Placenta-derived Adherent Cells Research Articles

Angiogenic properties of human placenta-derived adherent cells and efficacy in hindlimb ischemia

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored invivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Secreted trophic factors from human placenta-derived adherent cells promote endothelial cell survival, proliferation and tube formation via distinct signaling pathways

Secreted trophic factors from human placenta-derived adherent cells promote endothelial cell survival, proliferation and tube formation via distinct signaling pathways

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

Human placenta-derived adherent cells induce tolerogenic immune responses.

Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14+ monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells.

Abstract 10: Human Placenta-Derived Adherent Cell Treatment of Stroke Induces Angiogenesis, Increases Synaptic Plasticity and Improves Functional Outcome in Older-Adult Rats

Background and purpose: Human Placenta-Derived Adherent cells (PDAC TM ) are a novel adherent mesenchymal-like cell population derived from normal, full-term human placental tissue. PDA-001 (cenplacel-L) is a clinical formulation of PDAC TM developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in older-adult rats. Methods: Older-adult male Wistar rats (600-750g, 10-12 months) were subjected to 90 minutes of MCAo. To test the efficacy of PDA-001 treatment in older-adult rats, at 1 day after MCAo, randomly selected animals (n=12 per group) were injected, via a tail vein, with vehicle control (cell media), or with PDA-001 (4x10 6 or 8x10 6 cells). Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. All rats were sacrificed 29 days after MCAo, and lesion volumes were measured using hematoxylin and eosin (H&E). To assess cerebral tissue for angiogenesis and synaptogenesis, immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed, respectively. Results: PDA-001 treatment at the 4X10 6 and 8X10 6 doses significantly improved functional outcome after stroke in older-adult rats (p<0.05). The neurological benefit of PDA-001 treatment was associated with significant increases in the number of BrdU immunoreactive endothelial cells, increases in vascular density and perimeter in the ischemic brain (p<0.05), as well as significantly increased synaptic plasticity as measured by increased synaptophysin expression in the ischemic border zone (IBZ) (p<0.05). Conclusion: PDA-001 treatment significantly improved functional outcome after stroke in older-adult rats. The neurorestorative effects induced by PDA-001 treatment may be related to the increase in angiogenesis and synaptic plasticity.

Human Placenta-Derived Adherent Cell Treatment of Experimental Stroke Promotes Functional Recovery after Stroke in Young Adult and Older Rats

BackgroundHuman Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2–3 month old) and older rats (10–12 months old).MethodsTo evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×106, 4×106 or 8×106 PDA-001 cells (i.v.), vehicle or cell control. 4×106 or 8×106 PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed.ResultsIn young adult rats, treatment with 4×106 PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×106 and 8×106 treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05).ConclusionPDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and synaptic plasticity.

Neuroprotective Effect of Human Placenta-Derived Cell Treatment of Stroke in Rats

Human placenta-derived adherent (PDA001) cells are mesenchymal-like stem cells isolated from postpartum placenta. In this study, we tested whether intravenously infused PDA001 improves neurological functional recovery after stroke in rats. In addition, potential mechanisms underlying the PDA001-induced neuroprotective effect were investigated. Young adult male rats (2–3 months) were subjected to 2 h of middle cerebral artery occlusion (MCAo) and treated with PDA001 (4x10(6)) or vehicle controls [dextran vehicle or phosphate buffer saline (PBS)] via intravenous (IV) administration initiated at 4 h after MCAo. A battery of functional tests and measurements of lesion volume and apoptotic cells were performed. Immunostaining and ELISA assays for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and brain derived neurotrophic factor (BDNF) were performed in the ischemic brain to test the potential mechanisms underlying the neuroprotective effects of PDA001 cell treatment of stroke. PDA001 cell treatment at 4 h post stroke significantly improved functional outcome and significantly decreased lesion volume, TUNEL, and cleaved caspase 3-positive cell number in the ischemic brain, compared to MCAo-vehicle and MCAo-PBS control. Treatment of stroke with PDA001 cells also significantly increased HGF and VEGF expression in the ischemic border zone (IBZ) compared to controls. Using ELISA assays, treatment of stroke with PDA001 cells significantly increased VEGF, HGF, and BDNF levels in the ischemic brain compared to controls. When administered intravenously at 4 h after MCAo, PDA001 cells promoted neuroprotective effects. These effects induced by PDA001 cell treatment may be related to the increase of VEGF, HGF, and BDNF expression,and a decrease of apoptosis. PDA001 cells may provide a viable cell source to treat stroke.

