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Potential therapeutic applications of pluripotent stem cells Marius Wernig, MD, PhD Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology Stanford University School of Medicine fertilized egg early embryo Nuclear Transfer mid stage embryo Ectoderm Mesoderm adult tissues: Endoderm Germ cells Dolly‘s “mother“ Dolly early embryo “reprogramming” egg skin cells nucleus “Therapeutic” cloning transplantation differentiated donor cells embryonic stem cells early embryo egg ? skin cells nucleus Induced pluripotent stem (iPS) cells transplantation differentiated donor cells iPS cells blastocyst Oct4 Klf4 Sox2 c-Myc enucleated oocyte ? adult cells nucleus Gene repair in iPS cells transplantation differentiated donor cells repaired iPS cells gene targeting iPS cells Oct4 Klf4 Sox2 c-Myc adult cells Correction of sickle cell anemia (hbS/hbS) Correction of the sickle allele by gene targeting hbS hbA 18 16 * 14 * 12 hbA/hbS hbS/hbS treated 10 8 6 4 2 0 Hb (g/dl) RBC (x10^3/µl) Jacob Hanna, Rudolf Jaenisch DEB Dystrophic (scarring) Epidermolysis (destruction of skin) Bullosa (blistering) DEB RDEB Autosomal Recessive DDEB Autosomal Dominant RDEB Blisters present soon after birth Diagnosis is made by absence of type VII collagen in the upper most dermis at junction of epidermis Many years of painful, expanding wounds that never heal Death from malnutrition, infection or squamous cell carcinoma (SCC) Estimated incidence of RDEB is 1-2 infants per million births USA: 4 million annual births leads to 4-8 RDEB infants a year Worldwide: 75 million annual births leads to 75-150 RDEB infants a year RDEB: widespread blisters causing wounds that never heal Complications of RDEB Mitten hand deformity Squamous cell carcinoma Cutaneous basement membrane zone Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42 Gly-X-Y Gly-X-Y RDEB/DDEB mutations NC1 NC2 RDEB mutations Human type VII Collagen (Col7A1) Col7A1: 31,132bp at 3p21. 118exons (largest number yet), 8.9kb ORF, 2944 AA, ~300kDa prot. Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42 Christiano A.M. et al., Genomics. 21., 1994., p. 169-79 Ex Vivo genetic correction of RDEB Preclinical Data for FDA RDEB Viral transfer Keratinocytes LZRSE-Col7A1 From skin biopsy 6-12 days 3-7 days Epidermal Sheet Production Mouse Grafting + Pig Dermis 1 days 12-60 days Skin BMZ analyses Type VII Collagen expression in corrected RDEB human skin Anti-Type VII Collagen Mab Hoechst 33342 10x Anti-Dsg3 Mab Hoechst 33342 10x Type VII collagen at dermal-epidermal junction Human origin of the epidermis confirmed by human specific Desmoglein 3 protein staining Cell nuclear counterstain dye Hoechst 33342 Problems with retroviral gene transfer • Safety risk due to random integrations of retroviruses • Impossible to treat dominant forms of diseases • Uncontrolled expression of COL7A1 (throughout epidermis) iPS cell gene targeting approach differentiated donor cells repaired iPS cells iPS cells Oct4 Klf4 Sox2 c-Myc adult cells Bottlenecks for translation to human cells ✓ differentiated donor cells repaired iPS cells iPS cells Oct4 Klf4 Sox2 c-Myc adult cells 1. Published iPS cell derivation strategies a. integration-free Repeated plasmid transfections (regular, minicircles) Protein transduction Adeno viruses Sendai virus (RNA-based reproductive cycle) b. integration-based Standard Moloney viruses (retroviral silencing) Dox-inducible lentiviruses Transposase-mediated integration (Piggy-back) Excisable lentivirus (4F cassette) Derivation of EB-iPS cell lines Patient 1 1 118 3 1 118 15 Patient 2 1 14 118 1 117 118 Patient 3 1 2 118 1 54 118 Patient 4 1 1 3 118 86 118 Pei Wang, Thomas Leung, Seung Kim Zinc Finger Nucleases Two domains: • Zinc Finger domain DNA-binding domain • DNA-cleavage domain • High specificity: 18 bp Xenopous: >95% • C. elegans: 15% • Drosophyla: 15% • Human cells: 50% • Differentiation of hES cells into engraftable keratinocytes Guenou et al Lancet 2009 Testing function of iPSderived keratinocytes Cell autonomous expression in chimeric skin Callahan et al. Genes Dev 2004 Progenitor Assay in chimeric skin Sen et al. Nature 2010 Acknowledgements Sandra Melo Anthony Oro Vittorio Sebastiano Dana Ting Yeo Bahareh Haddad Jesse Karmazin Thomas Leung Pei Wang Seung Kim Zurab Siprashvili Andrea Tichy Al Lane Funding: California Institute for Regenerative Medicine
