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1. Potential applications of
germline-cell derived
pluripotent stem cells in
preclinical animal models
Fiorella Altruda
Molecular Biotechnology Center,
University of Turin
22 January 2011
3. Spermatogonial stem cells convert spontaneously to GPSCs in long term culture
*
ES-like
colony
Oct4
SSC to ES-like transition Nanog
SSEA-1
Sox2
Lin28
C-myc
Oct4
Sox2 SSC= Spermatogonial stem cells
Lin28 (Unipotent)
C-myc
GPSC= Germline-derived pluripotent
SSC colony stem cells (Pluripotent)
4. Characteristics of GPSCs
•Express Oct4, Nanog,SSEA-1, Lin28, c-myc
•Positive for Alkaline phosphatase
•Teratoma formation in vivo
•Chimera formation
Advantages of GPSCs versus ES cells and other adult stem cells
• GPSCs are already pluripotent (no “reprogramming” required)
• GPSCs can be passaged for over 30 times without loss in replicative
capacity or change in karyotype
• GPSCs can be frozen and thawed
• GPSCs are derived from adult tissues: Avoid ethical concerns over
embryo use for production of pluripotent stem cells
• And...
5. …GPSCs show high plasticity
Sarcomeric
cardiac troponin T
α-actinin
pan-cadherin Cx43
Immunostaining of cardiac clusters
Guan et al, Circ. Res 2007
8. Hepatocyte differentiation protocol
GPSC
Static
2 days
suspension
culture
Embryoid bodies
4 days
Plated
a-FGF + b-FGF (early)
on gelatin
or GF reduced- 4 days
matrigel
HGF (intermediate)
6 days
OSM/Dex/ITS (late)
Culture in
Collagen gel
Modified from Chinzei et al. Hepatology 2002
9. Morphological and molecular characterisation of GPSCs
during induced hepatocyte differentiation
A GPSC-derived hepatocytes
GPSC NT Day 7(gelatin) Day 21(gelatin) Day 21(collagen)
B Days of differentiation
GPSCNT 2 7 14 21 28 Liver
ALB
AFP
ALB: Albumin
TAT AFP: Alpha fetoprotein
Hx TAT: Tyrosine aminotransferase
Hx: Hemopexin
Hp
Hp: Haptoglobin
Oct4
Actin
Cyp7a1
C
Relative Quantity
10000
1000
Liver-specific 100
10
1 Cyp7a1: cytochrome P450, family 7,
subfamily A, polypeptide 1
0,1
NT 28 NT 28 liver
GPSC#1 GPSC#2 Fagoonee et al. Stem Cells and
Days of differentiation Development 2010
10. Percentage of hepatocytes in the embryoid bodies
GFP+ hepatocyte-
EB 21d like cell neuron-like cell
Lentivirus: pCCL.ET.GFP.Sin
Enhanced transthyretin promoter
Kind gift from L.Naldini
90 Undifferentiated GPSCs
hepatocytes (%)
80 Control Infected
GFP-positive
positive
70
60 0.04% 7.77%
50
FL2-H
FL2-H
40
30
20
10 R4 R4
0
NT 21 35 100 101 102 103 104 100 101 102 103 104
Days of differentiation FL1-H FL1-H
GPSC-derived hepatocytes
Control Day 21(infected) Day 35(infected)
0.03% 70.16% 82.68%
FL2-H
FL2-H
FL2-H
R3 R3 R3
0 1 2 3 4
100 101 102 103 104 100 101 102 103 104 10 10 10 10 10
FL1-H FL1-H FL1-H
11. Functional Analyses
Haptoglobin secretion
collagen
matrigel
Albumin secretion
gelatin
serum
Matrigel
Collagen gel Hp
Albumin (ng)/ mg protein
1000
Urea Synthesis
100
1000
Urea (µg mg protein
**a
* ***b
10
100 *
*
µg)/
***
1
7 11 14 18 24 27 PH 10
Days of differentiation
1
11 14 18 22 PH
Days of differentiation
Glycogen deposits and Indocyanine green uptake
PAS-staining Indocyanine green
GPSC-derived hepatocytes MEFs GPSC-derived hepatocytes
*
12. How far are we from in vivo-
derived hepatocytes?
