Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

3D innovative hiPSC-based models including the microenvironment for phenotypic screening - Application on metabolic diseases

44 views

Published on

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 instead of the classical 2D culture systems to enhance their maturation; 2. Adapt our 3D differentiation process to a 96 wells format to make it compatible for drug screening.

To reach our goals, we established a partnership with HCS Pharma, which has an expertise in high content phenotypic screening and produces an innovative 3D scaffold, BiomimesysTM. This scaffold is composed of hyaluronic acid that can be functionalized with extra cellular matric derivatives, with adjustable stiffness and porosity. We setup conditions for hiPSCs seeding and differentiation to reach a new protocol adapted to a 3D environment. Our preliminary data indicate that our procedure enhanced expression of hepatic markers such as transcription factors (FOXA2, FOXA3, HNF1a, HNF1b, HNF4a), cytochrome P450 (CYP450) family members (CYP3A4, CYP2A6, CYP7A1) or cholesterol metabolism regulators (PCSK9, Lipoprotein(a)). During our presentation, we will discuss our data hiPSCs differentiation in 3D, CYP450 activities and induction, as well as their application for the study of metabolic diseases.

Published in: Healthcare
  • Be the first to comment

  • Be the first to like this

3D innovative hiPSC-based models including the microenvironment for phenotypic screening - Application on metabolic diseases

  1. 1. BANDEAU TUTELLES L’unité de recherche de l’institut du thorax Inserm UMR 1087 / CNRS UMR 6291 Nantes, France 3D innovative hiPSC-based models including the microenvironment for phenotypic screening - Application on metabolic diseases Karim Si-Tayeb ELRIGfr Conference 23/24 mai 2018 Le Bischenberg, Bischoffsheim, France
  2. 2. 2 Familial hypercholesterolemia (FH) Facteurs de risque génétique Récepteur LDL Inconnus APOB PCSK9 Hypercholesterolemia: Impaired LDL-cholesterol uptake Modified from Norata et al. EHJ 2013 Identification of informative famillies displaying: -  A spontaneously low LDL-C level (FHBL) -  A hypercolesterolemia without cardiovascular disease (CIC Thorax, Matthieu Pichelin, Marie Marrec) Absence of mutations in known genes involved in FH (LDLR, PCSK9, APOB, ANGPTL3) (Jocelyne Magré, Jean-Jacques Schott)
  3. 3. 3 The Scientist Magazine April 2016 Si-Tayeb et al. Disease Models & Mechanisms 2016 Patient Urine-derived cells hiPS cells in vitro study of familial hypercholesterolemia Hepatocytes © l’institut du thorax Modeling FH in vitro from an urine sample - the power of hiPS cells
  4. 4. 4 From hiPS cells toward hepatocytes – back to developmental biology He: precardiac mesoderm End: endoderm Lb: liver bud E: endothelial cells STM: Septum transversum d0 d5 d10 d15 d20 Hepatocytes Definitif Endoderm Hepatic progenitors Specified Definitif Endoderm Human pluripotent stem cells Si-Tayeb et al. Developmental Cell 2010 Si-Tayeb et al. Hepatology 2010
  5. 5. 5 Fluorescent LDL uptake analyzed by flow cytometry Phenotype reproduced in vitro Familial hypercholesterolemia in a petri-dish Phenotype pharmacologicaly reversed in vitro Si-Tayeb, Idriss et al. Disease Models & Mechanims 2016
  6. 6. Hypercholestérolémie: Défaut d’internalisation du LDL-choléstérol
  7. 7. 7 A promising model that needs further maturation by adding a third dimension Nature review 2004
  8. 8. 8 Liver A promising model that needs further maturation by adding a third dimension Amandine Caillaud Méryl Roudaut
  9. 9. 9 A new differentiation protocol for a new environment Cytokines d0 d5 d10 d15 d20 Hepatocytes Definitif Endoderm Hepatic progenitors Specified Definitif Endoderm Human pluripotent stem cells Méryl Roudaut
  10. 10. 10 CYP450 basal activities and induction CYP450 2C9 (ibuprofen) Diclofenac -> 4OH-Diclofenac CYP450 1A2 (caffein) Phenacetin-> Acetaminophen CYP450 2A6 Nicotine-> Cotinine CYP450 2E1 (ethanol) Chlorzoxazone-> 6-0H-Chlorzoxazone Méryl Roudaut (nM) (nM) (nM)(nM)
  11. 11. 11 CYP450 basal activities and induction CYP450 3A4 Testosteron-> 6ß-OH-testosteron 0 100 200 300 400 500 0 10 20 30 40 50 60 70 nmol/L Minutes CYP3A4 activity (nmol/L/min/well) Basal activity Induced activity 0 50 100 150 200 250 Basal activity Induced activityActivitypercentage CYP3A4 activity * Méryl Roudaut (nM)
  12. 12. 12 Conclusion and perspectives hiPS cells -> Genetic variability Hepatic cells -> Phenotypic screening 3D 2D 6 puits 96 puits
  13. 13. 13 Conclusion and perspectives Liver NASH Fibrosis
  14. 14. www.umr1087@univ-nantes.fr L’unité de recherche de l’institut du thorax Inserm UMR 1087 / CNRS UMR 6291 Nantes, France Daniel Buren et Patrick Bouchain, Les Anneaux, Quai des Antilles, Nantes, création pérenne Estuaire 2007 © Martin Argyroglo/LVAN L’institut du thorax CARIOU Bertrand LE MAY Cédric MAGRE Jocelyne PRIEUR Xavier PICHELIN Matthieu ARNAUD Lucie AYER Audrey BLANCHARD Claire Wargny Matthieu MOREAU François CHADEUF Gilliane DIJK Wienecke THEDREZ Aurélie SI-TAYEB Karim IDRISS Salam CAILLAUD Amandine GIRARDEAU Aurore ROUDAUT Méryl CANTAT Pierre-Alexandre LORANT Victoria HCS Pharma MAUBON Nathalie MAUBON Gregory SOUGUIR Zied VANDENHAUTE Elodie

×