Placenta-derived adherent cells attenuate hyperalgesia and neuroinflammatory response associated with perineural inflammation in rats

Neuropathic pain is a debilitating condition of the somatosensory system caused by pathology of the nervous system. Current drugs treat symptoms but largely fail to target the underlying mechanisms responsible for the pathological changes seen in the central or peripheral nervous system. We investigated the therapeutic effects of PDA-001, a culture expanded placenta-derived adherent cell, in the rat neuritis model. Pain is induced in the model by applying carrageenan to the sciatic nerve trunk, causing perineural inflammation of the sciatic nerve. PDA-001, at doses ranging from 0.4×10(6) to 4×10(6) cells/animal, or vehicle control was intravenously administrated to assess the biological activity of the cells. A dose-dependent effect of PDA-001 on pain relief was demonstrated. PDA-001 at doses of 1×10(6) and 4×10(6), but not 0.4×10(6), reduced mechanical hyperalgesia within 24h following treatment and through day 8 after induction of neuritis. The mechanism underlying PDA-001-mediated reduction of neuroinflammatory pain was also explored. Ex vivo tissue analyses demonstrated that PDA-001 suppressed homing, maturation and differentiation of dendritic cells, thus inhibiting T-cell priming and activation in draining lymph nodes. PDA-001 also reduced interferon gamma and IL-17 in draining lymph nodes and in the ispilateral sciatic nerve, and increased the levels of IL-10 in draining lymph nodes and plasma, pointing to T-cell modulation as a possible mechanism mediating the observed anti-hyperalgesic effects. Furthermore, in the ipsilateral sciatic nerve, significantly less leukocyte infiltration was observed in PDA-001-treated animals. The results suggest that PDA-001may provide a novel therapeutic approach in the management of inflammatory neuropathic pain and similar conditions.

In Vitro Differentiation of Osteocytes and Adipocytes from Human Placenta-Derived Cells

To investigate the capability of human placenta-derived adherent cells to differentiate into osteocytes and adipocytes. Placenta-derived adherent cells were isolated by type IV collagenase digestion of a single freshly obtained human placenta and cultured under standard conditions. Cell surface markers of adherent cells from passages 3 - 9 were analysed by flow cytometry. Osteocytic differentiation was induced with β-glycerol phosphate, vitamin C and dexamethasone and confirmed by Alizarin red staining. Adipocytic differentiation was induced with dexamethasone and insulin and confirmed by oil red O staining. Placenta-derived adherent cells were positive for high levels of CD44 and CD105 and very low levels of CD29 but were negative for CD34, CD45 and CD19. This pattern of cell surface markers is identical to human mesenchymal stem cells. Alizarin red-positive cells were detected 10 days after the induction of osteocyte differentiation. Oil red O-positive cells were detected 7 days after the induction of adipocyte differentiation. Placenta-derived adherent cells can differentiate into either osteocytes or adipocytes in vitro. The human placenta may provide an alternative source of mesenchymal stem cells for basic research and clinical use.

Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)-rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.

Human Placenta-Derived Adherent Cells Delivered Intralesionally Inhibit Myeloma Bone Disease and Tumor Growth, While Intravenously Are Capable of Trafficking to Myelomatous Bone.

