Molecular control of cell fate decision in pluripotent … control of cell fate decision in...
Transcript of Molecular control of cell fate decision in pluripotent … control of cell fate decision in...
Molecular control of cell fate decision in pluripotent and adult stem cells: from basic science toward therapy
Gabriella Minchiotti
INBB, Roma 24 Ottobre 2014
Zygote Blastocyst Gastrula
ONCOGENESIS
Primary Tumor Metastasis
EMBRYOGENESIS
Tissue Regeneration
Adult Stem Cell Activation/Differantiation
Tissue Regeneration
Embryonic Stem CellsEmbryonic Stem Cells Cancer Stem CellsCancer Stem Cells
Adult Stem CellsAdult Stem Cells
Convergent pathways in embryogenesis, oncogenesis and tissue regeneration
TACs- Transit Amplifying Cells
Mechanisms of cell lineage specification in mammals
TACTAC
Adapted S. Pece et al., BBA 2011
Modified from Scadden D., Nature 2006
Stem cell
Role of local environmental cues in defining cell-type identity
Signaling pathways involved in cell fate specification
Munoz‐Sanjuan and Brivanlou, 2002
ActivinsNodalNodal-related Vg1TGF-1, 2, 3
Follistatincerberus
BMP2BMP4BMP7DDF6
Blastocyst Growth of ICM cells ES
cellsGerm layersspecification
criptoEGF-CFC
MF20 MF20
Parisi et al., JCB 2003
- Cripto
III tubulin
+ Cripto
III tubulin
+ Cripto - Cripto
Cripto redirects the neural fate of ESCs
Genetic or pharmacological inhibition of Cripto in mouse ESCs cells improves functional integration of ESCs and blocks tumor formation in Parkinsonian rats
% c
hang
e in
rota
tiona
l beh
avio
ur
Parish, et al., Stem Cells, 2005
wild-type ESCswild-type ESCs Cripto -/- ESCsCripto -/- ESCs
Lonardo, et al., Stem Cells, 2010
Cripto controls cell lineage specification in ESCs
CriptoCripto
APJ/ApelinAPJ/ApelinHIF-1HIF-1
Parish et al., Stem Cells, 2005
Therapeutic targetParkinsons’ disease Therapeutic target
Parkinsons’ disease
Lonardo et al., Stem Cells, 2010
APJ-Cerberus-Baf60CAPJ-Cerberus-Baf60C D’Aniello et al., Cardiovasc. Res., 2013
D’Aniello et al., Circ Res, 2010
Bianco et al., Am. J. Pathology, 2009
Patent- EP09166967
CRIPTOCRIPTOAdapted S. Pece et al., BBA 2011
Cripto regulates cell lineage commitment in pluripotent stem cells
PluripotentPluripotent
ADULT STEM CELLs??ADULT STEM CELLs??
Embryonic Stem Cells Adult Stem Cells
Cripto Cripto
Skeletal Muscle Regeneration
Common pathways in embryonic and adult stem cells
Myogenic satellite cells: physiology to molecular biology, T. J. Hawke, 2001
Skeletal muscle regeneration
Sequential expression of myogenic transcription factors
The Skeletal Muscle Satellite Cell: The Stem Cell That Came inFrom the Cold. Peter S. Zammit et al., 2006
Guardiola et al., PNAS 2012
Cripto is expressed in myoblasts and inflammatory cells in skeletal muscle regeneration
Cripto BFCripto Pax7 MyoD DAPI
T0
A B C D E
T24
F G H I J
K L M N O
T48
Cripto is expressed in myogenic precursor cells in isolated myofibers
Guardiola et al., PNAS 2012
Model of satellite cell self-renewal
Zammit P S et al. J Cell Biol 2004
*
*
*
**
**
**
**
T48 T72 T96
Num
ber o
f positive cells (%
)
Control ControlControlsCripto sCripto sCripto 0‐96h
sCripto 0‐48h
Committed myoblasts to differentiation (Pax7‐/MyoD+)
