Silke Glage

Generation of Induced Pluripotent Stem Cells from Human Cord Blood

Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. A critical issue concerning the clinical use of patient-specific iPSCs is the accumulation of mutations in somatic (stem) cells over an organisms lifetime. Acquired somatic mutations are passed onto iPSCs during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPSCs from cord blood (CB) as a juvenescent cell source. CBiPSCs show characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes. For future therapeutic production of autologous and allogeneic iPSC derivatives, CB could be routinely harvested for public and commercial CB banks without any donor risk. CB could readily become available for pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.

Induced pluripotent stem cell (iPSC)-derived Flk-1 progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute myocardial infarction

AIMS Induced pluripotent stem cell (iPSC)-derived cardiovascular progenitor cells represent a suitable autologous cell source for myocardial regeneration as they have the capability to form myocardial cells and to contribute to revascularization. As a first proof of concept we evaluated the potential of a murine iPSC-derived cardiovascular progenitor population, which expresses the surface marker foetal liver kinase-1 (Flk-1), to restore myocardial tissue and improve cardiac function after acute myocardial infarction (MI) in mice. METHODS AND RESULTS iPSC-derived Flk-1(pos) vs. Flk-1(neg) cells were selected by fluorescence activated cell sorting (FACS) and injected into the ischaemic myocardium of left anterior descending coronary artery (LAD)-ligated mice. Addressing safety aspects we used an octamer binding factor 4 (Oct4)-enhanced green fluorescent protein (eGFP) expressing iPSC clone from the transgenic Oct4-eGFP reporter mouse strain OG2 to enable FACS-based depletion of undifferentiated cells prior to transplantation. Infarcted animals were treated with placebo (phosphate-buffered saline, n = 13), Flk-1(neg) cells (n = 14), or Flk-1(pos) cells (n = 11; 5 × 10(5) cells each). Heart function was evaluated by magnetic resonance imaging and conductance catheter analysis 2 weeks postoperatively. Cardiovascular in vitro and in vivo differentiations were investigated by immunofluorescence staining. Treatment with Flk-1(pos) and Flk-1(neg) cells resulted in a favourable myocardial remodelling and improved left ventricular function. Engraftment and functional benefits were superior after transplantation of Flk-1(pos) compared with Flk-1(neg) cells. Furthermore, Flk-1(pos) grafts contained considerably more vascular structures in relation to Flk-1(neg) grafts. CONCLUSION iPSC-derived Flk-1(pos) progenitor cells differentiate into cardiovascular lineages in vitro and in vivo and improve cardiac function after acute MI. This proof of concept study paves the way for an autologous iPSC-based therapy of MI.

Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium.

Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES / hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.

Mesenchymal Stem Cells Directly Interact with Breast Cancer Cells and Promote Tumor Cell Growth In Vitro and In Vivo

Cellular interactions were investigated between human mesenchymal stem cells (MSC) and human breast cancer cells. Co-culture of the two cell populations was associated with an MSC-mediated growth stimulation of MDA-MB-231 breast cancer cells. A continuous expansion of tumor cell colonies was progressively surrounded by MSC(GFP) displaying elongated cell bodies. Moreover, some MSC(GFP) and MDA-MB-231(cherry) cells spontaneously generated hybrid/chimeric cell populations, demonstrating a dual (green fluorescent protein+cherry) fluorescence. During a co-culture of 5-6 days, MSC also induced expression of the GPI-anchored CD90 molecule in breast cancer cells, which could not be observed in a transwell assay, suggesting the requirement of direct cellular interactions. Indeed, MSC-mediated CD90 induction in the breast cancer cells could be partially blocked by a gap junction inhibitor and by inhibition of the notch signaling pathway, respectively. Similar findings were observed in vivo by which a subcutaneous injection of a co-culture of primary MSC with MDA-MB-231(GFP) cells into NOD/scid mice exhibited an about 10-fold increased tumor size and enhanced metastatic capacity as compared with the MDA-MB-231(GFP) mono-culture. Flow cytometric evaluation of the co-culture tumors revealed more than 90% of breast cancer cells with about 3% of CD90-positive cells, also suggesting an MSC-mediated in vivo induction of CD90 in MDA-MB-231 cells. Furthermore, immunohistochemical analysis demonstrated an elevated neovascularization and viability in the MSC/MDA-MB-231(GFP)-derived tumors. Together, these data suggested an MSC-mediated growth stimulation of breast cancer cells in vitro and in vivo by which the altered MSC morphology and the appearance of hybrid/chimeric cells and breast cancer-expressing CD90(+) cells indicate mutual cellular alterations.

