There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human ...hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient‐specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte‐like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)
Elevated levels of low‐density lipoprotein cholesterol (LDL‐C) in plasma are a major contributor to cardiovascular disease, which is the leading cause of death worldwide. Genome‐wide association ...studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL‐C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently, we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSCs). This finding raises the possibility of using patient‐specific iPSC‐derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach, we produced iPSCs from JD a patient with mutations in the low‐density lipoprotein receptor (LDLR) gene that result in familial hypercholesterolemia (FH). We demonstrate that (1) hepatocytes can be efficiently generated from FH iPSCs; (2) in contrast to control cells, FH iPSC‐derived hepatocytes are deficient in LDL‐C uptake; (3) control but not FH iPSC‐derived hepatocytes increase LDL uptake in response to lovastatin; and (4) FH iPSC‐derived hepatocytes display a marked elevation in secretion of lipidated apolipoprotein B‐100. Conclusion: Cumulatively, these findings demonstrate that FH iPSC‐derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient‐specific iPSC‐derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of cardiovascular disease. (HEPATOLOGY 2012)
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.
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.
UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.
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.
The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that ...facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate.
Loss of the nuclear hormone receptor hepatocyte nuclear factor 4α (HNF4α) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe ...disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4α, with expression of 567 genes reduced by ≥2.5‐fold (P ≤ 0.05) in Hnf4α−/− fetal livers. Although many of these genes are direct targets, HNF4α has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4α for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4α might reveal roles for HNF4α in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4α. Analyses of genomic DNA revealed an HNF4α binding site upstream of the CrebH coding sequence that was occupied by HNF4α in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH−/− mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)–stress response, CrebH−/− mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4α in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008.)
I 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 to
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract 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).
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