Diapositiva 1 - Dipartimento di Scienze veterinarie per la salute

Aspetti cellulari e molecolari che controllano
la pluripotenza ed il differenziamento delle cellule di
mammifero.
Tiziana A.L. BREVINI
Laboratorio di Embriologia Biomedica
•Cells of a multicellular organism are genetically identical but differentiate
into specific tissues.
•This is possible because each cell express, depending on the particular
cell type, different sets of genes
DNA methylation and histone modification are "epigenetic" mechanisms
for stably maintaining the activity state of genomic regions.
• These differences in gene expression arise
during development and are retained
through subsequent cell divisions.
• The epigenetic process allows for tissue
specificity determination in the cell
Life start with a very small bunch of cells that constitute an embryo.
These cells are able to develop into a perfect and complete organismthanks to their
capability to undergo epigenetic modifications.
Epigenetic landscape
by Conrad Waddington (1957)
Epigenetic landscape
by Conrad Waddington (1957)
Cellule pluripotenti
….in cima alla collina del differenziamento
…oocyte potency
Oocyte and embryonic stem
cell lines
Parthenogenetic embryonic stem
cells in animal models
Generation of human parthenotes
• We subjected to
parthenogenetic
activation 104 oocytes
• 61.5% of them cleaved
42-44 h later
• 12.8% developed to the
blastocyst stage
ethical permission granted from Comitato Etico Istituti Clinici di perfezionamento
Establishment of human
pluripotent parthenogenetic cells
• ICMs were isolated
and plated on
inactivated feederlayer
• 6 human
parthenogenetic (Hp)
lines were derived
Hp 1 cell line
ethical permission granted from Comitato Etico Istituti Clinici di perfezionamento
'Virgin birth' stem cells bypass ethical
objections
13:12 02 July 2006
NewScientist.com news service
Linda Geddes
Ethisch unbedenkliche
Stammzellenquelle vorgestellt
11-08-2006
Nueva forma de obtener células madre
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≥Î¥Ÿ¡ˆ
Cell lines derived without creating a viable embryo.
논란있는 배아생성없이 추출한 줄기세포라인
Pontifical Academy for Life
International Congress on "Stem cells: what future for therapy?"
Castelgandolfo, September 16, 2006
GeneChip Array
We used Affimetrix Human Genome plus array (54,000 probe sets).
The analysis included and compared HP1 and HP3 with three bi-parental human ES
cell lines (HES7, HES I-3 and HES I-6).
Data were normalized and analyzed by bioinformatics tools
Gene clustering analysis
Differenziamenti in vitro
Differentiation into
haematopoietic cells
Differentiation into
neural cells
Adult (4-6 years old) ovine ovaries (Sarda sheep) were
collected at local slaughterhouses
COCs were matured in vitro and activated with ionomycin/ 6DMAP.
Presumptive PA zygotes were cultured for 8 days and
transferred into the uterus of synchronized recipients .
At gestational day 27 sheep were slaughtered and
foetuses recovered
Cells were plated and immediately subjected to further studies
CONCLUSIONS 1
• Oocytes can be used as an alternative source
for establishing stable pluripotent cell lines
• These cell lines show high differentiation
ability in vitro, giving rise to cell types that
are derived from all three primary germ
layers (endoderm, mesoderm and ectoderm).
