Embryonic Stem Cellsand Therapy:

Promise, Problems, Reality

Rudolf JaenischWhitehead Institute and

Department of Biology, MIT

D. Melton

D. Melton

D. Melton

D. Melton

Melton 13Stem cells are crucial for tissue homeostasis

D. Melton

Melton 20

Pluripotent cellsMultipotent /

oligopotent cellsTotipotent cell:

The fertilized egg

Melton 22

Melton 21

Pluripotent cells Multipotent /oligopotent cells

D. Melton

What is the goal stemcell research?

Using the potential of ES cells

To provide matched cells for“customized” tissue repairin degenerative diseases

D. Melton

D. Melton

Year

Publications on stem cellsover the last 20 years

HumanES CellsIsolatedMouse

ES CellsIsolated

The interest in stem cells has grown exponentially

G. Daley

the quest for eternal youththe quest for eternal youth

Induction ofInduction of““PluripotencyPluripotency””

andand

Lucas Cranach, 1472-1553

Therapeutic Limitationsof Embryonic Stem Cells

• ES cells are derived from donatedembryos:

This causes immune rejection

One potential solution:– Nuclear cloning to create

“customized” ES cells

Embryonicstem cells

“Customized”embryonicstem cells

Sexuallyproducedembryo

Asexuallyproducedembryo

(from NIH web site)

Therapeutic Applications of Embryonic Stem Cells

“Customized” ES cells from cloned blastocysts: patient’s own cells

ES cells from IFV embryos: different from patient, immune rejections

Cell Cell 2002, 109: 17-272002, 109: 17-27

ClonedES Cells

Rag2-/-

Gene Correction

CorrectedES Cells

Egg Tail Tip CellβThalassemiaSickle cell anemiaFanconi’s anemiaLeukemia

G. Daley

I. Nuclear transferand ES cellderivation

II. Derivation of bone marrow

cells andtransplantationinto “patient”

Nuclear Cloning andNuclear Reprogramming:

Conclusion

• The egg must contain “reprogramming”factors that convert the adult nucleusinto an embryonic state

• The reprogrammed nucleus can generate– A cloned animal such as Dolly– A “customized” embryonic stem cell for

therapy

Problems withTherapeutic SCNT

1. Procedure too inefficient, costly forroutine treatment

2. Ethical objections to using humaneggs for therapy

Other options?

Alternatives to embryonic stemcells?

Do we need to use SCNT togenerate “customized”ES cells for therapy?

or

Such as Adult Stem cells?

Stem Cells:A developmental hierarchy B

T

Plts

WBC

RBC

Blood

Pluripotent All cell typesIn vitro

differentiation

Muscle

Bone

Fat

Mesenchymal-Connective

EmbryonicStem Cells

Liver/PancreasSkin, Testes, Gut

Neural

NeuronsOligoglia

Astroglia

Zygote(TOTIPOTENTIAL)

+

Blastocyst

?

?

Embryonic

Adult

G. Daley

Melton 21

Pluripotent cells Multipotent /oligopotent cells

Generation of patient-specific ES cells:Strategies

C. Cowan

Two complementing strategiesto induce reprogramming in

culture

1. Molecular circuitry of ES cells– What distinguishes pluripotent from

committed / differentiated cells

2. Forced expression of keyfactors in somatic cells

“Wiring Diagrams” of Developmental Processes

Davidson, et al. Science. 2002 Rothenberg and Anderson. Dev. Biol. 2002

Sox2

Oct4

Nanog

PcG proteins

ES cell

self-renewal

differentiatedcell

Regulation of Pluripotency inEmbryonic Stem Cells

Useful for induction of reprogramming?

A. MeissnerM. Wernig

R. Foreman

T. Brambrink

Oct4

Sox2c-Myc

Klf4

iPS cells(induced pluripotent

stem cells)

Derivation of iPS cells based onselection for reactivation of Oct4/Nanog

drug selection

resistant colonies

Oct4-iPSNanog-iPS

Embryonicand adult

fibroblasts

Nanog - neo

Oct4 - neo

Retrovirus mediatedgene transfer

Oct4-iPS

Nanog-iPS

Oct4/Nanogselected iPS cellshave• An ES-like morphology

• Identical molecularcharacteristics

ES cells

Key question:

In vitro reprogrammedcells:

Useful for therapy?Can they differentiate into

functional cells?

