1 Abstract Exploratory Generation of Murine mesenchymal Stem cells, And Investigation of Their Anti-Inflammatory Potential P.J. Mwaura1, Z. Ng’ang’a2, K. Gachuhi3 1 Jomo kenyatta University of Agriculture And Technology, Nairobi, kenya; 2 Kenya Medical Research Institute KEMRI ABSTRACT (revised) µg/mL METHODS Liver samples will be collected into PBS and fixed overnight in 40 g/L paraformaldehyde in PBS at 4 °C. Serial 5 μm sections of the right lobes of the livers will be stained with hematoxylin and eosin (HE) and will be examined histopathologically. The size and collagen content of the granulomas will be assayed. The Mean Granuloma Diameter (MGD) will be taken and compared within the different groups. Cytokines will be probed before and after administration of the MSCs. Isolation and culture of MSCs from Murine Bone Marrow The position paper by the International Society for Cellular Therapy (ISCT) defined internationally accepted minimal criteria for Mesenchymal stem cell identification as; 1. Adherence to plastic; 2. ≥95% of the MSC population must express CD73/5’-Nucleotidase, CD90/Thy1, and CD105/Endoglin as measured by flow cytometry; ≤2% of the MSC population must express CD34, CD45, CD11b/Integrin alpha M or CD14, CD79 alpha or CD19, and HLA Class II and 3. Multipotent differentiation potential as demonstrated by staining of in vitro differentiated cells. Isolation By plastic adherence Above protocol is adapted from Masoud Soleimani and Samad Nadri (2009) which is based on the plastic-adherence method, the isolation is therefore anchorage dependent. The protocol is based upon frequent media changes of a heterogenous bone marrow population containing haematopoietic stem cells hSCs and mesenchymal stem cells MSCs. The MSCs adhere to the surface of the plastic while the hSCs are not good adherers hence separation is easily achieved. Defining Flow Cytometry Panels for Sorting MSCs Positive markers : CD29PE, CD44PE-Cy7, CD90APC-Cy7, CD105APC, CD106FITC, Sca-1V450 Negative markers : CD11bV500, CD31PerCP-Cy5.5, CD45V500, Ter-119V500 The isolation protocols above will be compared in terms of yield of MSCs and presence of contaminating HSCs. Experimental design for in vitro experiment Experimental design for in vivo experiment Inflammation is now recognized as an overwhelming burden to the health status of our population and the underlying basis of a significant number of diseases that have a high morbidity and mortality rate. As of the moment, there are minimal efforts that have proven to be successful in the control of chronic inflammation. Novel strategies to control chronic inflammation are therefore vital. This research project proposes to generate murine mesenchymal stem cells and investigate their anti- inflammatory activity. The aim therefore is to generate murine mesenchymal stem cells (MSCs) and consequently induce granulomas in vitro and in vivo in mice. The mesenchymal stem cells will be co- cultured in-vitro with interleukin 1 conjugated artificial latex microparticles and macrophages from mice spleen to determine their ameliorative effect on the resulting granulomas. The MSCs will then be infused with phosphate buffered saline intravenously in the Schistosome infected Swiss Albino mice in vivo to determine their ameliorative potential on the liver granulomas. It is hypothesized that the co-cultured and infused mesenchymal stem cells will ameliorate the granuloma sizes and help in regeneration of damaged liver. The findings will advance the understanding of the anti-inflammatory activity of mesenchymal stem cells and there is potential to bring the same onto a therapeutic platform. ulsdhs PRELIMINARY RESULTS Mesenchymal Stem Cell Culture by Plastic Adherence negative controls Spindle shaped morphology characteristic of MSCs Quality Control of MSCs BACKGROUND A Mouse Mesenchymal Functional Identification Kit (Catalog #SC010 R&D Systems) will be procured. The Kit Contains an Adipogenic Supplement, Chondrogenic Supplement, Osteogenic Supplement-M, ITS Supplement, Anti-FABP4 (adipocyte marker), Anti-Collagen II (chondrocytes marker) and an Anti-Osteopontin (osteocyte marker). This kit will require media StemXVivo Mesenchymal Stem Cell Expansion Media (Catalog # CCM004). Induction of Granulomas In Vivo Several studies have shown that a majority of chronic diseases are a consequence of uncontrolled and persistent inflammation Scrivo et. al., 2011. Chronic inflammation has been linked with cardiovascular diseases, alzheimer’s disease, type 2 diabetes, tuberculosis, cancer, rheumatoid arthritis, joint pain, muscular pain, fibromyalgia, gum disease, cardiovascular diseases, eczema, neurodegenerative diseases, obesity, psoriasis, asthma, migraine, allergies, chronic bronchitis, sinusitis, IBS (irritable bowel syndrome), and gastritis Scrivo et. al., 2011) (Sonja VanderDol , 2008 Schistosomiasis is the model of chronic inflammation used, it is a common chronic helminthic infection of the liver that causes hepatic fibrosis and portal hypertension, contributing to the death of over half a million people a year. The egg induced granuloma formation is a Delayed Type Hypersensitivity (DTH or Type IV Hypersensitivity) reaction, and, although eventually resulting in severe pathology, appears to be a necessary protective host response against hepatotoxic components of Soluble Egg Antigen (SEA), secreted by the entrapped egg (Wynn et al, 2004). The ability of bone marrow derived mesenchymal stem cells (BMDMSC) to create a tolerogenic niche by direct interaction with immune cells and by secretion of regulatory molecules makes them attractive therapeutic candidates in the regulation of the inflammatory response to infection and injury (Smita et al. 2008). Both in vitro and in vivo studies have shown that MSCs are not inherently immunogenic and can surpass the Major histocompatibility complex barrier between genetically different individuals (Xi Chen et.al 2006). They have long been reported to be hypoimmunogenic or ‘immune privileged’. This property has enabled MSC transplantation across MHC barriers and the creation of off-the-shelf therapies consisting of MSCs grown in culture. The objective therefore, To generate murine mesenchymal stem cells and investigate their anti-inflammatory activity. Infection of mice with Schistosoma mansoni cercariae by the ring method Images of severely damaged mouse livers Spleen cells cultured with IL-1 tagged dextran microparticles No co-culture with MSCs Control A Co-culture with MSCs Control B CONCLUSIONS A Mouse mesenchymal Functional Identification kit will be procured from R&D systems to validate the stem cells. The flow cytometry panel will also be procured from BD in order to compare plastic adherence and FACS protocols. C. albicans Schistosome Egg Groups Control Groups Total Treatment with MSCS + PBS 6 rodents 12 Treatment with PBS only 24 REFERENCES Scrivo R, Vasile M, Bartosiewicz I, Valesini G (2011). Inflammation as "common soil" of the multifactorial diseases. Autoimmune Rev. Smita S Iyer, Mauricio (2008). Rojas Cell- & Tissue-based Therapy Anti-infl ammatory effects of mesenchymal stem cells: novel concept for future therapies Emory University, Department of Medicine, Atlanta, GA 30322, USA Sonja VanderDol (2008). Chronic Inflammation - The Unhappy Consequences of a Good Response Gone Bad Wynn TA, Thompson RW, Cheever AW, Mentink-Kane MM (2004). Immunopathogenesis of schistosomiasis. Immunol Rev; 201: 156–167.  Xi Chen, Marilyn Ann Armstrong and Gang Li (2006). Mesenchymal stem cells in immunoregulation, Immunology and Cell Biology 84, 413–421; Photograph: R. Lewis ACKNOWLEDGEMENTS Special thanks to Dr. Kimani Gachuhi Director CBRD who continues to support this work.