• Parthenogenetic cells display many
characteristics common to bi-parental cells
Laboratory of Biomedical Embryology
University of Milan
Fulvio Gandolfi
Valentina Tosetti
Mattia Crestan
Stefania Antonini
Fabiana Cillo
Georgia Pennarossa
Arianna Vanelli
Cell Factory
Ospedale Maggiore Policlinico
Lorenza Lazzari
Tiziana Montemurro
IRCCS Foundation
Infertility Unit
Ospedale Regina Elena
Guido Ragni
Alessio Paffoni
Stem Cells Unit
Silberman Institute of Life Sciences
Nissim Benvenisty
Amir Eden
DIVET
University of Milan
Eugenio Scanziani
Enrico Radaelli
Department of Animal Biology
University of Sassari
Paolo Rebulla
DBSM,
University of Insubria,
Magda deEguileor
Gianluca Tettamanti
Suggestions and discussion:
Alan Trounson
Bruno Peault
Stefania Corti
Azim Surani
Sergio Ledda
Luisa Bogliolo
Stefano Fois
Federica Ariu
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Paffoni A, Brevini TA, Somigliana E, Restelli L, Gandolfi F, Ragni G. In vitro development of human oocytes after parthenogenetic activation or intracytoplasmic
sperm injection. Fertil Steril. 2007 Jan;87(1):77-82.
Brevini TA, Tosetti V, Crestan M, Antonini S, Gandolfi F. Derivation and characterization of pluripotent cell lines from pig embryos of different origins.
Theriogenology. 2007 Jan 1;67(1):54-63.
Brevini TA, Antonini S, Cillo F, Crestan M, Gandolfi F. Porcine embryonic stem cells: Facts, challenges and hopes Theriogenology. 2007 Sep 1;68 Suppl 1:S206-13.
Epub 2007 Jun 19.
Brevini TA, Gandolfi F. Parthenotes as a source of embryonic stem cells. Cell Prolif. 2008 Feb;41 Suppl 1:20-30.
Brevini TA, Pennarossa G, Antonini S, Gandolfi F. Parthenogenesis as an approach to pluripotency: advantages and limitations involved. Stem Cell Rev. 2008
Sep;4(3):127-35.
Brevini TA, Antonini S, Pennarossa G, Gandolfi F. Recent progress in embryonic stem cell research and its application in domestic species. Reprod Domest Anim.
2008 Jul;43 Suppl 2:193-9.
Paffoni A, Brevini TA, Gandolfi F, Ragni G. Parthenogenetic activation: biology and applications in the ART laboratory. Placenta. 2008 Oct;29 Suppl B:121-5.
Brevini TA, Pennarossa G, Antonini S, Paffoni A, Tettamanti G, Montemurro T, Radaelli E, Lazzari L, Rebulla P, Scanziani E, de Eguileor M, Benvenisty N, Ragni G,
Gandolfi F.Cell lines derived from human parthenogenetic embryos can display aberrant centriole distribution and altered expression levels of mitotic spindle
check-point transcripts. Stem Cell Rev. 2009 Dec;5(4):340-52.
Brevini TA, Pennarossa G, Attanasio L, Vanelli A, Gasparrini B, Gandolfi F. Culture conditions and signalling networks promoting the establishment of cell lines
from parthenogenetic and biparental pig embryos.Stem Cell Rev. 2010 Sep;6(3):484-95.
Brevini TA, Pennarossa G, Gandolfi F.No shortcuts to pig embryonic stem cells. Theriogenology. 2010 Sep 1;74(4):544-50.
Brevini TA, Pennarossa G, deEguileor M, Tettamanti G, Ragni G, Paffoni A, Gandolfi F . Parthenogenetic cell lines: an unstable equilibrium between pluripotency
and malignant transformation. Curr Pharm Biotechnol. 2011 Feb 1;12(2):206-12.
Pennarossa G., Alessio Paffoni, Guido Ragni, Gandolfi F., Brevini T.A.L. Parthenogenesis in mammals: pros and cons in pluripotent cell derivation. Cent Eur J Biol.
2011, 6(5) • 2011 • 770-775 DOI: 10.2478/s11535-011-0047-3
Brevini TAL, Pennarossa G., Vanelli A., Maffei S. and Gandolfi F. Parthenogenesis in non-rodent species: developmental competence and differentiation
plasticity. Theriogenology, 2012 March, 77(4):766-772
Kyurkchiev S, Gandolfi F, Hayrabedyan S, Brevini TA, Dimitrov R, Fitzgerald JS, Jabeen A, Mourdjeva M, Photini SM, Spencer P, Fernández N, Markert UR. Stem
cells in the reproductive system. Am J Reprod Immunol. 2012 Jun;67(6):445-62
Brevini TA, Pennarossa G, Maffei S, Tettamanti G, Vanelli A, Isaac S, Eden A, Ledda S, de Eguileor M, Gandolfi F. Centrosome amplification and chromosomal
instability in human and animal parthenogenetic cell lines. Stem Cell Rev. 2012 Dec;8(4):1076-87. doi: 10.1007/s12015-012-9379-2.
