Download The Alchemy of Induced Pluripotent Stem Cells

The Alchemy of Induced
Pluripotent Stem Cells
Uma Ladiwala
UM-DAE Centre for Excellence in Basic
Sciences
Kalina Campus, Mumbai
ALCHEMY
Alchemists 300 years ago tried,
unsuccessfully, to turn base
LEAD into valuable GOLD
Cellular Alchemy:
Normally, stem cells give rise to somatic
cells of the adult organism
Recent developments have resulted in
reversing this process with the
production of stem cells from adult
somatic cells, eg. skin cells
These stem cells have been termed
“Induced Pluripotent Stem (iPS)
Cells”
What is a Stem Cell?- Properties
An unspecialized cell with a unique capacity for
- indefinite or prolonged self-renewal and
- ability to give rise to differentiated cells
What is Self Renewal? Differentiation?
Self-renewal - ability to undergo numerous cycles of
cell division while maintaining the
undifferentiated or unspecialized state
Clonality – ability of a single cell to form many
similar cells
Differentiation - process by which a less specialized
cell becomes a more specialized one.
Potency- the potential for differentiation to
specialized cell types
Potency of stem cells
Pluripotent – give rise to cells of all 3
germ layers (ectoderm, endoderm,
mesoderm and germ cells)
Multipotent – ability to differentiate into
many, related cell types
Progenitors –
oligopotent – few cell types
unipotent – one cell type but can self
renew
At the Molecular Level
Stem Cell
Pluripotency genes on
Differentiation genes off
Differentiated Cell
Pluripotency genes off
Differentiation genes on
Where are stem cells found?
Stem cells have been isolated
from the embryo, fetus and
adult
Embryonic stem (ES) cells:
derived from the inner cell
mass of the blastocyst (4-5
day embryo)
Adult stem cells : from adult
tissues
Stem cell types and origins
Adult
Embryonic
Stem cells-types and terminology
Can form all tissues
including placenta
Can form any
embryonic tissue but
not placenta
Division of Stem Cells
A : Stem cell
B : Progenitor cell
C : Differentiated cell
1 : Symmetric division
2 : Asymmetric division
3 : Progenitor cell division
4 : Terminal differentiation
Timeline of Stem Cell Research
1960s - Joseph Altman and Gopal Das present scientific evidence of adult
neurogenesis, ongoing stem cell activity in the brain; their reports are
largely ignored.
1978 - Haematopoietic stem cells in human cord blood.
1981 - Mouse embryonic stem cells are derived from the inner cell mass by
scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail
Martin is attributed for coining the term "Embryonic Stem Cell".
1996 - Cloning of Dolly the sheep by somatic cell nuclear transfer
1997 - Leukemia is shown to originate from a haematopoietic stem cell, the
first direct evidence for cancer stem cells.
1998 - James Thomson and coworkers derive the first human
embryonic stem cell line
2000s - Several reports of adult stem cell plasticity
2004-2005 - Korean researcher Hwang Woo-Suk human
embryonic stem cell line from unfertilised human oocytes
by SCNT. The lines were later shown to be fabricated.
August 2006 – Mouse Induced pluripotent stem cells: the journal
Cell publishes Takahashi and Yamanaka’s work.
What’s so special about Stem Cells?
They have the potential to replace cell
tissue that has been damaged or
destroyed.
They can replicate themselves over and
over for a very long time- nearly
inexhaustible source
Understanding how stem cells develop into
healthy and diseased cells will assist the
search for cures.
Drugs and chemicals can be screened and
tested on patients’ stem cells and their
differentiated tissues
Embryonic Stem (ES) cells
Derived from inner cell
mass of blastocyst
Capacity for almost
unlimited
symmetrical
divisions without
differentiation
Give rise to endoderm,
ectoderm, mesoderm
Clonogenic- derived
from a single ES cell
Capable of colonizing
germline and forming
egg and sperm cells
Cultivation of ES cells
Characterization of human and
mouse ES cells
Expression of cell surface markers –SSEA-3,SSEA-4
(hESC) SSEA-1 (mESC),TRA-1-60, TRA-1-81,
alkaline phosphatase, GTCM-2
- Pluripotency transcription factors – Oct-4, Sox-2,
Nanog, Rex1
- High Telomerase activity
- Karyotype- normal (46 XX or XY)
- In vitro pluripotency- embryoid body formation
- Teratoma formation in immune-incompetent micetumour contains tissues from all 3 germ layers
- Pluripotency in vivo – Chimera formation (mESC)
Derivation and Characterization of human
ES cells
AP
Oct4
TRA-1-60
TRA-1-81
Chen et al, 2007
SSEA3
SSEA4
Differentiation of human ES cells
In-Vitro
In-Vivo
EB
Neuronal cells
Intestine
Cardiac
Mesoderm
AFP
Germ
Respire
Sk muscle
Ova
Chen et al, 2007
Cartilage
http://www.isscr.org/video/beatingMyocytes.mpg
Neural tube
Embryonic or Adult Stem Cells for Cell
ReplacementTherapy: Advantages and
Disadvantages
Embryonic SC
“Pluripotent”
Adult SC
“Multipotent”
Stable. Can undergo many
cell divisions.