Are Oct-iPS and Nanog iPScells pluripotent?

Key criterion:

Can they makemice?

Germ line contribution?

Reprogramming of somatic cellsto pluripotency in the test tube

Sox2

Oct4

Klf4

c-Myc

A single skin cell gives rise to a full embryo upontransduction with the four factors

Reprogramming of somaticcells to pluripotency

Reprogramming process:

Fbx15 NanogOct4

Sequence of stochastic epigenetic events

Partiallyreprogrammed

Fullyreprogrammed

Somaticcell

Pluripotentcell

Sox2Oct4c-mycKlf4

Retroviruses expressed,endogenous Oct4 locus

not reprogrammed

Dnmt3a, b activated,retroviruses silenced,

endogenous Oct4/Nanogloci reprogrammed

In vitro reprogrammedhuman cells:

Two applications

1. Study of complex humandiseases in the test tube

2. Customized therapy

Generation of patient-specific ES cells:Strategies

C. Cowan

D. Melton

D. Melton

D. Melton

ES cell vs. iPS cells:

General implications for humanapplication?

• Does it replace the need for human EScells?

• Do we need nuclear transfer withhuman cells?

Translation to human systems:Issues

1. Screening for reprogrammed cells

2. Same factors as for mouse cellreprogramming?

3. Use of retroviruses, oncogenes: iPScells are genetically altered

• Risk of insertional mutations, cancer

It is crucial to isolate and geneticallymanipulate “normal” human ES cells

In vitro reprogramming:Replacement of need forembryonic stem cells?

Not at all:1. We do not known how long it will take to

achieve reprogramming with human cells

2. Patient specific genetically unmodifiedcell: so far derived only by NT

3. Successful in vitro reprogramming wasbased on work with ES cells, this is thegold standard for pluripotent cells

Why do we need newhuman ES cells?

Cells approved in Germany have to havebeen isolated prior to 2002. Isolationprocedures are known to affect thequality of the cells.

For example we need to knowhow culture conditions and passage

number affect the proliferation anddifferentiation potential of ES cells

The characteristics of ES cells depend on:The culture condition of IVF embryos and the

“history” of in vitro culture conditions

Fertilized Fertilized eggegg

IIII

II

BlastocystBlastocyst

In vitro selection forsurviving cells

ES cell lines

In vitroIn vitro culture: culture:condition Icondition I

Epigenetic /Epigenetic /genetic state Igenetic state I

In vitroIn vitro culture: culture:condition IIcondition II

Epigenetic /Epigenetic /genetic state IIgenetic state II

In vitro reprogramming:

Solution for some problemswith embryonic stem cells?

Some questionsBecause no embryo is involved in derivation of

iPS cells– Same moral value as a Hela cell?

• Does this relieve some of the concerns of working withhuman embryo derived cells?

• Permits/approvals required for certain uses (as with EScells)?

For example:One of the most interesting applicationsof patient specific ES cells:– Derivation of iPS cells from patient with AD or

Parkinson's: differentiation into neural precursors inculture dish

– Could in vitro reprogrammed cells be used forstudying the disease in vivo?

• Introduction of mutant neural precursors intodeveloping mouse brain

In vitro reprogrammed cells:Two applications

1. Study of complex humandiseases in the test tube

2. Customized therapy

Dedifferentiation and differentiation in the test tube:A strategy for cell based therapy

Somatic cellsFibroblasts,

Skin…

“Reprogrammed” ES cell

Cells frompatient

“Customized”cells for therapy

Differentiated cells for transplantationNeurons, Muscle, β cells...

Reprogrammingin petri dish

Differentiationin petri dish

A. MeissnerM. WernigT. BrambrinkR. Foreman

M. KuB. Bernstein

K. Hochedlinger(MGH)

M. KybaG. Daley