Prometheus vulture and the promise of stem cells

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Prometheus vulture and the promise of stem cells
Nadia Rosenthal Mouse Biology Programme EMBL-Monte
rotondo Rome
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Why cant we regenerate?
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Loss of regeneration capacity
Regeneration capacity
Evolutionary scale
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Regenerating fish hearts newt limb
regeneration?
From Poss et al Science 2002
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Heart regeneration in zebrafish is accompanied
by up-regulation of components of the Notch
pathway, followed by members of the Msx family,
which we show are also up-regulated during fin
regeneration Heart regeneration involves the
execution of a specific genetic program, rather
than redeployment of a cardiac development
program.
PNAS online Colloquium
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The scarless heart of the MRL mouse
C57/B6
MRL/mpj
3 months post-infarct
Leferovich et al, PNAS 2001
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Wound healing
Disruption of capillaries triggers activation of
platelets and clotting cascade Neutrophils and
leukocytes remove bacteria and release a variety
of growth factors and cytokines which function to
remodel the tissue Macrophages and keratinocytes
release factors inducing fibroblasts to produce
ECM components Formation of new blood vessels by
transdifferentiation of fibroblasts to
myofibroblasts Scar formation
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A controversial factor

• Insulin-like growth factor-1 (IGF-1)
• Important for fetal growth
• Synthesized in the liver after birth
• Endocrine form responsive to GH
• Autocrine/paracrine form transiently induced in
  response to local injury
• Promotes proliferation, differentiation,
  survival, angiogenesis, neurite outgrowth,
  neoplasia
• Reducing IGF-1 receptors extends lifespan!

..
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Igf-1 signalling in muscle (not the whole story)
IGF-1
IGF-1 RECEPTOR
CELL MEMBRANE
shc
Grb-2
RAS
SOS
PLC
IRS
RHO
IP3 DAG
MEKK1-4 RAF
p85
p110
ROCK
PI3K
Ca
PKC
MEK4/7 MEK1/2
CALCINEURIN
p70S6K
AKT
pNFATs NFATs
BAD
JNK1/23
ERK1/2
CREB
mTOR
GATAs AP1 MEF2
TRANSLATION
Bcl-2
PROLIFERATION
APOPTOSIS
4E-BP

• FIBRE TYPE
• MUSCLE HYPERTROPHY

activated
elF4E
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IGF-1 isoforms do matter
hGH
Class 1 isoforms

• Activated locally (muscle, brain, heart etc)
• Non-circulating
• Transiently expressed in damaged tissue

Class 2 isoforms

• Synthesized by liver
• Circulating
• Represses hGH

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Structure of the IGF-1 gene and isoforms
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
S
S
S
B
B
E
E
E
C
A
D
m
Local
Circulating
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Regulation of local IGF-1 isoforms
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
S
S
S
B
B
E
E
E
C
A
D
Induced by injury, sustained in repair
m
Class 1 IGF-1A
mIGF-1
B
E-35
C
A
D
S-48
MGF
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Changes in endogenous mouse IGF-1 isoform usage
1 2 3 4
- - -
- - -

-
1 - Wildtype muscle 2 - Exercised muscle 3 -
Ageing muscle (only in diaphragm) 4 - Dystrophic
muscle
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What is the effect of supplemental IGF-1
expression in skeletal muscle?
mIGF-1
MLC
SV40
MLC
transgene

• Skeletal muscle-specific expression
• Post-mitotic restriction of mIGF-1
• Active throughout life

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Local MLC/mIGF-1 transgene (Class I)

• INCREASES postnatal muscle mass and strength
• DECREASES fat
• AGING maintains muscle mass and function
• EXERCISE enhances anabolic effects
• DYSTROPHY counters muscle degeneration
• ALS delays paralysis, preserves muscle
  integrity
• CARDIAC CACHEXIA blocks it
• AngII MUSCLE ATROPHY blocks it
• Does not
• Induce cardiac hypertrophy
• Promote neoplasia

