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Effects of simulated microgravity on the frequency and function of marrow resident mesenchymal stem cells
Poster Abstracts
Methods: Lipoaspirate was separated from waste liquids and slowly infused with DMSO cryoprotectant (5% final) prior to controlled rate freezing. Thawed tissue was washed twice with lactated Ringer’s to remove DMSO prior to packaging and distribution to treatment site. Results: 26 adipose samples were cryopreserved and banked for greater than one year with the average length of storage being 573 days. Upon thaw the recovery yield of initial volume of cryopreserved tissue was 74.9%. The greatest number of sample thaws was for cosmetic use, followed by regenerative and reconstructive applications. Cellular viability of thawed tissue remained high at 97%. Discussion: 26 frozen and banked adipose samples stored for longer than one year have been requested for clinical use. Data demonstrates thawed tissue is viable and functional, implying no detrimental effects on MSC within the tissue. Thus, it is feasible to cryopreserve adipose tissue with minimal manipulation for later thawing and use in cosmetic, reconstructive, and tissue engineering applications.
218 THE INFLUENCE OF MESENCHYMAL STROMAL CELLS ON PLATELET ACTIVATION P. Netsch1, S. Elvers-Hornung1, S. Uhlig2, G. Rink1, H. Klueter1, P. Bugert1, K. Bieback1,2 1 Institute of Transfusion Medicine and Immunology, Mannheim, Europe, Germany, 2FlowCore Mannheim, Mannheim, Germany Mesenchymal stromal cells (MSCs) possess diverse promising therapeutic capacities like differentiation, immunomodulation or trophic support. Current clinical trials documented the safety of MSC injection with very few side effects. However, thromboembolic events after MSC infusion were reported. Recent in vitro studies focusing on plasmatic coagulation suggest both pro- and anticoagulatory activities of MSC. As more evidence is needed to ensure the safety of MSC infusion we aimed to investigate possible interactions between platelets and MSCs asking whether MSC can promote or inhibit platelet activation. Effects of MSC and MSC supernatant were studied by assessing platelet activation using flow cytometry (CD62P, CD63, PAC1-binding) and impedance aggregometry (Multiplate; Roche Diagnostics). Platelets were used unstimulated or stimulated with different agonists in the presence of MSCs (from lipo aspirate (LA), cord blood (CB) or bone marrow (BM)), human umbilical vein endothelial cells (HUVECs) or HeLa tumor cells as a platelet inhibiting or activation promoting population, respectively. In flow cytometry, HeLa cells induced activation of unstimulated platelets and increased activation marker expressed of stimulated platelets as expected. Interestingly, CB-MSCs showed similar trends. In contrast, BM-MSCs, LA-MSCs and HUVECs caused a dose related decrease in activation marker expression only of stimulated platelets independent of the agonist used. This was unrelated to cyclooxygenase and CD62p, as both indomethacin and CD62p blockade did not alter the inhibitory effect. In impedance aggregometry, we observed a dose dependent decrease in platelet aggregation independent of the cell type deployed, contradictory to the flow cytometry data suggesting the unsuitability of this assay to monitor cell-cell interactions. The data clearly demonstrate that LA- and BM-MSC are able to inhibit platelet activation in a dose-dependent manner. This non-activation and even inhibition of primary hemostasis supports the clinical safety data. Interestingly, whereas both LA- and BM-derived MSC depicted this anti-coagulatory phenotype, CBderived MSC differed in this respect. Further experiments are necessary to understand the underlying mechanisms to further increase safety of MSC therapies.
