Gabriela Kania

Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4+), and to a lesser extent Pdx1 (Pdx1+), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult β cells. In response to glucose, Pax4+ and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

Abnormal High-Density Lipoprotein Induces Endothelial Dysfunction via Activation of Toll-like Receptor-2

Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.

Somatic Stem Cell Marker Prominin-1/CD133 Is Expressed in Embryonic Stem Cell-Derived Progenitors

Prominin‐1/CD133 is a plasma membrane marker found in several types of somatic stem cells, including hematopoietic and neural stem cells. To study its role during development and with differentiation, we analyzed its temporal and spatial expression (mRNA and protein) in preimplantation embryos, undifferentiated mouse embryonic stem (ES) cells, and differentiated ES cell progeny. In early embryos, prominin‐1 was expressed in trophoblast but not in cells of the inner cell mass; however, prominin‐1 transcripts were detected in undifferentiated ES cells. Both ES‐derived cells committed to differentiation and early progenitor cells coexpressed prominin‐1 with early lineage markers, including the cytoskeletal markers (nestin, cytokeratin 18, desmin), fibulin‐1, and valosin‐containing protein. After spontaneous differentiation at terminal stages, prominin‐1 expression was downregulated and no coexpression with markers characteristic for neuroectodermal, mesodermal, and endodermal cells was found. Upon induction of neuronal differentiation, some prominin‐1–positive cells, which coexpressed nestin and showed the typical morphology of neural progenitor cells, persisted until terminal stages of differentiation. However, no coexpression of prominin‐1 with markers of differentiated neural cells was detected. In conclusion, we present the somatic stem cell marker prominin‐1 as a new parameter to define ES‐derived committed and early progenitor cells.

Mechanisms of Cardiac Fibrosis in Inflammatory Heart Disease

Heart injury from many causes can end up in a common final pathway of pathologic remodeling and fibrosis, promoting heart failure development. Dilated cardiomyopathy is an important cause of heart failure and often results from virus-triggered myocarditis. Monocytes and monocyte-like cells represent a major subset of heart-infiltrating cells at the injury site. These bone marrow-derived cells promote not only tissue injury in the short term but also angiogenesis and collagen deposition in the long term. Thus, they are critically involved in the typical tissue fibrosis, which evolves in the dilating ventricle during the process of pathologic remodeling. Recent findings suggest that heart-infiltrating monocyte-like cells indeed contain a pool of progenitors, which represent the cellular source both for accumulation of differentiated monocytes during the acute inflammatory phase and for transforming growth factor-beta-mediated myocardial fibrosis during the later chronic stages of disease. Obviously, a delicate balance of proinflammatory and profibrotic cytokines dictates the fate of bone marrow-derived heart-infiltrating progenitors and directly influences the morphologic phenotype of the affected heart. In this minireview, we provide an update on these mechanisms and discuss their significance in pathologic remodeling and heart failure progression after myocarditis.

Myeloid Differentiation Factor-88/Interleukin-1 Signaling Controls Cardiac Fibrosis and Heart Failure Progression in Inflammatory Dilated Cardiomyopathy

Rationale: The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self–antigen-presenting cells and promotes autoreactive CD4+ T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. Objective: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. Methods and Results: Using α-myosin heavy chain peptide (MyHC-α)–loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88−/− mice with similar distributions of heart-infiltrating cell subsets and comparable CD4+ T-cell responses. Injection of complete Freunds adjuvant (CFA) or MyHC-α/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88−/− mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow–derived cells was critical for development of cardiac fibrosis during progression to heart failure. Conclusions: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.

Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells.

Abstract We present a novel two-step protocol for the differentiation of embryonic stem (ES) cells into the hepatic lineage. Differentiated hepatocyte-like cells express genes and proteins characteristic for endodermal and hepatic cells and acquire a functional hepatic phenotype as demonstrated by albumin secretion and glycogen storage. During differentiation, α-fetoprotein, albumin, transthyretin, α-1-antitrypsin, cytochrome P450 subunits 2b9 and 2b13 and tyrosine aminotransferase transcripts are upregulated. Quantitative RT-PCR data revealed a fetal hepatic phenotype corresponding to day 13–14 of liver development. Terminally differentiated hepatocyte-like cells show a bi-nucleated, cuboidal morphology labeled by albumin, α-1-antitrypsin, liver amylase, dipeptidyl peptidase IV, c-met and cytokeratin 18. ES-derived intermediate cell types transiently and partially co-express nestin with albumin and α-fetoprotein, respectively, but not cytokeratin 19. This finding suggests an ES-derived potential hepatic progenitor cell type, which is partially nestin-, albumin- and α-fetoproteinpositive, but cytokeratin 19-negative.