Forelimb
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Supplementary circulating IGF-1 transgene (Class
2)

• Induces mild cardiac hypertrophy
• Promotes precocious neoplasia (1 year)
• Alters glucose metabolic pathways in muscle
• Does not
• Increase postnatal muscle mass and strength
• Minimise body fat
• Maintain aging muscle mass and function

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Local mIGF-1 enhances skeletal musculature
6 months
Musaro et al, Nature Genetics 2001
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AGEINGMaintenance of muscle mass in old mIGF-1
transgenic mice
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Enhanced regeneration in old mIGF-1 muscle
Higher magnif.
Centralised nuclei
Neonatal myosin
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Transgenic mIGF-1 blocks dystrophic muscle
degeneration and scarring
mdx x mIGF-1
mdx
Hydroxyproline, ug/mg
14 month sibs (diaphragm)
Barton et al, J.Cell. Biol. 2002
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ALS Can mIGF-1 attenuate it?
Neurons affected in ALS
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mIGF-1 delays paralysis and preserves muscle
regeneration in ALS mice
SODG93A/mIGF-1
SODG93A
early
early
early
late
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mIGF-1 prevents cardiac cachexia
10-12 weeks old FVB or transgenic mIGF-I/ mice
LAD-Ligation
Sham-OP
2 weeks Echocardiography
12 weeks Echocardiography sacrifice and tissue
analysis
C. Schulze and R. Lee, unpublished
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Transgenic mIGF-1 skeletal muscle mass is
preserved in MI-induced chronic heart failure
WT
mIGF-1
2000
1800
1600
CSA (?m2)
1400
1200
1000
800
600
400
200
0
EDL
Soleus
QUAD
EDL
Soleus
QUAD
MI
Sham
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Induction of Atrogin (muscle ubiquitin
E3-ligase) in skeletal muscles during chronic
heart failure
EDL
QUA
SOL

• RT-PCR

S MI S MI S MI
Atrogin
Actin
Northern
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Overexpression of IGF-1 reduces skeletal myosin
ubiquitination during chronic heart failure

• IP with anti-Ubiquitin Ab,
• Western against slow twitch/ventricular myosin

Heart
EDL
SOL
QUA
S MI S MI S MI S MI
Myosin ubiquination
Wildtype
Heart
EDL
SOL
QUA
S MI S MI S MI S MI
mIGF-I
Myosin ubiquination
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Does the increased regenerative response to
mIGF-1 involve stem cell recruitment?
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Cardiotoxin injury/regeneration model
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Stem cell markers
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Stem cells segregating as a side population (SP)
express MRD1
SP
Goodell et al,J.Exp.Med. 1996
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Stem cell lineage markers
CD45 c-kit Sca-1 Bone marrow SP
Muscle SP - -
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Stem cell lineage markersincrease in injured
mIGF-1 muscle
CD45 c-kit Sca-1 Bone marrow SP
Muscle SP
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New cell populations in injured mIGF-1 muscle
Control
CTX
0,5
13
Wildtype
1
0,2
0,2
3
cKit
4
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mIGF-1
12
4
28
21
Sca-1brightcells (express Flk1, Emx2)
Sca-1
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Emx2

• Vertebrate homeobox gene involved in CNS
  patterning
• Affects the symmetry of cell divisions and the
  fate of precursors from the cerebral cortex.
• Regulates the proliferation of stem cells in the
  adult mammalian central nervous system.
• Expression is graded along the developing newt
  limb axis
• Induced in regeneration blastema of amputated
  newt limbs
• Hypothesis
• newt programs are activated in the
  Sca-1bright population

