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Work Package 5 - a Genetically-Based Strategy to Identify New Targets in Ldl- C Metabolism

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Work Package 5 - a Genetically-Based Strategy to Identify New Targets in Ldl- C Metabolism WORK PACKAGE 5 - A GENETICALLY-BASED STRATEGY TO IDENTIFY NEW TARGETS IN LDL- C METABOLISM Task 3 : Functional characterization of new FHBL/ADH genes Méryl Roudaut Amandine Caillaud Karim SI-TAYEB Unité Inserm UMR 1087-CNRS UMR 6291 Transatlantic Network of Excellence 2014-2019 WORK PACKAGE 5 - A GENETICALLY-BASED STRATEGY TO IDENTIFY NEW TARGETS IN LDL- C METABOLISM Task 3 : Functional characterization of new FHBL/ADH genes Nouvelle cible FHBL UCell hypochol propositus’ sister (44386) Microscopie optique Native Ucell in culture UCell en cours de reprogrammation hiPS hypochol propositus (43899) Characterisation : Immunofluorescence OCT4 Tra1-60 Dapi Merge Clone 5 Clone Clone 7 Clone hiPS hypochol propositus (43899) Characterisation : flow cytometry Tra1-60 SSEA3 SSEA4 Clone 5 Clone Clone 7 Clone Work in progress… Propositus’ sister: - Clone picking - early characterization Propositus : - further characterization - hepatocyte differentiation (ongoing) What we need now? Hints from the genetic team to focus our functionnal exploration Developing a 3D model of Human iPS Cell-derived Hepatocytes Méryl Roudaut Jun, 29th 2017 Summary First part : generality Summary First part : generality Second part : first differentiation step Summary First part : generality Second part : first differentiation step Third part : second differentiation step Summary First part : generality Second part : first differentiation step Third part : second differentiation step Conclusion 3D gels and microenvironnement 3D compared to 2D culture : hepatic differentiation of UhiPS cells enhanced the expression of genes involved in cholesterol metabolism regulation Scaffolds are formed by crosslinking of Hyaluronic Acid with ADH (Adipic Acid Di-Hydrazide) to form reticulated chains that can be functionnalized. Institut du thorax - Human iPS Cell-derived Hepatocytes 2D protocol Days 0 2 5 Tissus ENDODERM Cytokins Activin A Activin A FGF2 BMP4 Institut du thorax - Human iPS Cell-derived Hepatocytes 2D protocol Days 0 2 5 10 15 Tissus ENDODERM HEPATIC ENDODERM HEPATIC PROGENITOR Cytokins Activin A Activin A FGF2 HGF FGF2 BMP4 BMP4 Institut du thorax - Human iPS Cell-derived Hepatocytes 2D protocol Days 0 2 5 10 15 20 Tissus ENDODERM HEPATIC ENDODERM HEPATIC PROGENITOR HEPATOCYTES Cytokins Activin A Activin A FGF2 HGF OSM FGF2 BMP4 BMP4 Second part : first differentiation step Experimentation plan Optimisation of seeding and the beginning of differentiation Days 0 2 5 Tissus ENDODERM Cytokins Activin A Activin A FGF2 BMP4 Experimentation plan Optimisation of seeding and the beginning of differentiation Seeding volume (ul) 10 20 Experimentation plan Optimisation of seeding and the beginning of differentiation Seeding volume (ul) 10 20 Cell number 50 000 100 000 200 000 … Experimentation plan Optimisation of seeding and the beginning of differentiation Seeding volume (ul) 10 20 Cell number 50 000 100 000 200 000 … … … Control condition Cytokins concentration (D 0) (compared to 2D) D 0-2 X 1 X 2 X 2 X 5 Experimentation plan Optimisation of seeding and the beginning of differentiation Seeding volume (ul) 10 20 Cell number 50 000 100 000 200 000 … … … Control condition Cytokins concentration (D 0) (compared to 2D) D 0-2 X 1 X 2 X 2 X 5 D 2-5 X 1 X 5 X 10 X 10 (A) (B) (C) (D) Results For the first step : 30 different conditions + controls conditions Only genes expression are seen in RT qPCR : Pluripotence genes OCT4 NANOG SOX2 Results For the first step : 30 different conditions + controls conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes OCT4 FOXA2 NANOG SOX17 SOX2 Results For the first step : 30 different conditions + controls conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes Hepatics progenitor genes OCT4 FOXA2 HNF4 NANOG SOX17 SOX2 Results For the first step : 30 different conditions + controls conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes Hepatics progenitor genes OCT4 FOXA2 HNF4 NANOG SOX17 SOX2 Best condition : 10 100 000 X 2 X 10 (C) Results Best condition OCT4 Control condition Results Best condition NANOG Control condition Results Best condition SOX2 Control condition Results FOXA2 Best condition Control condition Results SOX17 Best condition Control condition Results HNF4 Best condition Control condition Results Importance of the ARN quantity and quality Best condition Third part : second differentiation step Institut du thorax - Human iPS Cell-derived Hepatocytes 3D protocol Seeding volume 10 Cell number 100 000 Days 0 2 5 Tissus ENDODERM Cytokins Activin A Activin A FGF2 BMP4 X 2 X 10 Institut du thorax - Human iPS Cell-derived Hepatocytes 3D protocol Seeding volume 10 Cell number 100 000 Days 0 2 5 10 Tissus ENDODERM HEPATIC ENDODERM HEPATIC PROGENITOR Cytokins Activin A Activin A FGF2 FGF2 BMP4 BMP4 X 2 X 10 X ? Institut du thorax - Human iPS Cell-derived Hepatocytes 3D protocol FGF2 EGF PDGF RAC1 NKD1 WNT PATHWAY VEGF Institut du thorax - Human iPS Cell-derived Hepatocytes 3D protocol Seeding volume 10 Cell number 100 000 Days 0 2 5 10 Tissus ENDODERM HEPATIC ENDODERM Cytokins Activin A Activin A FGF2 FGF2 BMP4 BMP4 Synergy with FGF2 X 2 X 10 X ? + VEGF or PDGF or EGF Experimentation plan Optimisation of hepatic progenitor differentiation Seeding volume (ul) 10 Cell number 100 000 Cytokins concentration (compared to 2D) D 0-2 X 2 D 2-5 X 10 Experimentation plan Optimisation of hepatic progenitor differentiation Seeding volume (ul) 10 Cell number 100 000 Cytokins concentration (compared to 2D) D 0-2 X 2 D 2-5 X 10 D 5-10 X 5 X 10 Experimentation plan Optimisation of hepatic progenitor differentiation Seeding volume (ul) 10 Cell number 100 000 Cytokins concentration (compared to 2D) D 0-2 X 2 D 2-5 X 10 D 5-10 (E) X 5 (F) X 10 … VEGF PDGF EGF Experimentation plan Optimisation of hepatic progenitor differentiation D 5-10 (E) X 5 (F) X 10 … VEGF PDGF EGF Same to VEGF … C1 C2 C1 Experimentation plan Optimisation of hepatic progenitor differentiation D 5-10 (E) X 5 (F) X 10 … VEGF PDGF EGF Same to VEGF … C1 C2 C1 … … D F T D : Day 5 to 8 F : Day 8 to 10 T : Day 5 to 10 Results For the second step : 32 different conditions Only genes expression are seen in RT qPCR : Pluripotence genes OCT4 NANOG SOX2 Results For