Download Embryonic stem cells

The Pluripotency:
Lessons from
Embryonic Stem Cell
Properties
M. Saifur Rohman, M.D., Ph.D.
Cardiologist
Outline
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Regenerative medicine
Historical Perspective
Stem Cells
Embryonic Stem Cells (ESC)
Pluripotency induction (iPS): A future challenge
Adult Stem Cells (ASC): A comparison
The use stem cell in medicine
Regenerative Medicine
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Regenerative medicine aims to repair diseased or
damaged tissues by replacing the affected cells with
healthy, functional cells of the same type.
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The prospects of this discipline have been boosted by
the promise of ES cells, which are pluripotent (they
can differentiate into any cell type) & which can be
maintained in culture to “self-renewal” indefinitely.
Blelloch, Nature 2008.-
Historical Perspective
• Major Changes in regenerative medicine (replacement
of damaged or diseased cell tissues with new cells and
tissues) due to advances in stem cell technologies
• First successful bone marrow transplant done in1956
on leukemic patient. Bone marrow contains adult
derived hematopoietic stem cells, able to regenerate
tissues similar to the specialized tissues in which they
are found.
• Embryonic stem cells believed to have greater
potential. This line of stem cell research has been the
most controversial.
Historical Perspective
Isolation & Culture of Embryonic Stem Cells
(Human-1998; Mouse-1981)
Method patented
U.S. patent held by Univ.
Wisconsin
J. Thomson 1998
Advantages:
1) Proliferate indefinitely
2) Form any tissue
Why self-renew and differentiate?
1 stem cell
1 stem cell
Self renewal - maintains
the stem cell pool
4 specialized cells
Differentiation - replaces dead or damaged
cells throughout your life
Stem Cells: Definition
• Stem cells: Primitive cells that have the capacity for
extensive self-renewal, clonogenic, and the ability to
differentiate into multiple cell types
• Embryonic stem cells: Totipotent (pluripotent) cells
derived from the inner cell mass of the blastocyst;
they give rise to cells of all three germ layers
• Adult stem cells: present in all renewing tissues;
these cells divide for self-renewal and differentiate
into multiple progenitor cell types.
Stem Cells: Definition
• Progenitor cells: multipotential intermediate stem
cells that serve as the direct precursors for
tissue-specific mature cells.
• Endothelial progenitor cells: cells that are
present in blood and bone marrow; they are
involved in angiogenesis and postnatal
neovasculogenesis.
Stem Cells: Definition
• Mesenchymal stem cells: also referred to as
marrow stromal cells; these cells differentiate in
vitro along multiple pathways that include
cardiac myogenesis.
• Hemangioblasts: primitive embryonic cells that
give rise to both hemopoeitic stem cells and
endothelial progenitor cells; they may also exist
in adult bone marrow.
Mammalian development
Zygote
Oocyte
Developmental
potential
Sperm
Totipotent
Trophoblast
(extraembryonic)
Inner cell
mass
Blastocyst
Embryonic
Stem cells
Pluripotent
(in vitro)
Epiblast
Primitive
Multipotent
Primitive streak
Endoderm
(lung, liver,
pancreas, etc.)
Mesoderm
Ectoderm
(blood, heart, bone,(central and peripheral
skeletal muscle, etc.) nervous system,
epidermis, etc.)
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Emerging Technol Platform for SCs, 2010.-
Stem Cells
• Embryonic Stem Cell
• Adult Stem Cells
Human development : Stem cell perspective
totipotent
Loose definition
Strict definition
pluripotent
Generates every cell in the body including
the placenta and extra-embryonic tissues
Can form the entire human
being
Cannot form the entire
human being
Can generate every cell in the body except
placenta and extra-embryonic tissues
Become specific cell
types; may or may not
have plasticity
Characteristic of Stem Cells
• Undifferentiated cells with the capacity for
unlimited or prolonged self renewal that can give
rise to differentiated cells
• Metaplasia- the formation of one differentiated
cell type from another
• Slow cycling in cell division
• Contact-insensitive; deficient in gap junction
intercell. communication
• Specific gene expression
Embryonic Stem Cells (ESC)
Researchers extract stem cells from a 5-7 days old blastocyst.
Stem cells can divide in culture to form more of their own kind,
thereby creating a stem cell line.
The research aims to induce these cells to generate healthy tissue
needed by patients.
