XXV Convegno A.I.R.T.
Torino, 3-4 aprile 2014
Cellule staminali epatiche:
realtà attuale e sviluppi futuri
Renato Romagnoli
Chirurgia Generale 2 U - Centro Trapianto Fegato “E.S.Curtoni”
Direttore : Prof. M. Salizzoni
A.O.U. Città della Salute e della Scienza di Torino
Università degli Studi di Torino
Chirurgia Trapianto Fegato:
Prof. M. Salizzoni
R. Romagnoli
D. Patrono
N. Gilbo
P. Strignano
A. Brunati
Radiologia:
D. Righi
G. Isolato
C. Gazzera
MBC LAB: Prof. G. Camussi
M. B. Herrera
Malattie Metaboliche:
S. Bruno
M. Spada
F. Collino
V. Fonsato
CRRT Piemonte:
C. Cavallari
Prof. A. Amoroso
M.C. Deregibus
Oncoematologia Pediatrica: F. Antico
Fresenius Medical Care: Prof. C. Tetta
Prof.ssa F. Fagioli
Hepatocyte Transplantation
A ‘proof of concept’ for cell therapies in:
• inherited metabolic liver diseases
• acute liver failure
• acute-on-chronic liver failure
Hepatocyte Transplantation
in inherited metabolic liver diseases
Technique
Isolation and cryopreservation
of mature hepatocytes
from deceased donor organs
Repeated infusions of hepatocyte
suspensions in portal vein
(portal catheterization)
Average quantity of cells per
infusion: 100 X 106 / kg
Number of infusions per trial: 2  6
Hepatocyte Transplantation
in inherited metabolic liver diseases
Technique
Isolation and cryopreservation
of mature hepatocytes
from deceased donor organs
Repeated infusions of hepatocyte
suspensions in portal vein
(portal catheterization)
Average quantity of cells per
infusion: 100 X 106 / kg
Number of infusions per trial: 2  6
Results
40 patients treated
(1998-2012)
urea cycle disorders:
- CPS deficiency
- OTC deficiency
- ASS deficiency
- ASL deficiency
type I glycogenosis
type I Crigler-Najjar
hypercholesterolemia
Metabolic correction
Possible ‘bridge’ to organ
transplant
No portal thrombosis reported
Hepatocyte Transplantation
Drawbacks and Limits
• mature hepatocytes DO NOT proliferate in culture
• scarce availability and yield (cells usually isolated from
low quality livers, discarded from organ transplantation)
• poor viability and quality of cryopreserved cells
• need of immunosuppressive therapy
• limited durability of engrafted cells: 3  12 months
(for rejection or natural cell exhaustion and loss)
• need of repeated infusions
Properties of Stem Cells
• Cells capable of dividing and renewing
(proliferation and self-renewal)
• Cells that have the potential to give rise to
specialized cell types (differentiation)
• Cells that have the capacity to “repair”
damaged tissues (regeneration)
Types of Stem Cells
• Embryonic Stem Cells (ESC):
derived from 4-5 day-old
blastocyst
• Adult Stem Cells (ASC): identified after accomplished
organogenesis in many organs and tissues, such as bone marrow,
muscles, peripheral blood, umbilical cord blood, amniotic fluid, blood
vessels, skin, fat, teeth, heart, gut, liver, kidney, ovarian epithelium,
testis, etc
• Induced Pluripotent Stem Cells (iPSC):
fibroblasts could
be reprogrammed by viral transfection into a pluripotent state similar to
that observed in ESC through ectopic expression of four genes (Oct3/4,
Sox2, c-Myc, and Klf4)
Stem cells in liver diseases
ESC
Expansion
and/or
iPSC
ASC
Bioartificial liver
device
Differentiation
Transplant
Stem cells in liver diseases
ESC
Expansion
and/or
iPSC
ASC
Bioartificial liver
device
Differentiation
Transplant
Mesenchymal Stem Cells (MSC)
in acute liver injury
• Bone marrow MSCs
• Mesenchymal stem cell-derived molecules reverse fulminant hepatic
failure. Parekkadan B et al. PLoS One, 2007; 2 (9): e941
• Immediate intraportal transplantation of human bone marrow
mesenchymal stem cells prevents death from fulminant hepatic failure pigs.
