Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection - Inserm - Institut national de la santé et de la recherche médicale Accéder directement au contenu
Article Dans Une Revue Current Stem Cell Research & Therapy Année : 2019

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection

Résumé

BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells. RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis. CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.
Fichier principal
Vignette du fichier
s13287-019-1342-6.pdf (7.54 Mo) Télécharger le fichier
Loading...

Dates et versions

inserm-02337213 , version 1 (29-10-2019)

Identifiants

Citer

Jérôme Caron, Véronique Pène, Laia Tolosa, Maxime Villaret, Eléanor Luce, et al.. Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection. Current Stem Cell Research & Therapy, 2019, 10 (1), pp.221. ⟨10.1186/s13287-019-1342-6⟩. ⟨inserm-02337213⟩
45 Consultations
101 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More