Brevini T, Pennarossa G, Maffei S, Gandolfi F. Pluripotency network in porcine embryos and derived cell lines. Reprod Domest Anim. 2012 Aug;47 Suppl 4:86-91.
Gandolfi F, Pennarossa G, Maffei S, Brevini T. Why is it so difficult to derive pluripotent stem cells in domestic ungulates? Reprod Domest Anim. 2012 Aug;47
Suppl 5:11-7. doi: 10.1111/j.1439-0531.2012.02106.x.
…….a metà strada lungo la collina del differenziamento
progenitori
Sviluppo di modelli animali per
studi pre-clinici di medicina
rigenerativa del cuore
When it is necessary to move forward…
…an intermediate stop is useful
Similarities and differences
Body
mass
(g)
Longevity
(years)
Gestation
(gg)
Heart
weight
(g)
Lung
weight
(g)
Liver
weight
(g)
Human
50.000
-90.000
70
280
320
1200
1500
Pig
60.000150.000
25
114
370
1000
1500
Mouse
40
1-3
21
0,05
0,15
1,50
Human
Pig
Mouse
Brevini et al., 2011 (in press)
Extrapolation of murine systems, particularly after induction of
cardiovascular stress, has some limits that can be crucial the closer it
gets to a clinical application.
coronary architecture (bigger variability in human)
capillary density (higher in the mouse)
network di irrorazione endo-epicardica
molecular differences (suggests a possible difference of the
mechanical behavior of the myocardium between the two species)
Therefore, large animal models, like the pig, which more closely
approximate human physiology, function, and anatomy, are essential to
develop the discoveries from murine models into clinical therapies and
interventions for heart therapies
The isolation was carried out from the
hearts of 10 Large White pigs aged
between 4 and 8 months
Cells lines were obtained from 7 animals
ISOLATION OF ADULT CARDIAC PROGENITOR CELLS
FROM DIFFERENT REGIONS OF THE HEART
ATRIUM
AORTA
VENTRICLE
Aorta
L Ventr R Atr
Aorta
L Atr
Gata6
Oct-4
Mef2a
Nkx2.5
Mesp1
CD34
Mesp2
CD44
L Ventr
D Atr
L Atr
Tubulin
ANP
EXPRESSION OF GENES RELATED TO PLURIPOTENCY MESENCHIMAL AND CARDIAC
DIFFERENTIATION
RT-PCR ANALYSIS
AORTA
Oct-4
GATA6
CD34
CD44
Mef2a
Mesp1
Mesp2
Nkx2.5
ANP
Tubulin
+
+
+
+
+
+
+
+
+
+
Left
Ventricle
+
+
+
+
+
+
+
+
+
+
Right Atrium
Left Atrium
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
30
In vitro experiments: differentiation of
pig adult cardiac progenitors
31
DIFFERENTIATION EFFICIENCY OF
PIG CARDIAC PROGENITOR CELLS
Cardiomyocites
(Cx43)
Skeletal muscle
(Myosin)
Smooth muscle
actin (SMA)
Left Atrium
30%
1%
2%
Right Atrium
40%
1%
2%
Aorta
70%
1%
5%
Ventricle
60%
1%
5%
In vivo experiments
Immunosuppression
Cyclosporine treatment
(Sandimmune 5 mg/Kg)
Myocardial surgical damage models
Myocardial infarct
Ligation (left coronary A.)
Myocardial ischemia
Ameroid constrictor (left circumflex A.)