AbstractAbstract 3717Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by >37% (p<0.01 versus control) and inhibited MM growth in the 2 sets of experiments (p<0.04). The bone anabolic effect of PDAC was associated with increased number of osteoblasts (p<0.003) and reduced number of osteoclasts (p<0.004). Intralesional PDAC cytotherapy also promoted bone formation in nonmyelomatous SCID-rab mice. Intralesional but not subcutaneous engraftment of PDAC inhibited bone disease and tumor growth in SCID-rab mice. In contrast to intra-bone injection in SCID-rab mice, intra-tumor injection of PDAC had no effect on subcutaneous growth of the H929 myeloma cell line in SCID mice (8 mice/group). Live-animal imaging revealed that the majority of PDAC disappeared from the injected bones within 4 weeks. To test their systemic behavior, PDAC were intravenously injected into 18 SCID-rab mice engrafted with H929 myeloma cells. The presences of PDAC in various organs were evaluated 1, 2 and 7 days after injection. Ex vivo bioluminescence analysis of the implanted myelomatous bones detected PDAC in two of five bones on day 1, in four of four bones on day 2, and in four of nine bones on day 7. Intravenously injected PDAC were also detected in lungs not in any other murine tissues. Immunohistochemical staining for GFP in myelomatous bone sections detected GFP-expressing PDAC in rabbit marrow areas infiltrated with myeloma cells, supporting bioluminescence analysis. Our study suggest that PDAC stimulate bone formation by acting as bystander cells that increase endogenous osteoblastogenesis and inhibit osteoclastogenesis, and that alteration of the bone marrow microenvironment by PDAC attenuates growth of MM. PDAC cytotherapy is a promising therapeutic approach for myeloma bone disease. Disclosures:Khan:Celgene Cellular Therapeutics: Research Funding. Heidaran:Celgene Cellular Therapeutics: Employment. Pal:Celgene Cellular Therapeutics: Employment. Zhang:Celgene Cellular Therapeutics: Employment. He:Celgene Cellular Therapeutics: Employment. Zeitlin:Celgene Cellular Therapeutics: Employment. Abbot:Celgene Cellular Therapeutics: Employment. Faleck:Celgene Cellular Therapeutics: Employment. Hariri:Celgene Cellular Therapeutics: Employment.

In Vitro Differentiation of Human Placenta-derived Adherent Cells into Insulin-producing Cells

This study investigated the differentiation of placenta-derived adherent cells (PDACs) into insulin-producing cells (IPCs). PDACs were cultured and the cells characterized by analysis of cell surface markers using flow cytometry. The PDACs were then treated with induction media containing basic fibroblast growth factor (bFGF) and beta-mercaptoethanol (beta-ME). After induction, the presence of IPCs was demonstrated using dithizone staining, and the production of functional insulin was confirmed using immunocytochemistry and an enzyme-linked immunosorbent assay. Expression of the islet-associated genes PDX-1, Insulin 1, Insulin 2 and Glut 2 in the induced cells was measured using a reverse transcription-polymerase chain reaction; PDX-1 was expressed after 7 days of induction and PDX-1, Insulin 1 and Insulin 2 were all detected after 14 days. These results suggest that the placenta could be a new source of stem cells that can be induced to differentiate into IPCs following treatment with media containing bFGF and beta-ME.

Human Placenta-Derived Adherent Stem Cells Prevent Bone Loss and Stimulate Bone Formation in Myelomatous Bones, and Suppress Growth of Primary Multiple Myeloma

