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Phenotypic and functional characterization of mesenchymal stem cells from decidua parietalis of human term placenta
Abstracts / Placenta 36 (2015) A1eA60
lung structure and reduced pulmonary artery wall thickness (p<0.001) on PND14. Further more, hAEC treatment rescued the long-term outcome of lung injury through attenuating PH (p<0.05) and reduced RVAW thickness. Conclusion: This neonatal mouse lung injury model with intra-amniotic LPS injection and postnatal hyperoxia exposure characterised the clinical BPD with lung histological changes and associated PH. hAEC administration to BPD mouse pups normalised lung structure and reduced peripheral pulmonary artery muscularisation shortly after treatment. In our long term follow up, we also found an improvement in pulmonary hypertension and reduced right ventricular hypertrophy.
P1.47. PLACENTA DERIVED MESENCHYMAL STEM CELLS (PDMSCS) MODULATE HIF-1A, VEGF AND JUNB GENES IN OVARIAN CANCER CELLS. Domenica Giuffrida, Cristian Zenerino, Rossella Barrile, Anna Maria Nuzzo, Tullia Todros, Alessandro Rolfo. Dept. of Surgical Sciences, University of Turin, Turin, Italy Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers. We previously demonstrated that PDMSCs produce soluble factors able to inhibit OC cell cycle and growth through a pathway that still remains to be determined. Hypoxia Inducible Factor-1 alpha (HIF-1a), main player in cellular response to hypoxia, promotes the expression of VEGF, responsible for neo-angiogenesis. HIF-1a over-expression has been described in most human malignancies and it is linked to bad cancer prognosis. Activating Protein-1 (AP-1) molecules are pivotal regulators of OC cell cycle progression and metastatization. Herein, we investigated HIF-1a, VEGF and JunB expression in OC ES-2 and SKOV-3 cells treated by PDMSCs conditioned medium (CM) in order to verify our hypothesis that PDMSCs could act through the modulation of HIF-1a and anti-proliferative AP-1 molecules. Methods: PDMSCs were isolated from physiological human term placentae (n¼12). CM will be produced by incubating PDMSCs for 24-48 hours in DMEM medium. CM will be harvested and filtered to remove cellular debris. ES-2 and SKOV-3 cells were treated by 24/48h CMs for 24h or 48h and mRNA was isolated. Expression of HIF-1a, VEGF and JunB was assessed by Real Time PCR. Results: HIF-1a mRNA levels were significantly decreased in both ES-2 and SKOV-3 cells treated by 24/48h PDMSCs-CMs for 24/48h. HIF-1a decrease was accompanied by VEGF down-regulation at 48h in ES-2 and at 24h in SKOV-3 cells. Interestingly, CM promoted anti-proliferative JunB mRNA accumulation in ES-2 at 24h/48h, while it induced JunB down-regulation in SKOV-3 at both time points. Conclusions: We demonstrated, for the first time to our knowledge, that healthy PDMSCs-CM treatment exerts its anti-tumoral activity through HIF-1a pathway down-regulation and anti-proliferative JunB accumulation in malignant ES-2 OC cells. JunB down-regulation in moderately differentiated/less aggressive SKOV-3 PDMSCs-treated cells requires further investigation.