TRANSMYOCARDIAL INJECTION
After 3 week recovery period, transmyocardial injection of approximately
20 x 106 cells
35
EXPERIMENTAL END-POINTS
After 6 weeks evaluation of:
•
•
•
•
Re-perfusion
Angiogenesis
Regeneration of myocardium
Improvement of cardiac function
Problems encountered:
- Fibrillations => death
- Infections in the immunosuppressed animals => death
CLONING AS A POSSIBLE
ALTERNATIVE TO
IMMUNOSUPPRESSION IN
PIGS:
creation of syngenic animals
(genetically identical and
immunologically compatible)
Primary culture of pig adult/fetal fibroblasts
Vector transfection
Primary culture of
pig fibroblasts
GFP positive fibroblasts
GFP+
GFP negative fibroblasts
GFP-
GFP-
GFP-
Cloned syngenic pigs
GFP positive pig embryos
Transgenic pig fibroblasts GFP+
Brunetti et al., 2008
Pig fibroblasts GFP-
GFP positive pigs adult cardiac
progenitors
Pig cardiac progenitors can be isolated
FACS and RT-PCR demonstrate that these cells express many
markers that are common to the mouse and human
They can efficiently differentiate into cardiomyocites
We suggest they may represent a promising tool for the
development of pre-clinical studies addressed to myocardial
damage management in the pig
Laboratorio di Embriologia Biomedica
Università degli Studi di Milano
Fulvio Gandolfi
Vanelli Arianna
Georgia Pennarossa
Stefania Antonini
Stem Cell Research Institute Dibit, San Raffaele
Beatriz G. Galvez
Arianna Dellavalle
Giulio Cossu
VESPA
Università degli Studi di Milano
Fabio Acocella
Stefano Brizzola
Giovanni Tremolada
Alessio Vigani
AVANTEA Cremona
Cesare Galli
Giovanna Lazzari
Dario Brunetti
Istituto Mario Negri
Dipartimento di Ricerca Cardiovascolare
Roberto Latini
43
(2009) 8 (6): 610-614.
……..sulla collina del differenziamento
dopo Dolly e le iPS
Development and reprogramming in the
Waddington epigenetic landscape
Zhou & Melton 2008 CSC
DISEGNO SPERIMENTALE: indirizzare le cellule
dopo averne aumentato la plasticità della cromatina
Trattamento di cellule somatiche con modificatori
epigenetici
Differenziamento indotto in senso muscolare
CELLULE DI GRANULOSA UMANA
DIFFERENZIAMENTO MUSCOLARE IN VITRO:
COLTURA IN MEDIUM ADDIZIONATO CON VEGF
(5 ng/ml) PER 15 GIORNI
Keratin17
Desmin
MyoD
Myogenin
Myosin heavy
chain
GAPDH
DIFFERENZIAMENTO IN VITRO CON VEGF PER 15
GIORNI: marcatori delle cellule muscolari
Desmina
Miosina
MyoD
Cox-2, DAPI
MHC, DAPI
Desmin, DAPI
MyoD, DAPI
EFFICIENZA DI DIFFERENZIAMENTO INDOTTA DA
VEGF PER 15 GG
100
Media delle percentuali delle cellule
positive
90
80
70
60
50
40
30
20
10
0
Desmina
MyoD
Myf5
• induzione di uno stato di aumentata permissività nelle
cellule somatiche adulte umane
• up-regolazione dei geni di pluripotenza
• l’esposizione ad un medium di induzione specifico
permette di indirizzare le cellule di “aumentata
permissività” verso il differenziamento desiderato
(stesso foglietto embrionale)
DISEGNO SPERIMENTALE 3
Isolamento e caratterizzazione di fibroblasti cutanei di
suino
Trattamento di cellule somatiche con modificatori epigenetici
Differenziamento indotto in senso pancreatico
