- Email: [email protected]
Please refer to Oral Abstracts Section.
S106
Poster Abstracts
expression) for CD44, CD73, CD90, CD105 and CD166 and negative (<5% expression) for CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR. Cells harvested from the bioreactor and HYPERFlask tested negative for sterility (BacTAlert bottles) and Mycoplasma (PCR), and had endotoxin levels of <0.2 EU/mL. In summary, we demonstrated the feasibility for generating up to 375 million MSCs per production run with one Terumo Quantum® Cell Expansion System using a xeno- and serum-free media. 244 DIFFERENT CHARACTERISTICS OF MESENCHYMAL STEM CELLS ISOLATED FROM DIFFERENT LAYERS OF FULL TERM PLACENTA J. Kim1, C. Ha1, J. Rhim1, Y. Park2, W. Han1, S. Choi1, K. Lee1, H. Park1, H. Park1 1 Samsung Medical Center, Seoul, Republic of Korea, 2Chung-Ang University Hospital, Seoul, Republic of Korea Purpose: The placenta is very attractive source of mesenchymal stem cells (MSCs) for regenerative medicine due to readily availability, non-invasive acquisition and avoidance of ethical issues. Growing interest has developed to isolate MSCs from parts of placenta tissue because they are assumed to exhibit different proliferation and differentiation potentials due to the complex structure and functions of the placenta. The purpose of this study was to investigate the feasibility of MSCs isolation from different placental parts and comparing the characteristics of the isolated MSCs. Placenta was divided into amniotic epithelium (AE), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV), chorionic trophoblast without villi (CT-V) and decidua (DC), and whole placenta (Pla). Methods: Cells isolated from each layer were analyzed for their morphology, proliferation ability, surface markers, and multi-lineage differentiation potential, and the differences were compared. MSCs were isolated from all placental layers. The surface antigen phenotype and morphological and differentiation characteristics of the cells from all layers indicated that they exhibited properties of MSCs. Results: The MSCs from different placental layers had different proliferation rates and differentiation potential. MSCs from CM, CT-V, CV, and DC had better population doubling timeand multi-lineage differentiation potential. Conclusions: Our results indicate that MSCs with different characteristics can be isolated from all layers of term placenta. These finding suggest that it is necessary to appropriately select MSCs from different placental layers for successful and consistent outcomes in clinical application. 245 PLEASE REFER TO ORAL ABSTRACTS SECTION. 246 IDENTIFICATION OF NOVEL MESENCHYMAL STROMAL CELLDERIVED MIRNAS WITH ALLOSUPPRESSIVE POTENTIAL S. Kuci, Z. Kuci, C. Jordan, T. Klingebiel, P. Bader Hematology/Oncology, University Children’s Hospital, Frankfurt am Main, Germany Introduction: Preactivation of mesenchymal stromal cells (MSCs) with inflammatory cytokines (“licensing”) may improve suppression of the allogeneic response (allosuppression) through release of anti-inflammatory molecules. In this study, we assessed the allosuppressive potential of the miRNAs from preactivated MSCs. Materials and Methods: Bone marrow derived MSCs (P2) were preactivated with either IFN-gamma or TNF-alpha for 24 hours. Total RNA was extracted and used in a miScript miRNA PCR Array (Qiagen). miRNAs with the highest expression were assessed for their allosuppressive potential in mixed lymphocyte reaction (MLR). Results: miRNA-analysis of the licensed MSCs with IFN-gamma showed 15 miRNAs with a greater than a two-fold increase in expression and 17 miRNAs with more than a two-fold decrease in expression. miR-186-3p demonstrated the highest expression vs. control group (88.7-fold). MSCs licensed with TNF-alpha had 20 miRNAs with greater than a two-fold increase in expression and 25 miRNAs with more than a two-fold decrease in expression. miRNA146a-5p demonstrated the highest expression vs. control (94.7-fold). As licensed MSCs demonstrated a significantly higher allosuppressive effect compared to non-licensed MSCs, we assessed the effect of miR-186-3p and miRNA-146a-5p