SCAN – Stem Cell Action Network
Properties of Human ESC in Culture
• Pluripotent- able-to form any of 200 different
types of cells of the body
• Self renewing in vitro- can propagate or
proliferate indefinitely in the undifferentiated
state
• Express the enzyme telomerase (required to
maintain the end of chromosomes) and Oct4 ( a
master regulator of ESC pluripotency)
• Maintain normal chromosome structure and
complement even after long periods in culture (
unlike many other tissue cell lines)
Characteristics of Human ESC
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Normal Karyotypes
Express high telomerase activity
Express cell surface markers of primate ESC
Maintained undifferentiated proliferation for
4-5 months and development potential to
form thropoblast and, endoderm, mesoderm,
ectoderm
• Developed teratomas in immune deficient
mice
ESC: What they can do
differentiation
embryonic stem cells
PLURIPOTENT
all possible types of specialized cells
ESC: Challenges
skin
neurons
embryonic stem cells
blood
?
liver
Two Sources of
Embryonic Stem Cells
1. Excess fertilized eggs from IVF (in-vitro
fertilization) clinics
2. Therapeutic cloning (somatic cell nuclear
transfer)
SCAN – Stem Cell Action Network
Tens of thousands of
frozen embryos are
routinely destroyed
when couples finish their
treatment.
These surplus
embryos can be used to
produce stem cells.
Regenerative medical
research aims to
develop these cells into
new, healthy tissue to
heal severe illnesses.
SCAN – Stem Cell Action Network
Somatic Cell Nuclear
Transfer
The nucleus of a donated
egg is removed and
replaced with the nucleus
of a mature, "somatic cell"
(a skin cell, for example).
No sperm is involved in
this process, and no
embryo is created to be
implanted in a woman’s
womb.
The resulting stem cells
can potentially develop into
specialized cells that are
useful for treating severe
illnesses.
SCAN – Stem Cell Action Network
Pluripotency signature
• Pluripotent – distinct cellular marker and
functions
• Factors that are expressed in somatic cells
or tissue specific genes must be shut
down
• Expression of genes for pluripotency
associated factors : octamer binding
transcription (Oct4) and Nanog must be
initiated
Review : Epigenetic ?
Human ESC remain embryo because
of epigenetic factors
• Molecule central that
balancing act –
H3K4me3 &
H3K27me3
• Genes that modified
only by H3K4me3 
contain DNA recipe to
proliferate
• Genes that do not
carry both 
completely silenced in
ES
Science daily, Oct,8,2007
Embryonic stem cells regulations
• Maintained by 3
TF genes :
– Oct4
– Sox2
– Nanog
Specific mechanism
required to disrupt 
differentitation ( GCNF –
Germ Cell Nuclear factor)
• Feed-forward &
feedback  maintain
pluripotent gene
expression
• Oct4 – important TF regulate a pluripotent
gene expression in early
embryonic development
• During differentiation – the
expression is down
regulated
Yamanaka et al, Jaenish et al; Thomson et al,
1998
Oct-4 (octamer-binding transcription factor 4)
• Oct-4 (octamer-binding transcription factor 4)
also known as POU5F1 (POU domain, class 5,
transcription factor, is a protein that in humans is
encoded by the POU5F1 gene
• This protein is critically involved in the selfrenewal of undifferentiated embryonic stem
cells. As such, it is frequently used as a marker
for undifferentiated cells.
• Oct-4 expression must be closely regulated; too
much or too little will actually cause
differentiation of the cells.
