0134 : Using cardiomyocytes differentiated from urine-derived hiPSCs to recapitulate electrophysiological characteristics of LQT2 syndrome

RationaleHuman genetically inherited cardiac diseases have mainly been studied in heterologous systems or animal models, independently of the patients’ genetic background. Because sources for human cardiomyocytes are extremely limited, the use of urine samples to derive cardiomyocytes would be a non-invasive method to identify cardiac dysfunctions that lead to pathologies within the patients’ specific genetic background.ObjectiveCardiomyocytes differentiated from urine-derived pluripotent stem cells (UhiPS-CMs) were obtained from a patient with long QT syndrome and a mutation in hERG KCNH2 gene (p. A561P), and were characterized.Methods and ResultsCells obtained from urine samples from the A561P patient and his asymptomatic mother carrying no hERG mutation were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into cardiomyocytes using a modified matrix sandwich method. UhiPS-CMs showed proper expression of ventricular cytoskeletal proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials (APs) recorded using both high-throughput CellOptiq and patch-clamp techniques. Application of ajmaline, 4-aminopyridine, nifedipine, chromanol 293B or E-4031 to the UhiPS-CMs confirmed that INa, Ito, ICa, IKs and IKr currents, respectively, contributed to the APs. Comparing 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 a reduced IKr current. This phenotype led to APs prolongation that sometimes resulted in arrhythmias (early afterdepolarizations).ConclusionUrine-derived pluripotent stem cells from patients carrying ion channels mutations can be used as novel models to differentiate functional cardiomyocytes that recapitulate cardiac arrhythmia phenotypes

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

International audienceThe 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

Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

International audienceHuman embryonic stem cells can be differentiated along different lineages, providing the possibility of a precise analysis of genes profiles associated with specific commitments. Subtractive gene expression profiling between differentiated and undifferentiated cells provides lists of potential actors in this commitment. This combines, however, genes that are specifically associated with development and others that are over expressed because of nonlineage-specific differentiation systems. As a way to establish gene profiles associated with the neural and/or to the mesodermal commitments of human embryonic stem cells more precisely, we have carried out a 2-step analysis. We first performed a subtractive analysis of gene profiles of each of these lineages as compared to the undifferentiated stage. Then, we extended the analysis by comparing the 2 sets of results with each other. This strategy has allowed us to eliminate large numbers of genes that were over expressed in both sets of results and to uniquely associate different gene networks with either the neural or the mesodermal commitments

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

International audienceProprotein 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

International audienceBackground-—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

Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs

International audienceProprotein 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) β-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

PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development

Abstract The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D