The use of embryonic stem cells in regenerative medicine

Robert Lanza, MDVP Research & Scientific DevelopmentAdvanced Cell Technologyand Adjunct ProfessorWake Forest University School of Medicine

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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