Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum

<p>Abstract</p> <p>Background</p> <p>To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns, we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium.</p> <p>Results</p> <p>Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features, including an ability to differentiate into multiple cell lineages in teratoma assays. We, therefore, present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions.</p> <p>Conclusions</p> <p>We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP), which will be necessary for the clinical use of pluripotent stem cells and their derivatives.</p

Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

<p>Abstract</p> <p>Background</p> <p>The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.</p> <p>Results</p> <p>Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.</p> <p>Conclusions</p> <p>Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.</p

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

Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes

Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.Peer reviewe

Les myofibroblastes hépatiques dans la fibrose hépatique et le carcinome hépatocellulaire (source de protéases et cible thérapeutique)

Notre laboratoire a montré que les myofibroblastes hépatiques (MF) stimulent l'invasion in vitro des cellules de carcinome hépatocellulaire (CHC) via la sécrétion d'Hépatocyte Growth Factor. Dans ce travail, nous montrons que les MF et l'HGF stimulent l'expression de la Matrice Métalloprotéinase (MMP-3) par les cellules tumorales et que la MMP-3 est impliquée dans le mécanisme de l'invasion. Nous avons démontré que la MMP-3 présentait une localisation nucléaire. Nos données montrent que seule une forme maturée de MMP-3 est résente dans le noyau, que la MMP-3 possède un signal de localisation nucléaire qui assure son transport et que son activité protéolytique dans le noyau est corrélée avec l'apoptose. Finalement, nous avons étudié le mécanisme par lequel le trans-resvératrol, un polyphénol dérivé de la vigne, inhibe la prolifération des MF induite par l'Epidermal Growth Factor. Nous montrons qu'il agit en inhibant spécifiquement la phospho-inositide dependent kinase-1 (PDK-1).Our laboratory show that hepatic myofibroblasts (MF) in vitro induce hepatocellular carcinoma cells invasion by Hepatocyte Growth Factor secretion. In our study, we show that MF and HGF induce Matrix Metalloproteinase-3 (MMP-3) expression by tumor cells and that MMP-3 is involved in invasion mechanism. We demonstrated that MMP-3 shown a nuclear localization. Our data indicate that only a mature MMP-3 form is localized into nucleus, that MMP-3 contain a nuclear localization signal which allow its transport and its proteolytic activity in the nucleus is correlated with apoptosis. Finaly, we have studied the mechanism by which trans-resveratrol, a grappe-wined derived polyphenol, inhibit Epidermal Growth Factor induced MF proliferation. We show that it act by a specific inhibition of phospho-inositide-dependent kinase-1 (PDK-1).BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

PCSK9 Inhibition: Does Lipoprotein Size Matter?

