loaded on MP, and their influence on immune cell activation evaluated. MP stimulation of immune cell activation was tested in vitro and in vivo using a subcutaneous B16-F10 melanoma model induced in C57BL6 mice. Exposure to the TLR4 ligand lipopolysaccharide (LPS) bound to MP-induced acute inflammatory cytokine and chemokine activity in vitro and in vivo, with the elevation of CD45(+) leukocytes in particular GR-1(+) neutrophils and F4/80 macrophages in the TME. Nevertheless, LPS alone on MP was insufficient to significantly delay tumor progression. LPS combined with the C5aR ligand C5a-pep on the same MP resulted in a similar inflammation activation pattern. However, interleukin-10 levels were lower, and tumor growth was significantly delayed. Mixtures of these two ligands on separate MP did not yield the same cytokine activation pattern, demonstrating the importance of the cells' dual activation. The results suggest that combining inducers of distinct innate immune activation pathways holds promise for successful redirection of TME-residing IIC toward anti-tumoral activation.

A key issue in developing indirect cell based therapy for Multiple Sclerosis (MS) is determining the optimal route of cell delivery. Isolated and expanded human placental stromal cells (hPSC) represent a leading candidate for an off-the-shelf cell product with powerful immunomodulatory properties for allogeneic transplantation. We examined whether multiple intramuscular hPSC delivery is a feasible approach to treat neuroinflammatory diseases in a mouse model. Specifically, we examined both the fate of the implanted hPSC and their effect in experimental autoimmune encephaloemyelitis (EAE) model induced with MOG peptide. IM injection of hPSC at days 0 and 5 after EAE induction, prior to the appearance of clinical signs, caused only a mild delay in the onset of the disease (~2 days) and minor attenuation of the relapse following a repeated challenge. In another set of experiments, IM hPSC injection after the onset of disease (days 11 and 16 post EAE induction) did not affect disease severity at all. In-vitro proliferation assay showed no difference in the proliferative response of lymph node cells (LNCs) from transplanted versus control animals to the autoantigen MOG peptide. Transplanted hPSC elicited a strong recruitment of T cells and macrophage infiltration into the cell transplanted muscle, causing gradual hPSC rejection, with their almost complete elimination by ~60 days. We conclude that although peripheral IM injection of hPSC in animal models may be beneficial for time-limited effect, the immunomodulatory properties of the hPSC do not protect them from eventual rejection by the host immune system. These findings have important implications for defining the optimal route of delivery for immunomodulatory cell therapy in multiple sclerosis.

Tumor microenvironment (TME) harbors a multitude of immune cells which can comprise up to 50% of the tumor. The most abundant cells in the TME often are tumor associated macrophages (TAM) and tumor associated neutrophils (TAN). These cells potentially possess anti-tumoral cytotoxic effects, and have the potential to induce a broad range of cellular immune responses. However, in the developing tumor, due to elements secreted by the tumor cells, TAM and TAN are altered to act in a pro-tumor manner. The purpose of our study was the optimization and characterization of a treatment aimed at inducing an anti-tumoral response of innate immune cells in the TME. In a melanoma B16 mice model, micro-particles carrying targeting and induction components were injected intra-tumorally and the responses of the tumors, including immune cells populations were monitored. Without treatment the CD45+ cells ratio of total TME decreased as tumor progressed. Particles carrying Toll-like receptor ligands inhibited tumor growth, inducing significant elevation of CD45+ cells in the TME. Using different inducing molecules (inducers) or simultaneous use of several inducers enabled the stimulation of specific immune cell populations within the tumor. The current study suggests that by using tumor targeted particles with adequate inducers, one could change the tumor microenvironment innate immune cells toward an anti-tumor response. Citation Format: Ehud Shahar, Raphael Gorodetsky, Elina Aizenshtein, Jacob Pitcovski. Combination of innate immune activators stimulates leukocyte populations in the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2014-3633

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 2×106 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.