Publications by authors named "Karim Si-Tayeb"

26 Publications

  • Page 1 of 1

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

Stem Cell Reports 2021 Oct 26. Epub 2021 Oct 26.

Université de Nantes, CNRS, INSERM, l'institut du thorax, 44000 Nantes, France. Electronic address:

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) 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.
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http://dx.doi.org/10.1016/j.stemcr.2021.10.004DOI Listing
October 2021

[Hepatic organoids: What are the challenges?]

Med Sci (Paris) 2021 Oct 14;37(10):902-909. Epub 2021 Oct 14.

Inserm UMRS 1193, Université Paris-Saclay, 12-14 avenue Paul Vaillant Couturier, F-94800 Villejuif, France - Fédération hospitalo-universitaire Hépatinov, hôpital Paul Brousse, F-94800 Villejuif, France - Institut français de BioFabrication, hôpital Paul Brousse, F-94800 Villejuif, France.

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.
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http://dx.doi.org/10.1051/medsci/2021119DOI Listing
October 2021

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

Eur J Hum Genet 2020 09 17;28(9):1218-1230. Epub 2020 Feb 17.

Centre Hospitalier Universitaire de Nantes, Service de Génétique Médicale, 9 quai Moncousu, 44093, Nantes, France.

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.
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http://dx.doi.org/10.1038/s41431-020-0583-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609319PMC
September 2020

Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non-Integrative and Feeder-Free Reprogramming Strategy.

Curr Protoc Hum Genet 2017 01 11;92:21.7.1-21.7.22. Epub 2017 Jan 11.

Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil.

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cphg.26DOI Listing
January 2017

PCSK9 Inhibition: Does Lipoprotein Size Matter?

J Am Heart Assoc 2015 Nov 19;4(11). Epub 2015 Nov 19.

INSERM, UMR1087, l'institut du thorax, Nantes, France (K.S.T., B.C.) CNRS, UMR 6291, Nantes, France (K.S.T., B.C.) Université de Nantes, Nantes, France (K.S.T., B.C.) CHU Nantes, l'institut du thorax, CIC Endocrinology-Nutrition, Nantes, France (B.C.).

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http://dx.doi.org/10.1161/JAHA.115.002806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845216PMC
November 2015

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

Dis Model Mech 2016 Jan 19;9(1):81-90. Epub 2015 Nov 19.

INSERM, UMR1087, L'institut du thorax, Nantes F-44000, France CNRS, UMR 6291, Nantes F-44000, France Université de Nantes, Nantes F-44000, France CHU Nantes, L'institut du thorax, CIC Endocrinology-Nutrition, Nantes F-44000, France.

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.
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http://dx.doi.org/10.1242/dmm.022277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728336PMC
January 2016

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

J Am Heart Assoc 2015 Sep 1;4(9):e002159. Epub 2015 Sep 1.

Inserm, UMR 1087, l'institut du thorax, Nantes, France (M.J., K.S.T., Z.E.S.L., X.L., B.C., A.C., A.R., F.C., G.L., I.B., P.L., N.G.) CNRS, UMR 6291, Nantes, France (M.J., K.S.T., Z.E.S.L., X.L., B.C., A.C., A.R., F.C., G.L., I.B., P.L., N.G.) Université de Nantes, France (M.J., K.S.T., Z.E.S.L., X.L., B.C., A.C., A.R., F.C., G.L., I.B., P.L., N.G.) CHU Nantes, l'institut du thorax, Nantes, France (M.J., K.S.T., Z.E.S.L., X.L., B.C., A.C., A.R., F.C., G.L., I.B., P.L., N.G.).

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.
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http://dx.doi.org/10.1161/JAHA.115.002159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599503PMC
September 2015

Role of PCSK9 beyond liver involvement.

Curr Opin Lipidol 2015 Jun;26(3):155-61

aInserm, UMR1087-CNRS UMR6291, l'Institut du Thorax bUniversité de Nantes, Faculté de Médecine, Institut du Thorax cDepartment of Endocrinology, l'Institut du Thorax, University Hospital of Nantes, Nantes, France.

