Journée Hépatologie 2022

1. Top 5 in
Liver and bioengineering
Jean-Charles Duclos-Vallée
Centre Hépato-Biliaire
Paul-Brousse Hospital

2. Microtechnology and
applications in liver bioengineering
Microatterning
Co-culture
Encapsulation
Bioartificial/
Transplantable
bioengineered liver
Drug screening
Disease modelling

3. Microtechnology and
applications in liver bioengineering
Micropatterning
Co-culture
Encapsulation
Bioartificial/
Transplantable
bioengineered liver
Drug screening
Disease modelling
Microfluidics/
On chip devices
Biomaterials
Biomaterials
2D
Cell culture

4. Two main types of devices
Artificial Bio Artificial
Living component
A Necessity !
Figaro S, Pereira U, …, Legallais C et al. Int J Organs 2017

5. Global representation of the differentiation
protocol of hiPSCs into hepatocytes and
encapsulation into alginate beads
CHIR,
Activin A,
LY
FGF2,
BMP4,
FGF4, HGF
Definitive
endoderm (DE)
hiPSCs
Hepatoblasts
(iHBs)
Spheroid
HGF, OSM, Dex, SB, CE, Vk
iHBs spheroid encapsulation and
hepatocyte differentiation Encapsulated
spheroids
(E-3D)
Day 10-11
iHB self-
aggregation
iHBs differentiation
Day 26
Day 20
Day 16
Day 11
500 µm 500 µm 500 µm 500 µm
Encapsulation
Day 10
500 µm
Morphology of encapsulated spheroids
From Marwa Hussein

7. Distribution of PLL and elastic modulus of
the surface
PLL
Permeability of empty beads
(coated or not with PLL) to
fluorescent markers (albumin,
IgG, nanoparticles of different
diameters)

8. Distribution of PLL and elastic modulus of the
surface
PLL
Permeability of empty beads
(coated or not with PLL) to
fluorescent markers (albumin, IgG,
nanoparticles of different
diameters)
Metabolic activities of cells in
alginate‐PLL beads, measured on
Day 14, over 6 h' incubation in
equivalent pathological plasma
medium
Ammonia
detoxification rate
Lactate
detoxification rate
Albumin
production

10. Towards a functional
artificial liver
From Université Technologique de Compiègne

12. Definition of organoid: in vitro 3D
cellular cluster derived from tissue-
resident stem/progenitor cells,
embryonic stem cells (ESCs) or
induced pluripotent stem cells
(iPSCs) that recapitulates the
functionality of the tissue of origin
Lancaster MA and Knoblich JA. Science 2014
Huch M and Koo BK. Development 2015

13. Organogenesis and stages
for organoid progenitor isolation
Tremblay and Zaret, Dev Biol 2005
SI-Tayeb et al. Dev Cell 2010
Fiorotot et al. Acta 2018
Douarin Med Biol1975
Gualdi et al. Genes Dev 1996
Jung et al Science1999
Kamiya et al EMBO J 1999
Rossi et al; Genes Dev2001
Zaret et al Nat Rev Genet 2002
D’Amour Nat Biotechnol 2005
Han et al. Stem Cells 2011
Adapted from Prior N, Inacio P and Huch M Gut 2019
Activin/
Nodal
WNT
FGF
BMP
Hepatocytes
+
Cholangiocytes

14. Construction of liver organoids
Messina A et al. Cells 2022
500 µm 200 µm 200 µm 200 µm
hiPSCs iDE (day 5) iHE (day 8) iHB (day 10)
3D
0
1
2
3
4
5
6
7
d13 d16 d18 d20 d25
µg*millioncells/24h
2D 3D AFP secretion
0
0,2
0,4
0,6
0,8
1
d13 d16 d18 d20 d25
µg
*millioncells/24h
3D 2D
ALB secretion
ActivinA
hiPSC BMP4
DE
• FGF4
• EGF
• HGF
HE
• HGF
• OSM
• Dex
HB
• HGF
• OSM
• Dex
IH MH
1h 2 weeks
Phall
Nuclei
Confocal images of spheroid at day 25 of the differentiation protocol.
ALB HNF4α
Nuclei
200 µm
Self-assembling
3D configuration allows to generate hiPSC-derived liver
organoids with hepatic functions equivalent to PHHs

15. Bile acid secretion and bile canaliculi network
analysis of iHep-Orgs
Messina A et al. Cells 2022
Bile acid production and secretion by iHep-
Orgs in culture supernatants at day 20
Live imaging of the bile canaliculi network
formed by excretion of the fluorescent probe DCFA
in iHep-Org at day 38
Immunofluorescence staining for the apical
Membrane transporter BSEP in iHep-Org
at day 38

17. Liver-on-chip
HepaRG
Boul M Sci Reports 2021

18. Chip response to acetaminophen (APAP) exposure.
Cell viability in control and treated chips
(CTRL n = 3 chips, 2 mM APAP n = 2 chips)
Immunofluorescent images of
a control chip, a chip treated with APAP.
nuclei (blue), ZO-1 (green) and MDR3 (red)
Boul M Sci Reports 2021

22. At high-dose, the common
signature of the three
treatments reflected liver
steatosis profile highlighted by
the modulation
of genes related to PPAR
signaling, fatty acid, lipid
metabolism and
steroid biosynthesis.

23. Metabolomic, profiled
showed high level of fatty acids
and lipids (decanoic,
tetradecanoic and
palmitic acids), glucose,
fructose and tagatose

25. Schematic of key components of the
enterohepatic bile acid synthesis and
recycling pathways.

26. HLOs respond physiologically when physically
coupled to bile acid-treated ileal HIOs

28. Transcriptional profiling of primary cholangiocytes
Sampaziotis F et al. Science 2021

29. Cholangiocyte Organoid identity is
controlled by niche stimuli
Sampaziotis F et al. Science 2021

30. Cholangiocyte organoids (COs) rescue cholangiopathy
following transplantation and assume an identity
corresponding to the site of engraftment.
Sampaziotis F et al. Science 2021

31. Transplantation of cholangiocyte
organoids in human
Up regulation of intrahepatic
(SOX4) And loss of gallbladder Sox17
and improvement of bile properties
Absence of RFP cells
in the perfusate
Sampaziotis F et al. Science 2021

32. RHU
A building block approach towards a fully functional liver

33. Differentiation
of
liver cells
from
human pluripotent
stem cells
and
applications
Alexis Bernatets
Alan J Rajendran
Ana Mesic
Bruno
Le Pioufle
Sakina
Bensallem
Microfluidics/
Liver On chip devices
Anne Dubart-Kupperschmitt
Nassima Benzoubir
Mano Boul
Marwa Hussein
Eleanor Luce
Antonietta Messina

34. « Toute imagination humaine
vient du coeur ».
Paracelse