P1.48. AMNION CELL MEDIATED IMMUNE MODULATION DURING LUNG INJURY: CONTROLLING THE REGULATORY T CELL RESPONSE Jean Tan 1, Shawn Tan 1, Ruth Muljadi 1, Siow Teng Chan 1, Euan Wallace 1, 2, Rebecca Lim 1, 2. 1 MIMR-PHI Institute, Clayton, Victoria, Australia; 2 Monash University, Clayton, Victoria, Australia Background: Human amnion epithelial cells (hAECs) have protective and reparative properties when administered immediately and following established lung injury. hAECs can modulate macrophage and T cell activity and function to dampen inflammation and accelerate repair. However, the mechanisms by which hAECs mediate these effects through T cells and their Treg subtype have yet to be determined. Methods: The reliance of hAEC mediated lung repair on T cells was assessed by challenging Rag1-/- mice with bleomycin, followed by adoptive transfer of either Tregs or CD45+/FoxP3- cells. The extent of lung fibrosis and inflammation, and macrophage polarity and function were measured
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7 and 14 days later. The role of hAEC secretory factors involved in T cell activity and survival were also assessed in vitro. Results: Administration of hAECs to bleomycin challenged Foxp3-GFP knock in mice induced Treg expansion in the lungs. Further, lung repair in bleomycin challenged Rag1-/- mice was most significant in the cohort of animals administered hAECs and Tregs. This is coupled with hAEC-mediated polarization of macrophages towards an M2 anti-inflammatory phenotype. In vitro, hAECs directly induced FoxP3 transcription in naive CD4+ cells though TGF-b signalling. This is concomitant with suppression of T cell proliferation through PGE2. Conclusion: Interaction between hAECs and T cells contribute to their protective and reparative properties. Moreover, polarization of macrophages occurs as a consequence of this cellular interaction. Additionally, these mechanisms are shown to occur primarily through secretory factors.
P1.49. HUMAN PLACENTAL DECIDUA BASALIS (DBMSCS) MODULATE THE EXPRESSION OF RECEPTORS IMPORTANT IN MEDIATING THE IMMUNOSUPPRESSIVE FUNCTIONS OF MACROPHAGES IN CANCER Mohamed Abumaree 1, 2, Fawaz Abomaray 2, Khaled Al Saad 2, 3, Dunia Jawdat 2, Abdulaziz Al Khaldi 4, Ahmed Al Askar 2, Seham Al Harthy 5, Abdulmohsen Alkushi 1, 3, Bill Kalionis 6, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11481, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Division of Cardiac Surgery, Riyadh, 11426, Saudi Arabia; 5 King Abdulaziz City for Science and Technology, Riyadh, 11442, Saudi Arabia; 6 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells (MSCs) have a therapeutic potential in regenerative medicine because of their ability to differentiate into different cell types and to modulate the response of immune cells. In this study, we evaluated the ability of MSCs isolated from decidua basalis of human term placenta (DBMSCs) to alter the differentiation of human monocytes into macrophages. We used GM-CSF to induce monocyte differentiation into the M1 macrophage pathway and then co-cultured these cells with DBMSCs in the early stages of macrophage differentiation. We then assessed the influence on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry. The co-culture of DBMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway did not result in a shift to the antiinflammatory M2-like macrophage differentiation route as the cells morphologically did not take up the typical morphology of M2 macrophages, and the expression of cell surface markers including CD163, CD204 and CD206 did not increase, which are distinctive features of M2 macrophages. In contrast, DBMSCs significantly decreased the expression of CD163, CD204 and CD206 in macrophages. We have shown that DBMSCs cannot shift macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Instead, they down-regulated the expression of receptors known to be overexpressed in macrophages in cancer patients. Our findings suggest that DBMSCs could be employed in the treatment of cancer via acting on macrophages. However, more studies are needed to confirm the antitumor activity of DBMSCs in cancer treatments.