Purpose 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.
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http://dx.doi.org/10.1097/MOL.0000000000000180DOI Listing
June 2015

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

Am J Pathol 2014 Feb 21;184(2):332-47. Epub 2013 Nov 21.

INSERM, UMR 1087, the Institute of the Thorax, Nantes, France; CNRS, UMR 6291, Nantes, France; School of Medicine, University of Nantes, Nantes, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajpath.2013.09.026DOI Listing
February 2014

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

BMC Biol 2013 Jul 19;11:86. Epub 2013 Jul 19.

INSERM U 972, IFR 93, Bicêtre Hospital, and Paul Brousse Hospital, Villejuif F-94807, France.

Background: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.

Results: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.

Conclusions: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.
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http://dx.doi.org/10.1186/1741-7007-11-86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751548PMC
July 2013

Differential expression of E-cadherin, β-catenin, and Lewis x between invasive hydatidiform moles and post-molar choriocarcinomas.

Virchows Arch 2013 Jun 17;462(6):653-63. Epub 2013 May 17.

INSERM U972 Les Cellules Souches: de leurs niches aux applications thérapeutiques, Hôpital P. Brousse, Bâtiment Lavoisier, 14 avenue P.V. Couturier, 94800 Villejuif, France.

Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, β-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and β-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and β-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.
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http://dx.doi.org/10.1007/s00428-013-1427-zDOI Listing
June 2013

Comparison of cardiomyogenic potential among human ESC and iPSC lines.

Cell Transplant 2012 2;21(11):2523-30. Epub 2012 Aug 2.

Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA.

We recently reported that, following induction of clumps of pluripotent H1 human embryonic stem cells (hESCs) with activin-A and Bmp4 in defined medium for 5 days, widespread differentiation of rhythmically contracting cardiomyocytes occurs within 3-4 weeks. In this study, the same approach was used to assess whether human induced pluripotent stem cells (hiPSCs), which may theoretically provide an unlimited source of patient-matched cells for transplantation therapy, can similarly undergo cardiomyocyte differentiation. Differentiation of four pluripotent cell lines (H1 and H9 hESCs and C2a and C6a hiPSCs) was compared in parallel by monitoring rhythmic contraction, morphologic differentiation, and expression of cardiomyogenic genes. Based on expression of the cardiomyogenic lineage markers MESP1, ISL1, and NKX2-5, all four cell lines were induced into the cardiomyogenic lineage. However, in contrast to the widespread appearance of striations and rhythmic contractility seen in H9 and especially in H1 hESCs, both hiPSC lines exhibited poor terminal differentiation. These findings suggest that refined modes of generating hiPSCs, as well as of inducing cardiomyogenesis in them, may be required to fulfill their potential as agents of cardiac regeneration.
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http://dx.doi.org/10.3727/096368912X653165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522765PMC
November 2013

[The liver].

Bull Acad Natl Med 2011 Oct;195(7):1649-60

Chirurgie digestive et visérale, Hôpital Antoine-Béclère, 157 rue de la Porte-de-Trivaux - 92141 Clamart cedex.

Hepatocyte transplantation has not yet reached therapeutic status, as it has proven difficult to transplant a sufficient number of functional hepatocytes able to integrate and proliferate inside liver plates. It has recently been shown that whole livers can be decellularized by portal infusion of detergents, yielding a decellularized scaffold with a well preserved vascular network and specific liver matrix. Perfusion of different combinations of cells through the portal vein of these scaffolds results in reconstitution of a complete functional organ that can be transplanted in small animals. An auto-constructed human liver could be engineered from exogenous liver scaffolds seeded with various cell populations, including autologous cells derived from induced pluripotent stem cells. Auto-constructed livers might replace conventional liver grafts in future.
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October 2011

JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia.

Hepatology 2012 Dec 17;56(6):2163-71. Epub 2012 Sep 17.

