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ABSTRACT Introduction:We hypothesized extracellular vesicles (EVs) from preconditioned human-induced pluripotent stem cell–derived mesenchymal stem cells (iMSCs) attenuate LPS-induced acute lung injury (ALI) and endotoxemia.Methods:iMSCs were incubated with cell stimulation cocktail (CSC) and EVs were isolated. iMSC-EVs were characterized by size and EV markers. Biodistribution of intratracheal (IT), intravenous, and intraperitoneal injection of iMSC-EVs in mice was examined using IVIS. Uptake of iMSC-EVs in lung tissue, alveolar macrophages, and RAW264.7 cells was also assessed. C57BL/6 mice were treated with IT/IP iMSC-EVs or vehicle ± IT/IP LPS to induce ALI/acute respiratory distress syndrome and endotoxemia. Lung tissues, plasma, and bronchoalveolar lavage fluid (BALF) were harvested at 24 h. Lung histology, BALF neutrophil/macrophage, cytokine levels, and total protein concentration were measured to assess ALI and inflammation. Survival studies were performed using IP LPS in mice for 3 days.Results:iMSC-EV route of administration resulted in differential tissue distribution. iMSC-EVs were taken up by alveolar macrophages in mouse lung and cultured RAW264.7 cells. IT LPS-treated mice demonstrated marked histologic ALI, increased BALF neutrophils/macrophages and protein, and increased BALF and plasma TNF-α/IL-6 levels. These parameters were attenuated by 2 h before or 2 h after treatment with IT iMSC-EVs in ALI mice. Interestingly, the IT LPS-induced increase in IL-10 was augmented by iMSC-EVs. Mice treated with IP LPS showed increases in TNF-α and IL-6 that were downregulated by iMSC-EVs and LPS-induced mortality was ameliorated by iMSC-EVs. Administration of IT iMSC-EVs 2 h after LPS downregulated the increase in proinflammatory cytokines (TNF-α/IL-6) by LPS and further increased IL-10 levels.Conclusions:iMSC-EVs attenuate the inflammatory effects of LPS on cytokine levels in ALI and IP LPS in mice. LPS-induced mortality was improved with administration of iMSC-EVs.
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Administration of FVIII-Expressing Human Placental Cells to Juvenile Sheep Yields Multi- Organ Engraftment, Therapeutic Plasma FVIII Levels and Alter Immune Signaling Pathways to Evade FVIII Inhibitor Induction
Administration of FVIII-Expressing Human Placental Cells to Juvenile Sheep Yields Multi-Organ Engraftment, Therapeutic Plasma FVIII Levels and Alter Immune Signaling Pathways to Evade FVIII Inhibitor Induction 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster III Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Immune Mechanism, Diseases, Gene Therapy, Therapies, Adverse Events, Biological Processes, Transplantation Monday, December 13, 2021, 6:00 PM-8:00 PM We have previously reported that normal juvenile sheep that received weekly intravenous (IV) infusions of human (n=3) or an expression/secretion-optimized, bioengineered human/porcine hybrid (ET3) FVIII protein (n=3) for 5 weeks (20 IU/kg) developed anti-FVIII inhibitory antibodies (10-116 BU, and IgG titers of 1:20–1:245) by week 3 of infusion. By contrast, the IV infusion, or IP administration, of human placental mesenchymal cells (PLC) transduced with a lentiviral vector encoding a myeloid codon-optimized ET3 transgene (PLC-mcoET3) to produce high levels of ET3 protein (4.9-6IU/10^6 cells/24h) enabled the delivery of FVIII without eliciting antibodies, despite using PLC-mcoET3 doses that provided ~20-60 IU/kg ET3 each 24h to mirror the amount of FVIII protein infused. In addition, we showed that the route of PLC-mcoET3 administration (IP vs IV) did not impact the resultant plasma FVIII levels, with animals in these two groups exhibiting mean increases in FVIII activity (quantified by aPTT) of 30.9% and 34.2%, respectively, at week 15 post-treatment. Here, we investigated whether the sites and levels of PLC-mcoET3 engraftment were dependent upon the route of administration and performed s sheep-specific multiplexed transcriptomic analysis (NanoString) to define the immune signaling pathways that thwarted FVIII/ET3 protein immune response when ET3 was delivered through PLC. Tissue samples were collected from various organs at euthanasia and RT-qPCR performed using primers specific to the mcoET3 transgene, to the human housekeeping transcript GAPDH, and to sheep GAPDH, to quantify PLC-mcoET3 tissue engraftment, and normalize the results. RT-qPCR demonstrated PLC-mcoET3 engrafted, in both IP and IV groups, in all the organs evaluated (liver, lung, lymph nodes, thymus, and spleen). Animals that received PLC-mcoET3 via the IP route displayed higher overall levels of engraftment than their IV counterparts. The spleen was the preferential organ of engraftment for both IP and IV groups (IP:2.41±1.97%; IV: 0.64±0.54%). The IP group exhibited significantly higher engraftment in the left lobe of the liver (IP: 1.36±0.35%; IV: 0.041±0.022%), which was confirmed by immunohisto-chemistry (IHC) with an antibody to the human nuclear antigen Ku80 and ImageJ analysis (IP:5.24±3.36%; IV: 0±0). Of note is that the IP route resulted in higher levels of engraftment in the thymus, while IV infusion yielded higher levels of PLC-mcoET3 in lymph nodes. Analysis of H&E-stained tissues demonstrated they were devoid of any abnormal histologic changes and exhibited no evidence of hyperplasia or neoplasia, supporting the safety of the cell platform, irrespective of the route of administration. To date, NanoString analysis of PBMC collected at day 0, week 1, and week 5 post-infusion demonstrated that animals who received FVIII protein had upregulation of UBA5 and BATF, genes involved in antigen processing and Th17 signaling pathways, respectively. Although both IV and IP recipients of PLC-mcoET3 also had an increase in BATF, the IV group exhibited upregulation of BTLA, a gene involved in immune-tolerance, and downregulation of NOTCH and DDL1, involved in T cell differentiation, as well as MAPK12 and PLCG1, genes involved in proinflammatory cytokine regulation and T signaling within the Th17 signature. In IP recipients, BTLA, NOTCH, and DLL1 were all downregulated. Since ET3-reactive Th1 cells were not present in any of the treated animals, it is possible that the Th17 cells are responsible for the inhibitory antibodies seen in the juvenile sheep treated with FVIII/ET3 protein, while in animals receiving PLC-mcoET3, downregulation of genes involved in T cell differentiation and proinflammatory cytokine signaling keeps the immune system in check to avoid an immune response. Disclosures: Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.
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- Award ID(s):
- 1659663
- PAR ID:
- 10313915
- Date Published:
- Journal Name:
- ASH
- ISSN:
- 1296-5952
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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