219 EFFECTS OF SIMULATED MICROGRAVITY ON THE FREQUENCY AND FUNCTION OF MARROW RESIDENT MESENCHYMAL STEM CELLS C.N. Booker, S.V. Boregowda, D.G. Phinney Molecular Therapeutics, Scripps Florida, Jupiter, Florida, United States Exposure to microgravity induces a phenomenon known as disuse osteoporosis, which manifests as a significant loss in bone mineral density and cortical and trabecular bone volume. Several ex vivo studies have shown that mesenchymal stem cells (MSCs), which represent the main source of new bone formed
S93
in adult bone marrow, exhibit defects in cell proliferation and osteogenic differentiation following exposure to simulated microgravity. However, whether these cellular changes result in disuse osteoporosis in vivo remains indeterminate. To explore this question, we compared the frequency of MSC subtypes in bone marrow using FACS from normally housed mice and mice subjected to hind limb unloading (HU), which mimics to a large degree skeletal changes induced by microgravity. Subsequently, we performed a series of functional comparisons between primary MSCs isolated via immuno-depletion from the bone marrow of normally housed and HU mice. Importantly, primary MSCs were maintained in a closed, low oxygen system to preserve their stem/progenitor properties. HU-induced changes in trabecular bone volume, trabecular spacing, and marrow adiposity were confirmed by microCT analysis of long bones; these skeletal changes were correlated with changes in MSC yields, growth/survival, and differentiation potential. Functional alterations were further correlated with changes in key transcriptional regulators that are known to orchestrate stem and progenitor characteristics of MSCs. Together, these data implicate MSCs in the disuse osteoporosis phenotype seen in space flight and may also provide new mechanistic insight into the pathophysiology of age- and bedrest-related osteoporosis.
220 EFFECT OF HUMAN PLATELET LYSATE SUPPLEMENTED MEDIUM ON HUMAN MESENCHYMAL STEM CELL IDENTITY AND IMMUNOMODULATORY CAPACITY S. Cañadillas1,4, M.D. Carmona1,4, M.S. Gonzalez2, S. Nogueras1,4, V. Martín-Palanco4,5, R. Jimenez1,4, I. Lomas2, M. Luque1, R. Gutierrez1, L. Paco-Meza4,1, G. Carmona3,2, C. Herrera5,3,4 1 Andalusian Initiative for Advanced Therapies, Junta de Andalucia-Reina Sofia Hospital, Cordoba, Cordoba, Spain, 2Cell Therapy and Cell Reprogramming Unit, GMP network of the Andalusian Initiative for Advanced Therapies, Junta de Andalucia, Seville, Spain, 3Andalusian Initiative for Advanced Therapies, Junta de Andalucia, Seville, Spain, 4Cell Therapy, Maimonides Institute for Biomedical Research, Cordoba, Cordoba, Spain, 5 Haematology, Reina Sofia Hospital, Cordoba, Cordoba, Spain Background: Human Bone Marrow derived Mesenchymal stem cells (hMSCsMO) are promising candidates for cell-based therapies. Large scale in vitro expansion of human mesenchymal stem cells still involves the supplementation of culture media with Fetal Bovine Serum (FBS). However, in order to avoid the risks of contaminants by FBS and xenogenic compounds, respectively, clinical grade stem cell culture should turn to other options. Human platelet lysate (PL) represents an efficient alternative to fetal bovine serum for clinical-scale expansion of MSCs. Aim: We aimed to compare morphology (size and complexity), proliferation capacity, immunophenotype and immunomodulatory effect between hMSCs-MO cultured under both conditions, FBS and PL supplemented medium. Methods: hMSCs-MO were cultured under human platelet lysate or fetal bovine serum conditions and their biological characteristics were evaluated for cell therapy. A cell flow cytometer was used for determining cells size, cells complexity and immunophenotype. Proliferation rate were calculated and immunomodulatory effect was assessed by lymphocytes proliferation quantification using a cocultured system. Results: Under PL supplemented culture conditions, hMSCs-MO exhibited similar fibroblast-like morphology and expression patterns of surface markers than hMSCs-MO cultured under FBS supplemented medium conditions. Beside that, flow cytometer data analysis revealed significant differences in cells size and complexity, cultured with LP showed smaller size and higher complexity than hMSCs-MO cultured with FBS. hMSCs-MO had greater proliferative potential when culturing under PL supplemented medium. However, hMSCsMO cultured under PL conditions showed a lower immunomodulatory effect compared to those cells cultured under FBS conditions. Conclusions: hMSCs-MO under PL supplemented culture conditions, despite showed no change in immunophenotype, presented higher complexity and smaller size than hMSCs-MO cultured under FBS conditions. In adittion, hMSCs-MO incubated under PL supplemented medium had biological advantages in the proliferative capacity. However, hMSCs-MO cultured under FBS conditions seem to have advantages in immunomodulatory capacity compared to hMSCs-MO cultured under PL supplemented culture conditions.
