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