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Comparison of human platelet lysate and fetal bovine serum for optimal culture conditions of neonatal and adult mesenchymal stem cells
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Poster Abstracts
288 ENHANCED CELL RECOVERY RESULTING IN HIGHER YIELDS OF MSC WHEN USING THE COBE 2991 FOR CELL DENSITY SEPARATION OF BONE MARROW MATERIAL: A VALIDATION STUDY BETWEEN TWO MSC PRODUCTION CENTERS Tden Bleker-Grijsen1, L Carlee1, E Steeneveld2, I Lommerse1, H Roelofs2, J Zwaginga2, C Voermans1, D Thijssen-Timmer1 1 Laboratory for Cell Therapy, Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, Netherlands, 2Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, Netherlands Bone-marrow derived mesenchymal stromal cells are multi-potent cells which have immunomodulatory properties and the ability to migrate to sites of inflammation. MSC infusions have been shown to be effective in patients suffering from steroid refractory acute Graft versus Host Disease (GvHD), irrespective of donor origin. Currently a multi-center, randomized phase III trial (HOVON 113) is performed with MSC in steroid refractory GvHD patients. To this end we performed a validation study to compare cultureexpanded MSC generated at two production sites (Leiden and Sanquin). The protocol for clinical grade MSC production was developed in Leiden and implemented at Sanquin. Bone marrow of three healthy donors was split between the two production centers and cultured in parallel in the presence of FBS until passage 2. Sanquin used a fully closed cell density separation procedure (Cobe 2991 cell processor) as an alternative to a ficoll procedure in tubes, CellSTACKS with tubing systems instead of open flasks and recombinant TrypLE Select instead of Trypsine-Versene. All MSC cultures passed the release criteria as described in Le Blanc et al. 2008. Cell recovery after the density separation in the Cobe 2991 was two-fold higher compared to the manual separation. In addition 20% more MSC were obtained after seeding the same amount of mononuclear cells (MNC) obtained with the Cobe 2991. Equal amounts of MSC were harvested in the two centers when corrected for MSC numbers at Passage 0. No differences in viability were observed when TrypLE Select was used. In conclusion Sanquin has validated the production of clinical grade MSC in a more closed culture system and can now participate as a production center in the HOVON 113 study. The use of the Cobe 2991 increases the MNC recovery from bone marrow resulting in higher yields of MSC. 289 THE USE OF HUMAN BONE MARROW STEM CELLS REDUCES ENDOTOXIN-INDUCED LUNG INJURY IN SHEEP A Ting1, N Lehman1, N Cardenes2, E Kocyildirim3, M Romagnoli4, L Mroz2, E Carceres2, J Tedrow2, C Bermudez4, M Rojas2 1 Regnerative Medicine, Athersys, Inc, Cleveland, Ohio, United States, 2 Simmons Center for Interstitial Lung Disease, Division of Pulmonary, Allergy & Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 3McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 4 Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States The objective of this study was to assess the use of an adult bone marrow derived stem cell, MultiStemÒ, in an ovine model of Acute Respiratory Distress Syndrome or ARDS. ARDS is the cause of 10-15% ICU admissions, and is followed by poor survival and diminished quality of life. Although existing therapeutic approaches have decreased mortality, they generally aggravate lung injury. This presents the need for alternative therapeutic options. The infusion of mesenchymal stem cells (MSC) has been shown to be extremely beneficial in the treatment of different murine models of induced ARDS. Treatment with MSC improves lung function as measured by histology, decrease in lung water content and inflammatory cytokines in plasma and bronchoalveolar lavage that result in restoration of alveolar fluid clearance and increase in survival. However, the murine model is limited in that it is difficult to measure gas exchange performance, systemic/hemodynamics, and ventilation/perfusion mismatch. Using sheep for the study of ARDS allows for these measurements and sheep are similar to humans in global physiologic response and sensitivity to endotoxin, a common cause of ARDS. Therefore, the ovine model of ARDS has a larger clinical translational potential. Sheep were kept under anesthesia and the chest was open to facilitate visual evaluation of lung injury and sample biopsies at different time points. Sheep received a dose of
3mg/Kg E. coli endotoxin over 30 minutes. Pulmonary artery pressure (PAP) was measured continuously and arterial blood gases were measured every 30 minutes to determine the onset of the acute phase of pulmonary hypertension. The experimental group received 40 million human MultiStem by intratracheal delivery 30 minutes after the end of the infusion of endotoxin. Our results showed a significant recovery of PO2 and PCO2 levels in the group treated with MultiStem within 1.5 hours of maximum lung injury.