Methods: Lipoaspirate was separated from waste liquids and slowly infused with DMSO cryoprotectant (5% final) prior to controlled rate freezing. Thawed tissue was washed twice with lactated Ringer’s to remove DMSO prior to packaging and distribution to treatment site. Results: 26 adipose samples were cryopreserved and banked for greater than one year with the average length of storage being 573 days. Upon thaw the recovery yield of initial volume of cryopreserved tissue was 74.9%. The greatest number of sample thaws was for cosmetic use, followed by regenerative and reconstructive applications. Cellular viability of thawed tissue remained high at 97%. Discussion: 26 frozen and banked adipose samples stored for longer than one year have been requested for clinical use. Data demonstrates thawed tissue is viable and functional, implying no detrimental effects on MSC within the tissue. Thus, it is feasible to cryopreserve adipose tissue with minimal manipulation for later thawing and use in cosmetic, reconstructive, and tissue engineering applications.
218 THE INFLUENCE OF MESENCHYMAL STROMAL CELLS ON PLATELET ACTIVATION P. Netsch1, S. Elvers-Hornung1, S. Uhlig2, G. Rink1, H. Klueter1, P. Bugert1, K. Bieback1,2 1 Institute of Transfusion Medicine and Immunology, Mannheim, Europe, Germany, 2FlowCore Mannheim, Mannheim, Germany Mesenchymal stromal cells (MSCs) possess diverse promising therapeutic capacities like differentiation, immunomodulation or trophic support. Current clinical trials documented the safety of MSC injection with very few side effects. However, thromboembolic events after MSC infusion were reported. Recent in vitro studies focusing on plasmatic coagulation suggest both pro- and anticoagulatory activities of MSC. As more evidence is needed to ensure the safety of MSC infusion we aimed to investigate possible interactions between platelets and MSCs asking whether MSC can promote or inhibit platelet activation. Effects of MSC and MSC supernatant were studied by assessing platelet activation using flow cytometry (CD62P, CD63, PAC1-binding) and impedance aggregometry (Multiplate; Roche Diagnostics). Platelets were used unstimulated or stimulated with different agonists in the presence of MSCs (from lipo aspirate (LA), cord blood (CB) or bone marrow (BM)), human umbilical vein endothelial cells (HUVECs) or HeLa tumor cells as a platelet inhibiting or activation promoting population, respectively. In flow cytometry, HeLa cells induced activation of unstimulated platelets and increased activation marker expressed of stimulated platelets as expected. Interestingly, CB-MSCs showed similar trends. In contrast, BM-MSCs, LA-MSCs and HUVECs caused a dose related decrease in activation marker expression only of stimulated platelets independent of the agonist used. This was unrelated to cyclooxygenase and CD62p, as both indomethacin and CD62p blockade did not alter the inhibitory effect. In impedance aggregometry, we observed a dose dependent decrease in platelet aggregation independent of the cell type deployed, contradictory to the flow cytometry data suggesting the unsuitability of this assay to monitor cell-cell interactions. The data clearly demonstrate that LA- and BM-MSC are able to inhibit platelet activation in a dose-dependent manner. This non-activation and even inhibition of primary hemostasis supports the clinical safety data. Interestingly, whereas both LA- and BM-derived MSC depicted this anti-coagulatory phenotype, CBderived MSC differed in this respect. Further experiments are necessary to understand the underlying mechanisms to further increase safety of MSC therapies.
