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Dissecting self-renewal in stem
cells with RNA interference
Natalia Ivanova, Radu Dobrin, Rong Lu, Iulia Kotenko, John Levorse,
Christina DeCoste, Xenia Schafer,Yi Lun & Ihor R. Lemischka
Nature 442, 533-538 (3 August 2006(
Stem cells
In a developing embryo, stem cells can
differentiate into all of the specialized
embryonic tissues
 In adult organisms, stem cells replenish
specialized cells, and maintain the normal
turnover of regenerative organs (blood,
skin, intestinal tissues etc.)
 They can divide indefinitely, in contrast to
progenitor cells that also participate in
tissue repair

Embryonic stem cells

Embryonic
Most
adult
stemstem
cells are
cells
pluripotent
are lineage
restricted (multipotent)
Therapeutic potential


Therapies are currently in experimental
stages
(except
for
bone
marrow
transplantation)
One possible risk is that transplanted stem
cells could form tumors if cell division
continues uncontrollably
Somatic cell nuclear transfer

Breaking up human embryos offends
some people’s moral sensibilities
Induced pluripotent stem cells

Yamanaka and Takahashi showed that
pluripotency can be induced, first in
mouse fibroblasts and then in human
facial skin cells
Takahashi and Yamanaka, Cell 2006
Yamanaka factors
The Yamanaka team used the transcription
factors Oct4, Sox2, Myc, and Klf4
 Myc sometimes causes cancer as a sideeffects
 Yu et al. showed that a similar result can be
obtained using the TFS Oct4, Sox2, Nanog
and Lin28


They used human foreskin cells
iPSCs replace somatic cell nuclear
transfer
Ian Wilmut
announced that he
will abandon
somatic cell
nuclear transfer
 The iP technique is
more efficient and
less problematic to
accept socially

Control mechanism of pluripotency
and differentiation
Different cell types
have different gene
expression patterns
 Gene expression
patterns are
controlled by the
gene regulatory
network

Selection of genes that are downregulated during differentiation
Induce differentiation by the
addition of retinoic acid and
LIF removal
 High throughput expression
profiles for six days
 This results in 901 rapidly
downregulated genes
 Out of these 65 are known as
regulators or are unassigned
ESTs

Selection of genes whose downregulated causes differentiation



The 65 candidates are
regulators that were
down-regulated in the
experiment
A causal relationship
needs to be established
more rigorously
Knockout by shRNA is
transferred to daughter
cells
Knock-down
Silencing of genes that cause
differentiation
Recognizes
mRNA noncoding
region
Mix the transfected cells with w.t. cells
 If the transfected differentiate, their number will decrease because:
 Lack of essential differentiation growth factors
 Cell cycle slows
 Cell adhesions are not formed effectively
 Conditions for maintaining pluripotency provided to the culture

Results of “differentiation screening”
•
•
Two of the hits did not reduce alkaline
phosphatase activity or affect cell
morphology
Neither did knock-outs of the other genes
that were not selected by the screening
Activation of the MAPK signaling
pathway
•ERK
was hyper phosphorylated in some
knock-downs
•The MAPK pathway is activated in
trophectoderm
Removal of LIF
induces differentiation
Ruling out non-specific effects
•It
is still possible that the shRNA
experiment induced differentiation but
not due to the knock-down
•To rule out non-specific effects:
•Additional different shRNAs were
designed. This provided validation for
Nanog, Oct4, Sox2, Esrrb and Tcl1
•A rescue strategy was developed
Rescue strategy for TF knockdowns
•It
is still possible that the shRNA
experiment induced differentiation but
not due to the knock-down
shRNA that targets
Non-coding region
Only coding region
Doxycycline dependent self-renewal
•The
following knock-downs remained
similar to undifferentiated cells:
•Sox2
•Esrrb
•Tbx3
•Tcl1
•In
the other cases the results were not
that conclusive, e.g. Oct4 rescue cells
grow more slowly but retain ES
morphology
Doxycycline removal brings up
mature cell markers
Trophectodermal markers
The Trophoblast provides nutrients to the
embryo and develop into a large part of
the placenta
Commitment to specific lineages
•What
happens if a regulator is constantly turned on in
a developing embryo?
•Embryoid
an embryo
•Create
missing
body: an aggregate of ESCs that “mimics”
an embryoid body and check which lines are
Markers for all 3 lineages were expressed in NanogR cells
Colors=
two different clones
All 3 markers induced
Esrrb blocks mesoderm and
neuroectoderm
•Activation
of Esrrb in
the cells of the embryoid
body prevented
specification into these
tissues but not
endoderm
Expression patterns of silencingresponsive genes
•771
genes are upregulated or downregulated in
response to the shRNA treatments
•Pattern 1(771): Responsive to most shRNA
•Pattern 2(474): Responsive to Nanog,Oct4,Sox2
•Pattern 3(272): Responsive to Esrrb,Tbx3,Tcl1,
Dppa4
Differentiation inducers controlled
by self-renewal regulators
•Each
of 160
upregulated genes
was overexpressed
• In 18 cases cell
morphology
changed, Nanog
was down and
alkaine
phosphatase
activity was lost
Nanog compensate for loss of other
self-renewal regulators
•It
was previously
shown that
upregulation of Nanog
can prevent
differentiation even if
important substances
are removed from the
culture
•A Nanog expressing
vector was inserted
to knock-down cells
gene
shRNA
Morphology+
Alkaline phos. activity