P1.50. PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM DECIDUA PARIETALIS OF HUMAN TERM PLACENTA Mohamed Abumaree 1, 2, Najla Alshehri 2, Abdulaziz Almutairi 3, Fawaz Abomaray 2, Ahmed Al Askar 2, Abdulmohesen Alkushi 1, 4, Bill Kalionis 5, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, P.O. Box
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Abstracts / Placenta 36 (2015) A1eA60
3660, Mail Code 3124, Riyadh 11481, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11481, Saudi Arabia; 3 King Saud Bin Abdulaziz University for Health Sciences, College of Applied Medical Sciences, Riyadh, 11481, Saudi Arabia; 4 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11481, Saudi Arabia; 5 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells isolated from the decidua parietalis of the human term placenta (DPMSCs) have been reported as promising cells for use in regenerative medicine. However, the characteristics of DPMSCs are not fully elucidated and therefore, this study was conducted to isolate and characterize DPMSCs to reveal their potential use in stem cell-based therapy. DPMSCs were isolated from the decidua parietalis using the enzymatic digestion method and then were phenotypically characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation media as demonstrated by cytochemical staining. The expression profiles of genes and proteins of DPMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, the secretion of cytokines by DPMSCs was also evaluated using the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, DPMSCs were exposed to inflammatory stimuli to determine their proliferation and migration abilities. MSCs were isolated from the decidua parietalis of human term placenta and these DPMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these DBMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed a broad spectrum of adhesion molecules, chemokines and their receptors, growth factor receptors and cytokines and their receptors. Moreover, they secreted inflammatory and anti-inflammatory cytokines and they were able to proliferate and migrate in response to an inflammatory environment. In this study, we have shown that DPMSCs express a broad spectrum of biological factors which are possibly employed by these cells to mediate their cellular functions of adhesion, migration, homing, immune modulation and angiogenesis. Therefore, we propose that MSCs isolated from the decidua parietalis of human term placenta as an attractive source of MSCs for stem cell therapy.
P1.51. PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM DECIDUA BASILS OF HUMAN TERM PLACENTA Mohamed Abumaree 1, 2, Fawaz Abomaray 2, Khaled Al Saad 2,3, Dunia Jawdat 2, Abdulaziz Al Khaldi 4, Ahmed Al Askar 2, Seham Al Harthy 5, Abdulmohesen Alkushi 1, 3, Bill Kalionis 6, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11426, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Division of Cardiac Surgery, Riyadh, 11426, Saudi Arabia; 5 King Abdulaziz City for Science and Technology, Riyadh, 11442, Saudi Arabia; 6 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Australia Mesenchymal stem cells from decidua basalis (DBMSCs) of human placenta have been reported as a good source for stem cell-based therapy. However, these cells are not fully characterized and therefore our aim in this study is to isolate and characterize DBMSCs to demonstrate their potential application in stem cell transplantation. Cells were isolated from the decidua basalis using the enzymatic digestion method and then were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation media as demonstrated by cytochemical staining. The gene and protein expression profiles of DBMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by DBMSCs was also analysed using the sandwich enzyme-linked
immunosorbent assay (ELISA) technique. Moreover, the proliferation and migration potentials of DBMSCs were also determined. MSCs were isolated from the decidua basalis of human term placenta and these DBMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these DBMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed a broad spectrum of adhesion molecules, chemokines and their receptors, growth factor receptors and cytokines and their receptors. Moreover, they secreted some inflammatory and anti-inflammatory cytokines and they were able to proliferate and migrate in response to an inflammatory environment. We report that DBMSCs express many biological factors that may mediate their cellular functions such as adhesion, migration, homing, immune modulation and angiogenesis. Therefore, we suggest that MSCs isolated from the decidua basalis of human term placenta as an attractive source of MSCs for stem cell therapy.