Induction of osteolytic bone disease in multiple myeloma (MM) is caused by activation of osteoclastogenesis and suppression of osteoblastogenesis. Bone formation is reduced mainly through production of inhibitors of osteoblast differentiation by MM cells and by impaired osteogenic differentiation of endogenous mesenchymal stem cells (MSCs). Recently, human placenta has emerged as a potentially valuable source of progenitor cells for multiple therapeutic purposes, including bone repair and cancer (Parolini et al., Stem Cells 26:300–311, 2008). The aim of the study was to investigate the effects of human placenta-derived adherent cells (PDAC™) on MM bone disease and tumor growth in the SCID-rab mouse model for MM. PDAC™ are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Bone disease was evaluated by measurements of bone mineral density (BMD) and visualized by X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and live animal imaging. For in vivo tracking PDAC™ or our stroma-dependent BN MM cell line was transduced with a luciferase/GFP reporter in a lentiviral vector. In the first in vivo experiment, 10 SCID-rab mice were engrafted with a patient's MM cells. Following establishment of MM and detection of bone disease, luciferase-expressing PDAC™ (1×106 cells/bone) or phosphate-buffered saline (PBS) were injected directly into implanted myelomatous bones in SCID-rab mice (5 mice/group). At experiment's end (5 wk after cytotherapy) PDAC™ could be detected in mice by live animal imaging. Whereas in control mice, BMD of the implanted bone was reduced from pretreatment levels by 8±4%, administration of PDAC™ resulted in increased BMD of the implanted bone in all mice by 132±20% from pretreatment levels (p<0.0006). Levels of hIg in mice sera (tumor burden) at experiment's end were 202±56 μg/ml and 12±4 μg/ml in PBS- and PDAC™ -treated hosts, respectively (p<0.007). In the second in vivo experiment hosts engrafted with our luciferase-expressing BN MM line (Li et al., BJH 2007) were similarly injected with PDAC™ or PBS (8 mice/group). Six wk following treatment the BMD of the implanted bone in the control PBS group was reduced by 31±33% (p<0.0009) from pretreatment levels while in PDAC™ –treated group it was slightly reduced by 2±6% from pretreatment levels. Treatment with PDAC™ had no effect on in vivo growth of the BN MM cell line, indicating that prevention of bone disease by PDAC™ was not a consequence of reduced MM. In contrast to fetal MSCs, PDAC™ expressed high levels of OPG (>30 fold) and low levels of RANKL (<5 fold) as determined by qRT-PCR. Differentiation of osteoclast precursors in media supplemented with RANKL and M-CSF was reduced in the presence of PDAC™ or their conditioned media by 60±6% (p<0.004), an effect that was partially blocked by OPG neutralizing antibody (p<0.04). PDAC™ also induced apoptosis of osteoclast precursors as determined by annexin V/PI staining. We conclude that PDAC™ prevent bone loss and promote bone formation in myelomatous bone through simultaneous inhibition of osteoclastogenesis and stimulation of osteoblastogenesis, and that engraftment of PDAC™ inhibits growth of primary MM in vivo.

Placenta Derived Adherent Cells Modulate the Allogenic Mixed Lymphocyte Reaction Partially via an Indoleamine 2,3-Dioxygenase Mediated Mechanism.

Placenta Derived Adherent Cells (PDAC) are multipotent progenitor cells derived from human placental tissues. Previously we have reported that PDACs could suppress T-cell proliferation when added to in-vitro mixed lymphocyte reactions (PDAC-MLR) (Paludan C. et al, Blood. (ASH Annual Meeting Abstracts) 2006 108: Abstract 1737). Here we present aspects of the mechanism of this PDAC suppression property. We have found that PDACs modify cytokine production in the PDAC-MLR reaction in comparison to the MLR; TNF-α and IFN-γ are reduced 75% and 30% while TGF-β production is significantly increased. We have used a transwell assay system to investigate the cell-contact-dependency of the effects of PDACs on T-cell proliferation. The assay system comprised combinations of the MLR in the top chamber together with the PDAC-MLR, PDACs plus naive T cells or PDACs alone in the bottom chamber. Maximum inhibition of T cell proliferation of the MLR in the top insert could be achieved by placing the PDAC-MLR co-culture in the bottom chamber. Minimum suppression was obtained when placing PDACs plus naive T cells or PDACs alone in the bottom chamber. PDAC-MLR conditioned media could partially suppress the MLR reaction. Addition of L-tryptophan into the MLR with PDAC conditioned media completely abolished PDAC-induced suppression of T cell proliferation. Likewise, the addition of the 1-methyl tryptophan to the PDAC-MLR reaction could abolish the PDAC-induced suppression. These results suggested that the suppression of the MLR by PDACs was possibly due to the depletion of the essential amino acid L-tryptophan which could be due to up-regulation of indoleamine 2,3-dioxygenase (IDO). Quantitative RT-PCR analysis of IDO gene expression revealed that IDO was up-regulated by about 4000-fold when PDACs were co-cultured with activated T cells, but not when co-cultured with naive T cells. Experiments are ongoing to confirm the causative role of IDO, and other factors, in PDAC-suppression of T-cell proliferation. In summary, we believe that soluble factors including the production of pro-inflammatory cytokines may contribute to PDAC suppression of the MLR, that induction of soluble factors from PDACs is significantly augmented by T-cell activation and that IDO expression by PDACs during the PDAC-MLR reaction plays a significant and direct role in suppression of T cell proliferation by PDACs.

