1 / 30

Marius Wernig, MD, PhD Institute for Stem Cell Biology and Regenerative Medicine

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. Nuclear Transfer. fertilized egg. early embryo. mid stage embryo. Ectoderm.

maxwell-jordan
Download Presentation

Marius Wernig, MD, PhD Institute for Stem Cell Biology and Regenerative Medicine

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. Nuclear Transfer fertilized egg early embryo mid stage embryo Ectoderm Mesoderm Endoderm Germ cells adult tissues:

  3. Dolly‘s “mother“ Dolly early embryo “reprogramming” egg skin cells nucleus

  4. ? “Therapeutic” cloning transplantation differentiated donor cells embryonic stem cells early embryo egg skin cells nucleus

  5. Induced pluripotent stem (iPS) cells transplantation differentiated donor cells iPS cells Oct4 blastocyst Klf4 Sox2 c-Myc enucleated oocyte ? adult cells nucleus

  6. Gene repair in iPS cells transplantation differentiated donor cells repaired iPS cells genetargeting Oct4 Sox2 iPS cells Klf4 c-Myc adult cells

  7. hbA/hbS hbS/hbS * treated * Correction of sickle cell anemia (hbS/hbS) Correction of the sickle allele by gene targeting hbA/hbS #10 #11 #25 #3 hbS hbA Jacob Hanna, Rudolf Jaenisch

  8. DEB • Dystrophic (scarring) • Epidermolysis (destruction of skin) • Bullosa (blistering)

  9. DEB • RDEB • Autosomal Recessive • DDEB • Autosomal Dominant

  10. 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)

  11. 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

  12. RDEB: widespread blisters causing wounds that never heal

  13. Complications of RDEB Mitten hand deformity Squamous cell carcinoma

  14. Cutaneous basement membrane zone Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42

  15. Human type VII Collagen (Col7A1) RDEB mutations NC2 NC1 Gly-X-Y Gly-X-Y RDEB/DDEB mutations 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

  16. RDEB Keratinocytes From skin biopsy Viral transfer LZRSE-Col7A1 Epidermal Sheet Production Mouse Grafting + Skin BMZ analyses 6-12 days 3-7 days 1 days 12-60 days Pig Dermis Ex Vivo genetic correction of RDEB Preclinical Data for FDA

  17. Anti-Type VII Collagen Mab Anti-Dsg3 Mab Hoechst 33342 Hoechst 33342 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 Type VII Collagen expression in corrected RDEB human skin 10x 10x

  18. 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)

  19. iPS cell gene targeting approach differentiated donor cells repaired iPS cells Oct4 Sox2 iPS cells Klf4 c-Myc adult cells

  20. Bottlenecks for translation to human cells differentiation transplantation ✓ differentiated donor cells repaired iPS cells gene targeting safe reprogramming Oct4 Sox2 iPS cells Klf4 c-Myc adult cells

  21. 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)

  22. Derivation of EB-iPS cell lines

  23. Patient 1 118 1 3 118 1 15 Patient 2 1 14 118 1 117 118 Patient 3 1 2 118 1 54 118 Patient 4 1 3 118 1 86 118

  24. Pei Wang, Thomas Leung, Seung Kim

  25. Two domains: • Zinc Finger domain DNA-binding domain • DNA-cleavage domain • High specificity: 18 bp Zinc Finger Nucleases • Xenopous: >95% • C. elegans: 15% • Drosophyla: 15% • Human cells: 50%

  26. Differentiation of hES cells into engraftable keratinocytes Guenou et al Lancet 2009

  27. Testing function of iPS- derived keratinocytes Cell autonomous expression in chimeric skin Callahan et al. Genes Dev 2004 Progenitor Assay in chimeric skin Sen et al. Nature 2010

  28. Acknowledgements Sandra Melo Anthony Oro Thomas Leung Pei Wang Seung Kim Zurab Siprashvili Andrea Tichy Al Lane VittorioSebastiano Dana Ting Yeo Bahareh Haddad Jesse Karmazin Funding: California Institute for Regenerative Medicine

More Related