Heart-Infiltrating Prominin-1+/CD133+ Progenitor Cells Represent the Cellular Source of Transforming Growth Factor β–Mediated Cardiac Fibrosis in Experimental Autoimmune Myocarditis

Rationale: Myocardial fibrosis is a hallmark of inflammation-triggered end-stage heart disease, a common cause of heart failure in young patients. Objective: We used CD4+ T-cell–mediated experimental autoimmune myocarditis model to determine the parameters regulating cardiac fibrosis in inflammatory heart disease. Methods and Results: &agr;-Myosin heavy chain peptide/complete Freund’s adjuvant immunization was used to induce experimental autoimmune myocarditis in BALB/c mice. Chimeric mice, reconstituted with enhanced green fluorescence protein (EGFP)+ bone marrow, were used to track the fate of inflammatory cells. Prominin-1+ cells were isolated from the inflamed hearts, cultured in vitro and injected intracardially at different stages of experimental autoimmune myocarditis. Transforming growth factor (TGF)-&bgr;–mediated fibrosis was addressed using anti–TGF-&bgr; antibody treatment. Myocarditis peaked 21 days after immunization and numbers of cardiac fibroblasts progressively increased on follow-up. In chimeric mice, >60% of cardiac fibroblasts were EGFP+ 46 days after immunization. At day 21, cardiac infiltrates contained ≈30% of prominin-1+ progenitors. In vitro and in vivo experiments confirmed that prominin-1+ but not prominin-1− cells isolated from acutely inflamed hearts represented the cellular source of cardiac fibroblasts at late stages of disease, characterized by increased TGF-&bgr; levels within the myocardium. Mechanistically, the in vitro differentiation of heart-infiltrating prominin-1+ cells into fibroblasts depended on TGF-&bgr;–mediated phosphorylation of Smad proteins. Accordingly, anti–TGF-&bgr; antibody treatment prevented myocardial fibrosis in immunized mice. Conclusions: Taken together, heart-infiltrating prominin-1+ progenitors are the major source of subsequent TGF-&bgr;–triggered cardiac fibrosis in experimental autoimmune myocarditis. Recognizing the critical, cytokine-dependent role of bone marrow–derived progenitors in cardiac remodeling might result in novel treatment concepts against inflammatory heart failure.

Pluripotency of embryonic stem cells

Embryonic stem (ES) cells derived from pre-implantation embryos have the potential to differentiate into any cell type derived from the three germ layers of ectoderm (epidermal tissues and nerves), mesoderm (muscle, bone, blood), and endoderm (liver, pancreas, gastrointestinal tract, lungs), including fetal and adult cells. Alone, these cells do not develop into a viable fetus or adult animal because they do not retain the potential to contribute to extraembryonic tissue, and in vitro, they lack spatial and temporal signaling cues essential to normal in vivo development. The basis of pluripotentiality resides in conserved regulatory networks composed of numerous transcription factors and multiple signaling cascades. Together, these regulatory networks maintain ES cells in a pluripotent and undifferentiated form; however, alterations in the stoichiometry of these signals promote differentiation. By taking advantage of this differentiation capacity in vitro, ES cells have clearly been shown to possess the potential to generate multipotent stem and progenitor cells capable of differentiating into a limited number of cell fates. These latter types of cells may prove to be therapeutically viable, but perhaps more importantly, the studies of these cells have led to a greater understanding of mammalian development.

Selective In Vivo Visualization of Immune-Cell Infiltration in a Mouse Model of Autoimmune Myocarditis by Fluorine-19 Cardiac Magnetic Resonance

Background— The goal of this study was to characterize the performance of fluorine-19 (19F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by 19F-CMR. 19F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium. Methods and Results— Experimental autoimmune myocarditis was induced in BALB/c mice. After intravenous injection of 2×200 µL of a perfluorocarbon on day 19 and 20 (n=9) after immunization, in vivo 19F-CMR was performed at the peak of myocardial inflammation (day 21). In 5 additional animals, perfluorocarbon combined with FITC (fluorescein isothiocyanate) was administered for postmortem immunofluorescence and flow-cytometry analyses. Control experiments were performed in 9 animals. In vivo 19F-CMR detected myocardial inflammation in all experimental autoimmune myocarditis-positive animals. Its resolution was sufficient to identify even small inflammatory foci, that is, at the surface of the right ventricle. Postmortem immunohistochemistry and flow cytometry confirmed the presence of perfluorocarbon in macrophages, dendritic cells, and granulocytes, but not in lymphocytes. The myocardial volume of elevated 19F signal (r s=0.96; P<0.001), the 19F signal-to-noise ratio (r s=0.92; P<0.001), and the 19F signal integral (r s=0.96; P<0.001) at day 21 correlated with the histological myocarditis severity score. Conclusions— In vivo 19F-CMR was successfully used to visualize the inflammation specifically and robustly in experimental autoimmune myocarditis, and thus allowed for an unprecedented insight into the involvement of inflammatory cells in the disease process.