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SUPPRESSION OF ASYMMETRIC CELL KINETICS (“SACK”) A general method for selective and conditional expansion of tissue stem cells from ASYMMETREX Contact James L. Sherley, M.D., Ph.D. Director [email protected] Tissue Stem Cells 1. Rare cells in mature tissues 2. Responsible for tissue renewal 3. Responsible for tissue repair HUMAN CELL PRODUCTION NEED Routine, reliable, large scale production of renewable functional human cells for cell therapy medicine and drug development (e.g., hepatocytes) HUMAN CELL PRODUCTION NEED Routine, reliable, large scale production of renewable functional human cells? But “renewable functional human cells” is a tissue cell physiology oxymoron! The Cell Production Paradox Generally, mature functional human cells have limited multiplication capacity, i.e., they are unable to divide for their own self-renewal. Some Well Known Examples of the Cell Production Paradox… Small Intestines: Functional Transport Cells Stop Multiplying Replaced by Tissue Stem Cells Potten and Morris, 1988 Non-Dividing Mature Differentiated Cells “Streaming” Zajicek, JTB,1986 Potten et al., 2003 Dividing Tissue Stem Cells Blood Cell Production Similar Cell Multiplication Architecture:Similar Problem Terminal Arrest & DIVIDING TISSUE STEM CELL Death LIMITED DIVISION MATURE CELLS Similar Process In Hepatocyte Production Space of Disse Limited Division Mature Hepatocytes (Under normal conditions) Portal Triad Portal Triad Tissue Stem Cell Division * Death Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988 SOLUTION: 1) Mass produce tissue stem cells 2) Use to produce mature cells Portal Triad Tissue Stem Cell Division Space of Disse Limited Division Mature Hepatocytes Portal Triad * Death Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988 TISSUE STEM CELL EXPANSION BARRIER IN CULTURE Tissue Stem Cell Functional Cell Asymmetric Self-Renewal = Asymmetric Cell Kinetics The critical expansion barrier in culture? ASYMMETRIC CELL KINETICS time Linear Cell Production Kinetics ASYMMETRIC CELL KINETICS The critical ASC expansion barrier in culture: ASYMMETRIC CELL KINETICS time ASYMMETRIC CELL KINETICS With time, stem cells are lost due to dilution by the non-dividing differentiating cells they produce. TECHNOLOGICAL SOLUTION: SUPPRESSION OF ASYMMETRIC CELL KINETICS time Exponential Cell Kinetics! SYMMETRIC CELL KINETICS (Ideally, produce only tissue stem cells) “SACK” TECHNOLOGY and…REVERSIBLE SUPPRESSION OF ASYMMETRIC CELL KINETICS time ASYMMETRIC CELL KINETICS Kinetically “suppressed” expanded tissue stem cells retain the ability to revert back to asymmetric production of mature cells in vivo or in vitro. CURRENT SACK PROGRESS Species Rat Mouse Human SACK Clonal Cell Type Effect Strains TSCs hepatocyte √ √ √ bile epithelial √ √ √ lung ? √ inc. skeletal muscle √ √ inc. hair follicle √ √ √ pancreas √ √ √ hepatic √ √ √ skin √ √ inc. pancreas √ [√] √ kidney √ n/a n/a hematopoietic √ √ (TSCs = confirmed tissue stem cells expanded; inc., incomplete studies; n/a, not applicable) References Background Publications Lee et al. (2003) Biotech. & Bioeng. 83, 760-771 – SACK expansion, rat liver Paré and Sherley (2006) Curr. Topics Devel. Biol. 73, 141-171 – SACK principles Huh et al. (2011) Sci. Rep. 1, 175; DOI: 10.1038/srep00176 – Mouse hair follicle stem cells Paré and Sherley (2013) J. Stem Cell Res. & Therapy 149. DOI:104172/2157-7633.1000149 – Human pancreatic stem cells Patents 2010 2010 2010 2013 Methods for Ex Vivo Propagation of Adult Hepatic Stem Cells -US 7824912 Methods for Ex Vivo Propagation of Somatic Hair Follicle Stem Cells -US 7655465 Hepatocyte Precursor Cell Lines -US 7645610 Hepatocyte Precursor Cell Lines -US 8404481 Pending Patents 2014 Methods for Producing Human Pancreatic Islet Precursor Cells and Uses Thereof -PCT/US2012/042644 THE SACK TECHNOLOGY How does it work? Biochemical Control of Asymmetric Cell Kinetics IMPDH II gene AAAAAA mRNA p53 The p53 cancer gene regulates tissue stem cell kinetics state via guanine ribonucleotide (rGNPs) biosynthesis. IMPDH is the ratelimiting enzyme for this pathway. p53 expression reduces rGNPs, promoting asymmetric cell kinetics by tissue stem cells. IMP IMPDH Symmetric Cell Kinetics Liu et al., 1998, Mol. Biol. Cell 9, 15-28. Rambhatla et al., 2001, J. Biomed. & Biotech. 1, 28-37. XMP GMP Decreased rGNPS Asymmetric Cell Kinetics Purine Salvage Control of Asymmetric Cell Kinetics IMPDH II gene AAAAAA mRNA p53 IMP Hypoxanthine (Hx) Hx, Xs, and Xn are natural, non-toxic rGNP precursors that can be used to expand rGNP pools and bypass p53 XMP regulation of IMPDH to shift tissue stem cells from asymmetric cell kinetics to symmetric cell kinetics = “SACK GMP AGENTS” IMPDH Xanthosine (Xs), Xanthine (Xn) INCREASED rGNPS Symmetric Cell Kinetics Asymmetric Cell Kinetics Reversible Control of Asymmetric Cell Kinetics IMPDH II gene AAAAAA mRNA p53 IMP When SACK agents are no longer supplemented in the culture medium, expanded stem cells revert back to XMP asymmetric cell kinetics with production of mature cells, thereby achieving renewable functional human cells. GMP IMPDH …and reversible. Decreased rGNPS Symmetric Cell Kinetics Asymmetric Cell Kinetics SACK Strategy for Renewable Production of Mature Human Cells Suppress Asymmetric Cell Kinetics Asymmetric Cell Kinetics (initial tissue cell preparations) Differentiation Conditions SACK removal Increased Symmetric Asymmetric Stem Cell Kinetics Cell Kinetics For With Mass Production Production Of Tissue Stem Cells Of Mature Cells - RENEWABLE In vivo or in vitro SUMMARY AND CONCLUSIONS I.Asymmetrex’s patented SACK Technology provides a general method for expanding human tissue stem cells that can be developed as a renewable source of cells for cell therapy, drug development, and cell research. II.The SACK Technology can be used to produce renewable tissue stem cell strains from many different species (e.g., rat, mouse, human) and tissue types (e.g., liver, lung, kidney, hematopoietic, skin, pancreas, hair follicle) III.SACK expanded tissue stem cells can be clonally derived and stably genetically engineered.