Rescue from lethal acute radiation syndrome (ARS) with severe weight loss by secretome of intramuscularly injected human placental stromal cells.

BACKGROUND: Most 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: A 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.

Immunomodulatory placental-expanded, mesenchymal stromal cells improve muscle function following hip arthroplasty.

BACKGROUND: No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma-related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. METHODS: Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX-PAD or a placebo (n = 7) into the injured gluteus medius muscles. RESULTS: We did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as Week 6 in the treatment-groups. Surprisingly, until Week 26, the low-dose group outperformed the high-dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3 , 150 M: 237.4 ± 27.2 cm3 ]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T-cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement. CONCLUSIONS: Allogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.

Posttransplant Intramuscular Injection of PLX-R18 Mesenchymal-Like Adherent Stromal Cells Improves Human Hematopoietic Engraftment in A Murine Transplant Model.

Late-term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX)-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45+ cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p < 0.001) and 8 (7.3 vs. 13.1%, p < 0.05) and in the bone marrow at week 8 (28 vs. 40.0%, p < 0.01) in the PLX-R18 cohort. As measured by percentage of CD19+ cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8%) and 8 (10.1 vs. 4.1%). These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT.

Placenta-derived PLX-PAD mesenchymal-like stromal cells are efficacious in rescuing blood flow in hind limb ischemia mouse model by a dose- and site-dependent mechanism of action.

BACKGROUND: In peripheral artery disease (PAD), blockage of the blood supply to the limbs, most frequently the legs, leads to impaired blood flow and tissue ischemia. Pluristem's PLX-PAD cells are placenta-derived mesenchymal stromal-like cells currently in clinical trials for the treatment of peripheral artery diseases. METHODS: In this work, the hind limb ischemia (HLI) mouse model was utilized to study the efficacy and mechanism of action of PLX-PAD cells. ELISA assays were performed to characterize and quantitate PLX-PAD secretions in vitro. RESULTS: PLX-PAD cells administered intramuscularly rescued blood flow to the lower limb after HLI induction in a dose-dependent manner. While rescue of blood flow was site-dependent, numerous administration regimes enabled rescue of blood flow, indicating a systemic effect mediated by PLX-PAD secretions. Live PLX-PAD cells were more efficacious than cell lysate in rescuing blood flow, indicating the importance of prolonged cytokine secretion for maximal blood flow recovery. In vitro studies showed a multifactorial secretion profile including numerous pro-angiogenic proteins; these are likely involved in the PLX-PAD mechanism of action. DISCUSSION: Live PLX-PAD cells were efficacious in rescuing blood flow after the induction of HLI in the mouse model in a dose- and site-dependent manner. The fact that various administration routes of PLX-PAD rescued blood flow indicates that the mechanism of action likely involves one of systemic secretions which promote angiogenesis. Taken together, the data support the further clinical testing of PLX-PAD cells for PAD indications.

Placenta-Derived Adherent Stromal Cells Improve Diabetes Mellitus-Associated Left Ventricular Diastolic Performance.

Left ventricular (LV) diastolic dysfunction is among others attributed to cardiomyocyte stiffness. Mesenchymal stromal cells (MSC) have cardiac-protective properties. We explored whether intravenous (i.v.) application of PLacenta-eXpanded (PLX) MSC-like cells (PLX) improves LV diastolic relaxation in streptozotocin (STZ)-induced diabetic mice and investigated underlying mechanisms. Diabetes mellitus was induced by STZ application (50 mg/kg body weight) during five subsequent days. One week after the first STZ injection, PLX or saline were i.v. applied. Two weeks later, mice were hemodynamically characterized and sacrificed. At this early stage of diabetic cardiomyopathy with low-grade inflammation and no cardiac fibrosis, PLX reduced LV vascular cell adhesion molecule-1, transforming growth factor-ß1, and interferon-γ mRNA expression, induced the percentage of circulating regulatory T cells, and decreased the splenic pro-fibrotic potential in STZ mice. STZ + PLX mice exhibited higher LV vascular endothelial growth factor mRNA expression and arteriole density versus STZ mice. In vitro, hyperglycemic PLX conditioned medium restored the hyperglycemia-impaired tube formation and adhesion capacity of human umbelical vein endothelial cells (HUVEC) via increasing nitric oxide (NO) bioavailability. PLX further induced the diabetes-downregulated activity of the NO downstream protein kinase G, as well as of protein kinase A, in STZ mice, which was associated with a raise in phosphorylation of the titin isoforms N2BA and N2B. Concomitantly, the passive force was lower in single isolated cardiomyocytes from STZ + PLX versus from STZ mice, which led to an improvement of LV diastolic relaxation. We conclude that i.v. PLX injection improves diabetes mellitus-associated diastolic performance via decreasing cardiomyocyte stiffness. Stem Cells Translational Medicine 2017;6:2135-2145.

Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

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.

Mesenchymal 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 10 ng/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 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/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.

Cardioprotection by placenta-derived stromal cells in a murine myocardial infarction model.

BACKGROUND: Autologous cells for cell therapy of ischemic cardiomyopathy often display age- and disease-related functional impairment, whereas an allogenic immunotolerant cell product would allow off-the-shelf application of uncompromised donor cells. We investigated the cardiac regeneration potential of a novel, clinical-grade placenta-derived human stromal cell product (PLX-PAD). METHODS: PLX-PAD cells derived from human donor placentas and expanded in a three-dimensional bioreactor system were tested for surface marker expression, proangiogenic, anti-inflammatory, and immunomodulatory properties in vitro. In BALB/C mice, the left anterior descending artery was ligated and PLX-PAD cells (n = 10) or vehicle (n = 10) were injected in the infarct border zone. Four weeks later, heart function was analyzed by two-dimensional and M-mode echocardiography. Scar size, microvessel density, extracellular matrix composition, myocyte apoptosis, and PLX-PAD cell retention were studied by histology. RESULTS: In vitro, PLX-PAD cells displayed both proangiogenesis and anti-inflammatory properties, represented by the secretion of both vascular endothelial growth factor and angiopoietin-1 that was upregulated by hypoxia, as well as by the capacity to suppress T-cell proliferation and augment IL-10 secretion when co-cultured with peripheral blood mononuclear cells. Compared with control mice, PLX-PAD-treated hearts had better contractile function, smaller infarct size, greater regional left ventricular wall thickness, and less apoptosis after 4 wk. PLX-PAD stimulated both angiogenesis and arteriogenesis in the infarct border zone, and periostin expression was upregulated in PLX-PAD-treated hearts. CONCLUSIONS: Clinical-grade PLX-PAD cells exert beneficial effects on ischemic myocardium that are associated with improved contractile function, and may be suitable for further evaluation aiming at clinical pilot trials of cardiac cell therapy.

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.