290 MICRORNA PROFILING OF MESENCHYMAL STEM CELLS (MSCS) PROVIDES A PUTATIVE, GENERAL MSC SIGNATURE AND DISCRIMINATES CELLS DERIVED FROM DIFFERENT TISSUES D Mallison, D Olijnyk, S Paterson, S Ridha, D Dunbar, V O’Brien Sistemic Ltd, Glasgow, United Kingdom Background: Heterogeneity of MSCs, arising from the different tissue sources and culturing techniques, presents a significant challenge if manufacturers are to understand and preserve the favourable characteristics of these cells during their production. To address the challenge there is a need for reference standards and reliable assays to characterise MSCs and provide insight into their batch consistency and which are feasible ways to assess the potency of the final product at release. MicroRNA (miRNA) profiling is a highly-informative and broadly-applicable technique for cell characterisation: miRNA expression analyses provide sensitive assays of a stable analyte which can be used to assess the comparability of cell populations. Furthermore, alterations in miRNA expression can reveal deviations in cell phenotype which may crucially impact on the potency of manufactured cell populations. Methods: Total RNA was prepared using a column-based kit (Exiqon) and miRNA expression profiles were analysed using microarray slides (Agilent human miRNA 8*60K V16.0 of miRBase). Results: MicroRNA profiles where derived for human monocytes, leucocytes and MSC populations. The MSC cells were from three different tissue sources e adipose tissue, bone marrow and cord blood. In total, 55 datasets were generated from the microarray analysis. High-level visualisation demonstrated clustering of the cells based on lineage. Further analysis of the MSC cells revealed a high degree of homogeneity in miRNA profiles (>40% identity) between all MSCs irrespective of tissue origin. However, miRNA differences were also identified which define the tissue of origin. Conclusion: These data support the use of microRNA profiles to define: (1). a microRNA signature which describes an MSC population irrespective of tissue of origin and with potential as a standardisation assay; (2) miRNA biomarkers that permit the identification of the tissue of origin of the MSCs. 291 COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM FOR OPTIMAL CULTURE CONDITIONS OF NEONATAL AND ADULT MESENCHYMAL STEM CELLS CG Taylor, RN Dayment, MZ Albanna, EJ Woods Cook General BioTechnology, LLC, Indianapolis, Indiana, United States Fetal bovine serum (FBS) has significantly contributed to the large-scale expansion of animal and human mesenchymal stem/stromal cells (MSCs) and the rapid development of cell-based therapeutics; however, it poses several regulatory and species cross-contamination challenges hindering the clinical transition of most products. Serum-free media (SFM) supplemented with several growth factors has been proposed as an alternative approach to FBS; however, custom media development is often needed based on the cell type, source, and species. Also, the high cost of SFM makes it an impractical option for large-scale cell expansion. Hence, there is a pressing need to find a humanbased media additive. hPL is derived from human platelets and contains similar growth factors and cytokines found in FBS at comparable levels. It has been previously demonstrated that hPL supports the growth of various cells. The focus of this study was to evaluate the ability of a serum converted hPL that does not require the use of heparin (PL-NH) and standard hPL requiring heparin (PL-H) to support attachment, proliferation, and maintenance of multipotent properties of neonatal and adult MSCs from different tissues at different concentrations of media. Variants of hPL used in this study (COOK HPLÔ NH and H) are produced at an industrial scale (minimum lot size of 20 L) with high lot-to-lot consistency and purity. Preliminary results of both versions of hPL at different concentrations (2.5, 5, and 10%) supported cell
20th Annual ISCT Meeting