Immune Suppression by Placenta Derived Adherent Cells (PDAC) Correlate with Monocyte Chemoattractant Protein-1 and IL-2 Secretion.

The placenta is a readily available and ethically non-controversial source of large amounts of therapeutic stem cells. Placenta Derived Adherent Cells (PDACs) are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, placental cotyledons, or any combination thereof. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit defined phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70% and constitutively secrete IL-6, IL-8, and Monocyte Chemoattractant Protein-1 (MCP-1). PDACs demonstrate in vitro pluripotency in the adipogenic, osteogenic, and chondrogenic lineages. Furthermore, PDACs suppress T cell proliferation in certain Mixed Leukocyte Reaction (MLR) and the autologous EBV regression assays. Because secreted factors can powerfully modify immune responses and influence therapeutic use of cells, we report on the cytokine secretion in certain PDAC MLR and regression assays.Cytokines were measured on a Luminex system in supernatants from 6-day PDAC cultures, PDAC MLRs or PDAC regression assays. MLRs include PDACs, Dendritic Cells (DC)s, and T cells at DC/PDAC/T ratios 1/2/10. EBV regression assays included PDACs, EBV antigen-presenting cells (APC), and T cells at APC/PDAC/T ratios 1/2/10. Levels of IL-6 (11 ng/ml) and IL-8 (16 ng/ml) stayed constant in PDAC solo cultures, PDAC MLRs, and PDAC regression assays. MCP-1 concentration was 2 ng/ml in PDAC solo cultures, and non-suppressive control adherent cell MLRs and regression assays, but increased to 10 ng/ml in suppressed PDAC MLRs and PDAC regression assays. These values are consistent with reported MCP-1 serum levels.Interleukin-2 (IL-2) is both a T cell survival factor and an obligate factor for CD4+CD25+ T regulatory cells. T regulatory cells are not required for PDAC T cell suppression, but IL-2 levels consistently increase when MLR suppression by PDACs occurs. The CD4 MLR supernatants contained 65 pg/ml IL-2, and the CD8 MLR contained 35 pg/ml IL-2. In the 85% and 75% suppressed CD4 and CD8 PDAC MLRs, the IL-2 levels rose 5-fold to 331 pg/ml (CD4) and 2-fold to 67 pg/ml(CD8).These results indicate that IL-2 and MCP-1, traditionally known as stimulators of the immune response, may play a role in PDAC immune suppression. PDACs, which cause the secretion, may thus be useful therapeutic tools in the clinic.

Characterization of Placenta Derived Adherent Cells (PDAC): A Novel Type of Stem Cells Isolated from Human Placenta.

The placenta is a rich source of ethically non-controversial stem cells. In this work, we describe the development of procedures to isolate and culture adherent stem cells from a full term, postpartum human placenta (Placenta Derived Adherent Cells, PDACs). PDACs are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, cord, placental cotyledons, or any combination thereof. PDACs were established in a medium containing low concentrations of fetal calf serum and limited growth factors. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. We found that PDACs differentiate down the adipocyte, chondrocyte and osteocyte lineages. In an induction medium containing IBMX, insulin, dexamethasone and indomethacin, PDACs turned into fat laden adipocytes in 3 to 5 weeks. Under osteogenic induction culture conditions, PDACs were found to form bone nodules and have calcium depositions in their extracellular matrix. Chondrogenic differentiation of PDACs was performed in micropellets and was confirmed by formation of glycosaminoglycan in the tissue aggregates. In addition, we showed that PDACs suppress an MLR consisting of suitable effectors (T cells or NK cells) and target populations (allogeneic irradiated PBMCs or mature DCs). The range of suppression of T cell proliferation was 50 to 95 %. The observed suppression of the MLR is likely cell-to-cell contact dependent in the range of 20%. In summary, the PDACs described herein exhibit in vitro properties of pluripotent stem cells. The placenta is an excellent raw material for the isolation of stem cells.