Li J. Hepatology, 2012; 56: 1044-1052
• Amniotic fluid MSCs
• Therapeutic potential of a distinct population of human amniotic fluid
mesenchymal stem cells and their secreted molecules in mice with acute
hepatic failure. Zagoura DS et al. Gut, 2012; 61 (6): 894-906
• Amniotic-fluid-derived
mesenchymal
stem
cells
overexpressing
interleukin-1 receptor antagonist improve fulminant hepatic failure. Zheng
YB et al. PLoS One, 2012; 7 (7): e41392
• Umbilical Cord MSCs
• Systemic administration of a novel human umbilical cord mesenchymal
stem cells population accelerates the resolution of acute liver injury. Burra P
et al. BMC Gastroenterology, 2012; 12: 88
Problems inherent to MSC-based therapy
The heterogeneity of MSC populations may render
difficult:
• the evaluation of the potency of different MSC
preparations
• the comparison of different clinical trials
Potential complications:
• the intravenous administration of MSCs may induce
formation of pulmonary emboli or infarctions
• tumorigenesis and maldifferentiation
Problems inherent to MSC-based therapy
Maldifferentiation in experimental animal models:
- MSCs injected in the ischemic heart may in the long-term
differentiate into bone
Breitbach et al. Blood 2007
- In the anti-Ty-1-induced glomerulonephritis, MSCs in the longterm differentiate into adipocytes favoring development of a
chronic renal failure
Kunter et al. J Am Soc Nephrol 2007
- After lung irradiation, MSCs favor fibroblast and myofibroblast
accumulation in focal pulmonary areas
Epperly MW Am J Respir Cell Mol Biol 2003
The blue stars indicate sites at which there are high numbers of peribiliary glands,
the stem cell niches of the biliary tree
Adapted from Turner R et al, Hepatology, 2011; 53: 1035-1045
Intrahepatic maturational lineages
Adapted from Turner R et al, Hepatology, 2011; 53: 1035-1045
Stem cells in human liver
Human biliary tree stem cells (hBTSCs):
a multipotent cell
lineage capable of differentiating in cholangiocytes, hepatocytes and
pancreatic islets; EpCAM+, NCAM+, CD105-, CD133+, CK19+(?), AFP-,
ALB-
Human hepatic stem cells (hHpSCs): liver resident precursors
of hepatoblasts; EpCAM+, NCAM+, ICAM-1-, CD105-, CD133+, CK19+,
AFP-, ALB±
Hepatoblasts:
isolated by MACS (Magnetic-activated cell-sorting)
mostly from fetal and neonatal livers, but present also in adults; a
bipotential cell lineage capable of differentiating into both cholangiocytes
and hepatocytes; EpCAM+, NCAM-, CD105-, CD133+, CK19+, AFP+, ALB+
Human adult Liver derived Stem Cells (HLSCs): adult
liver resident MSCs; EpCAM-, NCAM-, CD105+, CD133- , CK19-, AFP+,
ALB+
The discovery
a-MEM/EBM
+ 10% FCS
+ hEGF + hFGF
Enzymatic
dissociation
Hepatocytes
15 days
Human Liver Stem Cells
Stem cell population from adult human liver
Hepatic markers ? YES
albumin
α FP
CK8
Mesenchymal Stem Cell markers ? YES
CK18
vimentin
nestin
musashi
Haemopoietic markers ? NO
Embryonic Stem Cell markers ? YES
nanog
SSEA4
pax2
octa4
Herrera et al., STEM CELLS 2006
Differentiation capabilities
Osteogenic differentiation
Calcium deposits
osteopontin
osteocalcin
Insulin-secreting cells
NO adipogenic
differentiation
Endothelial cells
Mature
Hepatocytes
Herrera et al., STEM CELLS 2006
Differentiation of HLSC into mature hepatocytes
Monolayer
or
+HGF/FGF4
15 days
α FP
CK8/CK18
Cytochrome P450
Incorporation and complete
release of Indocyanin green
albumin
urea
Herrera et al., STEM CELLS 2006, Fonsato et al., TISSUE ENG 2010
US patent number: 2010/0003752
Publications
Urea cycle disorders – Rare diseases
glutamate
Acetyl-Coa
N-acetylglutamate
NH3
HCO3
Orotic acid
+
2 ATP
CPS
carbamoylphosphate
2 ADP
L-ornithine
UREA
OTC
citrulline
ASS
L-arginine
Arginino-succinate
ASL
EU orphan designation
Investigational Medicinal Product Dossier (IMPD)
The Investigational Medicinal Product Dossier
is the basis for approval of clinical trials by the
competent authorities in the E.U.
An IMPD is requested whenever the performance of a clinical
study in any one of the EU Member States is intended.
