Embryonic Stem Cells and Therapy:
Transcript of Embryonic Stem Cells and Therapy:
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Embryonic Stem Cellsand Therapy:
Promise, Problems, Reality
Rudolf JaenischWhitehead Institute and
Department of Biology, MIT
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D. Melton
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D. Melton
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D. Melton
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D. Melton
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Melton 13Stem cells are crucial for tissue homeostasis
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D. Melton
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Melton 20
Pluripotent cellsMultipotent /
oligopotent cellsTotipotent cell:
The fertilized egg
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Melton 22
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Melton 21
Pluripotent cells Multipotent /oligopotent cells
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D. Melton
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What is the goal stemcell research?
Using the potential of ES cells
To provide matched cells for“customized” tissue repairin degenerative diseases
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D. Melton
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D. Melton
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Year
Publications on stem cellsover the last 20 years
HumanES CellsIsolatedMouse
ES CellsIsolated
The interest in stem cells has grown exponentially
G. Daley
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the quest for eternal youththe quest for eternal youth
Induction ofInduction of““PluripotencyPluripotency””
andand
Lucas Cranach, 1472-1553
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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
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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
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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”
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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
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Problems withTherapeutic SCNT
1. Procedure too inefficient, costly forroutine treatment
2. Ethical objections to using humaneggs for therapy
Other options?
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Alternatives to embryonic stemcells?
Do we need to use SCNT togenerate “customized”ES cells for therapy?
or
Such as Adult Stem cells?
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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
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Melton 21
Pluripotent cells Multipotent /oligopotent cells
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Generation of patient-specific ES cells:Strategies
C. Cowan
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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
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“Wiring Diagrams” of Developmental Processes
Davidson, et al. Science. 2002 Rothenberg and Anderson. Dev. Biol. 2002
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Sox2
Oct4
Nanog
PcG proteins
ES cell
self-renewal
differentiatedcell
Regulation of Pluripotency inEmbryonic Stem Cells
Useful for induction of reprogramming?
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A. MeissnerM. Wernig
R. Foreman
T. Brambrink
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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
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Oct4-iPS
Nanog-iPS
Oct4/Nanogselected iPS cellshave• An ES-like morphology
• Identical molecularcharacteristics
ES cells
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Key question:
In vitro reprogrammedcells:
Useful for therapy?Can they differentiate into
functional cells?
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Are Oct-iPS and Nanog iPScells pluripotent?
Key criterion:
Can they makemice?
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Germ line contribution?
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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
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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
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In vitro reprogrammedhuman cells:
Two applications
1. Study of complex humandiseases in the test tube
2. Customized therapy
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Generation of patient-specific ES cells:Strategies
C. Cowan
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D. Melton
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D. Melton
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D. Melton
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ES cell vs. iPS cells:
General implications for humanapplication?
• Does it replace the need for human EScells?
• Do we need nuclear transfer withhuman cells?
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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
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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
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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
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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
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In vitro reprogramming:
Solution for some problemswith embryonic stem cells?
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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
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In vitro reprogrammed cells:Two applications
1. Study of complex humandiseases in the test tube
2. Customized therapy
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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
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A. MeissnerM. WernigT. BrambrinkR. Foreman
M. KuB. Bernstein
K. Hochedlinger(MGH)
M. KybaG. Daley