Activated/proliferating satellite cells (Pax7+/Myod+)
Quiescent satellite cells (Pax7+/MyoD‐)
Isolated myofibers
Cripto promotes satellite cell lineage progression and proliferation
Guardiola et al., PNAS 2012
Cripto promotes satellite cell myogenic commitment antagonizing Myostatin
Myostatin+ Cripto
Myostatin
Control
Committed myoblasts (Pax7‐/MyoD+)
Activated/proliferating satellite cells
Quiescent satellite cells (Pax7+/MyoD‐) (Pax7+/Myod+)
Guardiola et al., PNAS 2012
CONDITIONAL SATELLITE CELL –SPECIFIC CRIPTO KO
Tg:Pax7-CreERT2::CriptoLoxP/-
CONDITIONAL SATELLITE CELL –SPECIFIC CRIPTO GOF
Tg:Pax7-CreERT2::CriptoGOF
Tg:Pax7-CreERT2::CriptoLoxP/-
Satellite cell-specific cripto ablation affects muscle regeneration
Guardiola et al., PNAS 2012
Impacts of Genetic modulation of Cripto signaling in the satellite cell compartment on
skeletal muscle regeneration
Toward Pharmacological modulation of Cripto signaling for skeletal muscle regeneration
96-channel headdisposable pipetting tips
The system executesProtocols for targeted differentiation of ES cellsScreening of compounds libraries
Advantage/benefitsHigh standard reliability, performance and flexibilityLow user interventionContamination risk reduction
Method standardization
Up to 4000 single compuonds simultaneously screened Co developed with Hamilton Robotics
Complete sterility
The Cell maker
+Cell-based Phenotypic Screening:
Cell Proliferation (36hrs)
Cell Colony Phenotype (5days)
Targeted Differentiation
(10-13 days)
• Metabolic Intermediates
• HDAC Inhibitors• FDA-approved
drugs
Small molecules
Small molecules
neuronsneuronscardiomyocytescardiomyocytes
Casalino et al., JMCB, 2011
Casalino et al., Molecular Biotechnology, 2011
Franci et al., Biol. Open, 2013Comes et al., Stem Cell Reports, 2013
L Proline –induced cells (PiCs):a novel metastable state of pluripotent stem cells
ESCs PiCsSensitivity to trypsin digestion ‐ +Alkaline phoshatase activity + ‐In vitro cardiac and neural differentiation + +Teratoma formation and blastocyst colonization + +
BF
E 11.5
EGFP
E 11.5
Casalino, Comes et al., JMCB, 2011
L-prolineControl
PiCs = Proline induced Cells
PiCs
L-Pro induces remodeling of the ESC transcriptome
Comes et al., Stem Cell Reports 2013
L-Proline -induced cytoskeletal rearrangements in ESCs
30 µm50 µm 20 µm
50 µm
50 µm 50 µm 50 µm
50 µm 50 µm
ESCs PiCs PiCs
Comes et al., Stem Cell Reports 2013
L-Pro induces a motile phenotype in ESCs
From Day 3 to Day 4 (images were collected every 5 minutes/ 20x objective)
Control ESCs L- proline -treated ESCs
invasion migration *
**
0
50
100
150
200
0 0 100ESCs PiCs
SDF-1(ng/ml)
Cel
l Mig
ratio
n / I
nvas
ion
(cel
ls p
er fi
eld)
PiCs are invasive and metastatic pluripotent stem cells
ES
Cs
PiC
s
EG
FP+
PiC
sB
16-B
L61x106 ESCs or PiCs injected into tail veins; mice were sacrificed 4 weeks after injection
0
20
40
60
80
100
120
140
15% 1-15% FBS
*
**C
ell M
igra
tion
(cel
ls p
er fi
eld)
ESCsPiCs
Comes et al., Stem Cell Reports 2013
A
E
B
Slug
E-cad
Snail
0 1 2
Relative Expression1 100 500
MMP-2
T
Vim
N-cad
Rel
ativ
e Ex
pres
sion N-cad
T
1
10
100
1000
Con
trol 2 3 4 5
+ L-Pro (day)
GADPH