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3− secretion in murine ileocolonic inflammation

Background: Ileocolonic luminal pH has been reported to be abnormally low in inflammatory bowel disease (IBD) patients, and one of the causative factors may be reduced epithelial HCOSymbol secretory rate (JSymbol). Disturbances in JSymbol may occur due to inflammation‐induced changes in the crypt and villous architecture, or due to the effect of proinflammatory cytokines on epithelial ion transporters. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Methods: To discriminate between these possibilities, the tumor necrosis factor alpha (TNF‐&agr;) overexpressing (TNF+/&Dgr;ARE) mouse model was chosen, which displays high proinflammatory cytokine levels in both ileum and colon, but develops only mild colonic histopathology and diarrhea. HCOSymbol secretion, mRNA expression, immunohistochemistry, and fluid absorptive capacity were measured in ileal and mid‐colonic mucosa of TNF+/&Dgr;ARE and wildtype (WT) (TNF+/+) mice in Ussing chambers, and in anesthetized mice in vivo. Symbol. No caption available. Results: The high basal JSymbol observed in WT ileal and mid‐colonic mucosa were luminal Cl−‐dependent and strongly decreased in TNF+/&Dgr;ARE mice. Downregulated in adenoma (DRA) mRNA and protein expression was strongly decreased in TNF+/&Dgr;ARE ileocolon, whereas cystic fibrosis transmembrane conductance regulator (CFTR), Na+/H+ exchanger 3 (NHE3), Na+/HCOSymbol cotransporter (NBC), and epithelial sodium channel (ENaC) expression was not significantly altered. This indicates that the severe defect in ileocolonic JSymbol was due to DRA downregulation. Fluid absorption was severely depressed in the ileum but only mildly affected in the mid‐distal colon, preventing the development of overt diarrhea. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Conclusions: Even mild ileocolonic inflammation may result in a decrease of epithelial HCOSymbol secretion, which may contribute to alterations in surface pH, intestinal flora, and mucus barrier properties. (Inflamm Bowel Dis 2011;) Symbol. No caption available.

A New Role of the Complement System: C3 Provides Protection in a Mouse Model of Lung Infection with Intracellular Chlamydia psittaci

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3−/− mice succumbing to infection after day 9. At a low infective dose, C3−/− mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3−/− mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

Opposite effects of mepyramine on JNJ 7777120-induced amelioration of experimentally induced asthma in mice in sensitization and provocation.

Background Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, JNJ 7777120, an antagonist at the histamine H4 receptor, reduces asthmatic symptoms, while the histamine H1 receptor-selective antagonist mepyramine is virtually without effect. In the present study, we analyzed the effect of combined antagonism at the histamine H1 and H4 receptors in a murine asthma model in relation to the timing of their application, i.e. sensitization or provocation. Methodology/Principal Findings Asthma was induced in mice by sensitization and provocation with ovalbumin. JNJ 7777120 and/or mepyramine were injected subcutaneously either during sensitization or during provocation, and typical asthma parameters were analyzed. JNJ 7777120, but not mepyramine, reduced serum concentrations of anti-OVA IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar-lavage (BAL)-fluids independently of the timing of application. Upon application of JNJ 7777120 plus mepyramine in combination during provocation, mepyramine inhibited the effects of JNJ 7777120. In contrast, when applied during sensitization, mepyramine enhanced the disease-ameliorating effects of JNJ 7777120. Conclusions/Significance Our study indicates that both histamine H1 and H4 receptors play important roles in the course of murine experimental asthma. Unexpectedly, the contribution of these receptors to the pathogenesis differs between the two phases, sensitization or provocation. Since in human asthma, repeated contact to the allergen is not only provocation but also a boost of sensitization, a combined pharmacological targeting of histamine H1 and H4 receptors could be taken into consideration as an option for the prevention of asthma and maybe other allergic diseases.

