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

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.

Pluripotent stem cell-derived bile canaliculi-forming hepatocytes to study genetic liver diseases involving hepatocyte polarity

“…Pluripotent HUES9 cells were differentiated toward hepatocyte-like cells (hiHeps) using a protocol based on embryonic hepatic development and previous studies on hepatocyte differentiation in culture [16,24,30] ( Figure 1A). Figure S1A).…”

“…Patientown cells are the ideal source to study endogenous mutant proteins. The ability to generate induced pluripotent stem cells (iPSC) from patients' cells and to differentiate these to hepatocytes has been a major step towards the study of endogenous mutant proteins, including ATP7B, in physiologically relevant cell types [16][17][18][19][20][21][22][23][24]. However, the potential of iPSC-derived hepatocytes to polarize and form bile canaliculi, and their potential to study the polarized trafficking of endogenously expressed mutant protein, is not known.…”

Bile‐Derived Organoids From Patients With Primary Sclerosing Cholangitis Recapitulate Their Inflammatory Immune Profile

“…(6)(7)(8) The ability to isolate and culture these stem cells may provide a sustainable source of cells to study cellular pathways and gene regulation in a disease-specific manner. Most exciting are recent reports that stem cells can be found in bodily fluids, such as urine, (9)(10)(11) thus providing a noninvasive source of cells to study disease. Therefore, we hypothesized that bile may also contain stem cells which could be obtained from patients with PSC who undergo endoscopic retrograde cholangiopancreatography (ERCP) procedures, providing access to biliary specimens well before end-stage liver disease and transplantation.…”

Focal adhesion kinase promotes BMP2‐induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways

“…Zhao et al, 2018). These cells can differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and in vivo (Si-Tayeb et al, 2016;S. Wu, Liu, Bharadwaj, Atala, & Zhang, 2011).…”