Human induced pluripotent stem cells-derived liver organoids grown on a Biomimesys® hyaluronic acid-based hydroscaffold as a new model for studying human lipoprotein metabolism.

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid-lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte-like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three-dimensional (3D) approach using Biomimesys®, a hyaluronic acid-based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28-day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low-density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC-derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a).

Generation of an induced pluripotent stem cell line (ITXi012-A) from a patient with genetically determined high-lipoprotein(a) plasma levels.

Elevated circulating lipoprotein(a) (Lp(a)) is a genetically determined risk factor for coronary artery disease and aortic valve stenosis (Tsimikas, 2017). Importantly, the LPA gene, which encodes the apolipoprotein(a) (protein-component of Lp(a)), is missing in most species, and human liver cell-lines do not secrete Lp(a). There is a need for the development of human in vitro models suitable for investigating biological mechanisms involved in Lp(a) metabolism. We here generated and characterized iPSCs from a patient with extremely high Lp(a) plasma levels genetically determined (Coassin et al., 2022). This unique cellular model offers great opportunities and new perspectives for investigations on biological mechanisms involved in Lp(a) metabolism.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.

This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).

Variants in the GPR146 Gene Are Associated With a Favorable Cardiometabolic Risk Profile.

BACKGROUND: In mice, GPR146 (G-protein-coupled receptor 146) deficiency reduces plasma lipids and protects against atherosclerosis. Whether these findings translate to humans is unknown. METHODS: Common and rare genetic variants in the GPR146 gene locus were used as research instruments in the UK Biobank. The Lifelines, The Copenhagen-City Heart Study, and a cohort of individuals with familial hypobetalipoproteinemia were used to find and study rare GPR146 variants. RESULTS: In the UK Biobank, carriers of the common rs2362529-C allele present with lower low-density lipoprotein cholesterol, apo (apolipoprotein) B, high-density lipoprotein cholesterol, apoAI, CRP (C-reactive protein), and plasma liver enzymes compared with noncarriers. Carriers of the common rs1997243-G allele, associated with higher GPR146 expression, present with the exact opposite phenotype. The associations with plasma lipids of the above alleles are allele dose-dependent. Heterozygote carriers of a rare coding variant (p.Pro62Leu; n=2615), predicted to be damaging, show a stronger reductions in the above parameters compared with carriers of the common rs2362529-C allele. The p.Pro62Leu variant is furthermore shown to segregate with low low-density lipoprotein cholesterol in a family with familial hypobetalipoproteinemia. Compared with controls, carriers of the common rs2362529-C allele show a marginally reduced risk of coronary artery disease (P=0.03) concomitant with a small effect size on low-density lipoprotein cholesterol (average decrease of 2.24 mg/dL in homozygotes) of this variant. Finally, mendelian randomization analyses suggest a causal relationship between GPR146 gene expression and plasma lipid and liver enzyme levels. CONCLUSIONS: This study shows that carriers of new genetic GPR146 variants have a beneficial cardiometabolic risk profile, but it remains to be shown whether genetic or pharmaceutical inhibition of GPR146 protects against atherosclerosis in humans.

Identification of a Gain-of-Function LIPC Variant as a Novel Cause of Familial Combined Hypocholesterolemia.

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.

Generation of a GPR146 knockout human induced pluripotent stem cell line (ITXi001-A-1).

Dyslipidemia is a key modifiable causal risk factor involved in the development of atherosclerotic cardiovascular disease. Recently, the G protein-coupled receptor 146 (GPR146), a member of the G-coupled protein receptors' family, has been shown to be a regulator of plasma cholesterol. Inhibition of hepatic GPR146 in mice displays protective effect against both hypercholesterolemia and atherosclerosis. Here, we characterize a genetically engineered human induced pluripotent stem cell (hiPSC) model invalidated for GPR146 (ITXi001-A-1) using CRISPR-Cas9 editing technology. Differentiation of ITXi001-A-1 towards hepatic fate will provide a suitable model for deciphering the molecular mechanisms sustaining the beneficial metabolic effects of GPR146 inhibition.

Generation of human induced pluripotent stem cell lines from four unrelated healthy control donors carrying European genetic background.