Oct4
• Upon differentiation  Oct4 is non
expressed
• GCNF is high expressed
• Methylation of Oct4 gene and histone
modifications  silencing Oct4 gene during
differentiation
• Loss of DNA methylation & chromatin
remodelling  no effect of repression
Oct4
• DNA methylation machinary :
DNA Methyltranferase &
Methyl DNA binding domain protein (MBDsMBD1 & MBD2)  MBD2 binds to CpG
dinucleotide (CpG" is shorthand for "—C—
phosphate—G—", that is, cytosine and guanine
separated by a phosphate)
MBD3 bind to unmethylated CpG
dinucleotide
Oct4 and Nanog
Cells
ESC manage their pluripotent status by
PcG mediated repressive histone lock
Lysine methylation  recrut spec.Binding prot HP1 to H3 lysine 9
Histone code
PRC1 (Protein regulator of cytokinesis 1)
t o methylated Histon H3 lysine 27
Acetylation  nucleosome looser, > accesible to transcription factors
Spivakov & Fischer, Nature, April 2007
Polycomb-group proteins
Pluripotency properties
• Embryoid body ) ball like embryo)embryoid bodies
• Chimeric mouse
• Promoter demethylationof oct3/4, Nanog
and Rex1,
• Histon demethylation
From pluripotent to differentiated cells
• Neural differentiation : AADC, DAT, ChAT,
LMX1b, MAP; b-tubulin, tyrosine hydrolase
• Cardiac differentiation : TnTc, MEF2C,
MYL2a, MyhcB , NKX2.5
• Teratoma formation – teratoma is landmark
for pluripotent
ESC in the lab, an example : mPer2 conditional
knock out generation
SalI
SacI-bl
Intron2
Exon2
Pgk-neo
SalI KpnI-bl
lox FLP
FLP lox
Saifur Rohman, et. Upublished data,
DT-A
Stem cell selection and implantation
Potential embryonic stem cell problems:
• Difficult to establish and maintain
• Difficult in obtaining pure cultures in the dish
• Potential for tumor formation and tissue destruction
• Questions regarding functional differentiation
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*Hansson M et al., Diabetes 53, 2603-2609, 2004
*Sipione S et al., Diabetologia 47, 499-508, 2004
*Rajagopal J et al.; Science 299, 363; 2003
*Zhang YM et al.; Circulation 106, 1294-1299; 2002
The Ethical Debate
In favor of ESCR:
Embryonic stem cell research (ESCR) fulfills the ethical
obligation to alleviate human suffering.
• Since excess IVF embryos will be discarded anyway, isn’t it
better that they be used in valuable research?
• SCNT (Therapeutic Cloning) produces cells in a petri dish,
not a pregnancy.
Against ESCR:
In ESCR, stem cells are taken from a human blastocyst,
which is then destroyed. This amounts to “murder.”
• There is a risk of commercial exploitation of the human
participants in ESCR.
• Slippery slope argument:
ESCR
will
lead to reproductive
SCAN – Stem
Cell Action
Network
cloning.
Cellular Plasticity
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The discovery of mammalian cellular plasticity
raises the possibility of reprogramming restricted
cell fate, & may provide an alternative to many of
the obstacles associated with using embryonic &
adult stem cells in clinical applications.-
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With a safe & efficient dedifferentiation process,
healthy, abundant & easily accessible adult cells
from a given individual could be used to generate
different functional cell types to repair damaged
tissues & organ.Lyssiatis et al, Emmerging Techno Platform for SCs, 2009.44
Induced pluripotent stem cells (iPS)
Pluripotent stem cell artificially
derived from a non pluripotent cell
by inducing a “forced “
expression of certain genes
Yamanaka et al, 2006, 2007,
Thomson et al, 2007, 2008
Induced Pluripotent Stem (iPS) Cells
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Yamanaka : cell lines with some of the properties of
ES cells by introducing just four transcription factors
associated with pluripotency – Oct3/4, Sox2, c-Myc
& Klf4 – into mouse skin fibroblast then selecting
cells that expressed a marker of pluripotency, Fbx15,
in response to these factors, these cells were called
iPS cells.-
Rossant, Nature 2007.-
Induced pluripotent stem cells (iPS cells)
‘genetic reprogramming’
= add certain genes to the cell
adult cell
induced pluripotent stem (iPS) cell
behaves like an embryonic stem cell
differentiation
culture iPS cells in the lab
Advantage: no need for embryos!