DIFFERENZIAMENTO PANCREATICO IN VITRO:
VALUTAZIONE MORFOLOGICA
Day 7
Day 10
Day 20
Day 36
DIFFERENZIAMENTO PANCREATICO IN VITRO:
GENI PANCREATICI
DEFINITIVE ENDODERM
PRIMITIVE
GUT TUBE
POSTERIOR
FOREGUT
PANCREATIC
ENDODERM
ENDOCRINE
PANCREATIC CELLS
SOX17
FOXA2
NES
HNF1
HNF4
PDX1
ONECUT
NKX6.1
PAX6
INS
GLC
SST
Day 0
(fibroblas
t)
-
-
-
-
-
-
-
-
-
-
-
-
Day 7
+++
+++
+++
++
++
+
+
-
-
-
-
-
Day 20
+
+
+
++
++
+++
+++
+++
++
+
+
+
Day 36
-
-
-
+
+
+++
++
++
+++
+++
+++
+++
DIFFERENZIAMENTO PANCREATICO IN VITRO:
MARCATORI PANCREATICI
SOX17PROPIDIUM
PAX6DAPI
FOXA2DAPI
Day 7
HNF4PROPIDIUM
ISL1DAPI
Day 20
C-PEPPDX1DAPI
Day 42
C-PEPNKX6.1DAPI
DIFFERENZIAMENTO PANCREATICO IN VITRO:
RILASCIO DI C-PEPTIDE DOPO STIMOLAZIONE
CON GLUCOSIO
Fold change
C-Peptide release after 1 h challange
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
42 days
102 days
5.5 mM DGlucose
20mM DGlucose
20mM L-Glucose
24h exp
EFFICIENZA DEL DIFFERENZIAMENTO
PANCREATICO evidenziata con FACS
39,2%
C-PEPTIDE
Esperimenti in corso con topi SCID STZ-treated
La valutazione delle potenzialità pancreatica di fibroblasti di cute suini dopo
differenziamento pancreatico è stata realizzata utilizzando un modello murino
in cui sono state distrutte le cellule beta (streptozotocina)
I topi sono stati iniettati nel sottocute con 5X106 cellule
Valutazione dei livelli di glucosio nel sangue
Blood glucose levels
Blood glucose (mg/dl)
700
Untreated fibroblasts
600
500
PCC
400
300
200
100
0
-6
-3
0
7
14 21 28 112 119 126 133
Time (days)
CONCLUSIONI
•E’ possibile modificare l’espressione di geni controllati per metilazione in cellule somatiche
adulte di mammifero,
•Questo fenomeno è di tipo transiente e reversibile, in quanto, nei successivi cicli di
replicazione del DNA la cellula ripristina la sua “memoria” epigenetica.
•La finestra temporale di maggiore plasticità può essere utilizzata al fine di indirizzare
opportunamente le cellule verso un lineage differente, utilizzando fattori opportunamente
addizionati al medium di coltura.
•In particolare, è stato possibile convertire
cellule umane di granulosa in cellule della muscolatura scheletrica, in grado di esprimere
fattori miogenici tipici di precursori e di cellule muscolari mature (conversione tra cellule
che derivano dallo stesso foglietto embrionale).
fibroblasti in cellule ti tipo pancreatico che producono attivamente insulina (conversione
tra cellule che derivano da foglietti embrionali diversi).
RINGRAZIAMENTI
UniMi, Laboratory of Biomedical Embryology
Fulvio Gandolfi
Georgia Pennarossa
Sara Maffei
Stefania Antonini
Mahbubur Rahman
Marino Campagnol
UniMi, Laboratoy of Stem Cell
Biology and Pharmacology of Neurodegenerative Diseases
Elena Cattaneo
Valentina Castiglioni
Infertility Unit
Ospedale Regina Elena
Guido Ragni
Alessio Paffoni
DBSM,
University of Insubria, Varese
Magda deEguileor
Gianluca Tettamanti
C-PEPPDX1DAPI
ATTIVITA' DI DIVULGAZIONE:
Nature "Science on the Solstice". 21 giugno 2006
BBC radio 4: The quest for virgin birth. 1 gennaio 2009
Radio 1 programma "Tornando a casa".
