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Safety and efficacy of placenta derived decidua stromal cells in experimental studies and clinical settings
Poster Abstracts
versus normoxia on the MSC secretome. We also analyzed the effects of MSC expansion in four different medium formulations, including three commercially available xenofree medium products: 5% human platelet lysate in αMEM, StemPro® MSC SFM XenoFree, StemMACS™ MSC Expansion Media XF, and Mesencult™-XF Medium. We used the Luminex bead platform to assess expression of 42 different cytokine and growth factors secreted into the conditioned medium by three different bone marrow-derived MSC donor cells at P1. We observed that growth of MSC in different media resulted in significant increase or decrease of 10 different cytokines (IL-10, MCP-3, MDC, PDGFBB, IL-15, IL-6, IL-7, IL-8, MCP-1 and VEGF) regardless of the medium. However, for 31 cytokines we observed variability in whether secretion was increased, decreased or unchanged when compared with the respective baseline media (Table Ia). Additionally, we showed that growth of MSC in hypoxic conditions (2% O2) resulted in variations in cytokine secretion when compared with cells grown in normoxia. This observation was most pronounced with MSC grown in platelet lysate, where the majority of analytes we measured were secreted significantly less by cells grown in hypoxic conditions. These analytes included G-CSF, GMCSF, IL-6 and VEGF, which have regenerative and growth promoting activity, IFNγ, IL-17A, IL-1α and TNFα, known to have MSC activating properties, and EGF, TGFα and IL-10, which promote differentiation and are immunosuppressive. For MSC grown in StemMACS medium, 19 different analytes were secreted significantly less in hypoxia, including G-CSF, IL-1α and TNFα. Similar to StemMACS, cells grown in Mesencult showed significantly lower levels of secretion of 16 cytokines in hypoxia compared with normoxia. Conversely, for cells grown in StemPro, 4 different cytokines were secreted in significantly greater amounts in hypoxic conditions: EGF, IL-12P70, PDGF-AA and IL-1B (Table Ib). In summary, the MSC secretome can be modulated by different culture conditions and growth media. More studies are required to further investigate the effects of these variables on MSC characteristics and how these effects might influence the therapeutic function of MSC products manufactured under different conditions.
271 SAFETY AND EFFICACY OF PLACENTA DERIVED DECIDUA STROMAL CELLS IN EXPERIMENTAL STUDIES AND CLINICAL SETTINGS B. Sadeghi1, G. Moretti1, A. Baygan1, B. Khoein1, G. Moll1, B. Gustafsson2, L. Kingspor3, M. Remberger4, M. Westgren2, O. Ringden1 1 Laboratory Medicine, TCR, Karolinska Institutet, Stockholm, Stockholm, Sweden, 2CLINTEC, Karolinska Institutet, Stockholm, Sweden, 3Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 4Oncology Pathology, Karolinska Institutet, Stockholm, Sweden We introduced bone marrow derived mesenchymal stromal cells (BM-MSCs) as novel therapy for acute GVHD. Not all patients responded to MSC therapy and some of them died due to invasive fungal infection. Decidua stromal cells (DSCs) are isolated from the fetal membrane and showed stronger immunosuppression compared to other MSCs. Toxicity was investigated in Balb/c mice. Human DSCs were infused IV in doses of 4, 20 and 40 × 10^6/kg. None of the animals died or showed acute toxicity or adverse reaction related to cell infusion both in short (+3 day) and long (+30 day) follow up. Blood biochemistry profiles related to liver, kidney, heart and blood were not influenced by DSC infusion. Coagulation factors as well as inflammatory indices were not affected. We also applied DSCs to treat GVHD in a full MHC mismatched model (B6 to Balb/c). Recipient mice were conditioned with 950cGy TBI and received DSCs. All mice receiving 40 × 10^6 DSC/ kg died from pulmonary embolism. However, those receiving lower doses had a lower GVHD score and a better weight compared to controls. We also evaluated stromal cell infusion on fungal infection in a pig model of septicemia. Pigs tolerated 1 × 10^6/kg DSCs or BM-MSCs well with no side effects and no enhanced risk of candida septicemia. In clinical settings, patients were treated with DSCs for severe acute GVHD (n = 40), hemorrhagic cystitis (11), chronic GVHD (4), polyneuropathies (2), ARDS (1). Median dose was 1,5 (0,7–2,8) × 10^6/kg, given from 1 up to 15 doses. DSCs were well tolerated and only three patients had transient infusion related toxicity. Adverse events using DSC were similar to retrospective controls, but with less death from acute GVHD. One-year survival for severe acute GVHD using DSCs was 72%, which was significantly better than 3% in historical controls (32) who received conventional therapy (P < 0,01).