P1.52. THE CONSEQUENCES OF THE INTERACTION BETWEEN CHORIONIC VILLOUS MESENCHYMAL STEM CELLS AND HUMAN NATURAL KILLER CELLS Mohamed Abumaree 1, 2, Abdulaziz Almutairi 3, Najla Alshehri 2, Fawaz Abomaray 2, Ahmed Al Askar 2, Abdulmohesen Alkushi 1, 4, Bill Kalionis 5, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11426, Saudi Arabia; 2 2King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Saud Bin Abdulaziz University for Health Sciences, College of Applied Medical Sciences, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 5 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells derived from the chorionic villi of human term placenta (pMSCs) have drawn a significant interest because of their potential to differentiate into different cell types and their immunomodulatory functions. These properties are essential for their use in stem cell transplantation and regenerative medicine. Recently, we revealed that pMSCs can induce an anti-inflammatory phenotype in human macrophages and exert an inhibitory effect on the differentiation, maturation and functions of dendritic cells, as well as on the proliferation of T cells. These data suggest that pMSCs can control the immune responses at multiple levels. However, the effect of pMSCs on natural killer (NK) cells is unknown yet. Our aim in this study is to show the consequences of the interaction between pMSCs and NK cell. pMSCs were isolated from the chorionic villi of human normal term placenta using an explant approach and then co-cultured with unstimulated or IL-2-stimulated NK cells isolated from peripheral blood of healthy donors. The proliferation and cytotoxicity functions of NK cells were then examined using proliferation and cytotoxicity assays, respectively. In addition the expression of receptors important in mediating the cytotoxicity functions of NK cells was also examined using flow cytometry. We demonstrated that pMSCs could significantly inhibit IL-2-induced proliferation of resting NK cells, while the proliferation of IL-2- stimulated NK cells was partially inhibited. We also demonstrated that IL-2-stimulated NK cells only could lyse pMSCs. In addition, the activating NK receptors NKp30, NKG2D, and NKp44 were also shown to be the major receptors mediating NK-mediating lysis of pMSCs. Importantly, the interaction between NK cells and pMSCs did not affect the cytotoxicity activity of NK cells on target cancer cells. These results should be considered when pMSCs is used in stem cell transplantation or regenerative medicine to develop an efficient therapeutic approach.
P1.53. IDENTIFICATION OF A TROPHOBLAST SIDE-POPULATION WITH LOW HOECHST STAINING IN HUMAN TERM PLACENTAE: ARE THESE TROPHOBLAST STEM CELLS? Teena Gamage, Larry Chamley, Jo James. The University of Auckland, Auckland, New Zealand
lung structure and reduced pulmonary artery wall thickness (p<0.001) on PND14. Further more, hAEC treatment rescued the long-term outcome of lung injury through attenuating PH (p<0.05) and reduced RVAW thickness. Conclusion: This neonatal mouse lung injury model with intra-amniotic LPS injection and postnatal hyperoxia exposure characterised the clinical BPD with lung histological changes and associated PH. hAEC administration to BPD mouse pups normalised lung structure and reduced peripheral pulmonary artery muscularisation shortly after treatment. In our long term follow up, we also found an improvement in pulmonary hypertension and reduced right ventricular hypertrophy.
P1.47. PLACENTA DERIVED MESENCHYMAL STEM CELLS (PDMSCS) MODULATE HIF-1A, VEGF AND JUNB GENES IN OVARIAN CANCER CELLS. Domenica Giuffrida, Cristian Zenerino, Rossella Barrile, Anna Maria Nuzzo, Tullia Todros, Alessandro Rolfo. Dept. of Surgical Sciences, University of Turin, Turin, Italy Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers. We previously demonstrated that PDMSCs produce soluble factors able to inhibit OC cell cycle and growth through a pathway that still remains to be determined. Hypoxia Inducible Factor-1 alpha (HIF-1a), main player in cellular response to hypoxia, promotes the expression of VEGF, responsible for neo-angiogenesis. HIF-1a over-expression has been described in most human malignancies and it is linked to bad cancer prognosis. Activating Protein-1 (AP-1) molecules are pivotal regulators of OC cell cycle progression and metastatization. Herein, we investigated HIF-1a, VEGF and JunB expression in OC ES-2 and SKOV-3 cells treated by PDMSCs conditioned medium (CM) in order to verify our hypothesis that PDMSCs could act through the modulation of HIF-1a and anti-proliferative AP-1 molecules. Methods: PDMSCs were isolated from physiological human term placentae (n¼12). CM will be produced by incubating PDMSCs for 24-48 hours in DMEM medium. CM will be harvested and filtered to remove cellular debris. ES-2 and SKOV-3 cells were treated by 24/48h CMs for 24h or 48h and mRNA was isolated. Expression of HIF-1a, VEGF and JunB was assessed by Real Time PCR. Results: HIF-1a mRNA levels were significantly decreased in both ES-2 and SKOV-3 cells treated by 24/48h PDMSCs-CMs for 24/48h. HIF-1a decrease was accompanied by VEGF down-regulation at 48h in ES-2 and at 24h in SKOV-3 cells. Interestingly, CM promoted anti-proliferative JunB mRNA accumulation in ES-2 at 24h/48h, while it induced JunB down-regulation in SKOV-3 at both time points. Conclusions: We demonstrated, for the first time to our knowledge, that healthy PDMSCs-CM treatment exerts its anti-tumoral activity through HIF-1a pathway down-regulation and anti-proliferative JunB accumulation in malignant ES-2 OC cells. JunB down-regulation in moderately differentiated/less aggressive SKOV-3 PDMSCs-treated cells requires further investigation.