Placenta Derived Stem Cells (PDAC) Suppress the Allo-MLR and the EBV Regression Assay.

The placenta is a readily available and ethically non-controversial source of large amounts of therapeutic stem cells. Placenta Derived Adherent Cells (PDACs) are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, placental cotyledons, or any combination thereof. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. Furthermore, PDACs demonstrate in vitro pluripotency. Here, we report on the immune suppressive capabilities of PDACs.PDACs were introduced in a six-day mixed leukocyte reaction, using purified CD8+ T cells as effectors and allogeneic dendritic cells (DC)s. The ratios were T/DC/PDAC=10/1/2. Alternatively, an EBV regression assay, measuring T cell proliferation against EBV expressing antigen presenting cells (APCs) generated from healthy donors was performed. Here, the T/APC/PDAC ratios were 10/1/2. In this system, PDACs inhibited the proliferation, as measured by CFSE dilution, of CD8+ T cells >50% (range: 0–95%, SEM 20%). The suppression was the same whether only CD4+ or CD8+ cells were used, or whether equal amounts of CD4+ and CD8+ T cells were added. When Natural Killer (NK) cells were used, with 50 U/ml IL-2, as effectors instead of T cells, the suppressive effect was 45% (range: 40–65%, SEM 5%). Different PDAC preparations suppressed the reactions equally well. Separation of certain PDACs and MLR by a transwell membrane reduced the suppression by 21%, suggesting that part of the suppression was contact dependent. Suppression by other types of PDACs, on the other hand, was 75% contact dependent. When CD4+CD25+ T regulatory cells were removed by magnetic bead separation, the suppression in the regression assay was unchanged.These results suggest that PDACs may have potential therapeutic use as universal immune suppressive donor cells. The suppression is independent of T regulatory cells.

Placenta Derived Adherent Cells (PDACs) Suppress Tumor Cells of Diverse Origin.

The placenta is a readily available and ethically non-controversial source of large amounts of therapeutic stem cells. We isolated adherent cells from the Umbilical Cord (UC) and the Amnion Chorion (AC) of term placentas. These Placenta Derived Adherent Cells (PDACs) displayed a cell surface phenotype of CD29+, CD44+, CD73+, CD90+, CD105+, CD200+, CD34−, and constitutively secreted IL-6, IL-8 and Monocyte Chemoattractant protein-1 (MCP-1). PDACs demonstrated in vitro pluripotency and suppressed T cell proliferation in both the allogeneic mixed leukocyte reaction, and the autologous EBV regression assay. Because progenitor cells have been described to inhibit tumor growth in some systems, the role of PDACs in tumor suppression was investigated.EBV transformed tumor cells were cultured either alone, or with AC or UC PDACs. After 6 days, live (7-AAD− Annexin-V−) tumor cells were counted on a flow cytometer. Free growing tumor cells numbered 40,000. When tumor cells were co-cultured with AC or UC PDACs, the growth was suppressed to 10,000 cells, a suppression of 75%. We designed and tested a panel of tumor cell lines that included retinoblastoma (RB), histiocytic lymphoma (HL), chronic myelogenous leukemia (CML), and colon adenocarcinoma (CAC). The tissue origin of the selected tumor cell lines included neuronal tissue (retinoblastoma), epithelium (carcinomas) and the hematopoietic lineage (lymphomas and CML). Co-culture experiments showed that the growth of all tumor lines was suppressed more than 40%. When a transwell was introduced separating PDACs and tumor cells, contact dependency of suppression was 12% for HL, 22% for CML, 42% for CAC, and 51% for EBV transformed tumor cells.These results indicate that PDACs may have therapeutic value with respect to certain cancers.