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We conclude that DSCs is a promising therapy for acute GVHD and toxicity/ inflammation after allogeneic stem cell transplantation with almost no side effects. 272 UMBILICAL CORD TISSUE DERIVED MESENCHYMAL STROMAL CELLS INHIBIT LYSOPHOSPHATIDYLCHOLINE MEDIATED ACTIVATION OF MICROGLIA IN ORGANOTYPIC CEREBELLAR CULTURES A. Saha1,2, L. Xu1, R. Franczak1, P. Noldner1, N. Meadows1, S. Buntz1, R. Storms1, A. Balber1, J. Kurtzberg1,2,3 1 Duke Clinical & Translational Science Institute, Durham, North Carolina, United States, 2Robertson Clinical and Translational Cell Therapy Program, Duke University Medical Center, Durham, North Carolina, United States, 3 Carolinas Cord Blood Bank, Durham, North Carolina, United States Umbilical cord tissue or Wharton’s Jelly is a rich source of rapidly proliferating mesenchymal stromal cells (human cord tissue MSCs; hCTMSC) that can be cultured on a large-scale. We are developing this MSC product for use in the treatment of children with autism spectrum disorders (ASDs) where increasing evidence points to a central role for immune dysregulation involving abnormal activation of microglial cells. We developed an organotypic brain slice culture model to ask whether hCTMSC inhibit microglial activation in vitro. We plan to use the assay to explore whether it could serve as a biomarker to predict the potential therapeutic effectiveness of various lots of MSCs in patients with ASDs. We utilized wildtype C57BL/6 mouse brain organotypic cerebellar slice cultures (OCSC) to create the model. Ex-vivo brain slices maintain many aspects of in vivo biology, including functional local synaptic circuitry with preserved brain architecture, while allowing good experimental access and precise control of the extracellular environment, making them ideal platforms for exploring function of individual cell types in the milieu of other neighboring cells. Treatment of OCSC with lysophosphatidylcholine (LPC) for 16 hours induced extensive microglial activation as identified by microglial morphometric analysis after immuno-histochemical staining. Seven hCTMSC lines generated from individual cord tissue donors using cGMP compatible methods were individually added to distinct LPC treated slice cultures, 48 hours after LPC was washed out. Two days later activation of CD68+ microglia was compared to control cultures maintained without hCTMSCs. Three out of seven hCTMSC lines significantly inhibited microglial activation. This preliminary finding supports the concept that hCTMSC products can be considered as therapeutic candidates for conditions associated with neuroinflammatory processes. However, different hCTMSC lines established using similar methods vary in biological activities that may be of therapeutic significance. We are now investigating the molecular basis underlying hCTMSC inhibition of microglial activation to help guide selection of appropriate cell lines to use for treatment of neuroinflammatory diseases like ASDs. Correlations with outcomes in clinical studies are also planned. 273 CHARACTERIZATION OF MSC DERIVED FROM UMBILICAL CORD TISSUES R. Storms, M. Lillich, R. Parrott, P. Noldner, N. Meadows, L. Cheatham, J. Kurtzberg Robertson Clinical and Translational Cell Therapy Program, Duke University, Durham, North Carolina, United States Our program has developed GMP-compliant methods for manufacturing a clinical grade mesenchymal stem cell product from human umbilical cord tissue (hCT-MSC). In the coming year, we plan to initiate a clinical trial using hCTMSC for the treatment of children with autism spectrum disorders. Sterile umbilical cords are obtained with maternal consent from full-term Caesarian deliveries. These tissues are digested using a cocktail of 4 proprietary enzymes, facilitated by continuous agitation in a GentleMACSTM Octo tissue dissociator (Miltenyi Biotec). The resulting cell suspension is used to initiate cultures in GMP-grade serum-free media (Irvine Scientific). The initial P0 cultures are typically maintained 10 to 14 days, and routinely yield greater than 20 million cells. The hCT-MSC reliably expand 30- to 40-fold with each subsequent passage. Therefore, clinically relevant cell yields can be achieved from a single umbilical cord within 2 passages. Importantly, this limits the hCT-MSC population doublings, which average 5.3 population doublings per passage. In addition, we have validated methods to cryopreserve the cells after each passage. Thus, the clinical cell product can be produced as needed, in stages.