the second step : 32 different conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes OCT4 FOXA2 NANOG SOX2 Results For the second step : 32 different conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes Hepatics progenitor genes OCT4 FOXA2 HNF4 NANOG HNF1b SOX2 FOXA3 TBX3 HHEX PPARA Results For the second step : 32 different conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes Hepatics progenitor Hepatocytes genes genes OCT4 FOXA2 HNF4 Albumine NANOG HNF1b AFP SOX2 FOXA3 TBX3 HHEX PPARA Results For the second step : 32 different conditions Only genes expression are seen in RT qPCR : Pluripotence genes Endoderm genes Hepatics progenitor Hepatocytes genes genes OCT4 FOXA2 HNF4 Albumine NANOG HNF1b AFP SOX2 FOXA3 TBX3 HHEX (E) PPARA Best condition : X 5 VEGF C2 T E VEGF T Results Results Results Results Results Results Conclusion Seeding volume 10 Cell number 100 000 Days 0 2 5 Tissus ENDODERM Cytokins Activin A Activin A FGF2 BMP4 X 2 X 10 Conclusion Seeding volume 10 Cell number 100 000 Days 0 2 5 8 10 Tissus ENDODERM HEPATIC ENDODERM Cytokins Activin A Activin A FGF2 FGF2 BMP4 BMP4 X 2 X 10 X 5 + VEGF C2 Conclusion Seeding volume 10 Cell number 100 000 Days 0 2 5 8 10 Tissus ENDODERM HEPATIC ENDODERM Cytokins Activin A Activin A FGF2 FGF2 BMP4 BMP4 X 2 X 10 X 5 + VEGF C2 Conclusion I am here Days 0 2 5 8 10 15 20 Tissus ENDODERM HEPATIC ENDODERM HEPATIC PROGENITOR HEPATOCYTE Conclusion Seeding volume 10 Cell number 100 000 Days 0 2 5 8 10 15 20 Tissus ENDODERM HEPATIC ENDODERM HEPATIC PROGENITOR HEPATOCYTES Cytokins Activin A Activin A FGF2 HGF OSM FGF2 BMP4 BMP4 X 2 X 10 X 5 X ? X ? + VEGF C2 Conclusion Days 20 Tissus HEPATOCYTE December 2017 Validation Patenting Thanks for your attention Any questions ? Hypochol : état des lieux juin 2017 Le recrutement : • 36 propositus Hypochol confirmés et prélevés pour la génétique • 4 avec des RDV prévus cet été • 10 en attente de prise de RDV • Nouveau screening / base biologie du CHU de Nantes • Démarche prospective pour les CES • Bioliance : courrier séparé du bilan en prospectif et en rétrospectif en fin de validation identitée Biologie mutatio N° N° sex nv LDL HDL chol trigly N° LD date de naissance envoi Diag n Rendu date bilan bio famille intégralis e g/L g/L tot g/L g/L connue 42578 42578 LD000M 09/12/1945 ? 04/12/2012 0,16 0,46 1,12 2,55 43899 43899 voir faF 03/05/1958 03/2017, absenceL 24/06/2014 0,51 0,73 1,33 0,44 44216 44216 LD000F 14/07/1967 à Lille,03/E - ; PCSK9 - 03/12/1014 0,21 0,84 1,09 0,19 44216 54935 LD002F 25/11/1965 12/09/2011 0,1 1,08 1,3 0,3 44216 54998 LD002F 09/06/1997 10/05/2017 1,08 0,64 1,83 0,55 Les résultats 45269 45269 LD000M 14/04/1943Lyon 3/17 01/04/2015 0,74 0,57 1,44 0,67 45420 45420 LD000M 27/04/1979Lyon 5/17 15/11/2014 0,38 0,35 0,87 0,72 / Lyon 45429 45429 LD000M 25/03/1977 non? 27/01/2014 0,46 0,99 1,64 0,97 47002 47002 LD000F 19/03/1988 7/15, résultats ? 01/07/2015 0,49 0,53 1,09 0,36 47260 47260 LD000M 10/05/1972 ats ? ; envoi diag Lyon 06/1 16/04/2015 0,51 0,37 1,38 2,49 47260 47511 LD000F 21/07/1970 23/09/2015 1,32 0,45 2,04 1,35 47490 47490 LD000F 18/09/1994on, 11/20Apo B à faire 28/10/2015 0,49 0,74 1,28 0,23 48554 48554 LD001F 25/02/1987
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