all possible types of
specialized cells
Induced pluripotent stem cells (iPS cells)
genetic reprogramming
pluripotent stem cell
(iPS)
adult cell (skin)
differentiation
Properties of iPS identic to natural
pluripotent stem cells
• Expression of certain stem cell genes and
protein
• Chromatin methylation patterns
• Doubling time
• Embryoid body formation
• Teratoma formation
• Viable chimera formation
• Potency differentiability
Identity of iPS
• Morphology : similar to ESC, sharp edged, flat,
tightly pcked
• Doubling time – same
• Stem cell markers: SSEA-3, SSEA-4, TRA-1-60,
TRA – 1 -81, TREA -2-49/6E, Nanog
• Telomerase activity:
• Stem cell genes: oct3/4, Nanog, GDF4, REXi, FGF4,
ESG1, DPPA2, DPPA4 and hTERT
Methode iPS - transfection
• Retrovirus ( Yamanaka et al, 2006)
• Mouse fibroblast  into iPS – by retroviral, 2007
– Harvard Team
• iPS from adult human (James Thomson,
Junying yu et al, 2008
• Adenovirus to transport 4 genes ( Konrad
Hochedlingler – Harvard Universitym 2008)
• Yamanaka 2009 – iPS without retrovirus but
plasmid
Genes of induction
1. Oct 3 , 4 - maintain pluripotency
2. Sox family – maintain pluripotency
3. Klf family – ( Thomson et al, Yamanaka )
controversial
4. Myc family – proto-oncogene
5. Nanog + Oct 3 , 4  promoting pluripotency
6. LIN28 : mRNA binding protein
Yamanaka et al, 2006, 2007, 2009
James Thomson 2006, 2007
Molecular mechanism of iPS generation
Molecular mechanism of iPS generation
Plasmid for cell reprogramming
iPS Cells generation steps
IPS cell generation ways
Application in animal
The use of iPS cells using patient fibroblasts
 Parkinson’s disease (Wernig and Jaenisch,
2008, Maehr and Melton PNAS 2009).
 Amyopathic Lateral Sclerosis, (Dimos and Eggan
Science 2008)
 Type I diabetes (Maehr and Melton PNAS 2009)
 Duchenne and Becker Muscular dystrophin,
Parkinson’s disease, Huntington disease, Down
syndrome, Lesch-Nyhan syndrome. (Park and
Daley Cell 2008).
The use of iPS cells using patient fibroblasts
• Blood cells (Loh and Daley 2009). B-cells
(Hanna and Jaenisch Cell 2008)
• Blood stem cells (Emiinli and Hochedlinger Nat
Genet 2009)
• Pancreatic b-cells (Stadtfeld and Hochedlinger
Boenjamin Setiawan, dr.,PhD
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Cell Stem Cell2008)
• Hepatic and gastric endoderm (Aoi and
Yamanaka Science 2008)
• Neural stem cells (Kim and Scholar, Nature
2008)
Adult stem cells
– Found in adult tissue
– Can self-renew many times
– Multipotent – they can differentiate to become
only the types of cells in the tissue they come
from.
• hematopoietic stem cells – give rise to blood
cells
• mesenchymal stem cells – give rise to cells
of connective tissues and bones
• umbilical cord stem cells – a rich source of
hematopoietic stem cells
• stem cells found in amniotic fluid – might be
more flexible than adult stem cells
Differentiation pathway
The differentiation pathways of adult stem cells.
– Hematopoietic stem cells give rise to all types
of blood cells: red blood cells, B lymphocytes, T
lymphocytes, natural killer cells, neutrophils,
basophils, eosinophils, monocytes, macrophages,
and platelets.
– Bone marrow stromal cells (mesenchymal stem
cells) give rise to a variety of cell types: bone
cells (osteocytes), cartilage cells (chondrocytes),
fat cells (adipocytes), and other kinds of
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connective tissue cells such as those in tendons.
Where are adult stem cells found and
what do they normally do?
• An adult stem cell is an undifferentiated cell
found among differentiated cells in a tissue
or organ, can renew itself, and can
differentiate to yield the major specialized
cell types of the tissue or organ.
Where adult stem cells are found?
Adult stem cells have been identified in many organs and
tissues. However, there are a very small number of stem
cells in each tissue.
• Stem cells are thought to reside in a specific area of each
tissue where they may remain quiescent (non-dividing,
STEM CELL NICHE) for many years until they are
activated by disease or tissue injury.
• The adult tissues reported to contain stem cells include
Brain, Bone marrow, Peripheral blood, Blood vessels,
Skeletal muscle, Skin, hUCB, Umbilical Cord, Amniotic
liquid, Adipose Tissue, liver etc.
Therapeutic Challenge
Embryonic
stem cells
Germ layers &
Tissue
differentiation
Reprogrammed
Adult stem cells
Tissue stem cells
Mesoderm
Ectoderm
Endoderm
Tissue
stem cells
Neurones
Muscle
Blood cells
Lung/Gut/Liver
DNA
transfection
Tissue
Engineering
Cell Transplantation
Gene Therapy
SC Technol, Basic Applic 2010.-
e.g., Neuronal bundles or
Pancreatic Islets, etc.
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