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HUMAN INDUCED PLURIPOTENT STEM CELLS AS A MODEL TO STUDY METABOLIC INHERITED LIVER DISEASES

Final Report Summary - IPSMILD (HUMAN INDUCED PLURIPOTENT STEM CELLS AS A MODEL TO STUDY METABOLIC INHERITED LIVER DISEASES)

The project IRG277188/IPSMILD focuses on the use of human induced pluripotent stem cells (hiPSC) to study the proprotein convertase subtilisin/kexin type 9 (PCSK9) during hepatocyte development in a human context and characterize its role in cholesterol homeostasis in normal and pathological conditions such as autosomal dominant hypercholesterolemia (ADH). While PCSK9 gain of function (GOF) mutations induces ADH, lost of function mutations (LOF) lead to a low level of circulating low-density lipoprotein-cholesterol (LDLc). This last features suggested that PCSK9 inhibition could results in a cardio-protective effects. Therefore, therapeutic strategies aiming to inhibit PCSK9 with siRNA or specific antibodies, we designed in order to improve ADH, and recent phase III clinical trials confirmed the benefit of such strategies. However, because of limitation of current model (mice models, tumor cell lines, heterologous surexpression systems), unknown functions and roles of PCSK9 are yet to discover. IPSMILD main goal was to establish an original model to enhance our understanding of PCSK9 functions, by using human induced pluripotent stem cells (hiPSC) reprogrammed from somatic cells of patients carrying PCSK9 GOF and LOF mutations and differentiated them into hepatocyte-like cells (HLC). We setup culture conditions for the isolation and amplification of progenitor cells from human urine samples and their reprogramming into hiPSC (UhiPSC). We obtain UhiPSC from healthy donors as well as patients with the GOF mutation PCSK9-S127R and LOF mutations PCSK9-R104C/V114A. We verified that PCSK9 was expressed during hiPSC differentiation into HLC by quantitative PCR and Elisa assay. Patient-derived UhiPSC carrying the GOF mutation S127R on one allele, expressed a mutated form of PCSK9 that is not correctly processed and secreted. We were able to show that, while there was no difference at the mRNA level, secreted PCSK9 was 2 fold lower in HLC carrying the mutation compare to controls, confirming that our model will be suitable for the study of PCSK9 on ADH. Therefore their ability to uptake LDL has been evaluated and our experiments demonstrated that the S127R mutation led to a significant decrease of LDL internalization compared to control HLC. In addition, both HLC were submitted to a statin treatment, the current treatment against ADH and we observed that while control HLC displayed an increase of LDL uptake in presence of statin, HLC carrying the PCSK9-S127R showed an even greater response that brought the level of internalized LDL at the same level of untreated control HLC. Together, our data showed that this model is relevant to model PCSK9-mediated ADH and test potential therapeutic molecules. Finally, in line with the LOF mutation R104C/V114R of PCSK9 resulting in a very low level of circulating LDLc, our preliminary data showed that upon differentiation, HLC carrying the PCSK9 LOF mutations displayed a higher level of LDL uptake, which strengthen the pertinence of our model.
In parallel, to study the effect of the loss of PCSK9 on HLC differentiation and function, we generated and characterized three hiPSC lines expressing shRNA directed against PCSK9 and analyzed their effects.
The conduction of this project and the development of cellular tools to achieve it allowed me to strongly integrate the host laboratory (L’institut du thorax, UMR INSERM1087/CNRS6291, IRS-UN Nantes) and setup several collaborations with local, national and international research teams. This environment is particularly favorable for the development and the emergence of my research team as well as its visibility at the international level. I am currently applying for a tenure track position in order to sustain my research activities in the field within my current host laboratory.