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Do Sca-1 cells represent early muscle precursors
or just monocyte infiltration?
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Cell cycle blockade (5-FU) ablates recruitment of
CD45/Sca-1 cells to injured muscle
5FU, CTX
Control
Bone marrow
0,11
( CTX alone 0.6)
CD45
5.7
18
6.5
6.9
(CD45 cells still present)
Muscle
Sca-1
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Pax7 is required for mesenchymal cell commitment
to a myogenic fate
MyoD Myf5
Myogenin MRF4
Pax7
Progenitor cell
Satellite cell
Myogenic precursor cell
Myotube
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Pax7 protein is increased in regenerating muscle
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Overlapping Sca-1 and Pax7 expression in
regenerating mIGF-1 muscle
Hoechst
Sca-1
Pax7
Overlay
Blood vessel
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Can stem cells acquire a myogenic phenotype in
culture?
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Sca-1/c-kit cells from mIGF-1 muscle do NOT
become myogenic in culture
CD45
HSC phenotype
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..unless they are co-cultured with
myoblasts(environment dictates fate decisions)
E
on Wt myoblasts
on mIGF-1 myoblasts
Wt
mIGF-1
Whole bone marrow (or c-kit/Sca-1cells) from a
desmin/LacZ transgenic mouse
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Sca-1/c-kit - cells from mIGF-1 muscle undergo
efficient myogenesis in culture
wildtype
mIGF-1
Desmin 3 days GM
MyHC 2 days DM
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..although cells expressinglate myogenic markers
do not co-express Sca-1
MLC3/LacZ
Sca-1
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Sca-1 sorted cells from muscle are multipotent
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Where do the Sca-1 cells come from?
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Increased SP cells in the bone marrow 48 hrs
after muscle injury
Control
CTX
Wildtype
0,2
0,5
mIGF-1
0,6
0,17
(no increase in liver or spleen)
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Does local mIGF-1 increase bone marrow cell
recruitment to injured muscle?
1.Deplete host bone marrow (irradiation) 2.
Reconstitute host with bone marrow SP from
a ckit/GFP transgenic mouse (tail vein injection
of 2000 cells) 3. After 10 days, injure
muscle (C TX injection) 4.Analyze muscle by FACS
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Enhanced recruitment of transplanted bone marrow
cells to mIGF-1 injured muscles
wildtype
MLC/mIGF-1
4.2
2.1
Control muscle
CD45
Ctx injected muscle
cKit-GFP
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SKELETAL MUSCLE
mIgf-1
Early myogenic progenitor Sca-1
(Sca-1bright)
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Enhanced recruitment of c-kit cells to mIGF-1
injured muscles without irradiation

Wt
mIGF-1

Necrotic zone
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Whats downstream of mIGF-1?
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Calcineurin activity mediates mIGF-1 activity in
cell culture
IGF-1
IGF-1 RECEPTOR
CELL MEMBRANE
shc
Grb-2
RAS
SOS
PLC
IRS
RHO
IP3 DAG
MEKK1-4 RAF
p85
p110
ROCK
PI3K
Ca
PKC
MEK4/7 MEK1/2
p70S6K
AKT
BAD
CALCINEURIN
JNK1/23
pNFATs NFATs
ERK1/2
CREB
mTOR
TRANSLATION
Bcl-2
GATAs AP1 MEF2
PROLIFERATION
APOPTOSIS
4E-BP
activated
elF4E
MYOCYTE HYPERTROPHY
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Calcineurin A isoform structure
Calmodulin binding
Autoinhibitory domain
Catalytic domain
CnB binding sites
CnA alpha
CnA active
(artificial truncation)
CnA beta2
CnA beta1
Unknown Domain
CnB
Isoform used in all previous in vivo studies
Calcium binding sites
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Tissue distribution of CnA isoforms
Wildtype
Tg MLC/IGF-1
Soleus
Gast
Thymus
Triceps
Spleen
Soleus
Triceps
Gsst
Testis
Thigh
Mass
Heart
Brain
Thigh
Mass
TA
TA
CnA alpha
7.5 Kb
CnA beta2
4.4 Kb
CnA beta1
3.6 Kb
CnA gamma
2.5 Kb
28s
18s
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What is the effect of supplemental native CnA
alpha isoform expression in skeletal muscle?
CnA
MLC
SV40
MLC
transgene