Less Stable. Capacity for
self-renewal is limited.
Easy to obtain but
blastocyst is destroyed
(Ethics)
No ethical concerns
Difficult to isolate in adult
tissue.
Possibility of immune
rejection
Host rejection minimized
or absent
High potential for tumours
Less tumorigenic potential
The Ideal Stem cell – the “Holy
Grail” of Cell Replacement Therapy
- Ability to differentiate into many cell types
- Easily accessible
- Individual-specific i.e. personalized or nonimmunogenic
- Vastly renewable
- Demonstrably safe
- Non-tumorigenic
The Induced Pluripotent
Stem (iPS) Cell : A Likely
Candidate?
Re-programming the nucleus
Stem cell
Differentiation is not an irreversible commitment
Differentiated cell
Stem cell
Nuclear reprogramming - functional or molecular changes
in cells undergoing fate changes
Reprogramming by somatic cell
nuclear transfer and cell fusion
Dolly the Sheep
Transcription factors for
reprogramming
Transcription factors are proteins that
bind to DNA and regulate gene
expression
Oct3/4 and Sox2: transcription factors
that function in maintaining
pluripotency in both early embryos and
ES cells.
c-Myc and Klf4: transcription factors that
modify chromatin structure so that
Oct3/4 and Sox2 can bind to their
target; proto-oncogenes
The making of iPS cells
Cell trapping strategy: selection of
Fbx15-neomycin-resistant cells
What is fbx15 ? - a transcription
factor in ES cells and early embryo
but not essential for maintainence
of pluripotency
Takahashi and Yamanaka, Cell, Aug 25, 2006
24 candidate
genes for
pluripotency
factors:
Ecat1,
Dpp5(Esg1),
Fbx015,
Nanog, ERas,
Dnmt3l, Ecat8,
Gdf3, Sox15,
Dppa4, Dppa2,
Fthl17, Sall4,
Oct4, Sox2,
Rex1, Utf1,
Tcl1,
Dppa3, Klf4,
b-cat, cMyc,
Stat3, Grb2
Takahashi and Yamanaka, Cell, Aug 25, 2006
Takahashi and Yamanaka, Cell, Aug 25, 2006
Takahashi and Yamanaka, Cell, Aug 25, 2006
Were these iPS cells identical to the
ES cells?
NO
- The transcriptional profile was somewhere
between fibroblasts and ES cells
- No live chimeras produced
So these iPS cells were somewhat similar but
not identical to ES cells
WHY?
Because fbx15 was selected for. Fbx15 is a
factor that is expressed in ES cells but is not
essential for the maintainence of pluripotency
Is there a way to improve this and
get ES –like iPS cells?
Okita K et al, Nature, 2007
Nanog-selected iPS cells
-Expressed all markers and
characteristics of ES cells
-Chimera formation when injected into
blastocysts
but
20% of the mice developed tumours
Proposed explanation for the
difference
Reprogramming: 2-stage process
Vit C
ESC morphology
Some pluripotent
markers
Loss of somatic
markers
LIF unresponsive
No chimeras
Germline incompetent
ESC morphology
All pluripotent
markers
No somatic
markers
LIF responsive
Chimera forming
Germline
competent
Mechanism of ES cell pluripotency
Oct4, Sox2m and Nanog form an interconnected autoregulatory network
Proposed mechanism of iPS cell
reprogramming
Exogenous Oct4 and
Sox2 reactivate
endogenous Oct4,
Sox2 and Nanog and
the auto-regulatory
loop then becomes
self-sustaining.