in suppression of alloreaction in MLR by using their specific inhibitors. The inhibitor of miR-186-3p was able to reverse the allosuppressive effect of not only non-licensed MSCs (p < 0.0001), but also the effect of licensed MSCs with IFN-gamma (P < 0.0001) (n = 5). Interestingly, this effect was comparable or even better to the effect of indomethacin (IM) as a specific inhibitor of PGE2 synthesis. When combined with IM, the inhibitor of miR-186-3p amplified the reversal effect, suggesting that PGE2 may be a target molecule of this miRNA. The downstream activation pathways including specific gene targets of miRNA186-3p remain to be elucidated. However, the use of specific inhibitor against miRNA-146a-5p was unable to reverse the allosuppressive effect of MSCs, indicating that this miRNA may not be involved in their immunomodulatory potential. Conclusion: Our results demonstrate that miR-186-3p may serve as a novel mediator molecule for the strikingly high allosuppressive potential of preactivated MSCs. 247 MESENCHYMAL STROMAL CELLS GENERATED FROM PATIENTS WITH NON-MALIGNANT HEMATOPOIETIC DISEASES Z. Kuci1, C. Jordan1, S. Wehner1, J. Greil2, T. Klingebiel1, P. Bader1, S. Kuci1 1 Hematology/Oncology, University Children’s Hospital, Frankfurt am Main, Germany, 2Hematology/Oncology, University Children’s Hospital, Heidelberg, Germany Introduction: Mesenchymal stromal cells (MSCs) of healthy donors are wellcharacterized as to their immunomodulatory and regenerative potential. There are, however, only few reports on the functionality of MSCs from patients with non-malignant hematopoietic diseases. In this study, we assessed function of MSCs of these patients before transplantation as a possible autologous cell therapy for the treatment of post-transplant complications such as osteonecrosis and improvement of the engraftment. Materials and Methods: To generate MSCs we used bone marrow samples from eight pedriatic patients. Three of them had thalassemia major (TM), three suffered from sickle cell disease (SCD), one had severe congenital neutropenia (SCN) and one suffered from very severe aplastic anemia (VSAA). As control group served MSCs generated from eight pediatric donors. We assessed the frequency of progenitor cells for MSCs (CFU-Fs) per 1x10e6 BM-MNCs, their phenotype, proliferation capacity (population doublings-PD, and doubling timeDT) as well as their differentiation potential in osteoblasts, adipocytes and chondrocytes. Their immunosuppressive effect was evaluated by means of mixed lymphocyte reaction (MLR). Results: SCD patients showed a significantly higher number of CFUs (301 ± 84) compared to TM patients (53 ± 21) (P > 0.05). Despite the higher number of CFU-Fs in SCD patients this difference versus control group (183 ± 42) was not significant. The VSAA patient had the lowest number of CFU-Fs (15 CFUs). MSCs of patients showed a normal phenotype but a slightly prolonged proliferation (one PD per 2.23 ± 0.31 days) compared to control group (1.8 ± 0.09 days), whereby the VSAA showed the longest doubling time (4,4 days). Patients’ MSCs differentiated in three lineages as the control group. In the MLR, the immunosuppressive potential of patients’ MSCs was lower, compared to control group (44,1 ± 5.3% vs. 52 ± 3,9%), but not significantly different. MSCs of the patient with VSAA showed again the lowest immunosuppressive effect (23%). Indomethacin as an inhibitor of PGE2 synthesis, was able to partially reverse the effect of MSCs. In MLR MSCs of all patients were as efficient as the control MSCs in expansion of T-regulatory cells. Conclusion: Our results indicate that MSCs of all patients with exception of MSCs from a VSAA patient, possess the same potential as the MSCs of healthy donors. Therefore, they may be used as an autologous cell therapy in posttransplant complications. 248 CELL CULTURE CONFLUENCY LEVEL AND THE VARIABILITY OF PLATELET LYSATE POOLS IMPACT THE READOUT OF THE OSTEGENIC DIFFERENTIATION ASSAY OF MESENCHYMAL STROMAL CELLS A. Laitinen, T. Kaartinen, S. Oja, M. Korhonen, K. Alfthan, J. Nystedt Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland Mesenchymal stromal cells (MSCs) are widely used for research and clinical purposes. These cells are preferably cultured in xeno-free culture conditions
