Vision Research Symposium Conference Presentation
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Transcript of Vision Research Symposium Conference Presentation
The use of embryonic stem cells in regenerative medicine
Robert Lanza, MDVP Research & Scientific DevelopmentAdvanced Cell Technologyand Adjunct ProfessorWake Forest University School of Medicine
Alzheimer’s
Dwarfism
Parkinson’s
Strokes
Epilepsy
Hemophilia
Kidney failure
Chronic pain
CancerInfertility
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
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Year
Nu
mb
er o
f P
ati
en
ts (i
n th
ou
san
ds)
Waiting List
Organs Transplanted
Anatomy & Function of RPE
• Immune barrier • Absorption of stray light• Vit A metabolism & transport• Phagocytosis of shed photoreceptor segments
FUNCTIONS OF RPE
Diseases Associated with RPE dysfunction
• Age-related macular degeneration• Retinitis pigmentosa• Stargardt's disease• Best's vitelliform macular dystrophy• Leber's congenital amaurosis
Animal models of RPEdysfunction
• Royal College of Surgeons (RCS) rat (MERTK mutation,phagocytosis-impaired)
• Mouse models: RPE65 -/-; rd-mouse (cGMP-phosphodiesterase mutation, loss of rods)
• Dog (Briard, RPE65 mutation)
• Monkey (rhesus monkey with naturally occurring maculardegeneration)
Transplantation of RPE in Humans
Associated problemsSources of RPE cells
* autologous tissue
* cell lines
* donor tissue (adult, fetal) safety ethical Batch-to-batchvariation
may have impaired function limited supply
Potential tumorigenicity
Advantages of ECS-derived Tissues for RegenerativeMedicine
• 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
[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
x400
x200
hES-RPE express RPE markers (bestrophin &CRALBP)
-- 32
-- 46
-- 78
CRALBP
Mw
a b c
bestrophin
bestrophin
CRALBP
Immunostaining Western blot
PEDF RPE65
RT-PCR
1 2 1 2
1 – fetal RPE2 – hES-RPE
hES-RPE express RPE65 and PEDF
DB
X15,200
x7000
Phagocytosis of latex beads (electron microscopy)
Latex beadspigment
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
hES-RPE vs. its in vivo counterpart
RPE hES-RPEcobblestone, pigmented
transdifferentiation-differentiation
phagocytosis
molecular markers
RPE65
CRALBP
bestrophin
PEDF
MERTK
Gene expression profiling of hES-RPE vs. their in vivocounterpart
RPE in animal studies
RPE transplantation into subretinal space of RCS rats (in collaboration with Raymond Lund, University of Utah)
RCS rats naturally become blind in several weeksdue to RPE degeneration and photoreceptor death
Study designcell line RPE (H9) Control: culture medium
Tests: head tracking (behavior)electroretinogram (ERG) histology
In vitro assessment:molecular markers of RPEmorphology and behavior
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
RPE65
bestro
phin
CRALBP
PEDFPax
6
GAPDH
H9 RPE used for transplantation in RCS rats
conehES-RPE
ERG at P60
Am
plitu
de (u
V)
40
a-hES-RPE a-sham b-hES-RPE b-sham cone b-sham
180
020
6080
120140160
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
Rel
ativ
e ac
uity
(c/d
)
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)
hES-RPE: ongoing pre-clinical studies and research goals
• functional studies in animal models with different batches of cells (more and less differentiated, different passages, different lines)
• finding reliable markers for predicting therapeutic value of newly generated cells
• studies of hES-RPE survival on Bruch’s membrane
• production of hES-RPE under GMP conditions
• dosage and safety studies of hES-RPE in animal models
Generation of ES cells using parthenogenesis
WBC colony from cloned stem cells
•Cardiovascular disease costs the US $329 billion annually
Is it possible to generate ES cells without destroyingembryos?
• The most basic objection to ES cell research is that it deprivesembryos
of any further potential to develop into complete human beings
• For a decade, PGD has been used successfully to remove a singlecell (blastomere) for genetic testing without interfering with thedevelopmental potential of the biopsied embryo. Over 2,000healthy babies have been born using this procedure
• Question: Can such a biopsied cell be used to generate ES cells?
Biopsy Procedure
Live Young Biopsied 23/47 (49%)Non-Biopsied 38/75 (51%)
Oct-4
Alk Phos
Oct-4
Alk Phos
SSEA-1 Troma-1
Laz-Z Lac-Z
b III tubulin Ectoderm
Smooth muscle actin Endoderm
alpha feto-protein Endoderm
Biopsy Procedure (Human Embryo)
Blastocyst Biopsied 6/8 (75%) Non-Biopsied 1/4 (75%)
Derivation of hES Cells From Single Blastomeres
Blastomere biopsy
GFP hESsFeeders
Feeders
1 or 2 single blastomeres biopsiedand co-cultured with parent embryo
Multiple single blastomeresbiopsied and co-cultured together
Blastomere divided outgrowth first passage
second passage established line
Stages of Derivation of hES Cells From Single Blastomere
Markers of Pluripotency
Alkaline phosphatase
TRA I-81 SSEA-4
TRA 1-60Oct-4
SSEA-3
Endoderm - Cdx2 (intestine) Ectoderm – nestin (neural tissue) Mesoderm - smooth muscle actin
Kidney tissue
teratoma
Teratoma Formation in NOD-SCID Mice
In Vitro Differentiation Into Cells of Specific Therapeutic Interest
RPECapillary structures
Ac-LDL
Bestrophin
MA01 (blastomere-derived hESC line) generated hematopoietic progenitors 5-10 timesmore efficiently than H9 and 3-5 times more efficiently than H1
Ac-LDL
MA09 (blastomere-derived hESC line) generated vascular/endothelial progenitors 1-2 times more efficiently than H1 and H9
MA01 & MA09 (blastomere-derived hESC lines) generated neural progenitors without theneed forEB-intermediates, stromal feeder layers, or low-density passaging
MA01 MA09
karyotypeCharacterization of Single Blastomere-Derived hES Cell Lines
Mar
kers
H1
MA
01
MA
09
Mar
kers
H1
MA
01
MA
09
No Presence of Y Chromosome or GFP Gene Setected by PCR
X
YGFP
FES primerpair WA31 primer pair ds526
216 220 224 228 142 151 154 192 196 200 204 230 238 242 250
H1 1
H9 2
ACT 4 3
MA01 4
MA09 5
MA04 7
BLANK 8
ds592 ds417
170 178 182 186 190 173 177 181
H1 1
H9 2
ACT 4 3
MA01 4
MA09 5
MA04 7
BLANK 8
Checkerboard Fingerprints
H1
H1
MA01MA09
MA09MA01