versus normoxia on the MSC secretome. We also analyzed the effects of MSC expansion in four different medium formulations, including three commercially available xenofree medium products: 5% human platelet lysate in αMEM, StemPro® MSC SFM XenoFree, StemMACS™ MSC Expansion Media XF, and Mesencult™-XF Medium. We used the Luminex bead platform to assess expression of 42 different cytokine and growth factors secreted into the conditioned medium by three different bone marrow-derived MSC donor cells at P1. We observed that growth of MSC in different media resulted in significant increase or decrease of 10 different cytokines (IL-10, MCP-3, MDC, PDGFBB, IL-15, IL-6, IL-7, IL-8, MCP-1 and VEGF) regardless of the medium. However, for 31 cytokines we observed variability in whether secretion was increased, decreased or unchanged when compared with the respective baseline media (Table Ia). Additionally, we showed that growth of MSC in hypoxic conditions (2% O2) resulted in variations in cytokine secretion when compared with cells grown in normoxia. This observation was most pronounced with MSC grown in platelet lysate, where the majority of analytes we measured were secreted significantly less by cells grown in hypoxic conditions. These analytes included G-CSF, GMCSF, IL-6 and VEGF, which have regenerative and growth promoting activity, IFNγ, IL-17A, IL-1α and TNFα, known to have MSC activating properties, and EGF, TGFα and IL-10, which promote differentiation and are immunosuppressive. For MSC grown in StemMACS medium, 19 different analytes were secreted significantly less in hypoxia, including G-CSF, IL-1α and TNFα. Similar to StemMACS, cells grown in Mesencult showed significantly lower levels of secretion of 16 cytokines in hypoxia compared with normoxia. Conversely, for cells grown in StemPro, 4 different cytokines were secreted in significantly greater amounts in hypoxic conditions: EGF, IL-12P70, PDGF-AA and IL-1B (Table Ib). In summary, the MSC secretome can be modulated by different culture conditions and growth media. More studies are required to further investigate the effects of these variables on MSC characteristics and how these effects might influence the therapeutic function of MSC products manufactured under different conditions.
271 SAFETY AND EFFICACY OF PLACENTA DERIVED DECIDUA STROMAL CELLS IN EXPERIMENTAL STUDIES AND CLINICAL SETTINGS B. Sadeghi1, G. Moretti1, A. Baygan1, B. Khoein1, G. Moll1, B. Gustafsson2, L. Kingspor3, M. Remberger4, M. Westgren2, O. Ringden1 1 Laboratory Medicine, TCR, Karolinska Institutet, Stockholm, Stockholm, Sweden, 2CLINTEC, Karolinska Institutet, Stockholm, Sweden, 3Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 4Oncology Pathology, Karolinska Institutet, Stockholm, Sweden We introduced bone marrow derived mesenchymal stromal cells (BM-MSCs) as novel therapy for acute GVHD. Not all patients responded to MSC therapy and some of them died due to invasive fungal infection. Decidua stromal cells (DSCs) are isolated from the fetal membrane and showed stronger immunosuppression compared to other MSCs. Toxicity was investigated in Balb/c mice. Human DSCs were infused IV in doses of 4, 20 and 40 × 10^6/kg. None of the animals died or showed acute toxicity or adverse reaction related to cell infusion both in short (+3 day) and long (+30 day) follow up. Blood biochemistry profiles related to liver, kidney, heart and blood were not influenced by DSC infusion. Coagulation factors as well as inflammatory indices were not affected. We also applied DSCs to treat GVHD in a full MHC mismatched model (B6 to Balb/c). Recipient mice were conditioned with 950cGy TBI and received DSCs. All mice receiving 40 × 10^6 DSC/ kg died from pulmonary embolism. However, those receiving lower doses had a lower GVHD score and a better weight compared to controls. We also evaluated stromal cell infusion on fungal infection in a pig model of septicemia. Pigs tolerated 1 × 10^6/kg DSCs or BM-MSCs well with no side effects and no enhanced risk of candida septicemia. In clinical settings, patients were treated with DSCs for severe acute GVHD (n = 40), hemorrhagic cystitis (11), chronic GVHD (4), polyneuropathies (2), ARDS (1). Median dose was 1,5 (0,7–2,8) × 10^6/kg, given from 1 up to 15 doses. DSCs were well tolerated and only three patients had transient infusion related toxicity. Adverse events using DSC were similar to retrospective controls, but with less death from acute GVHD. One-year survival for severe acute GVHD using DSCs was 72%, which was significantly better than 3% in historical controls (32) who received conventional therapy (P < 0,01).
