ESC-Derived Cells For Restoration of Tissues & Organs

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

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

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

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

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

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 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)

Hemangioblasts derived from hESC

Hemangioblasts Generated from Human ES Cells

40X 10X

• 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

Generation of ES cells using parthenogenesis

WBC colony from cloned stem cells

•Cardiovascular disease costs the US $329 billion annually

10

20

30

40

50

60

70

80

90

100

19

88

19

89

19

90

19

91

19

92

19

93

19

94

19

95

19

96

19

97

19

98

19

99

20

00

20

01

Year

Nu

mb

er o

f P

ati

ents

(i

n th

ou

san

ds)

Waiting List

Organs Transplanted

End-stage renal disease will cost US $1 trillion during the comingdecade