attachment and proliferation of amniotic-, bone marrow-, adipose-, and cord blood-derived MSCs to comparable levels to FBS at all concentrations. The multipotency of MSCs expanded in hPL was maintained throughout several passages. This study demonstrates the ability of using hPL as an alternative media additive to FBS for large-scale expansion of adult and neonatal stem cells for cell-based therapeutics. 292 COMPARISON OF CLINICALLY APPROVED HUMAN PLATELET LYSATES FOR CULTIVATION OF MESENCHYMAL STROMAL CELLS FROM BONE MARROW AND ADIPOSE TISSUE J Tratwal, B Follin, RH Søndergaard, M Juhl, A Ekblond, J Kastrup, M Haack-Sørensen Cardiology Stem Cell Centre, Rigshospitalet, Copenhagen, Denmark Background: New developments and progress in stem cell technology in recent years has given rise to new therapeutic strategies for different degenerative diseases. Mesenchymal stromal cells (MSCs) have gained much attention for regenerative medicine because they are capable of self-renewal, can differentiate to a variety of cell lineages, have trophic and immunosuppressive effects, and have been shown clinically to alleviate symptoms of several diseases. Clinical translation of MSC-based approaches often requires in vitro cultureexpansion to achieve sufficient number of cells. Methods: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different human platelet lysates (hPL), manufactured differently, but each fulfilling tracking criteria imposed by good manufacturing practice. BMSCs and ASCs were cultured in 5% PLT-Max (Mill Creek), PL-H and PL-NH (Cook HPLTM) with Minimum Essential Medium Eagle-alpha, and were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, chromosomal stability and lineage differentiation of BMSCs and ASCs were analysed. Results: BMSCs and ASCs cultured in all three hPL-media showed a significant increase in proliferation capacity compared to FBS culture. In general, BMSCs and ASCs fulfilling the ISCT criteria regarding BMSC and ASC phenotype. Comparative genomic hybridization was performed to assess the level of genomic stability of the BMSCs and ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs were induced to differentiate into osteogenic, adipogenic or chondrogenic lineages, and both BMSCs and ASCs from all four original culture conditions were able to differentiate toward the three lineages. Conclusion: All three clinically approved human platelet lysates accelerate proliferation of BMSCs and ASCs and meet the ISCT requirements without exhibiting chromosomal aberrations. 293 WILL NOT BE PRESENTED 294 STANDARDIZED HUMAN PLATELET LYSATE SUPPLEMENT DEMONSTRATES TO BE AN EFFECTIVE, SERUM-FREE, XENO-FREE, FBS REPLACEMENT FOR CULTURING AT-/BM-/ AND UC-MESENCHYMAL STEM CELLS Y Tseng1, K Liu1, C Ku1, T Burnouf2,3 1 GwoWei Technology Inc., Vancouver, British Columbia, Canada, 2Human Protein Process Sciences, Lille, France, 3Institute of Biomaterials and Tissue Engineering, Taipei Medical Universit, Taipei, Taiwan Fetal bovine serum (FBS) is not recommended for ex vivo culture of human cells for transplantation into patients as there is risk of immunological reactions and of transmitting bovine pathogens. FBS in clinical stem cell culture is prohibited in Germany and may be prohibited in Europe, USA and other countries in the future. Human platelet lysates contain a natural mixture of growth factors including (PDGF-AA, -AB, and -BB), vascular endothelial growth factor (VEGF), transforming growth factor-b (TGF-b1 and TGF-b2), epidermal growth factor (EGF), basic fibroblast growth factor (FGF), and can successfully replace FBS for enhanced cell culture of mesenchymal stem cells (MSC) and other human cells. Gwowei R&D has applied their proprietary platelet fractionation process to produce a standardized human platelet-derived growth factors supplement to replace FBS supplement for human cell culture. Gwowei’s process fractions allogeneic platelets (supplied by ARC & CBS in
S85
NA) to produce a functionally consistent human Platelet Lysate (hPL) supplement, which contains a mixture of primary growth factors, including: PDGF-BB, PDGF-AB, FGF, TGF-b1. From collaborators in North America, Europe and China, Gwowei R&D has extensive testing data demonstrating that human AT-MSC, BM-MSC, and UC-MSC isolated and grown with this standardized hPL supplement achieves; a sub-confluent layer (1), exhibit typical spindle morphology (2) and maintain multi-potent capability (3). Gwowei’s 5% hPL (v/v) supplement (i.e. 4 lots) consistently demonstrated superior performance compared to 10-20% defined FBS in both primary and expansion culture. This new 5% hPL (UltraGROÔ) is a promising, scaleable and standardized supplement to consider for replacing 10-20% FBS in medium for growing and producing mesenchymal stem cells derived from adipose tissue, bone marrow and umbilical cords.