219 EFFECTS OF SIMULATED MICROGRAVITY ON THE FREQUENCY AND FUNCTION OF MARROW RESIDENT MESENCHYMAL STEM CELLS C.N. Booker, S.V. Boregowda, D.G. Phinney Molecular Therapeutics, Scripps Florida, Jupiter, Florida, United States Exposure to microgravity induces a phenomenon known as disuse osteoporosis, which manifests as a significant loss in bone mineral density and cortical and trabecular bone volume. Several ex vivo studies have shown that mesenchymal stem cells (MSCs), which represent the main source of new bone formed
S93
in adult bone marrow, exhibit defects in cell proliferation and osteogenic differentiation following exposure to simulated microgravity. However, whether these cellular changes result in disuse osteoporosis in vivo remains indeterminate. To explore this question, we compared the frequency of MSC subtypes in bone marrow using FACS from normally housed mice and mice subjected to hind limb unloading (HU), which mimics to a large degree skeletal changes induced by microgravity. Subsequently, we performed a series of functional comparisons between primary MSCs isolated via immuno-depletion from the bone marrow of normally housed and HU mice. Importantly, primary MSCs were maintained in a closed, low oxygen system to preserve their stem/progenitor properties. HU-induced changes in trabecular bone volume, trabecular spacing, and marrow adiposity were confirmed by microCT analysis of long bones; these skeletal changes were correlated with changes in MSC yields, growth/survival, and differentiation potential. Functional alterations were further correlated with changes in key transcriptional regulators that are known to orchestrate stem and progenitor characteristics of MSCs. Together, these data implicate MSCs in the disuse osteoporosis phenotype seen in space flight and may also provide new mechanistic insight into the pathophysiology of age- and bedrest-related osteoporosis.
220 EFFECT OF HUMAN PLATELET LYSATE SUPPLEMENTED MEDIUM ON HUMAN MESENCHYMAL STEM CELL IDENTITY AND IMMUNOMODULATORY CAPACITY S. Cañadillas1,4, M.D. Carmona1,4, M.S. Gonzalez2, S. Nogueras1,4, V. Martín-Palanco4,5, R. Jimenez1,4, I. Lomas2, M. Luque1, R. Gutierrez1, L. Paco-Meza4,1, G. Carmona3,2, C. Herrera5,3,4 1 Andalusian Initiative for Advanced Therapies, Junta de Andalucia-Reina Sofia Hospital, Cordoba, Cordoba, Spain, 2Cell Therapy and Cell Reprogramming Unit, GMP network of the Andalusian Initiative for Advanced Therapies, Junta de Andalucia, Seville, Spain, 3Andalusian Initiative for Advanced Therapies, Junta de Andalucia, Seville, Spain, 4Cell Therapy, Maimonides Institute for Biomedical Research, Cordoba, Cordoba, Spain, 5 Haematology, Reina Sofia Hospital, Cordoba, Cordoba, Spain Background: Human Bone Marrow derived Mesenchymal stem cells (hMSCsMO) are promising candidates for cell-based therapies. Large scale in vitro expansion of human mesenchymal stem cells still involves the supplementation of culture media with Fetal Bovine Serum (FBS). However, in order to avoid the risks of contaminants by FBS and xenogenic compounds, respectively, clinical grade stem cell culture should turn to other options. Human platelet lysate (PL) represents an efficient alternative to fetal bovine serum for clinical-scale expansion of MSCs. Aim: We aimed to compare morphology (size and complexity), proliferation capacity, immunophenotype and immunomodulatory effect between hMSCs-MO cultured under both conditions, FBS and PL supplemented medium. Methods: hMSCs-MO were cultured under human platelet lysate or fetal bovine serum conditions and their biological characteristics were evaluated for cell therapy. A cell flow cytometer was used for determining cells size, cells complexity and immunophenotype. Proliferation rate were calculated and immunomodulatory effect was assessed by lymphocytes proliferation quantification using a cocultured system. Results: Under PL supplemented culture conditions, hMSCs-MO exhibited similar fibroblast-like morphology and expression patterns of surface markers than hMSCs-MO cultured under FBS supplemented medium conditions. Beside that, flow cytometer data analysis revealed significant differences in cells size and complexity, cultured with LP showed smaller size and higher complexity than hMSCs-MO cultured with FBS. hMSCs-MO had greater proliferative potential when culturing under PL supplemented medium. However, hMSCsMO cultured under PL conditions showed a lower immunomodulatory effect compared to those cells cultured under FBS conditions. Conclusions: hMSCs-MO under PL supplemented culture conditions, despite showed no change in immunophenotype, presented higher complexity and smaller size than hMSCs-MO cultured under FBS conditions. In adittion, hMSCs-MO incubated under PL supplemented medium had biological advantages in the proliferative capacity. However, hMSCs-MO cultured under FBS conditions seem to have advantages in immunomodulatory capacity compared to hMSCs-MO cultured under PL supplemented culture conditions.