marius wernig, md, phd institute for stem cell biology and regenerative medicine and department of...
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Marius Wernig, MD, PhDInstitute for Stem Cell Biology and Regenerative
Medicineand Department of Pathology
Stanford University School of Medicine
Potential therapeutic applications of pluripotent stem cells
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mid stage embryo
early embryo
fertilized egg
Ectoderm Mesoderm Endoderm Germ cells
Nuclear Transfer
adult tissues:
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nucleus
skin cells
egg
early embryo
Dolly
Dolly‘s “mother“
“reprogramming”
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nucleus
skin cells
egg
embryonic stem cells
differentiated donor cells
early embryo
?
“Therapeutic” cloning
transplantation
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nucleus
adult cells
enucleated oocyte
iPS cells
differentiated donor cells
transplantation
blastocyst
?
Induced pluripotent stem (iPS) cells
Oct4Oct4Sox2Sox2Klf4Klf4
c-Mycc-Myc
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adult cells
repaired iPS cells
differentiated donor cells
transplantation
Gene repair in iPS cells
Oct4Oct4Sox2Sox2Klf4Klf4
c-Mycc-MyciPS cells
gene targeting
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Correction of sickle cell anemia (hS/hS)
Correction of the sickle allele by gene targeting
hS
hA
hA/hS
#3 #25#10
#11
0
2
4
6
8
10
12
14
16
18
Hb (g/dl) RBC (x10^3/µl)
hA/hShS/hStreated
**
Jacob Hanna, Rudolf Jaenisch
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DEBDystrophic
(scarring)
Epidermolysis(destruction of skin)
Bullosa (blistering)
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DEBRDEB
Autosomal Recessive
DDEB
Autosomal Dominant
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RDEBBlisters 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)
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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
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RDEB: widespread blisters causing wounds that never
heal
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Complications of RDEB
Mitten hand deformitySquamous cell
carcinoma
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Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42
Cutaneous basement membrane zoneCutaneous basement membrane zone
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Human type VII Collagen (Col7A1)
R
DE
Bm
uta
tion
sR
DE
B/D
DE
Bm
uta
tion
s
Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42
Col7A1: 31,132bp at 3p21. 118exons (largest number yet), 8.9kb ORF, 2944 AA, ~300kDa prot.
Christiano A.M. et al., Genomics. 21., 1994., p. 169-79
Gly-X-YGly-X-Y Gly-X-YGly-X-YNC1 NC
2
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Ex Vivo genetic correction of RDEB Preclinical Data for FDA
+ Skin BMZ analyses
Epidermal SheetProduction
RDEBKeratinocytesFrom skin biopsy
Viral transferLZRSE-Col7A1
MouseGrafting
6-12 days6-12 days 3-7 days3-7 days 1 days1 days 12-60 12-60 daysdays
Pig Dermis
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Type VII Collagen expression in corrected RDEB human skin
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
10x10x 10x10xAnti-Dsg3 MabAnti-Dsg3 MabHoechst 33342Hoechst 33342
Anti-Type VII Collagen MabAnti-Type VII Collagen MabHoechst 33342Hoechst 33342
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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)
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adult cells
repaired iPS cells
differentiated donor cells
iPS cell gene targeting approach
Oct4Oct4Sox2Sox2Klf4Klf4
c-Mycc-MyciPS cells
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adult cells
repaired iPS cells
differentiated donor cells
Bottlenecks for translation to human cells
Oct4Oct4Sox2Sox2Klf4Klf4
c-Mycc-MyciPS cells
gene
targ
etin
g
safe reprogramming
differentiation transplantation✓
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1. Published iPS cell derivation strategies
a. integration-free
Repeated plasmid transfections (regular, minicircles)Protein transductionAdeno virusesSendai 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)
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Derivation of EB-iPS cell lines
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11 33
11 1414
11 22
118118
118118
118118
11 11811833
Patient 1
Patient 2
Patient 3
Patient 4
11 1515 118118
11 117117 118118
11 5454 118118
11 8686 118118
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Pei Wang, Thomas Leung, Seung Kim
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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%
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Differentiation of hES cells into engraftable keratinocytes
Guenou et al Lancet 2009
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Testing function of iPS- derived keratinocytes
Cell autonomous expression in chimeric skin
Progenitor Assay in chimeric skin Callahan et al. Genes Dev 2004
Sen et al. Nature 2010
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Vittorio Sebastiano
Dana Ting Yeo
Bahareh Haddad
Jesse Karmazin
Acknowledgements
Sandra MeloAnthony Oro
Thomas LeungPei WangSeung Kim
Zurab SiprashviliAndrea TichyAl Lane
Funding: California Institute for Regenerative Medicine