E-CAD
Control 3 4 5
+ L-Pro (day)
Rel
ativ
e E-
CAD
Lev
el
0
1
Control 3 4 5+ L-Pro (day)
-120
-36
kDa
N-CAD
GADPH
Control 3 4 5
+ L-Pro (day)
T
kDa
-135
-50
-36
Relative Expression
day
5
E-CAD/Hoechst
day
3
Control + L-Pro
+ L-Pro (day)
+ L-Pro (day)
L-Proline induces a fully reversible EMT –like transition in embryonic stem cells: embryonic stem to Mesenchymal Transition (esMT)
L-Proline induces a fully reversible EMT –like transition in embryonic stem cells: embryonic stem to Mesenchymal Transition (esMT)
Comes et al., Stem Cell Reports 2013
compactedstem cells
scatteredstem cells
L-Proline
esMT
MesT
E-cadherinE-cadherin
invasiveness
teratoma
Vitamin C improves cell riprogramming (Shi et al., Cell Stem Cell2009) by modulating H3K9 and H3K36 methylation (Chen J. et alNature Genet., 2012)
Vitamin C is a key cofactor of the reactions driven byhistone demethylases of the JMJ family
Vitamin C
L-Proline is a genome-wide inducer of H3K9 and H3K36 methylation L-Proline is a genome-wide inducer of H3K9 and H3K36 methylation
Comes et al., Stem Cell Reports 2013
L-Pro triggers an esMT reminiscent of the EMT that occurs at theinvasive front of the tumor, and contributes to the acquisition of cellplasticity and invasiveness.
L-Pro–induced esMT is fully reversible (MesT) and is accompanied bya global remodeling of H3K9 and H3K36 methylation status
compactedstem cells
scatteredstem cells
L-ProlineesMT
Vitamin CMesT
E-cadherinE-cadherin
invasiveness
teratoma
External collaborations
Ombretta Guardiola
Gennaro Andolfi
Stefania Comes
IGB FacilitiesIntegrated MicroscopyAnimal House
Lab membersIGB collaborations
Eduardo J. Patriarca Maria MatarazzoDario De CesareLaura Casalino
Alessandro Fiorenzano
Annalisa Fico
Giorgio Scita, IFOM, Milan
Nicola Laprano
Carolina Prezioso
Thanks to
Claudia Angelini, Institute for Applied Mathematics, CNR, Naples
Ann Zeuner, Ruggero de Maria, ISS, Rome
FACS
Past members
Cristina D’Aniello
Salvatore Iaconis
Silvia Brunelli, HSR, Milan
Sharhaghim Tajbakhsh, Pasteur Institute, Paris
TIGEM Bioinformatic Core Facility,, Naples
Michael Shen, Columbia University, NY
Dror Seliktar, Technion, Haifa, Israel
NGS
EMBO WORKSHOP ON Stem Cell Mechanobiology in Development and Disease October 18-21, 2015 Capri, Naples
The�IGB�Meeting�Coordinators���Maria�R.�Matarazzo,�Maria�G.�Miano�
Gabriella Minchiotti, Institute of Genetics and Biophysics ‘A. Buzzati Traverso’, (IGB), Naples, Italy Paolo A. Netti, Italian Institute of Technology (IIT), Naples, Italy Viola Vogel, Laboratory of Applied Mechanobiology, ETH, Zürich, Switzerland
Yohanns Bellaïche, France Françoise Brochard-Wyart, France Chen Christofer S., USA Giulio Cossu, UK George Q. Daley, USA Carl-Philipp Heisenberg, Austria Donald E. Ingber, USA Benoît Ladoux, France David A. Lee, UK Ohad Medalla, Switzerland Christine Mummery, The Netherlands
Paolo Netti, Italy Graziella Pellegrini, Italy Stefano Piccolo, Italy Mattieu Piel, France Giorgio Scita, Italy Dror Sellktar, Israel GV Shivashankar, Italy Craig A. Simons, Canada Molly Stevens, UK Viola Vogel, Switzerland Fiona Watt, UK
Workshop��Secretariat�Anna�Maria�Aliperti,�Federica�Staempfli