P1.48. AMNION CELL MEDIATED IMMUNE MODULATION DURING LUNG INJURY: CONTROLLING THE REGULATORY T CELL RESPONSE Jean Tan 1, Shawn Tan 1, Ruth Muljadi 1, Siow Teng Chan 1, Euan Wallace 1, 2, Rebecca Lim 1, 2. 1 MIMR-PHI Institute, Clayton, Victoria, Australia; 2 Monash University, Clayton, Victoria, Australia Background: Human amnion epithelial cells (hAECs) have protective and reparative properties when administered immediately and following established lung injury. hAECs can modulate macrophage and T cell activity and function to dampen inflammation and accelerate repair. However, the mechanisms by which hAECs mediate these effects through T cells and their Treg subtype have yet to be determined. Methods: The reliance of hAEC mediated lung repair on T cells was assessed by challenging Rag1-/- mice with bleomycin, followed by adoptive transfer of either Tregs or CD45+/FoxP3- cells. The extent of lung fibrosis and inflammation, and macrophage polarity and function were measured
A27
7 and 14 days later. The role of hAEC secretory factors involved in T cell activity and survival were also assessed in vitro. Results: Administration of hAECs to bleomycin challenged Foxp3-GFP knock in mice induced Treg expansion in the lungs. Further, lung repair in bleomycin challenged Rag1-/- mice was most significant in the cohort of animals administered hAECs and Tregs. This is coupled with hAEC-mediated polarization of macrophages towards an M2 anti-inflammatory phenotype. In vitro, hAECs directly induced FoxP3 transcription in naive CD4+ cells though TGF-b signalling. This is concomitant with suppression of T cell proliferation through PGE2. Conclusion: Interaction between hAECs and T cells contribute to their protective and reparative properties. Moreover, polarization of macrophages occurs as a consequence of this cellular interaction. Additionally, these mechanisms are shown to occur primarily through secretory factors.
P1.49. HUMAN PLACENTAL DECIDUA BASALIS (DBMSCS) MODULATE THE EXPRESSION OF RECEPTORS IMPORTANT IN MEDIATING THE IMMUNOSUPPRESSIVE FUNCTIONS OF MACROPHAGES IN CANCER Mohamed Abumaree 1, 2, Fawaz Abomaray 2, Khaled Al Saad 2, 3, Dunia Jawdat 2, Abdulaziz Al Khaldi 4, Ahmed Al Askar 2, Seham Al Harthy 5, Abdulmohsen Alkushi 1, 3, Bill Kalionis 6, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11481, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Division of Cardiac Surgery, Riyadh, 11426, Saudi Arabia; 5 King Abdulaziz City for Science and Technology, Riyadh, 11442, Saudi Arabia; 6 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells (MSCs) have a therapeutic potential in regenerative medicine because of their ability to differentiate into different cell types and to modulate the response of immune cells. In this study, we evaluated the ability of MSCs isolated from decidua basalis of human term placenta (DBMSCs) to alter the differentiation of human monocytes into macrophages. We used GM-CSF to induce monocyte differentiation into the M1 macrophage pathway and then co-cultured these cells with DBMSCs in the early stages of macrophage differentiation. We then assessed the influence on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry. The co-culture of DBMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway did not result in a shift to the antiinflammatory M2-like macrophage differentiation route as the cells morphologically did not take up the typical morphology of M2 macrophages, and the expression of cell surface markers including CD163, CD204 and CD206 did not increase, which are distinctive features of M2 macrophages. In contrast, DBMSCs significantly decreased the expression of CD163, CD204 and CD206 in macrophages. We have shown that DBMSCs cannot shift macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Instead, they down-regulated the expression of receptors known to be overexpressed in macrophages in cancer patients. Our findings suggest that DBMSCs could be employed in the treatment of cancer via acting on macrophages. However, more studies are needed to confirm the antitumor activity of DBMSCs in cancer treatments.