Department of Cell Biology, Neurobiology and Anatomy, Program in Regenerative Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Unlabelled: 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).
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http://dx.doi.org/10.1002/hep.25871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900031PMC
December 2012

Hepatocyte-like cells differentiated from human induced pluripotent stem cells (iHLCs) are permissive to hepatitis C virus (HCV) infection: HCV study gets personal.

J Hepatol 2012 Sep 5;57(3):689-91. Epub 2012 May 5.

INSERM Unité 972, IFR93 Hospital Bicêtre, F-94276 Kremlin Bicêtre, France.

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http://dx.doi.org/10.1016/j.jhep.2012.04.012DOI Listing
September 2012

HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells.

Development 2011 Oct 18;138(19):4143-53. Epub 2011 Aug 18.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

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.
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http://dx.doi.org/10.1242/dev.062547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171218PMC
October 2011

Induction of cardiomyogenesis in human embryonic stem cells by human embryonic stem cell-derived definitive endoderm.

Stem Cells Dev 2012 Apr 10;21(6):987-94. Epub 2011 Aug 10.

Department of Cell Biology, Neurobiology, and Anatomy and the Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.
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http://dx.doi.org/10.1089/scd.2011.0161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315759PMC
April 2012

Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

Anesthesiology 2010 Oct;113(4):906-16

Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

Background: Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning.

Methods: H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters.

Results: Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening.

Conclusions: APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.
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http://dx.doi.org/10.1097/ALN.0b013e3181eff6b7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945423PMC
October 2010

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

BMC Dev Biol 2010 Aug 3;10:81. Epub 2010 Aug 3.

Department of Cell Biology, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Background: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1186/1471-213X-10-81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923111PMC
August 2010

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

BMC Dev Biol 2010 Jun 2;10:60. Epub 2010 Jun 2.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Background: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1186/1471-213X-10-60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896937PMC
June 2010

Organogenesis and development of the liver.

Dev Cell 2010 Feb;18(2):175-89

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Embryonic 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.
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http://dx.doi.org/10.1016/j.devcel.2010.01.011DOI Listing
February 2010

Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.

Hepatology 2010 Jan;51(1):297-305

Department of Cell Biology, Division of Pediatric Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Unlabelled: 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.
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http://dx.doi.org/10.1002/hep.23354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946078PMC
January 2010

Hepatocyte nuclear factor 4alpha is implicated in endoplasmic reticulum stress-induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H.

Hepatology 2008 Oct;48(4):1242-50

Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Unlabelled: Loss of the nuclear hormone receptor hepatocyte nuclear factor 4alpha (HNF4alpha) 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 HNF4alpha, with expression of 567 genes reduced by > or =2.5-fold (P < or = 0.05) in Hnf4alpha(-/-) fetal livers. Although many of these genes are direct targets, HNF4alpha has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4alpha for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4alpha might reveal roles for HNF4alpha 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 HNF4alpha. Analyses of genomic DNA revealed an HNF4alpha binding site upstream of the CrebH coding sequence that was occupied by HNF4alpha 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 HNF4alpha in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH.
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http://dx.doi.org/10.1002/hep.22439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717709PMC
October 2008

Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis.

Am J Pathol 2006 Oct;169(4):1390-401

INSERM E362, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France.