The IMPD includes information related to the
• quality
• manufacture
• control of the Investigational Medicinal Product
• data from non-clinical studies and from its clinical use
• an overall risk-benefit assessment
• critical analyses of the non-clinical and clinical data in relation to the
potential risks and benefits of the proposed study
Pre-clinical studies for IMPD
In vitro
PHARMACODYNAMICS
To assess the
pharmacological effect
of HLSC in liver damage
In vivo
PHARMACOKINETICS
In vivo
To evaluate the
biodistribution of HLSC
after injection
Disease models Healthy models
(SCID mice)
(SCID and nude
mice and rats)
TOXICOLOGY
To find a safe first dose, to identify
potential target organs for toxicity
and to assess potential
tumorigenicity
Good manufacturing practice
(GMP)-grade production of HLSC
• GalN/LPS induced mouse
model of hepatic failure
• N-Acetyl-p-aminophen
induced mouse model of
acute liver injury
Different administration routes:
• Intravenous
• Intra-parenchyma
• Intra-portal
In vivo
Different doses and modality of
administration
Pharmacodynamics
Regenerative potential of HLSC in a model of acute liver injury
(induced by N-acetyl-p-aminophen) in SCID mice
7 days
35 days
Human HLA-I
Human HLA-I
Mouse β-μglob
Human HLA-I
Human HLA-I
FISH
Human HLA-I
Control serum
35 days
FISH
35 days
Not injured liver + HLSC
Herrera al, STEM CELLS, 2006
Pharmacodynamics
T=0
GalN/LPS
after 30’
2x106 (6x0.33)
sacrifice
•
•
•
•
•
7 hours
24 hours
3 days
7 days
21 days
0.2-0.5 x106
Herrera et al., HEPATOLOGY 2013
Pharmacodynamics
Herrera et al., HEPATOLOGY 2013
Pharmacodynamics
APOPTOSIS
PROLIFERATION
Herrera et al., HEPATOLOGY 2013
Pharmacodynamics
In vivo differentiation of HLSC
Timepoint
n° HLA+
cells/hpf
% of
HLA+/CK8+
CK18+ cells
24
hours
20.1±16.1
2.5%
7 days
16.8±8.9
88%
21 days
12.6±3.3
48%
Herrera et al., HEPATOLOGY 2013
Pharmacokinetics
BIODISTRIBUTION
model
N-Acetyl-p-aminophen
species
SCID mice with FHF
Healthy SCID mice
GalN/LPS
SCID mice with FHF
Rats
Healthy animals
SCID mice
Nude mice
dose and route
n° animals
0.2x106 i.v.
6
PBS i.v.
6
0.2x106 i.v.
6
2x106 i.v.
9
PBS i.v.
9
0.2x106 i. par.
4
0.5x106 i. par.
14
1x106 i. par.
4
PBS i. par.
2
1.8x106 i.v. (OI)
8
PBS i.par.
1
5x106 i.par. (OI)
5
PBS i.v.
2
1.8x106 i.v. (OI)
10
PBS i.par.
3
1x106 i. par. (OI)
10
Pharmacokinetics
BIODISTRIBUTION: i.v. injection of CFSE labeled HLSC
Liver
24 h
7d
21 d
Spleen
Lung
Pharmacokinetics
BIODISTRIBUTION: intra-parenchymal injection of HLSC
Liver
Spleen
Lung
6d
14 d
HLA
HLA+
35
cells
30
25
20
HLA
15
10
5
0
1.000.000
500.000
6D
200.000
500.000
500.000
14 D
21 D
BIODISTRIBUTION: Optical Imaging (OI)
Pharmacokinetics
Toxicology
Aims:
To find a safe first dose for dose escalation studies in man
To identify potential target organs for toxicity
To exclude the development of tumors
Toxicology
To find a safe first dose for dose escalation studies in man
To identify potential target organs for toxicity
model
GalN/LPS
specie
SCID mice
with FHF
dose and route
results
1x106 i.v.
From the highest to lowest dose, one single injection resulted in animal
death by pulmonary occlusion.
0.75x106 i.v.
0.5x106 i. v.
0.35x106 i.v. x 6
No signs of toxicity recorded after 48 hours, 7 and 21 days
10x106 intra-portal
The surgical procedures is feasible. Liver necrosis in small regions and
signs of thrombosis indicate that probably the amount of injected cells
should be reduced and heparin should be added
5x106 intra-portal
All rats appeared in good health conditions . Macroscopic observation of
collected organs (liver spleen lung) did not show signs of necrosis or
vascular thrombosis. Histological evaluation of liver, lung, spleen sections
showed a normal morphology
5x106 i.par.
All rats appeared in good heath conditions without signs of suffering.
Macroscopic observation of collected organs (liver spleen lung) did not
show signs of necrosis or vascular thrombosis. Histological evaluation of
liver, lung, spleen sections showed a normal morphology.
0.5x106 i.par. x 2
Steatosis was observed in all samples, probably due to anaesthetics and
to the operation procedure. AST and ALT levels in the blood showed
similar liver functions in all mice 14 days after the second injection.