P1.50. PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM DECIDUA PARIETALIS OF HUMAN TERM PLACENTA Mohamed Abumaree 1, 2, Najla Alshehri 2, Abdulaziz Almutairi 3, Fawaz Abomaray 2, Ahmed Al Askar 2, Abdulmohesen Alkushi 1, 4, Bill Kalionis 5, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, P.O. Box
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Abstracts / Placenta 36 (2015) A1eA60
3660, Mail Code 3124, Riyadh 11481, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11481, Saudi Arabia; 3 King Saud Bin Abdulaziz University for Health Sciences, College of Applied Medical Sciences, Riyadh, 11481, Saudi Arabia; 4 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11481, Saudi Arabia; 5 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells isolated from the decidua parietalis of the human term placenta (DPMSCs) have been reported as promising cells for use in regenerative medicine. However, the characteristics of DPMSCs are not fully elucidated and therefore, this study was conducted to isolate and characterize DPMSCs to reveal their potential use in stem cell-based therapy. DPMSCs were isolated from the decidua parietalis using the enzymatic digestion method and then were phenotypically characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation media as demonstrated by cytochemical staining. The expression profiles of genes and proteins of DPMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, the secretion of cytokines by DPMSCs was also evaluated using the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, DPMSCs were exposed to inflammatory stimuli to determine their proliferation and migration abilities. MSCs were isolated from the decidua parietalis of human term placenta and these DPMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these DBMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed a broad spectrum of adhesion molecules, chemokines and their receptors, growth factor receptors and cytokines and their receptors. Moreover, they secreted inflammatory and anti-inflammatory cytokines and they were able to proliferate and migrate in response to an inflammatory environment. In this study, we have shown that DPMSCs express a broad spectrum of biological factors which are possibly employed by these cells to mediate their cellular functions of adhesion, migration, homing, immune modulation and angiogenesis. Therefore, we propose that MSCs isolated from the decidua parietalis of human term placenta as an attractive source of MSCs for stem cell therapy.
P1.51. PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM DECIDUA BASILS OF HUMAN TERM PLACENTA Mohamed Abumaree 1, 2, Fawaz Abomaray 2, Khaled Al Saad 2,3, Dunia Jawdat 2, Abdulaziz Al Khaldi 4, Ahmed Al Askar 2, Seham Al Harthy 5, Abdulmohesen Alkushi 1, 3, Bill Kalionis 6, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11426, Saudi Arabia; 2 King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Division of Cardiac Surgery, Riyadh, 11426, Saudi Arabia; 5 King Abdulaziz City for Science and Technology, Riyadh, 11442, Saudi Arabia; 6 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Australia Mesenchymal stem cells from decidua basalis (DBMSCs) of human placenta have been reported as a good source for stem cell-based therapy. However, these cells are not fully characterized and therefore our aim in this study is to isolate and characterize DBMSCs to demonstrate their potential application in stem cell transplantation. Cells were isolated from the decidua basalis using the enzymatic digestion method and then were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation media as demonstrated by cytochemical staining. The gene and protein expression profiles of DBMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by DBMSCs was also analysed using the sandwich enzyme-linked
immunosorbent assay (ELISA) technique. Moreover, the proliferation and migration potentials of DBMSCs were also determined. MSCs were isolated from the decidua basalis of human term placenta and these DBMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these DBMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed a broad spectrum of adhesion molecules, chemokines and their receptors, growth factor receptors and cytokines and their receptors. Moreover, they secreted some inflammatory and anti-inflammatory cytokines and they were able to proliferate and migrate in response to an inflammatory environment. We report that DBMSCs express many biological factors that may mediate their cellular functions such as adhesion, migration, homing, immune modulation and angiogenesis. Therefore, we suggest that MSCs isolated from the decidua basalis of human term placenta as an attractive source of MSCs for stem cell therapy.