• Skeletal muscle-specific expression
• Post-mitotic restriction of CnA

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Wt MLC/CnA alpha
MLC/CnA alpha transgenic muscle regenerates
faster than wild-type after cardiotoxin induced
injury
Day2
Day 4
Day 6
Day 8
A. Paul, unpublished
Day 10
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CnAb isoform switch in regenerating muscle
2 day
5 day
testes
brain
- CTX - CTX
CnAb1
CnAb2
28S
18S
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Time course of muscle regeneration
Control
1-Day
2-Days
5-Days
10-Days
testes
Brain
CnAb1
28s
18s
K. McCullagh, unpublished
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The mIGF-1 transgene induces CnA beta 1
expression in ALS muscle
RNA
Protein
C. Giacinti, unpublished
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How does mIGF-1 get the right cells to the right
place?
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Chemokines the good, the bad and the
under-utilised
Enhance their attractive role in stem cell
homing to tissue damage
Block their activity in metastatic tumour
progression
63
Increased CXC receptors in bone marrow of mIGF-1
injured muscle
Cont.
Bone marrow
Skeletal muscle
64
CC Chemokine induction in muscle injury
Treatment only
30
25
20
15
10
5
0
Scya2
Scya6
Scya8
mean frequency
65
Transient activation of CC chemokine receptors in
injured muscle is enhanced by mIGF-1
CTX 2d 5d 2d 5d
Cnt 2d 5d 2d 5d
CCR1
CCR4
CCR5
L32
Cont.
GAPDH
CTX
Cont.
CTX
wildtype
mIGF-1
wt
Tg IGF-1
66

Biphasic model of response to injury
Circulating cells home to repair more severely
damaged tissues
Resident cells repair minor tissue damage
Damage
Injury
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Newts do it, why cant we?
The critical hypothesis.. is that regeneration
is a basic, primordial attribute of
metazoanslost secondarily for reasons which are
not understood.
- Jeremy Brockes
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Enhanced myogenesis of transgenic mIGF-1 primary
skeletal muscle cultures
Myosin
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Long term survival of mIGF-1 myocyte cultures
Wildtype mIGF-1 transgenic
2 months in serum-free media
70
Serum restimulation of mIGF-1 myocyte
proliferation
(20 FCS)
MyoD colony
MyoD- colony
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Modes of repair

• Circulating stem cells home to injured tissue
• (survival factors)

2. Resident progenitor cells proliferate in
response to injury
3. Dedifferentiated cells proliferate in response
to injury
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Improving recruitment and recycling of our own
stem cells

• Enhance bone marrow stem cell mobilization and
  homing
• (growth factors, chemokines)

• Activate local progenitor cell reserves, inducing
  controlled
• dedifferentiation and repair in adult tissues

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U. Roma La Sapienza Antonio Musarò Cristina
Giacinti Gabriella Dobrowolny Laura
Pelosi Marcello Coletta IRCCS Roma Giovanna
Borsellino Luca Battistini SCRI Milano Guilio
Cossu Genetics Institute Jennifer Moran
Harvard Med. School Richard Lee Christian
Schulze U.Texas Eric Olson Frank
Naya U.Pennsylvania Lee Sweeney Elisabeth
Barton U.Western Australia Miranda
Grounds Jason White London Harfield
Hospital Magdi Yacoub Pak Bhavsar
LAB Antonio Musarò Karl McCullagh Lana
Tsao Frederic Depreux Serafima Zaltsman Angelika
Paul Michele Pelosi Faye Mourki Nadine Winn Maria
Paola Santini Olivier Mirabeau Katya
Semenova Enrique Lara José Gonzales Esfir
Slonimsky
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