Poster Abstracts
expression) for CD44, CD73, CD90, CD105 and CD166 and negative (<5% expression) for CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR. Cells harvested from the bioreactor and HYPERFlask tested negative for sterility (BacTAlert bottles) and Mycoplasma (PCR), and had endotoxin levels of <0.2 EU/mL. In summary, we demonstrated the feasibility for generating up to 375 million MSCs per production run with one Terumo Quantum® Cell Expansion System using a xeno- and serum-free media. 244 DIFFERENT CHARACTERISTICS OF MESENCHYMAL STEM CELLS ISOLATED FROM DIFFERENT LAYERS OF FULL TERM PLACENTA J. Kim1, C. Ha1, J. Rhim1, Y. Park2, W. Han1, S. Choi1, K. Lee1, H. Park1, H. Park1 1 Samsung Medical Center, Seoul, Republic of Korea, 2Chung-Ang University Hospital, Seoul, Republic of Korea Purpose: The placenta is very attractive source of mesenchymal stem cells (MSCs) for regenerative medicine due to readily availability, non-invasive acquisition and avoidance of ethical issues. Growing interest has developed to isolate MSCs from parts of placenta tissue because they are assumed to exhibit different proliferation and differentiation potentials due to the complex structure and functions of the placenta. The purpose of this study was to investigate the feasibility of MSCs isolation from different placental parts and comparing the characteristics of the isolated MSCs. Placenta was divided into amniotic epithelium (AE), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV), chorionic trophoblast without villi (CT-V) and decidua (DC), and whole placenta (Pla). Methods: Cells isolated from each layer were analyzed for their morphology, proliferation ability, surface markers, and multi-lineage differentiation potential, and the differences were compared. MSCs were isolated from all placental layers. The surface antigen phenotype and morphological and differentiation characteristics of the cells from all layers indicated that they exhibited properties of MSCs. Results: The MSCs from different placental layers had different proliferation rates and differentiation potential. MSCs from CM, CT-V, CV, and DC had better population doubling timeand multi-lineage differentiation potential. Conclusions: Our results indicate that MSCs with different characteristics can be isolated from all layers of term placenta. These finding suggest that it is necessary to appropriately select MSCs from different placental layers for successful and consistent outcomes in clinical application. 245 PLEASE REFER TO ORAL ABSTRACTS SECTION. 246 IDENTIFICATION OF NOVEL MESENCHYMAL STROMAL CELLDERIVED MIRNAS WITH ALLOSUPPRESSIVE POTENTIAL S. Kuci, Z. Kuci, C. Jordan, T. Klingebiel, P. Bader Hematology/Oncology, University Children’s Hospital, Frankfurt am Main, Germany Introduction: Preactivation of mesenchymal stromal cells (MSCs) with inflammatory cytokines (“licensing”) may improve suppression of the allogeneic response (allosuppression) through release of anti-inflammatory molecules. In this study, we assessed the allosuppressive potential of the miRNAs from preactivated MSCs. Materials and Methods: Bone marrow derived MSCs (P2) were preactivated with either IFN-gamma or TNF-alpha for 24 hours. Total RNA was extracted and used in a miScript miRNA PCR Array (Qiagen). miRNAs with the highest expression were assessed for their allosuppressive potential in mixed lymphocyte reaction (MLR). Results: miRNA-analysis of the licensed MSCs with IFN-gamma showed 15 miRNAs with a greater than a two-fold increase in expression and 17 miRNAs with more than a two-fold decrease in expression. miR-186-3p demonstrated the highest expression vs. control group (88.7-fold). MSCs licensed with TNF-alpha had 20 miRNAs with greater than a two-fold increase in expression and 25 miRNAs with more than a two-fold decrease in expression. miRNA146a-5p demonstrated the highest expression vs. control (94.7-fold). As licensed MSCs demonstrated a significantly higher allosuppressive effect compared to non-licensed MSCs, we assessed the effect of miR-186-3p and miRNA-146a-5p
