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Autologous mesenchymal stromal cells to support allogeneic hematopoietic stem cell engraftment in sickle cell disease
S46
Poster Abstracts
158 HUMAN PLATELET LYSATE-ITS ROLE IN THE CRYOPRESERVATION OF MESENCHYMAL STEM CELLS EJ Woods1,2, S Thirumala1, C Taylor1, B King1 1 Cook Regentec, Indianapolis, Indiana, United States, 2Indiana University School of Medicine, Indianapolis, Indiana, United States Cryopreservation is widely used to generate large stocks or cell banks that can be stored for short and long periods ready to be recovered in a functionally potent state and used as required in a cell therapy application. Animal based reagents are usually employed in cryopreservation media as a source of nutrients and other perceived benefits during the post-thaw period. Current evidence suggest that the animal serum proteins used in cryopreservation media are difficult to remove during post-thaw washing, and any residual proteins in cells may trigger adverse reactions in the patients who receive cell infusions or transplants. Therefore, demand is increasing for cryopreservation media to be devoid of any animal derived components, especially when cells are intended for cell therapy application. Human platelet lysate (HPL) provides an animal component free option without the risks and regulatory challenges associated with animal derived components. In this study we investigated HPL for its ability to recover functionally potent cells following thawing and subsequent culture in cryopreservation of human derived mesenchymal stem cells (MSC). The results indicated HPL can improve cell recovery and functionality following thawing and provides an opportunity to develop animal protein free regulatory compliant cryopreservation media for cell therapy applications.
159 AUTOLOGOUS MESENCHYMAL STROMAL CELLS TO SUPPORT ALLOGENEIC HEMATOPOIETIC STEM CELL ENGRAFTMENT IN SICKLE CELL DISEASE E Stenger, S Yuan, M Garcia, IB Copland, J Galipeau Emory University, Atlanta, Georgia, United States Introduction: Allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for sickle cell disease (SCD), but for the majority of patients who lack a matched sibling donor, hematopoietic stem cell (HSC) engraftment remains a significant barrier. Based upon their dual function in promoting hematopoiesis and immunomodulation, mesenchymal stromal cells (MSCs) are an attractive cell-based therapy to modulate immunity and engraftment post-HCT. Studies have revealed limitations in approaches using cryopreserved, random donor MSCs expanded in FBS. This suggests that fresh, autologous MSCs expanded in human platelet-lysate could circumvent these limitations, thus increasing the potential of MSCs to enhance engraftment. Methods: MSCs were expanded ex vivo in human platelet-lysate from bone marrow (BM) samples obtained from pediatric SCD patients. BM was obtained fresh from 3 patients and frozen (post-ficoll) from 2 patients with mean starting volumes of 7.832.02 ml and 2 ml, respectively. Results: For fresh BM samples, a mean of 0.570.45 mononuclear cells (MNCs) were required to achieve 1 MSC after being seeded for 13.300.58 days. For frozen BM samples, a mean of 0.100.04 MNCs were required to achieve 1 MSC after being seeded for 15.503.54 days. Using patient weight on day of HCT, this corresponded to a mean MSC dose of 3.430.32x106/kg and 1.850.07x106/kg from fresh and frozen BM samples, respectively. Conclusions: In pediatric patients with SCD, autologous BM-derived MSCs can be expanded ex vivo in human platelet-lysate in a clinically relevant timeframe. MSC expansion was efficient from either fresh or frozen (postficoll) samples. Future studies are ongoing to confirm that the phenotype and function of MSCs from individuals with SCD are comparable to control MSCs prior to the use of autologous MSCs to enhance HSC engraftment in SCD patients. 160 EXPOSURE OF MESENCHYMAL STEM CELLS TO DMSO POSTTHAW AFFECTS CELL VIABILITY AND SIGNALING SL Gibb1,2, M Matthay2, F Nizzi3, M Marlowe3, B Jones3, S Pati1,2 1 Blood Systems Research Institute, San Francisco, California, United States, 2UCSF, San Francisco, California, United States, 3Blood Systems Laboratories, Tempe, Arizona, United States