15. GPSC-
Pearson with E16 Hepatocytes
0.74
0.75
0.76
0.77
0.78
0.79
0.80
0.81
0.82
0.83
0.84
ES nt
ES EB day 2
ES EB day 7
ES EB day 14
ES EB day 21
ES EB day 28
GPSC1 nt
GPSC1 EB day 2
GPSC1 EB day 7
GPSC1 EB day 14
GPSC1 EB day 21
GPSC1 EB day 28
GPSC2 nt
GPSC2 EB day 2
than adult primary hepatocytes
GPSC2 EB day 7
GPSC2 EB day 14
GPSC2 EB day 21
GPSC2 EB day 28
GPSC-derived hepatocytes are closer to E16 (fetal) primary hepatocytes
Pearson correlation
16. Conclusions
GPSCs can be induced to differentiate into
functional hepatocytes in vitro :
• they express liver specific genes
• they show functional properties of hepatocytes
• they show high efficiency (80%) of differentiation
• they are closer to fetal hepatocytes
Fagoonee et al., Stem Cells and Development 2010
18. Cell therapy and correction of genetic liver diseases
Mouse models : Glycogen storage disease type 1a (G-6-Pase KO
mice from S.Eva (Gaslini hospital, Genova))
Hemochromatosis (Hfe-null mice from E.Tolosano)
Fulminant hepatic failure (galactosamine/LPS)
Ongoing Experimental set-up
set-
Hepatocytic differentiation of GPSCs
Intrasplenic or
Intraparenchymal
injection
Partially hepatectomised, monocrotaline-treated female mice
Determine presence of Y chromosome (FISH) in female livers (collaborator: G. Inghirami)
Isolation of human SSC and derivation of GPSCs
19. Glycogen Storage Disease Type 1a
(GSD-1a)
• Autosomal recessive disorder
• Caused by mutations in the catalytic subunit of the
glucose-6-phosphatase
• glucose-6-phosphatase is a key enzyme in the glucose
metabolism
• glucose-6-phosphatase is expressed primarily in the liver
and kidney
• GSD-1°patients manifest hypoglycemia, growth
retardation, hepatomegaly and other metabolic disorders
• No complete cure have been reported (liver
transplantation)
20. Perl’s Staining 5 days post injection
1 2
Hfe-null mice
3 4
1-2 negative control (mice treated with monocrotaline, hepatectomised)
3-4 positive (mice treated with monocrotaline, hepatectomised and GPSC injection)
22. Do GPSCs differentiate into functional renal cells?
•Optimise existing protocols
•Determine which renal cell types formed
•Functional analyses
•Animal models: renal ischemia and reperfusion injury
glycerol-induced acute renal injury
Autosomal Recessive Polycystic Kidney Disease
Collaborators: G. Camussi
S. Bruno
23. Find novel genes involved in
pluripotency maintenance
• Genes
• miRNAs
Pluripotency assays
Collaborators: Ferdinando Di Cunto
Paolo Provero
Rosario Piro
24. Acknowledgements
MBC COLLABORATORS
BIDMC, Boston Anatomia Patologica, Turin
Sharmila Fagoonee Giorgio Inghirami
Pier Paolo Pandolfi
Robin Hobbs Cristina Abele
Letizia De Chiara
Francesca Barbieri MBC
San Raffaele, Milan
Marco Zuin Giovanni Camussi
Luigi Naldini
Alessio Cantore Stefania Bruno
Emanuela Tolosano
Lorenzo Silengo Paolo Provero
IRCC, Candiolo
Enzo Medico Ferdinando Di Cunto
Daniela Cantarella Rosario Piro
Gaslini, Genova
Sandra Eva