metabolic diseases upon hepatocyte-like cell (HLC) differentiation. In particular, HLCs have been used to model cholesterol metabolism regulation, by mimicking the main disease features in vitro. Human iPSCs can be generated from urine samples of patients with a well-described phenotype and carrying specific genotypes. This non-invasive approach allowed the study of LDLR- and PCSK9-mediated autosomal dominant hypercholesterolemia (ADH) as well as PCSK9-mediated familial hypobetalipoproteinemia (FHBL). While the direct link between hiPSCs and patients, as well as the abundance of HLCs provide promising advantages of such strategy, it is impaired mainly by the neonatal characteristic of HLCs as well as the difficulty to perform high throughput studies for pharmacological investigations.

1. 1. L'institut du thorax, Inserm UMR1087 – CNRS UMR 6291, Université de Nantes, Nantes, France.
2. HCS Pharma, Lille.
3. Plateforme de Spectrométrie de Masse, CRNHO, Inra UMR 1280, Nantes France
A single procedure to generate functional hiPSCs-derived liver organoids - Towards an
innovative tools suitable for drug screening
Méryl Roudaut1,2, Amandine Caillaud1, Aurélie Thédrez1, Wieneke Dijk1, Aurore Girardeau1, Matthieu Pichelin1, Lucie Arnaud1,
Mikaël Croyal3, Cédric Le May1, Elodie Vandenhaute2, Zied Souguir2, Nathalie Maubon2, Bertrand Cariou1, Karim Si-Tayeb1
INTRODUCTION
We previously showed that human pluripotent stem cells (hiPSCs) provide a suitable model to study metabolic diseases
upon hepatocyte-like cell (HLC) differentiation. In particular, HLCs have been used to model cholesterol metabolism
regulation, by mimicking the main disease features in vitro. Human iPSCs can be generated from urine samples of
patients with a well-described phenotype and carrying specific genotypes. This non-invasive approach allowed the study
of LDLR- and PCSK9-mediated autosomal dominant hypercholesterolemia (ADH) as well as PCSK9-mediated familial
hypobetalipoproteinemia (FHBL). While the direct link between hiPSCs and patients, as well as the abundance of HLCs
provide promising advantages of such strategy, it is impaired mainly by the neonatal characteristic of HLCs as well as the
difficulty to perform high throughput studies for pharmacological investigations.
Therefore, to overcome these burdens, we choose to :
1. Differentiate hiPSCs into HLCs in a 3D environment to enhance their maturation;
2. Adapt our 3D differentiation process to a 96 wells format to make it compatible for drug
screening.
CONCLUSIONS
Karim SI-TAYEB, L’unité de recherche de l’institut du thorax
karim.si-tayeb@univ-nantes.fr
+ 33 (2) 28080176
Comparaison between 2D and 3D hepatic differentiation by RNAseq
www.umr1087.univ-nantes.fr
A 3D environment for hiPS cells differentiation
Liver
Porosity & stiffness
ECM compounds
(Zanger and Schwab, 2013)
(Brunton L and al., 2018)
Testosteron
disappearence
6ß-OH-Testosteron
apparition
Functional test - Cytochrome P450 activities and induction of hiPSC-derived liver organoids (MS)
Liver
Bile
Fatty
acid
Cholesterol
Lipoprotein
Glucose
Insulin
Cytochrome
Xenobiotic
Hepatocyte
ADH
Hepatocyte
FHBL
Control
Control
ADH
FHBL
A newly designed differentiation protocol
Undifferentiated
hiPS
cells
Hepatocyte
differentiation
2D
3D
2D
3D
Undifferentiated
hiPS cells
Hepatocyte
differentiation
2D
3D
2D
3D
Hepatocyte
differentiation
Undifferentiated
hiPS cells
2D
3D 2D
3D 2D
3D Volcano plot 3D hepatocytes (red) vs 2D hepatocytes (green)
Benjamini & Hochberg method (7448 /57905 DE genes)
Log2 (fold change)
CYP1A2 (caffein)
Phenacetin -> Acetaminophen
CYP2D6
Dextromethorphan -> Dextrorphan
CYP2C9 (ibuprofen)
Diclofenac-> 4OH-Diclofenac
CYP2E1 (ethanol)
Chlorzoxazone -> 6OH-Chlorzoxazone
CYP3A4
Testosteron -> 6ßOH-Testosteron
Induced activity
Basale activity
The BiomimesysTM Liver provides a suitable model for hiPSC differentiation into liver organoids that displayed mature features at the
gene expression and functional level. A 384 well format is being optimized for high throughput phenotypic screening.
Toward liver organoids Functional tests in liver organoids
DAPI 2-Deoxy-D-glucose Merge
Untreated
Insulin
6nM
50µm 50µm 50µm
50µm 50µm 50µm
DAPI LDL-bodipy Merge
Untreated
Mevastatin
50nM
50µm 50µm 50µm
50µm 50µm 50µm
Liver organoid
Albumin
Desmin
DAPI
Merge
3D 2D 3D 2D 3D 2D 3D 2D
Amiodarone or ethanol-
induced lipid accumulation
Mevastatin-induced LDL-
bodipy internalization
Insulin-induced 2-Deoxy-D-
glucose internalization
50µm 50µm 50µm
DAPI Nile Red Merge
Untreated
50µm 50µm 50µm
Amiodarone
20µM
50µm 50µm 50µm
Ethanol
200nM
Immunofluorescence characterization
Merge
DAPI ZO-1 Merge
50µm 50µm 50µm
50µm 50µm 50µm 50µm
DAPI CD31 LHX2 Merge
50µm 50µm 50µm 50µm
DAPI OATP1B1 LYVE1 Merge
BiomimesysTM
scaffolds are formed
by crosslinking
Hyaluronic Acid with
ADH (Adipic Acid Di-
Hydrazide) to form
reticulated chains. Liver organoid - collagen I (bleu)
Hydroscaffold Liver organoid - cytoskeleton : Phalloidin
(green)
Induced activity
Basale activity
Induced activity
Basale activity