Rats
Healthy animals
SCID mice
0.25x106 i.par. x 2
Toxicology
To exclude the development of tumors
n° of
nude
mice
cells
n° of cells
injected
Duration of
monitoring
10
HLSC
1x107 sub-cut
16 weeks
10
HeLa (POSITIVE CTR)
1x107 sub-cut
16 weeks
5
DPBS (NEGATIVE CTR)
0.2 ml
16 weeks
Validity
The test resulted
VALID
The test is valid if:
• at least 90% of the positive control group develops growing tumors
• none of the negative control group develops growing tumors
HLSC did not cause tumour formation
Toxicology
Karyotype and Tumorigenicity
Skin
Genetically stable: 2 months
14th passage
Growth in agar
Liver
No tumour formation 6 months after
s.c. inoculum in SCID mice
Banking in Areta International (GMP)
1. Feasibility studies: HLSC from 3 different adult liver biopsies
•
•
•
Isolation
Expansion
Characterization: FACS analysis (CD73, CD29, CD105, and Albumin), and
Potency test in RCCS (rotary cell culture system ) for 4 days
2. Production:
•
•
•
isolation, expansion and characterization of HLSC for
creation of a master cell bank (MCB)
MCB production (isolation and expansion)
MCB characterization (FACS analysis and Potency test)
MCB release (Sterility test, tumor in vivo formation and karyotype
analysis)
3. Validation:
•
•
•
First validation run
Second validation run
Third validation run
Validation run: defrost a HLSC vial containing 50x106 HLSCs for freezing in
bags
Where are we now in Turin ?
Orphan Drug designation granted by EMA to HLSC in OTC and CPS (urea cycle
disorders) and in Acute Liver Failure
First Clinical Protocol  Final Authorization for 3 patients
HLSC in patients suffering from inborn liver metabolic diseases causing lifethreatening neonatal onset of hyperammonemic encephalopathy
Study code: HLSC 01-11; EudraCT: 2012-002120-33
Principal Investigator: Dr. Marco Spada
Co-Investigators: Dr. Renato Romagnoli – Dr. Dorico Righi
ISS and AIFA (Rome) : June 2013
COMITATO ETICO (Turin) : December 2013
ACTIVE ENROLMENT FEASIBLE: February 2014
Piemonte – Valle d’Aosta : 4.500.000 abitanti - 40.000 neonati/anno
TORINO
Clinica Pediatrica, Università di Torino – Ospedale Infantile Regina Margherita
Azienda Ospedaliera Città della Salute e della Scienza di Torino
Unità Operative:
- Malattie Metaboliche Ereditarie
- Epatologia Pediatrica
- Nefrologia pediatrica  dialisi CVVHD nel neonato
- Terapia Intensiva Pediatrica
- Laboratorio Terapie Cellulari in Ematologia Pediatrica
- Centro Trapianti di Fegato pediatrico e dell’adulto
- Radiologia Interventistica «epatica»
- Immunologia dei Trapianti
- Laboratorio Biochimica Metabolica (esami 2° livello)
TRAPIANTO DI CELLULE STAMINALI EPATICHE
Origine delle cellule
 Frammenti di tessuto epatico prelevati dall’equipe chirurgica da fegati
che vengono utilizzati per trapianto
 Nessun danno anatomico e funzionale all’organo trapiantato
(macrobiopsie)
 Donatori d’organo classificati a “rischio standard”, ovvero casi in cui
dal processo di valutazione non emergano fattori di rischio per malattie
trasmissibili
TRAPIANTO DI CELLULE STAMINALI EPATICHE
NELLE MALATTIE METABOLICHE EREDITARIE
Vantaggi clinici attesi
 Possibilità di produzione GMP e banking di cellule
perenne disponibilità di cellule ABO-compatibili rapidamente
trapiantabili
 Utilizzo di organi “standard” rispetto al trapianto di epatociti
 Terapia immunosoppressiva non prevista dal protocollo
(attività immunomodulante delle HLSC come le MSC)
 Stabilizzazione metabolica e clinica dei pazienti, con possibilità di
dilazionare o addirittura evitare il trapianto d’organo
Where are we now in Turin ?
Orphan Drug designation granted by EMA to HLSC in OTC and CPS (urea cycle
disorders) and in Acute Liver Failure
First Clinical Protocol  Final Authorization
HLSC in patients suffering from inborn liver metabolic diseases causing lifethreatening neonatal onset of hyperammonemic encephalopathy
Study code: HLSC 01-11; EudraCT: 2012-002120-33
ISS and AIFA (Rome) : June 2013
COMITATO ETICO (Turin) : December 2013
ACTIVE ENROLMENT FEASIBLE: February 2014
Second Clinical Protocol in preparation
HLSC in patients with acute liver failure arising in a previously healthy liver
Final remarks
 The ‘stem cell bench’ is stepping into the clinic, especially
in diseases without architectural distortion of the liver
 Nearly every field of hepatology will be touched in the
mid-term, but solid organ transplantation as destination
therapy for cirrhosis will still hold its place for a while
 Multidisciplinarity in translational research is a valuable
and essential model for progress
 Funding remains a major issue. In this context, creation of
European multinational Consortia should be encouraged
Grazie per l’attenzione !