International audienceI n order to assess the risk of cardiovascular diseases in patients, fasting plasma lipids levels are usually measured as total cholesterol, triglycerides, and high-density lipopro-tein cholesterol (HDL-C) and combined to estimate low-density lipoprotein cholesterol (LDL-C) levels using the Friedewald formula. While this indirect measure is strongly correlated with the risk of cardiovascular diseases in many epidemiological studies, it lacks information related to inter-individual differences and patient's pathophysiological status. 1 Indeed, 2 individuals carrying the same amount of LDL-C could display different numbers of LDL particles (LDL-P) or size. As the correlation between LDL-P and risks of cardiovascular diseases may be stronger than LDL-C, the debate persists on the type of measure that could more accurately predict the best prognosis. 2 Such discrepancies seem to be particularly relevant in patients with metabolic syndrome and diabetes. The proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as a key factor involved in lipoprotein metabolism regulation since its characterization as the third gene of autosomal-dominant hypercholes-terolemia in 2003 (ADH). 3 PCSK9 acts as a chaperone protein that binds the LDL receptor (LDLR) at the cell membrane and induces LDLR lysosomal degradation rather than recycling. 4 PCSK9 gain-of-function mutations increase LDLR degradation leading to autosomal-dominant hyperc-holesterolemia. 5 The identification of the link between PCSK9 loss-of-function mutations, low level of plasmatic LDL-C, and increased protection against cardiovascular diseases has sustained the concept of PCSK9 inhibition as a new therapeutic strategy in hypercholesterolemia. 6 Among PCSK9 inhibitors, the most advanced ones are based on humanized monoclonal antibodies (mAb) targeting extracel-lular PCSK9 4,7 and 2 of them (alirocumab: Praluent â ; evolocumab: Repatha â) have been recently approved by the U.S. Food and Drug Administration and European Medicines Evaluation Agency. The overall outcome of these studies indicated that PCSK9 mAb injections every 2 to 4 weeks led to up to 60% decrease of plasma LDL-C concentrations, either assessed by indirect calculation or by direct measurement. 7 It should be reminded here that LDL-C estimation with Friedewald calculation underestimates true LDL-C values in the lowest ranges (<1.8 mmol/L), a range that it is often achieved with PCSK9 inhibitors. 8 However, little is known about the effect of PCSK9 inhibition on qualitative modifications of LDL-P. In this issue of JAHA, Koren et al 9 investigated the effect of the human PCSK9 mAb alirocumab (150 mg Q2W) on the concentration and size of LDL-P by nuclear magnetic resonance spectroscopy in hypercholesterolemic patients under a stable dose of atorvastatin, who were previously included in a phase II, placebo-controlled, randomized clinical trial. 10 Upon a 12 weeks treatment, the authors showed that concomitantly to LDL-C and HDL-C, LDL-P and HDL-P plasmatic concentrations decreased and increased, respectively , with the same trends in patients treated with alirocumab compared to placebo. The decrease of LDL-P concentration occurred in all subclasses in the alirocumab group, including large (À71.3% versus À21.8% in placebo group) and small LDL-P (À54.0% versus +17.8% in placebo group). Interestingly, alirocumab promoted a substantial increase of large rather than small or medium HDL-P. A decrease of very low-density lipoprotein (VLDL) particles has been also observed in the alirocumab group, which mainly reflected a reduction in medium and small VLDL-P. However, an increased concentration of large VLDL-P was significantly observed in alirocumab-treated patients. A previous study showed that the level of plasma PCSK9 was negatively correlated with lipoprotein sizes in patients with stable coronary artery disease and without statin treatment. 11 Although indirect, these results indicated a sex effect with a lack of relation between PCSK9 level and lipoprotein size in women. It would be interesting t

Role of PCSK9 beyond liver involvement

International audiencePURPOSE OF REVIEW: Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9. RECENT FINDINGS: The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons. SUMMARY: Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors

Organogenesis and Development of the Liver

SummaryEmbryonic development of the liver has been studied intensely, yielding insights that impact diverse areas of developmental and cell biology. Understanding the fundamental mechanisms that control hepatogenesis has also laid the basis for the rational differentiation of stem cells into cells that display many hepatic functions. Here, we review the basic molecular mechanisms that control the formation of the liver as an organ

From Human-Induced Pluripotent Stem Cells to Liver Disease Modeling: A Focus on Dyslipidemia

International audienceSince the reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) became a reality, numerous advances have been made for the reprogramming process itself, cell differentiation and disease modeling. While differentiation procedures of hiPSCs into hepatocyte-like cells are under continuous investigations in order to generate fully mature and functional hepatocytes, current models already showed great promises in terms of modeling liver pathologies including metabolic diseases. This review provides an overview of the reprogramming, hepatic differentiation, and application aspects of patient-derived hepatocyte-like cells, with a more focused attention on the modeling of cholesterol metabolism defects

Future Challenges in the Generation of Hepatocyte-Like Cells From Human Pluripotent Stem Cells

International audienceThe purpose of this review is to provide an updated perspective on directing human pluripotent stem cells (hPSCs) to hepatocyte-like-cells (HLCs) and the associated challenges. Recent advances in th