Preconditioning human natural killer cells with chorionic villous mesenchymal stem cells stimulates their expression of inflammatory and anti-tumor molecules.

BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human placentae (pMSCs) produce a unique array of mediators that regulate the essential cellular functions of their target cells. These properties make pMSCs attractive candidates for cell-based therapy. Here, we examined the effects of culturing human natural killer (NK) cells with pMSCs on NK cell functions. METHODS: pMSCs were cultured with IL-2-activated and non-activated NK cells. NK cell proliferation and cytolytic activities were monitored. NK cell expression of receptors mediating their cytolytic activity against pMSCs, and the mechanisms underlying this effect on pMSCs, were also investigated. RESULTS: Our findings show that IL-2-activated NK cells, but not freshly isolated NK cells, efficiently lyse pMSCs and that this response might involve the activating NK cell receptor CD69. Interestingly, although pMSCs expressed HLA class I molecules, they were nevertheless lysed by NK cells, suggesting that HLA class I antigens do not play a significant role in protecting pMSCs from NK cell cytolytic activity. Co-culturing NK cells with pMSCs also inhibited NK cell expression of receptors, including CD69, NKpG2D, CD94, and NKp30, although these co-cultured NK cells were not inhibited in lysing cancer cells in vitro. Importantly, co-cultured NK cells significantly increased their production of molecules with anti-tumor effects. CONCLUSIONS: These findings suggest that pMSCs might have potential applications in cancer therapy.

Human chorionic villous mesenchymal stem/stromal cells modify the effects of oxidative stress on endothelial cell functions.

Mesenchymal stem/stromal cells derived from chorionic villi of human term placentae (pMSCs) produce a unique combination of molecules, which modulate important cellular functions of their target cells while concurrently suppressing their immune responses. These properties make MSCs advantageous candidates for cell-based therapy. Our first aim was to examine the effect of high levels of oxidative stress on pMSC functions. pMSCs were exposed to hydrogen peroxide (H2O2) and their ability to proliferate and adhere to an endothelial cell monolayer was determined. Oxidatively stressed pMSCs maintained their proliferation and adhesion potentials. The second aim was to measure the ability of pMSCs to prevent oxidative stress-related damage to endothelial cells. Endothelial cells were exposed to H2O2, then co-cultured with pMSCs, and the effect on endothelial cell adhesion, proliferation and migration was determined. pMSCs were able to reverse the damaging effects of oxidative stress on the proliferation and migration but not on the adhesion of endothelial cells. These data indicate that pMSCs are not only inherently resistant to oxidative stress, but also protect endothelial cell functions from oxidative stress-associated damage. Therefore, pMSCs could be used as a therapeutic tool in inflammatory diseases by reducing the effects of oxidative stress on endothelial cells.

Phenotypic and Functional Characterization of Mesenchymal Stem/Multipotent Stromal Cells from Decidua Basalis of Human Term Placenta.

Mesenchymal stem cell (MSC) therapies for the treatment of diseases associated with inflammation and oxidative stress employ primarily bone marrow MSCs (BMMSCs) and other MSC types such as MSC from the chorionic villi of human term placentae (pMSCs). These MSCs are not derived from microenvironments associated with inflammation and oxidative stress, unlike MSCs from the decidua basalis of the human term placenta (DBMSCs). DBMSCs were isolated and then extensively characterized. Differentiation of DBMSCs into three mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed. Real-time polymerase chain reaction (PCR) and flow cytometry techniques were also used to characterize the gene and protein expression profiles of DBMSCs, respectively. In addition, sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect proteins secreted by DBMSCs. Finally, the migration and proliferation abilities of DBMSCs were also determined. DBMSCs were positive for MSC markers and HLA-ABC. DBMSCs were negative for hematopoietic and endothelial markers, costimulatory molecules, and HLA-DR. Functionally, DBMSCs differentiated into three mesenchymal lineages, proliferated, and migrated in response to a number of stimuli. Most importantly, these cells express and secrete a distinct combination of cytokines, growth factors, and immune molecules that reflect their unique microenvironment. Therefore, DBMSCs could be attractive, alternative candidates for MSC-based therapies that treat diseases associated with inflammation and oxidative stress.

Phenotypic and Functional Characterization of Mesenchymal Stem/Multipotent Stromal Cells From Decidua Parietalis of Human Term Placenta.