Puntata del 2 aprile 2009
Rai1. Uno Mattina. Puntata dedicata al Nobel per la Medicina. 12 ottobre
2012.
III Convegno nazionale del Gruppo dei Ricercatori Italiani sulle Cellule
Staminali Embrionali "Finanziare la ricerca sulle cellule staminali embrionali ",
1 luglio 2008, Sala di Refettorio di Palazzo San Macuto, Camera dei Deputati,
Roma
MeetMetonight. 29 settembre 2012
Collaborazioni Scientifiche:
-Istituto Zooprofilattico della Lombardia e dell'Emilia Romagna, D.ssa Maura Ferrari
-Cell Factory, Ospedale Policlinico di Milano, D.ssa Lorenza Lazzari
-Centro di Sterilità, Ospedale Policlinico di Milano, Dr. Guido Ragni
-Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, Copenhagen, DK,, Prof. Poul Hyttel
-The Hebrew University, Jerusalem, Israel, Prof Nissim Benvenisty
-Dipartimento di Biologia, Università degli Studi di Milano, Prof Giulio Cossu
-Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, Dr. Ivan Torrente
-The Macaulay Institute, Aberdeen, UK, Prof. Stuart Rhind
-California Institute for Reproductive Medicine, Prof. Alan Trounson
-Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, AU, Prof Alan Mackay-Sim
-AVANTEA, Cremona, Prof. Cesare Galli
-Dipartimento di Morfofisiologia Veterinaria, Università di Torino, Prof. Luca Bonfanti
- Centro di Biotecnologie della Riproduzione, Università Vita-Salute, D.ssa Lucia De Santis
- Università degli Studi dell'Insubria, Dipartimento di Biotecnologie e Scienze della Vita, Varese.Prof.ssa Magda deEguileor
- Università degli Studi di Milano. Dipartimento di Scienze Farmacologiche. Prof.ssa Elena Cattaneo
- Departement des Sciences Animales University of Laval, Ste-Foy, and Embryogene Network, Quebec, (Canada). Prof. Marc-Andre Sirard
- Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center, Kansas City, KS (USA). Prof. David
Albertini
-Dipartimento di Biologia Animale, Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari, Universita degli Studi di Sassari,
Facolta di Medicina Veterinaria, Sassari (Italy). Prof. Sergio Ledda
- Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide (Australia). Prof. David T. Armstrong
- Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, Università degli Studi di Napoli Federico II. Dr.ssa Bianca Gasparrini
-Imprinting and Cancer Group, Epigenetics and Cancer Biology Program, Bellvitge Institute for Biomedical Research (IDIBELL), Barcelona
(Spain). Prof. David Monk
- Área de Genética y Reproducción. Centro de Biotecnología Animal. SERIDA (Spain). Prof. Enrique Gomez e Prof.ssa Marta Monoz
- University of Murcia Department of Physiology. Faculty of Veterinary. Murcia (Spain). Prof.ssa Pilar Coy
- Universidade Federal do Ceará, Campus Sobral, Biotecnologia (Brazil). Prof. José Roberto Viana Silva.
C-PEPPDX1DAPI
Grazie per l’attenzione!
CLONING AS A POSSIBLE ALTERNATIVE TO
IMMUNOSUPPRESSION IN PIGS
Immunosuppression
CLONING
Pregnant sows
Adult male ear biopsy
40-day-old foetuses
Primary culture of pig
adult fibroblasts
Primary culture of pig
fetal fibroblasts
Brunetti et al., 2008
CLONING AS A POSSIBLE ALTERNATIVE TO
IMMUNOSUPPRESSION IN PIGS
Primary
culture of pig
fibroblasts
Cloned syngenic pigs
CLONING AS A POSSIBLE
ALTERNATIVE TO
IMMUNOSUPPRESSION IN
PIGS:
Creation of Green Fluorescent
Protein positive pig adult cardiac
progenitors