Exogenous factors are
silenced by
DNA methylation
Scheper, Copray,
2009
Induced pluripotency : the two-stage
switch
Stage 1
Stage 2
Downregulation of lineage genes
Activation of auto-regulatory loop
Activation of specific ES genes
Full reactivation of ES cell
transcriptional network
Chromatin remodelling
Completion of transgene silencing
iPS Cells- Starting cells
Mouse
Embryonic fibroblasts
Adult tail fibroblasts
Hepatocytes
Gastric epithelial cell
Pancreatic cell
Neural stem cell
B lymphocyte
Keratinocyte
Human
Skin fibroblast
Keratinocyte
Bone marrow stem cell
Peripheral blood cell
Efficiency of re-programming is poor
Hochdelinger and Plath, 2009
Derivation of human iPS cells
In human cells efficiency of reprogramming ranges
between 0.02% to 0.002%
Potentials of iPS cells
- Ability to differentiate into many cell
types
- Easily accessible
- Individual-specific i.e. personalized or
non-immunogenic
- Vastly renewable
- Useful for studying mechanisms of
disease
- Useful for drug, toxicity testing
iPS cell reprogramming: Problems
Use of viral vectors for induction
Low efficiency of reprogramming
Risk of tumour formation
Efficient differentiation protocols required
Further work towards “safer” and more
efficient generation of iPS cells
Reduced number of transcription factor
used:
No myc: Nakagawa and Yamanaka, Nat Biotechnol 2008,
Wernig and Jaenisch, Cell Stem Cell 2009
No Sox2: by adding GSK-3 inhibitor, Zhou and Ding,
Stem cell 2009, in neural stem cell, Kim and Scholer
Nature 2008
No Klf4/myc, by addition of Valproic acid : Huangfu and
Melton, Nat Biotech 2008
No Myc and Sox2, by addition of BIX01294 and
PD0325901 (Zhou and Ding, Cell Stem Cell 2008).
Klf4 only by adding Kenpaullone (Lyssiotis and Jaenisch,
PNAS 2009)
Specific pathways:
TGF-β inhibitor replaces Sox2 and cMyc and
induce Nanog (Maherali and Hochedlinger,
Curr Biol 2009, Ichida and Eggan 2009 )
p53 inhibition augments iPS efficiency (Hong
and Yamanaka, Nature 2009,Utikal and
Hochedlinger Nature 2009, Marion and
Blastco Nature 2009, Li and Serrano Nature
2009, Kawamura and Belmonte 2009)
Hypoxia stimulates iPS generation – Yoshida and
Yamanaka Cell Stem Cell 2009
Wnt signaling stimulates reprogramming
efficiency (Marsonm, Jaenisch Cell Stem Cell
2008)
Better vectors:
Drug Inducible vectors (Wernig and Jaenisch, Nat
Biotechnol 2008, Hockemeyer and Jaenisch, Cell
Stem Cell 2008)
Non-integrating vectors adenovirus in hepatocyte
(Stadtfeld and Hochedlinger, Science 2008)
Multi-cistronic vectors: single lentiviral cassette ( Carey
and Jaenisch, PNAS 2009, Sommer and Mostoslavsky,
Stem Cell 2009)
Vector free (episomes, Yu and Thomson, Science 2009;
direct transfection, Okita and Yamanaka Science
2008)
Direct protein induction: poly arginine modification of
recombinant protein (Zhou and Ding, Cell Stem Cell
2009),
Parallels between regeneration and
reprogramming
Natural dedifferentiation occurs during
regeneration in teleost fish, amphibians
C-Myc, Sox2, Klf-4 expressed during limb
regeneration in newts (Maki et al, 2009)
Oct4, Sox2 required for normal fin
regeneration in zebrafish, but levels not as
high as in pluripotent cells (Christen et al,
2010)
If iPS cells are shown to be safe,
non-tumorigenic and
efficiently differentiated
then
“Lead will be turned into Gold”
Work Plans-overview
Generation of adult human neural stem cells and
differentiated progeny from adult somatic cells by
non-retroviral reprogramming
(Collaborator: Dr. Jacinta D’Souza)


MEFs
Adult human fibroblast/keratinocyte
iPS cell or pre-iPS cell better ?
Can pre-iPS cells give rise to multipotent stem cells?
Most efficient method for induction?
Efficient differentiation ?

Three-dimensional cultures on synthetic scaffolds
Thank You