Mesenchymal Stem Cells (MSCs) have been shown to have therapeutic potential in a number of disease states. As the number of clinical trials involving the use of adult bone marrow derived MSCs and potential for commercialization increases, the need to optimize cryopreservation and post-thaw handling of cells is critical. Cryopreservation of MSCs in liquid nitrogen storage units requires the employment of storage formulations that routinely include Dimethyl sulphoxide (DMSO). Although DMSO is essential for preventing the formation of destructive ice crystals, the effects on cells post-thaw have not been studied. We hypothesized that exposure of MSCs to DMSO post-thaw may alter cell viability and function and lead to a loss of therapeutic benefit. Our results using in vitro assays show a clear effect of DMSO on MSC viability that is both dose-dependent and time-dependent. We found a 20% loss of MSC viability after 1hour of exposure to 10% DMSO compared to control non-exposed cells. To determine if DMSO exposure could alter cell signaling in MSCs, we investigated its effects on NF-kB activation. NF-kB is a signaling pathway that has been shown to regulate inflammation and cytokine production by MSCs. Our results show that as little as 1% DMSO exposure can lead to significant increases in NF-kB activation. Furthermore, washing of cells immediately post-thaw to remove DMSO content prevented activation of NF-kB. Our results highlight the importance of expedited washing of MSCs post-thaw to remove all residual DMSO and maintain the viability and function of the MSCs. 161 ALLOGENEIC TRANSPLANTATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS (UC-MSC) FOR A SINGLE MALE PATIENT WITH DUCHENNE MUSCULAR DYSTROPHY (DMD) A Patel1, N Riordan2 1 University of Utah, Salt Lake City, Utah, United States, 2Amniotic Therapies, Dallas, Texas, United States Duchenne Muscular Dystrophy (DMD) is a degenerative X-linked genetic disorder affecting skeletal and cardiac muscles, and leading to eventual death. Currently, the major therapeutic strategies for DMD patients are pharmacological agents, gene transfer or modification, and cell therapies still in development. Pharmacological agents have not shown favorable results in clinical trials. Only corticosteroids manage to delay the progression of the disease but carry numerous adverse effects. Cell therapy is a possible treatment for DMD. The regenerative and antiinflammatory properties of MSC position them as a possible treatment option for DMD. A 28-year-old male who was diagnosed with DMD at the age of 3 was treated with human umbilical cord MSC on 7 occasions intravenous and intramuscular injections at the Stem Cell Institute in Panama City, Panama. After each treatment the patient and his primary physician reported an improved quality of life, with each treatment effect lasting approximately 5-6 months and no significant side effects. A single patient IND was filed and approved by US FDA. This is the report of the results of the first treatment of a patient with DMD in the USA with expanded human umbilical cord MSCs. 162 MANUFACTURING OF AUTOLOGOUS BM-MSCS FOR THE TREATMENT OF CROHN’S DISEASE: VALIDATION A FROM PHASE I CLINICAL TRIAL (NCT01659762) M Garcia2, J Galipeau1,3,4, IB Copland1,3,4 1 Hematology and Oncology, Emory University, Atlanta, Georgia, United States, 2Stem Cell Transplantation and Immunotherapy, Emory Healthcare, Atlanta, Georgia, United States, 3Pediatrics, Emory University, Atlanta, Georgia, United States, 4Winship Cancer Institute, Emory University, Atlanta, Georgia, United States Background: Mesenchymal stromal cells (MSCs) have emerged as a promising therapy for immune mediated ailments, however, conclusive Phase III data has remained elusive. We have identified allogeneic source, cyrobanking and xenogeneic growth supplements as areas of concern. In an effort to overcome these therapeutic obstacles, we developed a clinical manufacturing protocol to support IND14825 where autologous bone