P1.52. THE CONSEQUENCES OF THE INTERACTION BETWEEN CHORIONIC VILLOUS MESENCHYMAL STEM CELLS AND HUMAN NATURAL KILLER CELLS Mohamed Abumaree 1, 2, Abdulaziz Almutairi 3, Najla Alshehri 2, Fawaz Abomaray 2, Ahmed Al Askar 2, Abdulmohesen Alkushi 1, 4, Bill Kalionis 5, Mohammed Al Jumah 2. 1 King Saud Bin Abdulaziz University for Health Sciences, College of Science and Health Professions, Riyadh, 11426, Saudi Arabia; 2 2King Abdullah International Medical Research Center, Riyadh, 11426, Saudi Arabia; 3 King Saud Bin Abdulaziz University for Health Sciences, College of Applied Medical Sciences, Riyadh, 11426, Saudi Arabia; 4 King Abdulaziz Medical City, Department of Pathology, Riyadh, 11426, Saudi Arabia; 5 University of Melbourne, Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia Mesenchymal stem cells derived from the chorionic villi of human term placenta (pMSCs) have drawn a significant interest because of their potential to differentiate into different cell types and their immunomodulatory functions. These properties are essential for their use in stem cell transplantation and regenerative medicine. Recently, we revealed that pMSCs can induce an anti-inflammatory phenotype in human macrophages and exert an inhibitory effect on the differentiation, maturation and functions of dendritic cells, as well as on the proliferation of T cells. These data suggest that pMSCs can control the immune responses at multiple levels. However, the effect of pMSCs on natural killer (NK) cells is unknown yet. Our aim in this study is to show the consequences of the interaction between pMSCs and NK cell. pMSCs were isolated from the chorionic villi of human normal term placenta using an explant approach and then co-cultured with unstimulated or IL-2-stimulated NK cells isolated from peripheral blood of healthy donors. The proliferation and cytotoxicity functions of NK cells were then examined using proliferation and cytotoxicity assays, respectively. In addition the expression of receptors important in mediating the cytotoxicity functions of NK cells was also examined using flow cytometry. We demonstrated that pMSCs could significantly inhibit IL-2-induced proliferation of resting NK cells, while the proliferation of IL-2- stimulated NK cells was partially inhibited. We also demonstrated that IL-2-stimulated NK cells only could lyse pMSCs. In addition, the activating NK receptors NKp30, NKG2D, and NKp44 were also shown to be the major receptors mediating NK-mediating lysis of pMSCs. Importantly, the interaction between NK cells and pMSCs did not affect the cytotoxicity activity of NK cells on target cancer cells. These results should be considered when pMSCs is used in stem cell transplantation or regenerative medicine to develop an efficient therapeutic approach.
P1.53. IDENTIFICATION OF A TROPHOBLAST SIDE-POPULATION WITH LOW HOECHST STAINING IN HUMAN TERM PLACENTAE: ARE THESE TROPHOBLAST STEM CELLS? Teena Gamage, Larry Chamley, Jo James. The University of Auckland, Auckland, New Zealand