295 MULTIPLE INTRA-ARTICULAR TRANSPLANTATIONS ENHANCES THE BENEFIT OF DENTAL TISSUE DERIVED STEM CELLS THERAPY FOR THE TREATMENT OF CHRONIC OSTEOARTHRITIS R Bootcha1, J Temvilitr2, P Sriwattanakul3, S Petchdee4 1 Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom, Thailand, 2Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand, 3 BioEden Asia Tooth cell bank, Bangkok, Thailand, 4Large Animal and Wildlife Clinical Sciences Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom, Thailand There are several types of osteoarthritis treatments in dogs. However, the results of many surgical and medical treatments are still unsatisfactory. Cell transplantation of dental tissue derived stem cell is a new treatment that could restore the degenerated articular cartilage in dog with osteoarthritis (OA). In this study, the production of canine dental derived stem cells and their possible application in cellular therapy for dogs were evaluated. We hypothesize that multiple doses of puppy deciduous teeth stem cells (pDSCs) might improve clinical outcome of OA and can be replaced the drug therapy. The clinical effects of multiple intra-articular injections of 5 millions pDSCs were evaluated on 10 dogs with lameness associated with OA of the coxofemoral joints lasting on average 3 months. The second doses of pDSCs were delivered within 28 days after the first transplantation. Clinical outcomes were evaluated by radiographic evidence, gait changes including persistent lameness at walk or trot, limited range of motion with pain and the responses to questionnaire from the owners. Multiple injections of pDSCs showed that OA of coxofemoral joints markedly improved with time. These findings suggest that multiple doses of allogeneic dental tissue derived stem cells transplantations provide a significant potential for clinical uses in the treatment of lameness, and pDSCs might be a novel strategy in the cell therapy for OA. 296 THE ACTIVATION OF DIRECTIONAL STEM CELL MOTILITY BY GREEN LIGHT-EMITTING DIODE IRRADIATION J Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2center for stem cell research, Wan Fang Medical Center, Taipei Medical University, Taipei, Taiwan Light-emitting diode (LED) irradiation is potentially a photostimulator to manipulate cell behavior by opsin-triggered phototransduction and thermal energy supply in living cells. Directional stem cell motility is critical for the efficiency and specificity of stem cells in tissue repair. We explored that green LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to migrate away from the LED light source through activation of extracellular signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of these kinases as well as green LED irradiation-induced cell migration was facilitated by increasing adenosine triphosphate (ATP) production in OFSCs after green LED exposure, and which was thermal stressindependent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from human orbital fat tissue, constitutionally express three opsins, i.e. retinal pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin (OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to green LED irradiation-induced OFSC migration. In conclusion, stem cells are
Poster Abstracts
