Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia

Karim Si-Tayeb, Salam Idriss, Benoite Champon, Amandine Caillaud, Matthieu Pichelin, Lucie Arnaud, Patricia Lemarchand, Cédric Le May, Kazem Zibara, Bertrand Cariou, Karim Si-Tayeb, Salam Idriss, Benoite Champon, Amandine Caillaud, Matthieu Pichelin, Lucie Arnaud, Patricia Lemarchand, Cédric Le May, Kazem Zibara, Bertrand Cariou

Abstract

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19 ± 0.77-fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.

Keywords: Autosomal dominant hypercholesterolemia; Hepatocyte differentiation; Human induced pluripotent stem cells; PCSK9; Urine-derived somatic cells.

Conflict of interest statement

Competing interests

B.Cariou has received advisory board fees from Amgen and Sanofi/Regeneron Pharmaceuticals. The other authors have nothing to disclose.

© 2016. Published by The Company of Biologists Ltd.

Figures

Fig. 1.
Fig. 1.
Ucell characterization. (A) Urine-derived progenitor cell (Ucell) morphology after isolation and amplification. (B) Cell marker expression analysis (percentage of positive cells) of Ucells, bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells by flow cytometry. (C) Osteoblastic and chondrogenic differentiation of Ucells detected by Alizarin Red and Alcian Blue staining, respectively. Scale bars: 100 µm.
Fig. 2.
Fig. 2.
UhiPSCcharacterization. (A) Detection of OCT3/4 and Tra1-60 expression by fluorescent immunostaining on control UhiPSCs and UhiPSCs carrying the PCSK9-S127R or PCSK9-R104C/V114A mutations. (B) Representative flow-cytometry analysis for the detection of SSEA4, SSEA3 and TRA1-60 expression by control (Ct) UhiPSCs. (C) Global gene-expression profile comparison between Ucell control (ct), Ucell S127R, UhiPSC control and UhiPSCs carrying the PCSK9-S127R mutation. (D) Hematoxylin- and eosin-stained histological section of teratomas showing, from left to right, ectoderm-derived neurons, mesoderm-derived cartilage and endoderm-derived intestinal-like tissue for UhiPSC-Control (Ct), and ectoderm-derived retinal cells, mesodermal-derived bone tissue and endodermal-derived exocrine pancreatic glands for UhiPSC PCSK9-S127R. Scale bars: 100 µm.
Fig. 3.
Fig. 3.
UhiPSCdifferentiation intoHLCs. (A) Pictures of HLCs carrying the S127R or R104C/V114A mutations. Left: HLC morphology observed by brightfield microscopy (the lower panel represents a magnification of the upper panel). Right: detection of the hepatic markers FOXA2, AFP, HNF4α and albumin by fluorescent immunostaining (nuclei were stained in blue in merged pictures). Scale bars: 100 µm. (B) Gene expression analysis by RT-qPCR of albumin, HNF4a, SREBF2, LDLR, PCSK9 and HMGCR in control, S127R and R104C/V114A HLCs (one clone per UhiPSC line; control n=6 differentiations, PCSK9-S127R n=3 differentiations, PCSK9-R104C/V114A n=3 differentiations). (C) Secreted PCSK9 detection by ELISA assay (one clone per UhiPSC line; control n=9 differentiations, PCSK9-S127R n=9 differentiations, PCSK9-R104C/V114A n=14 differentiations). (D,E) Quantification by flow cytometry of incorporated LDL from HLCs (D) PCSK9-S127R and (E) R104C/V114A compared to controls. MFI, mean fluorescence intensity. *P<0.05, **P<0.01, ***P<0.001.
Fig. 4.
Fig. 4.
Differentiated cell response to pravastatin treatment. (A,B) Gene expression analysis by RT-qPCR of albumin, HNF4a, SREBF2, LDLR, PCSK9 and HMGCR in an untreated condition (black bars) and after 24 h of pravastatin treatment at 10 µM (white bars) of control and S127R HLCs (one clone per UhiPSC line; control n=6 differentiation, PCSK9-S127R n=3 differentiation). (C) Secreted PCSK9 detection by ELISA assay (one clone per UhiPSC line; control n=6 differentiations, PCSK9-S127R n=6 differentiations). (D) Detection of incorporated LDL by flow cytometry (one clone per UhiPSC line; control n=6 differentiations, PCSK9-S127R n=3 differentiations). MFI, mean fluorescence intensity. *P<0.05, **P<0.01.
Fig. 5.
Fig. 5.
Comparative analysis of the response of control and PCSK9-S127R HLCs to pravastatin treatment, normalized to untreated conditions. (A) Gene expression analysis by RT-qPCR of SREBF2, LDLR, PCSK9 and HMGCR (three clones per UhiPSC line; n=3 differentiations per clone). (B) Secreted PCSK9 detected by ELISA assay (one clone per UhiPSC line; n=9 differentiations per clone). (C) Detection of incorporated LDL by flow cytometry (three clones per UhiPSC line; n=3 differentiations per clone). **P<0.01.

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