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.

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
Development and automation of 3D innovative hiPSC-based liver organoids including
the microenvironment for phenotypic screening - Application on metabolic diseases
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)
LiverBile
Fatty
acid
CholesterolLipoprotein
GlucoseInsulin
CytochromeXenobiotic
Hepatocyte
ADH
Hepatocyte
FHBL
Control
Control
ADH
FHBL
A newly designed differentiation protocol
Undifferentiated
hiPScells
Hepatocyte
differentiation
2D3D2D3D
Undifferentiated
hiPS cells
Hepatocyte
differentiation
2D3D2D3D
Hepatocyte
differentiation
Undifferentiated
hiPS cells
2D3D 2D3D 2D3D 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
Basale activity
Induced activity
Basale activity
Induced activity
Basale activity
Induced 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
20 µm
20 µm20 µm
20 µm20 µm
DAPI Albumin
Desmin Albumin
Desmin
Albumin
Desmin
DAPI - Amiodarone + Amiodarone
Functional test - Amiodarone-induced lipid accumulation in liver organoids