288 ENHANCED CELL RECOVERY RESULTING IN HIGHER YIELDS OF MSC WHEN USING THE COBE 2991 FOR CELL DENSITY SEPARATION OF BONE MARROW MATERIAL: A VALIDATION STUDY BETWEEN TWO MSC PRODUCTION CENTERS Tden Bleker-Grijsen1, L Carlee1, E Steeneveld2, I Lommerse1, H Roelofs2, J Zwaginga2, C Voermans1, D Thijssen-Timmer1 1 Laboratory for Cell Therapy, Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, Netherlands, 2Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, Netherlands Bone-marrow derived mesenchymal stromal cells are multi-potent cells which have immunomodulatory properties and the ability to migrate to sites of inflammation. MSC infusions have been shown to be effective in patients suffering from steroid refractory acute Graft versus Host Disease (GvHD), irrespective of donor origin. Currently a multi-center, randomized phase III trial (HOVON 113) is performed with MSC in steroid refractory GvHD patients. To this end we performed a validation study to compare cultureexpanded MSC generated at two production sites (Leiden and Sanquin). The protocol for clinical grade MSC production was developed in Leiden and implemented at Sanquin. Bone marrow of three healthy donors was split between the two production centers and cultured in parallel in the presence of FBS until passage 2. Sanquin used a fully closed cell density separation procedure (Cobe 2991 cell processor) as an alternative to a ficoll procedure in tubes, CellSTACKS with tubing systems instead of open flasks and recombinant TrypLE Select instead of Trypsine-Versene. All MSC cultures passed the release criteria as described in Le Blanc et al. 2008. Cell recovery after the density separation in the Cobe 2991 was two-fold higher compared to the manual separation. In addition 20% more MSC were obtained after seeding the same amount of mononuclear cells (MNC) obtained with the Cobe 2991. Equal amounts of MSC were harvested in the two centers when corrected for MSC numbers at Passage 0. No differences in viability were observed when TrypLE Select was used. In conclusion Sanquin has validated the production of clinical grade MSC in a more closed culture system and can now participate as a production center in the HOVON 113 study. The use of the Cobe 2991 increases the MNC recovery from bone marrow resulting in higher yields of MSC. 289 THE USE OF HUMAN BONE MARROW STEM CELLS REDUCES ENDOTOXIN-INDUCED LUNG INJURY IN SHEEP A Ting1, N Lehman1, N Cardenes2, E Kocyildirim3, M Romagnoli4, L Mroz2, E Carceres2, J Tedrow2, C Bermudez4, M Rojas2 1 Regnerative Medicine, Athersys, Inc, Cleveland, Ohio, United States, 2 Simmons Center for Interstitial Lung Disease, Division of Pulmonary, Allergy & Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 3McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 4 Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States The objective of this study was to assess the use of an adult bone marrow derived stem cell, MultiStemÒ, in an ovine model of Acute Respiratory Distress Syndrome or ARDS. ARDS is the cause of 10-15% ICU admissions, and is followed by poor survival and diminished quality of life. Although existing therapeutic approaches have decreased mortality, they generally aggravate lung injury. This presents the need for alternative therapeutic options. The infusion of mesenchymal stem cells (MSC) has been shown to be extremely beneficial in the treatment of different murine models of induced ARDS. Treatment with MSC improves lung function as measured by histology, decrease in lung water content and inflammatory cytokines in plasma and bronchoalveolar lavage that result in restoration of alveolar fluid clearance and increase in survival. However, the murine model is limited in that it is difficult to measure gas exchange performance, systemic/hemodynamics, and ventilation/perfusion mismatch. Using sheep for the study of ARDS allows for these measurements and sheep are similar to humans in global physiologic response and sensitivity to endotoxin, a common cause of ARDS. Therefore, the ovine model of ARDS has a larger clinical translational potential. Sheep were kept under anesthesia and the chest was open to facilitate visual evaluation of lung injury and sample biopsies at different time points. Sheep received a dose of
3mg/Kg E. coli endotoxin over 30 minutes. Pulmonary artery pressure (PAP) was measured continuously and arterial blood gases were measured every 30 minutes to determine the onset of the acute phase of pulmonary hypertension. The experimental group received 40 million human MultiStem by intratracheal delivery 30 minutes after the end of the infusion of endotoxin. Our results showed a significant recovery of PO2 and PCO2 levels in the group treated with MultiStem within 1.5 hours of maximum lung injury.
