Induced Pluripotent Stem

Cells (iPSC)

Stem Cells
Stem cells are master cells with two important
characteristics
Unspecialized cells capable of their own renewal
Ability to differentiate into different cell types

The stem cells may have various differentiation

potentials

Totipotent
Pluripotent
Multipotent
Unipotent

Pluripotent Stem Cells

Types of Pluripotent Stem

Cells
Embryonic stem cells
Embryonic carcinoma cells
Embryonic germ cells

Origins of pluripotent cells

Embryonic Carcinoma Cells

Teratomas

Adult

Fetus

Abnormal karyotypes,
Germline transmission unlikely

Zygote

Blastocyst

Primordial germ Cells Inner cell mass (ICM)

Embryonic Germ Cells

Embryonic Stem Cells

Undergo spontaneous
Pluripotent cells
differentiation
Partially
Unable to support normal
understood
development
due to epigenetic modifications Great potential
during PGCs

Criteria for pluripotency

Immortality
Undifferentiation
Clonality
Broad developmental potential

Demonstration of
pluripotency

In vitro
a) Differentiation triggered when grown in suspension,
Embryoid Body formation
b) Different cells obtained spontaneously
c) Specific growth factors can be used to direct the
differentiation of ES cells into specific cells

In vivo
a) Teratoma formation when injected into nude
mouse
b) When injected into host blastocysts, the ES cells
integrate, proliferate and differentiate into all
germ lineages including germ cells

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)
Possibility of immune
rejection
High potential for tumours

No ethical concerns
Difficult to isolate in adult
tissue.
Host rejection minimized or
absent
Less tumorigenic potential

Regulation of pluripotency in ES cells

Pluripotency is maintained by
promotion of proliferation and
Inhibiting differentiation

Epigenetic modifications
ES Cells pluripotency

Transcription Factors

Stage specific
functioning

Require other
processes

Target genes/
receptors required

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

Factors Required
Nanog
Oct 3/4
Sox 2
LIF
c-Myc
Klf4
Zic3

Oct3/4
POU-domain transcription factor
Maintains pluripotency (ESCs, EGCs, ECCs, GSCs)
Tightly regulated transcription factor, associated with a number of
target genes implicated in pluripotency maintenance
Regulatory elements in target genes are in close vicinity of Sox2binding sites
Key factor in the transcriptional framework of self-renewing stem
cells

Sox2
Member of HMG-domain DNA-BP family
Necessary for embryonal development and to prevent ES cell
differentiation
Many ES cell pluripotency-associated genes are co-regulated
by Sox2 and Oct3/4
A ternary complex formed with Oct4 or Oct1 on enhancer
sequence of Fgf4 is must for functioning
Cooperate with other TFs, e.g. Nanog to activate transcription
of pluripotency markers

Nanog
Transcription factor containing homeobox domain
Downstream effectors of signals of LIF and BMP
Elevated levels excludes inclusion of LIF and feeder layer
Works with other key factors including Oct4 and Sox2

Klf4
Member of the quartet, a Kruppel-type zinc finger
transciption factor
Can act as an oncogene and as a tumor suppressor protein
Over expression inhibits differentiation of ES cells
Klf4 upregulates, in concert with Oct3/4, but the role as cofactor for Oct3/4 may be limited to only a few targets
Can repress p53, a negative regulator of Nanog

Zic3
Oct4

Sox2

Nanog

Zic3

Sox17, PDGFRA
Gata4, Gata6
Foxa2, Sox7

Endoderm

Zic3 contributes to the maintenance of pluripotency by operating

downstream of Oct4, Nanog, and Sox2 to inhibit endoderm
lineage specification as characterized by endodermal markers
Sox17, PDGFRA, Gata4, Gata6, Foxa2, and Sox7
The presence of Zic3 also maintains the expression of the
homeodomain protein Nanog, a key regulator of pluripotency in
embryonic stem cells

c-Myc
Helix-loop-helix/leucine zipper transcription factor
Takes part in a variety of cellular functions
Downstream effecter of STAT3 in LIF receptor signaling
pathway
c-Myc is a substrate for GSK3b in Wnt signalling pathway
Compensates anti-proliferative effects of Klf4, e.g. in iPS
cells

Leukaemia inhibitory factor,

LIF
Interleukin-6 cytokine family
Essential for maintaining pluripotency in vitro in the presence
of serum
Binds to a heterodimeric receptor comprising of LIF-receptor
(LIFR) and gp130 on cell membrane
Binding results in the activation of Jak/ Stat signal
transduction pathway
Activated Stat3 maintains pluripotency

Combinatorial signaling pathways

(involving LIF and master regulator genes)
in maintaining mouse ESC pluripotency

Boiani and Scholer, 2005

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

Pluripotent lineages in the mouse embryo

ll

Pluripotent cells form the ICM of the blastocyst

After giving rise to the primitive endoderm on the surface
of the ICM, pluripotent stem cells form the
epiblast and start to proliferate rapidly after implantation
They then form the primitive ectoderm, a monolayer
epithelium that has restricted pluripotency
which goes on to give rise to the germ cell lineage and to
the somatic lineages of the embryo
Certain key transcription factors (blue) are required for
the differentiation of the various embryonic
lineages

Differentiation of mouse ES cells

Differentiation of mouse ES cells

ES cells differentiate into three cell types
primitive endoderm
trophectoderm (TE)
primitive ectoderm
mimicking the differentiation potential of pluripotent stem cells in
preimplantation embryos
In the absence of LIF and in the presence of an excess of Oct3/4,
ES cells differentiate into primitive endoderm-like cells
In the absence of Nanog and in the presence of Gata6, they
differentiate into parietal endoderm-like cells
Removing Oct3/4 and adding Cdx2 to, ES cell culture induces TElike differentiation.
MEFc, mouse embryonic fibroblast conditioned medium

Regulation of proliferation of mouse ES

cells

Regulation of proliferation of mouse ES

cells
Pluripotent transcription factors activate the
expression of certain effectors that drive
ES cell proliferation
Eras and Tcl1 stimulate the phosphoinositide-3kinase (PI3K)/Akt signaling pathway to
promote the cell cycle, whereas b-Myb and
cMyc activate the progression of the cell
cycle
directly

Epigenetic regulations of ES
cells pluripotency

Characteristics of the pluripotent

epigenome

The nucleus shrinks and the distribution of

electrondense areas (mainly heterochromatin)
changes dramatically when ES cells are induced to
differentiate into primitive endoderm by the ectopic
expression of Gata6

Epigenetic features of the pluripotent

cell nucleus

Small regions of perinuclear heterochromatin exist, but most of

the chromatin exists as euchromatin, bearing histone marks
associated with transcriptional activity
The hyperdynamics of chromatin proteins (green) might contribute
to the maintenance of euchromatin
Bivalent domains are also a feature of the pluripotent epigenome,
in which active histone marks (such as H3K4me) are flanked by
transcriptionally repressive histone marks (such as H3K9me)

Transcriptional regulation of the mouse

Oct3/4 gene
A