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We conclude that DSCs is a promising therapy for acute GVHD and toxicity/ inflammation after allogeneic stem cell transplantation with almost no side effects. 272 UMBILICAL CORD TISSUE DERIVED MESENCHYMAL STROMAL CELLS INHIBIT LYSOPHOSPHATIDYLCHOLINE MEDIATED ACTIVATION OF MICROGLIA IN ORGANOTYPIC CEREBELLAR CULTURES A. Saha1,2, L. Xu1, R. Franczak1, P. Noldner1, N. Meadows1, S. Buntz1, R. Storms1, A. Balber1, J. Kurtzberg1,2,3 1 Duke Clinical & Translational Science Institute, Durham, North Carolina, United States, 2Robertson Clinical and Translational Cell Therapy Program, Duke University Medical Center, Durham, North Carolina, United States, 3 Carolinas Cord Blood Bank, Durham, North Carolina, United States Umbilical cord tissue or Wharton’s Jelly is a rich source of rapidly proliferating mesenchymal stromal cells (human cord tissue MSCs; hCTMSC) that can be cultured on a large-scale. We are developing this MSC product for use in the treatment of children with autism spectrum disorders (ASDs) where increasing evidence points to a central role for immune dysregulation involving abnormal activation of microglial cells. We developed an organotypic brain slice culture model to ask whether hCTMSC inhibit microglial activation in vitro. We plan to use the assay to explore whether it could serve as a biomarker to predict the potential therapeutic effectiveness of various lots of MSCs in patients with ASDs. We utilized wildtype C57BL/6 mouse brain organotypic cerebellar slice cultures (OCSC) to create the model. Ex-vivo brain slices maintain many aspects of in vivo biology, including functional local synaptic circuitry with preserved brain architecture, while allowing good experimental access and precise control of the extracellular environment, making them ideal platforms for exploring function of individual cell types in the milieu of other neighboring cells. Treatment of OCSC with lysophosphatidylcholine (LPC) for 16 hours induced extensive microglial activation as identified by microglial morphometric analysis after immuno-histochemical staining. Seven hCTMSC lines generated from individual cord tissue donors using cGMP compatible methods were individually added to distinct LPC treated slice cultures, 48 hours after LPC was washed out. Two days later activation of CD68+ microglia was compared to control cultures maintained without hCTMSCs. Three out of seven hCTMSC lines significantly inhibited microglial activation. This preliminary finding supports the concept that hCTMSC products can be considered as therapeutic candidates for conditions associated with neuroinflammatory processes. However, different hCTMSC lines established using similar methods vary in biological activities that may be of therapeutic significance. We are now investigating the molecular basis underlying hCTMSC inhibition of microglial activation to help guide selection of appropriate cell lines to use for treatment of neuroinflammatory diseases like ASDs. Correlations with outcomes in clinical studies are also planned. 273 CHARACTERIZATION OF MSC DERIVED FROM UMBILICAL CORD TISSUES R. Storms, M. Lillich, R. Parrott, P. Noldner, N. Meadows, L. Cheatham, J. Kurtzberg Robertson Clinical and Translational Cell Therapy Program, Duke University, Durham, North Carolina, United States Our program has developed GMP-compliant methods for manufacturing a clinical grade mesenchymal stem cell product from human umbilical cord tissue (hCT-MSC). In the coming year, we plan to initiate a clinical trial using hCTMSC for the treatment of children with autism spectrum disorders. Sterile umbilical cords are obtained with maternal consent from full-term Caesarian deliveries. These tissues are digested using a cocktail of 4 proprietary enzymes, facilitated by continuous agitation in a GentleMACSTM Octo tissue dissociator (Miltenyi Biotec). The resulting cell suspension is used to initiate cultures in GMP-grade serum-free media (Irvine Scientific). The initial P0 cultures are typically maintained 10 to 14 days, and routinely yield greater than 20 million cells. The hCT-MSC reliably expand 30- to 40-fold with each subsequent passage. Therefore, clinically relevant cell yields can be achieved from a single umbilical cord within 2 passages. Importantly, this limits the hCT-MSC population doublings, which average 5.3 population doublings per passage. In addition, we have validated methods to cryopreserve the cells after each passage. Thus, the clinical cell product can be produced as needed, in stages.