290 MICRORNA PROFILING OF MESENCHYMAL STEM CELLS (MSCS) PROVIDES A PUTATIVE, GENERAL MSC SIGNATURE AND DISCRIMINATES CELLS DERIVED FROM DIFFERENT TISSUES D Mallison, D Olijnyk, S Paterson, S Ridha, D Dunbar, V O’Brien Sistemic Ltd, Glasgow, United Kingdom Background: Heterogeneity of MSCs, arising from the different tissue sources and culturing techniques, presents a significant challenge if manufacturers are to understand and preserve the favourable characteristics of these cells during their production. To address the challenge there is a need for reference standards and reliable assays to characterise MSCs and provide insight into their batch consistency and which are feasible ways to assess the potency of the final product at release. MicroRNA (miRNA) profiling is a highly-informative and broadly-applicable technique for cell characterisation: miRNA expression analyses provide sensitive assays of a stable analyte which can be used to assess the comparability of cell populations. Furthermore, alterations in miRNA expression can reveal deviations in cell phenotype which may crucially impact on the potency of manufactured cell populations. Methods: Total RNA was prepared using a column-based kit (Exiqon) and miRNA expression profiles were analysed using microarray slides (Agilent human miRNA 8*60K V16.0 of miRBase). Results: MicroRNA profiles where derived for human monocytes, leucocytes and MSC populations. The MSC cells were from three different tissue sources e adipose tissue, bone marrow and cord blood. In total, 55 datasets were generated from the microarray analysis. High-level visualisation demonstrated clustering of the cells based on lineage. Further analysis of the MSC cells revealed a high degree of homogeneity in miRNA profiles (>40% identity) between all MSCs irrespective of tissue origin. However, miRNA differences were also identified which define the tissue of origin. Conclusion: These data support the use of microRNA profiles to define: (1). a microRNA signature which describes an MSC population irrespective of tissue of origin and with potential as a standardisation assay; (2) miRNA biomarkers that permit the identification of the tissue of origin of the MSCs. 291 COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM FOR OPTIMAL CULTURE CONDITIONS OF NEONATAL AND ADULT MESENCHYMAL STEM CELLS CG Taylor, RN Dayment, MZ Albanna, EJ Woods Cook General BioTechnology, LLC, Indianapolis, Indiana, United States Fetal bovine serum (FBS) has significantly contributed to the large-scale expansion of animal and human mesenchymal stem/stromal cells (MSCs) and the rapid development of cell-based therapeutics; however, it poses several regulatory and species cross-contamination challenges hindering the clinical transition of most products. Serum-free media (SFM) supplemented with several growth factors has been proposed as an alternative approach to FBS; however, custom media development is often needed based on the cell type, source, and species. Also, the high cost of SFM makes it an impractical option for large-scale cell expansion. Hence, there is a pressing need to find a humanbased media additive. hPL is derived from human platelets and contains similar growth factors and cytokines found in FBS at comparable levels. It has been previously demonstrated that hPL supports the growth of various cells. The focus of this study was to evaluate the ability of a serum converted hPL that does not require the use of heparin (PL-NH) and standard hPL requiring heparin (PL-H) to support attachment, proliferation, and maintenance of multipotent properties of neonatal and adult MSCs from different tissues at different concentrations of media. Variants of hPL used in this study (COOK HPLÔ NH and H) are produced at an industrial scale (minimum lot size of 20 L) with high lot-to-lot consistency and purity. Preliminary results of both versions of hPL at different concentrations (2.5, 5, and 10%) supported cell
20th Annual ISCT Meeting