Poster Abstracts
158 HUMAN PLATELET LYSATE-ITS ROLE IN THE CRYOPRESERVATION OF MESENCHYMAL STEM CELLS EJ Woods1,2, S Thirumala1, C Taylor1, B King1 1 Cook Regentec, Indianapolis, Indiana, United States, 2Indiana University School of Medicine, Indianapolis, Indiana, United States Cryopreservation is widely used to generate large stocks or cell banks that can be stored for short and long periods ready to be recovered in a functionally potent state and used as required in a cell therapy application. Animal based reagents are usually employed in cryopreservation media as a source of nutrients and other perceived benefits during the post-thaw period. Current evidence suggest that the animal serum proteins used in cryopreservation media are difficult to remove during post-thaw washing, and any residual proteins in cells may trigger adverse reactions in the patients who receive cell infusions or transplants. Therefore, demand is increasing for cryopreservation media to be devoid of any animal derived components, especially when cells are intended for cell therapy application. Human platelet lysate (HPL) provides an animal component free option without the risks and regulatory challenges associated with animal derived components. In this study we investigated HPL for its ability to recover functionally potent cells following thawing and subsequent culture in cryopreservation of human derived mesenchymal stem cells (MSC). The results indicated HPL can improve cell recovery and functionality following thawing and provides an opportunity to develop animal protein free regulatory compliant cryopreservation media for cell therapy applications.
159 AUTOLOGOUS MESENCHYMAL STROMAL CELLS TO SUPPORT ALLOGENEIC HEMATOPOIETIC STEM CELL ENGRAFTMENT IN SICKLE CELL DISEASE E Stenger, S Yuan, M Garcia, IB Copland, J Galipeau Emory University, Atlanta, Georgia, United States Introduction: Allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for sickle cell disease (SCD), but for the majority of patients who lack a matched sibling donor, hematopoietic stem cell (HSC) engraftment remains a significant barrier. Based upon their dual function in promoting hematopoiesis and immunomodulation, mesenchymal stromal cells (MSCs) are an attractive cell-based therapy to modulate immunity and engraftment post-HCT. Studies have revealed limitations in approaches using cryopreserved, random donor MSCs expanded in FBS. This suggests that fresh, autologous MSCs expanded in human platelet-lysate could circumvent these limitations, thus increasing the potential of MSCs to enhance engraftment. Methods: MSCs were expanded ex vivo in human platelet-lysate from bone marrow (BM) samples obtained from pediatric SCD patients. BM was obtained fresh from 3 patients and frozen (post-ficoll) from 2 patients with mean starting volumes of 7.832.02 ml and 2 ml, respectively. Results: For fresh BM samples, a mean of 0.570.45 mononuclear cells (MNCs) were required to achieve 1 MSC after being seeded for 13.300.58 days. For frozen BM samples, a mean of 0.100.04 MNCs were required to achieve 1 MSC after being seeded for 15.503.54 days. Using patient weight on day of HCT, this corresponded to a mean MSC dose of 3.430.32x106/kg and 1.850.07x106/kg from fresh and frozen BM samples, respectively. Conclusions: In pediatric patients with SCD, autologous BM-derived MSCs can be expanded ex vivo in human platelet-lysate in a clinically relevant timeframe. MSC expansion was efficient from either fresh or frozen (postficoll) samples. Future studies are ongoing to confirm that the phenotype and function of MSCs from individuals with SCD are comparable to control MSCs prior to the use of autologous MSCs to enhance HSC engraftment in SCD patients. 160 EXPOSURE OF MESENCHYMAL STEM CELLS TO DMSO POSTTHAW AFFECTS CELL VIABILITY AND SIGNALING SL Gibb1,2, M Matthay2, F Nizzi3, M Marlowe3, B Jones3, S Pati1,2 1 Blood Systems Research Institute, San Francisco, California, United States, 2UCSF, San Francisco, California, United States, 3Blood Systems Laboratories, Tempe, Arizona, United States