in suppression of alloreaction in MLR by using their specific inhibitors. The inhibitor of miR-186-3p was able to reverse the allosuppressive effect of not only non-licensed MSCs (p < 0.0001), but also the effect of licensed MSCs with IFN-gamma (P < 0.0001) (n = 5). Interestingly, this effect was comparable or even better to the effect of indomethacin (IM) as a specific inhibitor of PGE2 synthesis. When combined with IM, the inhibitor of miR-186-3p amplified the reversal effect, suggesting that PGE2 may be a target molecule of this miRNA. The downstream activation pathways including specific gene targets of miRNA186-3p remain to be elucidated. However, the use of specific inhibitor against miRNA-146a-5p was unable to reverse the allosuppressive effect of MSCs, indicating that this miRNA may not be involved in their immunomodulatory potential. Conclusion: Our results demonstrate that miR-186-3p may serve as a novel mediator molecule for the strikingly high allosuppressive potential of preactivated MSCs. 247 MESENCHYMAL STROMAL CELLS GENERATED FROM PATIENTS WITH NON-MALIGNANT HEMATOPOIETIC DISEASES Z. Kuci1, C. Jordan1, S. Wehner1, J. Greil2, T. Klingebiel1, P. Bader1, S. Kuci1 1 Hematology/Oncology, University Children’s Hospital, Frankfurt am Main, Germany, 2Hematology/Oncology, University Children’s Hospital, Heidelberg, Germany Introduction: Mesenchymal stromal cells (MSCs) of healthy donors are wellcharacterized as to their immunomodulatory and regenerative potential. There are, however, only few reports on the functionality of MSCs from patients with non-malignant hematopoietic diseases. In this study, we assessed function of MSCs of these patients before transplantation as a possible autologous cell therapy for the treatment of post-transplant complications such as osteonecrosis and improvement of the engraftment. Materials and Methods: To generate MSCs we used bone marrow samples from eight pedriatic patients. Three of them had thalassemia major (TM), three suffered from sickle cell disease (SCD), one had severe congenital neutropenia (SCN) and one suffered from very severe aplastic anemia (VSAA). As control group served MSCs generated from eight pediatric donors. We assessed the frequency of progenitor cells for MSCs (CFU-Fs) per 1x10e6 BM-MNCs, their phenotype, proliferation capacity (population doublings-PD, and doubling timeDT) as well as their differentiation potential in osteoblasts, adipocytes and chondrocytes. Their immunosuppressive effect was evaluated by means of mixed lymphocyte reaction (MLR). Results: SCD patients showed a significantly higher number of CFUs (301 ± 84) compared to TM patients (53 ± 21) (P > 0.05). Despite the higher number of CFU-Fs in SCD patients this difference versus control group (183 ± 42) was not significant. The VSAA patient had the lowest number of CFU-Fs (15 CFUs). MSCs of patients showed a normal phenotype but a slightly prolonged proliferation (one PD per 2.23 ± 0.31 days) compared to control group (1.8 ± 0.09 days), whereby the VSAA showed the longest doubling time (4,4 days). Patients’ MSCs differentiated in three lineages as the control group. In the MLR, the immunosuppressive potential of patients’ MSCs was lower, compared to control group (44,1 ± 5.3% vs. 52 ± 3,9%), but not significantly different. MSCs of the patient with VSAA showed again the lowest immunosuppressive effect (23%). Indomethacin as an inhibitor of PGE2 synthesis, was able to partially reverse the effect of MSCs. In MLR MSCs of all patients were as efficient as the control MSCs in expansion of T-regulatory cells. Conclusion: Our results indicate that MSCs of all patients with exception of MSCs from a VSAA patient, possess the same potential as the MSCs of healthy donors. Therefore, they may be used as an autologous cell therapy in posttransplant complications. 248 CELL CULTURE CONFLUENCY LEVEL AND THE VARIABILITY OF PLATELET LYSATE POOLS IMPACT THE READOUT OF THE OSTEGENIC DIFFERENTIATION ASSAY OF MESENCHYMAL STROMAL CELLS A. Laitinen, T. Kaartinen, S. Oja, M. Korhonen, K. Alfthan, J. Nystedt Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland Mesenchymal stromal cells (MSCs) are widely used for research and clinical purposes. These cells are preferably cultured in xeno-free culture conditions