<!-- wp:paragraph --> <p>We previously showed that human pluripotent stem cells (hiPSCs) provide a suitable model to study<br>metabolic diseases upon hepatocyte-like cell (HLC) differentiation. With a non-invasive approach, hiPSCs can be generated from urine samples of patients and HLCs have been used to model cholesterol metabolism regulation, by the study of LDLR- and PCSK9-mediated autosomal dominant hypercholesterolemia (ADH) as well as PCSK9-mediated familial hypobetalipoproteinemia (FHBL). This model provides promising advantages with a direct link to the patient and with an unlimited source of HLCs. But like all models, there are limitations, mainly by the neonatal characteristic of HLCs lead to difficulties for pharmacological investigations.</p> <!-- /wp:paragraph --> <!-- wp:paragraph --> <p>Therefore, to overcome these burdens, we chose to 1. Differentiate hiPSCs into HLCs in an innovative<br>3D <a href="https://hcs-pharma.com/biomimesys/">hyaluronic acid-based hydroscaffold</a>, BIOMIMESYS® produces by HCS Pharma to enhance their maturation. 2. Adapt our 3D differentiation process to a 96-well format to make it compatible for drug screening. 3. Characterization of the 3D HLCs model by metabolism tests and compare to primary human hepatocyte (PHH).</p> <!-- /wp:paragraph --> <!-- wp:paragraph --> <p>We gathered 3’ SRP data all along the differentiation process and RNAseq has been performed by comparing 2D and 3D differentiation conditions to characterize hiPSCs differentiation into liver organoids. We observed an enhanced expression of most hepatic genes and genes expressed by non-parenchymal cells such as stellate cells. Immunofluorescence data confirmed the co-localization of albumin-positive<br>hepatocytes, desmin-positive stellate cells and LYVE1-positive endothelial cells in liver organoids. Finally, at a functional level, several CYP activities including CYP3A4 were detected at the basal level and successfully induced. Liver organoids responded to pharmacological treatments as shown by their ability to accumulate lipids upon amiodarone treatment or uptake LDL-bodipy upon statin treatment.</p> <!-- /wp:paragraph --> <!-- wp:paragraph --> <p>Altogether, our development gave rise to functional liver organoids generated with a unique and common procedure, in a process of automating for future high throughput screening.</p> <!-- /wp:paragraph -->

1. 1. L'institut du thorax, Inserm UMR1087 – CNRS UMR 6291, Université de Nantes, Nantes, France
2. HCS Pharma, Lille, France
3. Plateforme de Spectrométrie de Masse, CRNHO, INRAE, UMR 1280, Nantes France
DEVELOPMENT OF INNOVATIVE HIPSC-BASED MODELS INCLUDING AN INNOVATIVE 3D
MODIFIED HYALURONIC ACID HYDROSCAFFOLD FOR PHENOTYPIC SCREENING
Méryl Roudaut2, Amandine Caillaud1, Aurore Girardeau1, Matthieu Pichelin1, Mikaël Croyal3, Elodie Vandenhaute2, Zied
Souguir2, Nathalie Maubon2, Bertrand Cariou1, Karim Si-Tayeb1
INTRODUCTION
We previously showed that human induced pluripotent stem cells (hiPSCs) provide a suitable model to study
metabolic diseases upon hepatocyte-like cells (HLCs) 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) (see right).
While the direct link between hiPSCs and patients, as well as the abundance of HLCs provide promising
advantages of such a 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 chose to:
1. Differentiate hiPSCs into HLCs in a 3D physiological environment to enhance their maturation
2. Adapt our 3D differentiation process to 96-well format to make it compatible for drug screening
CONCLUSION
Méryl Roudaut, HCS Pharma
meryl.roudaut@hcs-pharma.com
+ 33 (0)769 999 137
Comparison between 2D and 3D hepatic differentiation by RNAseq
www.umr1087.univ-nantes.fr
A 3D environment for hiPSC differentiation into liver organoids
Porosity & stiffness
ECM components
(Zanger and Schwab, 2013)
(Brunton L and al., 2018)
Testosteron
disappearence
6ß-OH-Testosteron
production
Functional tests: Cytochrome P450 activities and induction of hiPSC-derived liver organoids (MS)
Liver
Bile
Fatty
acid
Cholesterol
Lipoprotein
Glucose
Insulin
Cytochrome
Xenobiotic
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
BIOMIMESYS® Liver matrix for iPSCs provides a suitable support for hiPSC differentiation into liver organoids that displayed mature features at the
gene expression and functional levels. The presence of a physiological matrix and the different liver cell types as well as a long-lasting model will
help to better mimic a model of (N)ASH ((Non) Alcoholic Steato Hepatitis).
Toward liver organoids Functional tests in liver organoids grown in BIOMIMESYS®
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
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
BIOMIMESYS® hydroscaffoldsTM are
formed by crosslinking Hyaluronic Acid and
ECM components (collagens) with ADH
(Adipic Acid Di-Hydrazide) to form
reticulated chains. This is not a hydrogel!
Hydroscaffold (Scanning Electron Microscopy)
Liver organoid - collagen I (blue)
Liver organoid - cytoskeleton : Phalloidin
(green)
Induced activity
Basale activity
Induced activity
Basale activity
Long-term maintenance
Day 0
Day 2
Day 5
Day 7-10
Day 13-16
Day 19
Day 22
Day 25
Day 28-49
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***
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Internalization of fluorescent LDL
Basal phenotype
reproduced in vitro
Pharmacological
phenotype
reproduced in vitro
Si-Tayeb, Idriss et al. Disease
Models & Mechanims 2016
Internalization of
fluorescent LDL
(FACS analysis)
Control patient Hypercholesterolemic
patient
Control
patient
Hypercholesterolemic
patient
Control
Patient
Hypercholesterolemic
patient
Liver organoid
HydroscaffoldsTM
HydroscaffoldsTM
Liver organoid surface