Mesenchymal Stem Cells (MSCs) have been shown to have therapeutic potential in a number of disease states. As the number of clinical trials involving the use of adult bone marrow derived MSCs and potential for commercialization increases, the need to optimize cryopreservation and post-thaw handling of cells is critical. Cryopreservation of MSCs in liquid nitrogen storage units requires the employment of storage formulations that routinely include Dimethyl sulphoxide (DMSO). Although DMSO is essential for preventing the formation of destructive ice crystals, the effects on cells post-thaw have not been studied. We hypothesized that exposure of MSCs to DMSO post-thaw may alter cell viability and function and lead to a loss of therapeutic benefit. Our results using in vitro assays show a clear effect of DMSO on MSC viability that is both dose-dependent and time-dependent. We found a 20% loss of MSC viability after 1hour of exposure to 10% DMSO compared to control non-exposed cells. To determine if DMSO exposure could alter cell signaling in MSCs, we investigated its effects on NF-kB activation. NF-kB is a signaling pathway that has been shown to regulate inflammation and cytokine production by MSCs. Our results show that as little as 1% DMSO exposure can lead to significant increases in NF-kB activation. Furthermore, washing of cells immediately post-thaw to remove DMSO content prevented activation of NF-kB. Our results highlight the importance of expedited washing of MSCs post-thaw to remove all residual DMSO and maintain the viability and function of the MSCs. 161 ALLOGENEIC TRANSPLANTATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS (UC-MSC) FOR A SINGLE MALE PATIENT WITH DUCHENNE MUSCULAR DYSTROPHY (DMD) A Patel1, N Riordan2 1 University of Utah, Salt Lake City, Utah, United States, 2Amniotic Therapies, Dallas, Texas, United States Duchenne Muscular Dystrophy (DMD) is a degenerative X-linked genetic disorder affecting skeletal and cardiac muscles, and leading to eventual death. Currently, the major therapeutic strategies for DMD patients are pharmacological agents, gene transfer or modification, and cell therapies still in development. Pharmacological agents have not shown favorable results in clinical trials. Only corticosteroids manage to delay the progression of the disease but carry numerous adverse effects. Cell therapy is a possible treatment for DMD. The regenerative and antiinflammatory properties of MSC position them as a possible treatment option for DMD. A 28-year-old male who was diagnosed with DMD at the age of 3 was treated with human umbilical cord MSC on 7 occasions intravenous and intramuscular injections at the Stem Cell Institute in Panama City, Panama. After each treatment the patient and his primary physician reported an improved quality of life, with each treatment effect lasting approximately 5-6 months and no significant side effects. A single patient IND was filed and approved by US FDA. This is the report of the results of the first treatment of a patient with DMD in the USA with expanded human umbilical cord MSCs. 162 MANUFACTURING OF AUTOLOGOUS BM-MSCS FOR THE TREATMENT OF CROHN’S DISEASE: VALIDATION A FROM PHASE I CLINICAL TRIAL (NCT01659762) M Garcia2, J Galipeau1,3,4, IB Copland1,3,4 1 Hematology and Oncology, Emory University, Atlanta, Georgia, United States, 2Stem Cell Transplantation and Immunotherapy, Emory Healthcare, Atlanta, Georgia, United States, 3Pediatrics, Emory University, Atlanta, Georgia, United States, 4Winship Cancer Institute, Emory University, Atlanta, Georgia, United States Background: Mesenchymal stromal cells (MSCs) have emerged as a promising therapy for immune mediated ailments, however, conclusive Phase III data has remained elusive. We have identified allogeneic source, cyrobanking and xenogeneic growth supplements as areas of concern. In an effort to overcome these therapeutic obstacles, we developed a clinical manufacturing protocol to support IND14825 where autologous bone