attachment and proliferation of amniotic-, bone marrow-, adipose-, and cord blood-derived MSCs to comparable levels to FBS at all concentrations. The multipotency of MSCs expanded in hPL was maintained throughout several passages. This study demonstrates the ability of using hPL as an alternative media additive to FBS for large-scale expansion of adult and neonatal stem cells for cell-based therapeutics. 292 COMPARISON OF CLINICALLY APPROVED HUMAN PLATELET LYSATES FOR CULTIVATION OF MESENCHYMAL STROMAL CELLS FROM BONE MARROW AND ADIPOSE TISSUE J Tratwal, B Follin, RH Søndergaard, M Juhl, A Ekblond, J Kastrup, M Haack-Sørensen Cardiology Stem Cell Centre, Rigshospitalet, Copenhagen, Denmark Background: New developments and progress in stem cell technology in recent years has given rise to new therapeutic strategies for different degenerative diseases. Mesenchymal stromal cells (MSCs) have gained much attention for regenerative medicine because they are capable of self-renewal, can differentiate to a variety of cell lineages, have trophic and immunosuppressive effects, and have been shown clinically to alleviate symptoms of several diseases. Clinical translation of MSC-based approaches often requires in vitro cultureexpansion to achieve sufficient number of cells. Methods: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different human platelet lysates (hPL), manufactured differently, but each fulfilling tracking criteria imposed by good manufacturing practice. BMSCs and ASCs were cultured in 5% PLT-Max (Mill Creek), PL-H and PL-NH (Cook HPLTM) with Minimum Essential Medium Eagle-alpha, and were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, chromosomal stability and lineage differentiation of BMSCs and ASCs were analysed. Results: BMSCs and ASCs cultured in all three hPL-media showed a significant increase in proliferation capacity compared to FBS culture. In general, BMSCs and ASCs fulfilling the ISCT criteria regarding BMSC and ASC phenotype. Comparative genomic hybridization was performed to assess the level of genomic stability of the BMSCs and ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs were induced to differentiate into osteogenic, adipogenic or chondrogenic lineages, and both BMSCs and ASCs from all four original culture conditions were able to differentiate toward the three lineages. Conclusion: All three clinically approved human platelet lysates accelerate proliferation of BMSCs and ASCs and meet the ISCT requirements without exhibiting chromosomal aberrations. 293 WILL NOT BE PRESENTED 294 STANDARDIZED HUMAN PLATELET LYSATE SUPPLEMENT DEMONSTRATES TO BE AN EFFECTIVE, SERUM-FREE, XENO-FREE, FBS REPLACEMENT FOR CULTURING AT-/BM-/ AND UC-MESENCHYMAL STEM CELLS Y Tseng1, K Liu1, C Ku1, T Burnouf2,3 1 GwoWei Technology Inc., Vancouver, British Columbia, Canada, 2Human Protein Process Sciences, Lille, France, 3Institute of Biomaterials and Tissue Engineering, Taipei Medical Universit, Taipei, Taiwan Fetal bovine serum (FBS) is not recommended for ex vivo culture of human cells for transplantation into patients as there is risk of immunological reactions and of transmitting bovine pathogens. FBS in clinical stem cell culture is prohibited in Germany and may be prohibited in Europe, USA and other countries in the future. Human platelet lysates contain a natural mixture of growth factors including (PDGF-AA, -AB, and -BB), vascular endothelial growth factor (VEGF), transforming growth factor-b (TGF-b1 and TGF-b2), epidermal growth factor (EGF), basic fibroblast growth factor (FGF), and can successfully replace FBS for enhanced cell culture of mesenchymal stem cells (MSC) and other human cells. Gwowei R&D has applied their proprietary platelet fractionation process to produce a standardized human platelet-derived growth factors supplement to replace FBS supplement for human cell culture. Gwowei’s process fractions allogeneic platelets (supplied by ARC & CBS in
S85