Mesenchymal stem/multipotent stromal cells (MSCs) from the human placenta show stem cell-like properties useful for regenerative medicine. Previously, we reported that MSCs isolated from the fetal part of human term placentae have characteristics, which make them a potential candidate for regenerative medicine. In this study, we characterized MSC isolated from the maternal part of human term placenta. The MSCs were isolated from the decidua parietalis (DPMSCs) of human placenta using a digestion method and characterized by colony-forming unit assay and the expression of MSC markers by flow cytometry technique. In addition, DPMSC differentiation into the 3 mesenchymal lineages was also performed. Moreover, the gene and protein expression profiles of DPMSCs were identified by real-time polymerase chain reaction and flow cytometry techniques, respectively. Furthermore, proteins secreted by DPMSCs were detected by sandwich enzyme-linked immunosorbent assays. Finally, the proliferation and migration potentials of DPMSCs were also determined. The DPMSCs were positive for MSC markers and negative for hematopoietic and endothelial markers, as well as costimulatory molecules and HLA-DR. Functionally, DPMSCs formed colonies and differentiated into chondrocytes, osteocytes, and adipocytes. In addition, they proliferated and migrated in response to different stimuli. Finally, they expressed and secreted many biological and immunological factors with multiple functions. Here, we carry out an extensive characterization of DPMSCs of human placenta. We report that these cells express and secrete a wide range of molecules with multiple functions, and therefore, we suggest that these cells could be an attractive candidate for cell-based therapy.

Human Chorionic Villous Mesenchymal Stem Cells Modify the Functions of Human Dendritic Cells, and Induce an Anti-Inflammatory Phenotype in CD1+ Dendritic Cells.

BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human term placenta (pMSCs) have drawn considerable interest because of their multipotent differentiation potential and their immunomodulatory capacity. These properties are the foundation for their clinical application in the fields of stem cell transplantation and regenerative medicine. Previously, we showed that pMSCs induce an anti-inflammatory phenotype in human macrophages. In this study, we determined whether pMSCs modify the differentiation and maturation of human monocytes into dendritic cells (DCs). The consequences on dendritic function and on T cell proliferation were also investigated. METHODS: Interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) were used to stimulate the differentiation of monocytes into immature dendritic cells (iDCs), which were subsequently co-cultured with pMSCs. Lipopolysaccharide (LPS) was used to induce maturation of iDCs into mature dendritic cells (mDCs). Flow cytometry and enzyme-linked immunosorbent assays (ELISA) were used to quantify the effect pMSC co-culturing on DC differentiation using CD1a, a distinctive marker of DCs, as well as other molecules important in the immune functions of DCs. The phagocytic activity of iDCs co-cultured with pMSCs, and the effects of iDCs and mDC stimulation on T cell proliferation, were also investigated. RESULTS: Monocyte differentiation into iDCs was inhibited when co-cultured with pMSCs and maturation of iDCs by LPS treatment was also prevented in the presence of pMSCs as demonstrated by reduced expression of CD1a and CD83, respectively. The inhibitory effect of pMSCs on iDC differentiation was dose dependent. In addition, pMSC co-culture with iDCs and mDCs resulted in both phenotypic and functional changes as shown by reduced expression of costimulatory molecules (CD40, CD80, CD83 and CD86) and reduced capacity to stimulate CD4(+) T cell proliferation. In addition, pMSC co-culture increased the surface expression of major histocompatibility complex (MHC-II) molecules on iDCs but decreased MHC-II expression on mDCs. Moreover, pMSC co-culture with iDCs or mDCs increased the expression of immunosuppressive molecules [B7H3, B7H4, CD273, CD274 and indoleamine-pyrrole 2,3-dioxygenase (IDO). Additionally, the secretion of IL-12 and IL-23 by iDCs and mDCs co-cultured with pMSCs was decreased. Furthermore, pMSC co-culture with mDCs decreased the secretion of IL-12 and INF-γ whilst increasing the secretion of IL-10 in a T cell proliferation experiment. Finally, pMSC co-culture with iDCs induced the phagocytic activity of iDCs. CONCLUSIONS: We have shown that pMSCs have an inhibitory effect on the differentiation, maturation and function of DCs, as well as on the proliferation of T cells, suggesting that pMSCs can control the immune responses at multiple levels.

Human placental mesenchymal stem cells (pMSCs) play a role as immune suppressive cells by shifting macrophage differentiation from inflammatory M1 to anti-inflammatory M2 macrophages.

BACKGROUND: Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions. METHODS: We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied. RESULTS: The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Co-culture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1ß, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors. CONCLUSIONS: We have shown that pMSCs can transition macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair.

Phenotypic and functional characterization of mesenchymal stem cells from chorionic villi of human term placenta.

BACKGROUND: Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy. METHODS: A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined. RESULTS: pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1). CONCLUSIONS: We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.