Matrix metalloproteinase (MMP)-3 is a protease involved in cancer progression and tissue remodeling. Using immunofluorescence and immunoelectron microscopy, we identified nuclear localization of MMP-3 in several cultured cell types and in human liver tissue sections. Western blot analysis of nuclear extracts revealed two immunoreactive forms of MMP-3 at 35 and 45 kd, with the 35-kd form exhibiting caseinolytic activity. By transient transfection, we expressed active MMP-3 fused to the enhanced green fluorescent protein (EGFP/aMMP-3) in Chinese hamster ovary cells. We showed that EGFP/aMMP-3 translocates into the nucleus. A functional nuclear localization signal was demonstrated by the loss of nuclear translocation after site-directed mutagenesis of a putative nuclear localization signal and by the ability of the MMP-3 nuclear localization signal to drive a heterologous protein into the nucleus. Finally, expression by Chinese hamster ovary cells of EGFP/aMMP-3 induced a twofold increase of apoptosis rate, compared with EGFP/pro-MMP-3, which does not translocate to the nucleus. Increased apoptosis was abolished by site-directed mutagenesis of the catalytic site of MMP-3 or by using the MMP inhibitor GM6001. This study elucidates for the first time the mechanisms of nuclear localization of a MMP and shows that nuclear MMP-3 can induce apoptosis via its catalytic activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1780186PMC
http://dx.doi.org/10.2353/ajpath.2006.060005DOI Listing
October 2006

The grape-derived polyphenol resveratrol differentially affects epidermal and platelet-derived growth factor signaling in human liver myofibroblasts.

Int J Biochem Cell Biol 2006 28;38(4):629-37. Epub 2005 Nov 28.

INSERM, E362, Bordeaux, F-33076 France.

The grape-derived polyphenol resveratrol is anti-proliferative for human liver myofibroblasts, which may be beneficial for the treatment of liver fibrosis. However, its mechanism of action is ill understood. Here, we have studied how resveratrol interfered with signaling pathways used by epidermal or platelet-derived growth factors to induce the proliferation of these cells. We found that resveratrol inhibited epidermal growth factor or platelet-derived growth factor-induced DNA synthesis. Resveratrol did not, however, decrease epidermal growth factor receptor autophosphorylation or activation of extracellular regulated kinases, but strongly inhibited the phosphorylation of Akt and of its substrate forkhead related transcription factor. This suggested that resveratrol inhibited epidermal growth factor-induced mitogenic signaling through inhibition of the phosphatidylinositol 3-kinase /Akt pathway. The phosphatidylinositol 3-kinase inhibitor LY 294002, also, inhibited epidermal growth factor-dependent DNA synthesis and Akt phosphorylation but did not decrease extracellular regulated kinases phosphorylation. In contrast, resveratrol inhibited platelet-derived growth factor-stimulated receptor autophosphorylation and every subsequent signaling step. Resveratrol did not directly inhibit phosphatidylinositol 3-kinase activity measured on immunoprecipitates from epidermal growth factor-stimulated myofibroblasts, but it strongly reduced the autophosphorylation of the phosphatidylinositol 3-kinase downstream target phospho-inositide-dependent kinase-1 that phosphorylates Akt. We, thus, show that resveratrol has growth factor-specific effects: it inhibits platelet-derived growth factor signaling via reduced receptor activation, whereas it reduces epidermal growth factor-dependent DNA synthesis via inhibition of the phosphatidylinositol 3-kinase/Akt pathway, possibly through inhibition of phospho-inositide-dependent kinase-1 activity.
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http://dx.doi.org/10.1016/j.biocel.2005.11.001DOI Listing
June 2006

Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells.

Int J Cancer 2002 Jan;97(2):157-62

Groupe de Recherches pour l'Etude du Foie (GREF), INSERM E9917, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.

Intra-hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF-induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose-dependently decreased HGF-induced invasion of HepG2 cells with a maximum of 82.7 +/- 13.3% at 20 microM. TIMP-2, a natural inhibitor, decreased invasion up to 51.2 +/- 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT-PCR. MMPs 1, 7-9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP-2 and MMP-13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP-3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF-stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose-dependently stimulated the secretion of pro-MMP-3 in the culture medium. The role of MMP-3 in HGF-induced invasion was directly confirmed by using an antibody to MMP-3, that blocked invasion. Finally, RT-PCR demonstrated MMP-3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP-3, mediate HGF-induced invasion of HCC cells. The in vivo expression of MMP-3 in HCC suggests a role for this protease in HCC progression.
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http://dx.doi.org/10.1002/ijc.1595DOI Listing
January 2002
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