NA) to produce a functionally consistent human Platelet Lysate (hPL) supplement, which contains a mixture of primary growth factors, including: PDGF-BB, PDGF-AB, FGF, TGF-b1. From collaborators in North America, Europe and China, Gwowei R&D has extensive testing data demonstrating that human AT-MSC, BM-MSC, and UC-MSC isolated and grown with this standardized hPL supplement achieves; a sub-confluent layer (1), exhibit typical spindle morphology (2) and maintain multi-potent capability (3). Gwowei’s 5% hPL (v/v) supplement (i.e. 4 lots) consistently demonstrated superior performance compared to 10-20% defined FBS in both primary and expansion culture. This new 5% hPL (UltraGROÔ) is a promising, scaleable and standardized supplement to consider for replacing 10-20% FBS in medium for growing and producing mesenchymal stem cells derived from adipose tissue, bone marrow and umbilical cords.
295 MULTIPLE INTRA-ARTICULAR TRANSPLANTATIONS ENHANCES THE BENEFIT OF DENTAL TISSUE DERIVED STEM CELLS THERAPY FOR THE TREATMENT OF CHRONIC OSTEOARTHRITIS R Bootcha1, J Temvilitr2, P Sriwattanakul3, S Petchdee4 1 Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom, Thailand, 2Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand, 3 BioEden Asia Tooth cell bank, Bangkok, Thailand, 4Large Animal and Wildlife Clinical Sciences Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom, Thailand There are several types of osteoarthritis treatments in dogs. However, the results of many surgical and medical treatments are still unsatisfactory. Cell transplantation of dental tissue derived stem cell is a new treatment that could restore the degenerated articular cartilage in dog with osteoarthritis (OA). In this study, the production of canine dental derived stem cells and their possible application in cellular therapy for dogs were evaluated. We hypothesize that multiple doses of puppy deciduous teeth stem cells (pDSCs) might improve clinical outcome of OA and can be replaced the drug therapy. The clinical effects of multiple intra-articular injections of 5 millions pDSCs were evaluated on 10 dogs with lameness associated with OA of the coxofemoral joints lasting on average 3 months. The second doses of pDSCs were delivered within 28 days after the first transplantation. Clinical outcomes were evaluated by radiographic evidence, gait changes including persistent lameness at walk or trot, limited range of motion with pain and the responses to questionnaire from the owners. Multiple injections of pDSCs showed that OA of coxofemoral joints markedly improved with time. These findings suggest that multiple doses of allogeneic dental tissue derived stem cells transplantations provide a significant potential for clinical uses in the treatment of lameness, and pDSCs might be a novel strategy in the cell therapy for OA. 296 THE ACTIVATION OF DIRECTIONAL STEM CELL MOTILITY BY GREEN LIGHT-EMITTING DIODE IRRADIATION J Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2center for stem cell research, Wan Fang Medical Center, Taipei Medical University, Taipei, Taiwan Light-emitting diode (LED) irradiation is potentially a photostimulator to manipulate cell behavior by opsin-triggered phototransduction and thermal energy supply in living cells. Directional stem cell motility is critical for the efficiency and specificity of stem cells in tissue repair. We explored that green LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to migrate away from the LED light source through activation of extracellular signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of these kinases as well as green LED irradiation-induced cell migration was facilitated by increasing adenosine triphosphate (ATP) production in OFSCs after green LED exposure, and which was thermal stressindependent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from human orbital fat tissue, constitutionally express three opsins, i.e. retinal pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin (OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to green LED irradiation-induced OFSC migration. In conclusion, stem cells are