Four human induced pluripotent stem cell (hiPSC) lines have been generated from healthy control European donors, and validated. This resource represents a useful tool for stem cell-based research, as references for developmental studies and disease modeling linked to any type of human tissue and organ, in an ethnical-, sex- and age-matched context. They providea reliable in-vitro model for single cell- and tissue-based investigations, and are also a valuable tool for genome editing-based studies.

Generation of three human induced pluripotent stem cell lines with IRX5 knockout and knockin genetic editions using CRISPR-Cas9 system.

Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) ß-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.

[Hepatic organoids: What are the challenges?] / Les organoïdes hépatiques - Quels sont les enjeux ?

The study and understanding of liver organogenesis have allowed the development of protocols for pluripotent stem cells differentiation to overcome the lack of primary cells, providing an almost unlimited source of liver cells. However, as their differentiation in conventional 2D culture systems has shown serious limits, hepatic organoids derived from human pluripotent stem cells represent a promising alternative. These complex and organized structures, containing one or more cell types, make it possible to recapitulate in vitro some of the organ functions, thus enabling numerous applications such as the study of the liver development, the mass production of functional liver cells for transplantation or the development of bioartificial livers, as well as the in vitro modeling of hepatic pathologies allowing high throughput applications in drug screening or toxicity studies. Economic and ethical issues must also be taken into account before using these organoids in therapeutic applications.

TITLE: Les organoïdes hépatiques - Quels sont les enjeux ? ABSTRACT: L'étude et la compréhension de l'organogenèse du foie ont permis le développement de protocoles de différenciation des cellules souches pluripotentes afin de pallier le manque de cellules primaires, offrant ainsi une source quasi illimitée de cellules hépatiques. La différenciation de ces cellules dans des systèmes de culture conventionnels en deux dimensions (2D) ayant cependant montré ses limites, des organoïdes hépatiques ont été dérivés de cellules souches pluripotentes humaines et représentent désormais une alternative prometteuse. Ces structures 3D, complexes et organisées, intégrant un ou plusieurs types cellulaires, permettent de reproduire in vitro une ou plusieurs fonctions de l'organe, et ouvrent ainsi la voie à de nombreuses applications, comme l'étude du développement du foie, la production en masse de cellules hépatiques fonctionnelles pour la transplantation ou le développement de foies bioartificiels, sans oublier la modélisation de pathologies hépatiques permettant le criblage à haut débit de médicaments ou des études de toxicité. Des enjeux économiques et éthiques doivent également être pris en considération avant une utilisation de ces organoïdes pour des applications thérapeutiques.

A dominant vimentin variant causes a rare syndrome with premature aging.

Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer's or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient's features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.

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

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.

Toward Personalized Medicine: Using Cardiomyocytes Differentiated From Urine-Derived Pluripotent Stem Cells to Recapitulate Electrophysiological Characteristics of Type 2 Long QT Syndrome.

BACKGROUND: Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes (CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. METHODS AND RESULTS: Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias. CONCLUSIONS: UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.

Human induced pluripotent stem cells in hepatology: beyond the proof of concept.

The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.

Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay.

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01 ng Pacific CTX-1B g(-1) of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally.

Update on methodologies available for ciguatoxin determination: perspectives to confront the onset of ciguatera fish poisoning in Europe.

Ciguatera fish poisoning (CFP) occurs mainly when humans ingest finfish contaminated with ciguatoxins (CTXs). The complexity and variability of such toxins have made it difficult to develop reliable methods to routinely monitor CFP with specificity and sensitivity. This review aims to describe the methodologies available for CTX detection, including those based on the toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting any of these methodological approaches for routine monitoring of ciguatera may be dependent upon the applicability of the method. However, identifying a reference validation method for CTXs is a critical and urgent issue, and is dependent upon the availability of certified CTX standards and the coordinated action of laboratories. Reports of CFP cases in European hospitals have been described in several countries, and are mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTX-producing tropical genus Gambierdiscus in the eastern Atlantic Ocean of the northern hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in Europe [1]. The question of the possible contribution of climate change to the distribution of toxin-producing microalgae and ciguateric fish is raised. The impact of ciguatera onset on European Union (EU) policies will be discussed with respect to EU regulations on marine toxins in seafood. Critical analysis and availability of methodologies for CTX determination is required for a rapid response to suspected CFP cases and to conduct sound CFP risk analysis.