Enriched protein screening of human bone marrow mesenchymal stromal cell secretions reveals MFAP5 and PENK as novel IL-10 modulators

The secreted proteins from a cell constitute a natural biologic library that can offer significant insight into human health and disease. Discovering new secreted proteins from cells is bounded by the limitations of traditional separation and detection tools to physically fractionate and analyze samples. Here, we present a new method to systematically identify bioactive cell-secreted proteins that circumvent traditional proteomic methods by first enriching for protein candidates by differential gene expression profiling. The bone marrow stromal cell secretome was analyzed using enriched gene expression datasets in combination with potency assay testing. Four proteins expressed by stromal cells with previously unknown anti-inflammatory properties were identified, two of which provided a significant survival benefit to mice challenged with lethal endotoxic shock. Greater than 85% of secreted factors were recaptured that were otherwise undetected by proteomic methods, and remarkable hit rates of 18% in vitro and 9% in vivo were achieved

Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells

Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation

Prostaglandin E2 produced by alginate-encapsulated mesenchymal stromal cells modulates the astrocyte inflammatory response

Astroglia are well known for their role in propagating secondary injury following brain trauma. Modulation of this injury cascade, including inflammation, is essential to repair and recovery. Mesenchymal stromal cells (MSCs) have been demonstrated as trophic mediators in several models of secondary CNS injury, however, there has been varied success with the use of direct implantation due to a failure to persist at the injury site. To achieve sustained therapeutic benefit, we have encapsulated MSCs in alginate microspheres and evaluated the ability of these encapsulated MSCs to attenuate neuro-inflammation. In this study, astroglial cultures were administered lipopolysaccharide (LPS) to induce inflammation and immediately co-cultured with encapsulated or monolayer human MSCs. Cultures were assayed for the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-[Formula: see text] produced by astroglia, MSC-produced prostaglandin E2, and expression of neurotrophin-associated genes. We found that encapsulated MSCs significantly reduced TNF-[Formula: see text] produced by LPS-stimulated astrocytes more effectively than monolayer MSCs, and this enhanced benefit commences earlier than that of monolayer MSCs. Furthermore, in support of previous findings, encapsulated MSCs constitutively produced high levels of PGE2, while monolayer MSCs required the presence of inflammatory stimuli to induce PGE2production. The early, constitutive presence of PGE2significantly reduced astrocyte-produced TNF-[Formula: see text], while delayed administration had no effect. Finally, MSC-produced PGE2was not only capable of modulating inflammation, but appeared to have an additional role in stimulating astrocyte neurotrophin production. Overall, these results support the enhanced benefit of encapsulated MSC treatment, both in modulating the inflammatory response and providing neuroprotection.</jats:p

Symposium: Advances in Biomaterials and Devices, and Their Financing

My name is Professor Michael Baram and I direct the Center for Law and Technology here at the law school. Today\u27s meeting is the third annual Technology Law Symposium to be held here, sponsored by the high technology law firm of Testa, Hurwitz & Thibeault, LLP and the Center for Law and Technology. Our meeting today is focused on an exciting area of research and product development. This area involves the use of conventional as well as new genetically engineered biomaterials in new medical device configurations for implantation and with the purpose of restoring bodily functions, regenerating tissue, bone, cartilage, even organs, and limbs someday. This is a field that has great promise for improving health care in the United States. Some people have even said it offers the great promise of perpetual life

Secreted Factors from Bone Marrow Stromal Cells Upregulate IL-10 and Reverse Acute Kidney Injury

Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.National Human Genome Research Institute (U.S.) (Grant number T32 HG002295)National Institutes of Health (U.S.) (Grant K01DK087770)National Institutes of Health (U.S.) (Grant R21DK085267)National Institutes of Health (U.S.) (Grant DK39773)National Institutes of Health (U.S.) (Grant DK72381)Shriners Hospitals for Childre

Symposium: Advances in Biomaterials and Devices, and Their Financing

My name is Professor Michael Baram and I direct the Center for Law and Technology here at the law school. Today\u27s meeting is the third annual Technology Law Symposium to be held here, sponsored by the high technology law firm of Testa, Hurwitz & Thibeault, LLP and the Center for Law and Technology. Our meeting today is focused on an exciting area of research and product development. This area involves the use of conventional as well as new genetically engineered biomaterials in new medical device configurations for implantation and with the purpose of restoring bodily functions, regenerating tissue, bone, cartilage, even organs, and limbs someday. This is a field that has great promise for improving health care in the United States. Some people have even said it offers the great promise of perpetual life

Enriched Protein Screening of Human Bone Marrow Mesenchymal Stromal Cell Secretions Reveals MFAP5 and PENK as Novel IL-10 Modulators

The secreted proteins from a cell constitute a natural biologic library that can offer significant insight into human health and disease. Discovering new secreted proteins from cells is bounded by the limitations of traditional separation and detection tools to physically fractionate and analyze samples. Here, we present a new method to systematically identify bioactive cell-secreted proteins that circumvent traditional proteomic methods by first enriching for protein candidates by differential gene expression profiling. The bone marrow stromal cell secretome was analyzed using enriched gene expression datasets in combination with potency assay testing. Four proteins expressed by stromal cells with previously unknown anti-inflammatory properties were identified, two of which provided a significant survival benefit to mice challenged with lethal endotoxic shock. Greater than 85% of secreted factors were recaptured that were otherwise undetected by proteomic methods, and remarkable hit rates of 18% in vitro and 9% in vivo were achieved. © 2014 The American Society of Gene and Cell Therapy.National Human Genome Research Institute (U.S.) (Grant T32 HG002295