"ESC-Derived Cells for Restoration of Tissues & Organs" 10/16/2006

1. ESC-Derived Cells For Restoration of Tissues & Organs
Robert Lanza, MD
VP Research & Scientific Development
Advanced Cell Technology
and Adjunct Professor
Wake Forest University School of Medicine

2. Alzheimer’s
Dwarfism
Parkinson’s
Strokes
Epilepsy
Hemophilia
Kidney failure
Chronic pain
Cancer
Infertility
Burns
AIDS
Muscular dystrophy
ALS
Affective disorders
Macular degeneration
Hypoparathyroidism
Heart disease
Liver failure
Enzymatic defects
Diabetes
Osteoarthritis
Multiple sclerosis
Huntington’s
Hypocholesterolemia
Rheumatoid
arthritis
Atherosclerosis
Ulcers
Spinal cord
injuries

4. Anatomy & Function of RPE
• Immune barrier
• Absorption of stray light
• Vit A metabolism & transport
• Phagocytosis of shed
photoreceptor segments
FUNCTIONS OF RPE

5. Diseases Associated with RPE dysfunction
• Age-related macular degeneration
• Retinitis pigmentosa
• Stargardt's disease
• Best's vitelliform macular dystrophy
• Leber's congenital amaurosis

6. Transplantation of RPE in Humans
Associated problemsSources of RPE cells
* autologous tissue
* cell lines
* donor tissue (adult, fetal) safety ethical Batch-to-batch
variation
may have impaired function limited supply
Potential tumorigenicity

7. Advantages of ECS-derived Tissues for Regenerative
Medicine
• Unlimited supply
• Can be derived under GMP conditions pathogen-
free
• Can be produced with minimal batch to batch
variation
• Can be thoroughly characterized to ensure optimal
performance

9. [All hES cell lines studied reproducibly generated RPE lines
that could be passaged, characterized, and expanded]
•WiCell hES cell lines (23 RPE lines generated)
WA01 WA09
WA07
•Harvard hES cell lines (22 RPE lines generated)
HUES1 HUES6
HUES2 HUES7
HUES3 HUES8
HUES5 HUES10
•ACT hES cell lines (25 RPE lines generated)
MA01 MA03 MAJ1
MA04 MA09
MA14 MA40
RPE can be generated from hES cells

10. Stages of RPE isolation from spontaneously differentiating hES cells
35 mm plate one of the clustersone of the clusters cell suspension at plating
4 days
x100x200
x200
7 days
x200
Passage 1 -- 25 daysPassage 1 -- 25 days
x200
x0.75

11. x400
x200
hES-RPE express RPE markers (bestrophin &
CRALBP)
-- 32
-- 46
-- 78
CRALBP
Mw
a b c
bestrophin
bestrophin
CRALBP
Immunostainin
g
Western blot

12. PEDF RPE65
RT-PCR
1 2 1 2
1 – fetal RPE
2 – hES-RPE
hES-RPE express RPE65 and PEDF

13. DB
X15,200
x7000
Phagocytosis of latex beads (electron microscopy)
Latex beads
pigment

14. hES-RPE vs. its in vivo counterpart
RPE hES-RPE
cobblestone, pigmented
transdifferentiation-differentiation
phagocytosis
molecular markers
RPE65
CRALBP
bestrophin
PEDF
MERTK

15. Gene expression profiling of hES-RPE vs. their in vivo
counterpart

17. RPE transplantation into subretinal space of RCS rats
(in collaboration with Raymond Lund, University of Utah)
RCS rats naturally become blind in several weeks
due to RPE degeneration and photoreceptor death
Study design
cell line RPE (H9)
Control: culture medium
Tests:
head tracking (behavior)
electroretinogram (ERG)
histology
In vitro assessment:
molecular markers of RPE
morphology and behavior

18. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
R
PE65
bestrophin
C
R
A
LB
P
PED
F
Pax6
G
A
PD
H
H9 RPE used for transplantation in RCS rats

19. cone
hES-RPE
ERG at P60
Amplitude(uV)
40
a-hES-RPE a-sham b-hES-RPE b-sham cone
b-sham
180
0
20
60
80
120
140
160
100
hES-RPE transplantation into subretinal space of RCS rats
Optomotor at P100
hES-RPE Sham Untreated
0.5
0.3
0.4
0.2
0
0.1
Relativeacuity(c/d)

21. Summary
• hES-RPE is similar to its in vivo counterpart by multiple parameters
(morphology, behavior, phagocytosis, molecular markers)
• hES-RPE can be reproducibly generated from hES cells
• hES-RPE attenuates photoreceptor loss in animal model of retinal degeneration
hES-RPE advantages
• Minimize batch-to-batch variation
• Can be derived under GMP conditions
• Can be produced from feeder-free hES cells
• Can be easily generated in large quantities
(for pathogen & safety assessment, and pre-clinical & clinical studies)

22. Hemangioblasts derived from hESC

23. Hemangioblasts Generated from Human ES Cells
40X 10X

24. • Established an efficient method to generate
hemangioblasts from multiple hESC lines
• Cells can be easily & reproducibly scaled up
• Hemangioblasts derived from hESC form
functional hematopoietic & endothelial cells
in vitro
• Rapidly repair damaged vasculature in vivo
(ischemic limbs, retina, and myocardium)
Summary

27. Generation of ES cells using parthenogenesis

29. WBC colony from cloned stem cells

30. •Cardiovascular disease costs the US $329 billion annually

31. 10
20
30
40
50
60
70
80
90
100
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
Year
NumberofPatients(inthousands)
Waiting List
Organs Transplanted

35. End-stage renal disease will cost US $1 trillion during the coming
decade