Serotonin 5-HT7 receptor is critically involved in acute and chronic inflammation of the gastrointestinal tract.

Background:Intestinal inflammation is often associated with an increased level of serotonin (5-HT), an important gastrointestinal signaling molecule involved in gut homeostasis through stimulation of specific receptors. In this study, we investigated the role of 5-HT7 receptor (5-HT7R) in the induction and development of intestinal inflammation using a mouse model of acute and chronic colitis and human patients with Crohns disease (CD). Methods:Acute colitis was induced through administration of dextran sodium sulfate to wild-type, 5-HT7R–deficient mice and hematopoietic bone marrow chimera. Chronic colitis was induced in interleukin 10–deficient mice. The role of 5-HT7R in gut inflammation was assessed using agonist/antagonist treatment. We investigated expression and distribution of 5-HT7R, extent of gut inflammation with magnetic resonance imaging and histological analysis, survival rate, and disease activity index. Finally, biopsies from the large intestine of patients with CD were analyzed. Results:Under basal conditions, 5-HT7R is expressed both in enteric neurons and CD11c+ cells of the large intestine. Expression of 5-HT7R significantly increased after induction of colitis in mice and in inflamed intestinal regions of patients with CD in CD11c/CD86 double-positive cells. Pharmacological blockade or genetic ablation of 5-HT7R resulted in increased severity of both acute and chronic dextran sodium sulfate-induced colitis, whereas receptor stimulation showed an anti-inflammatory effect. Analysis of bone marrow chimera indicated importance of 5-HT7R expressed by hematopoietic cells in intestinal inflammation. Conclusions:The 5-HT7R expressed on CD11c/CD86-positive myeloid cells modulates the severity of intestinal inflammation in an acute and chronic colitis and thus represents a potential therapeutic target for the treatment of inflammatory disorders such as CD.

Efficient Hematopoietic Redifferentiation of Induced Pluripotent Stem Cells Derived from Primitive Murine Bone Marrow Cells

Heterogeneity among induced pluripotent stem cell (iPSC) lines with regard to their gene expression profile and differentiation potential has been described and at least partly linked to the tissue of origin. Here, we generated iPSCs from primitive [lineage negative (Lin(neg))] and nonadherent differentiated [lineage positive (Lin(pos))] bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and embryonic stem cells (ESC). In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction toward the hematopoietic lineage. All 3 BM-iPSC lines derived from undifferentiated Lin(neg) cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41 and in 2 of these lines high proportions of CD41+/ CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in 4 Lin(pos) BM-iPSC and 3 Fib-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in 2 of the Lin(neg) BM-iPSCs only. Thus, in conclusion, our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for Lin(neg) BM-iPSC lines, thereby supporting the notion of an epigenetic memory in iPSCs.

Primate iPS cells as tools for evolutionary analyses

Induced pluripotent stem cells (iPSCs) are regarded as a central tool to understand human biology in health and disease. Similarly, iPSCs from non-human primates should be a central tool to understand human evolution, in particular for assessing the conservation of regulatory networks in iPSC models. Here, we have generated human, gorilla, bonobo and cynomolgus monkey iPSCs and assess their usefulness in such a framework. We show that these cells are well comparable in their differentiation potential and are generally similar to human, cynomolgus and rhesus monkey embryonic stem cells (ESCs). RNA sequencing reveals that expression differences among clones, individuals and stem cell type are all of very similar magnitude within a species. In contrast, expression differences between closely related primate species are three times larger and most genes show significant expression differences among the analyzed species. However, pseudogenes differ more than twice as much, suggesting that evolution of expression levels in primate stem cells is rapid, but constrained. These patterns in pluripotent stem cells are comparable to those found in other tissues except testis. Hence, primate iPSCs reveal insights into general primate gene expression evolution and should provide a rich source to identify conserved and species-specific gene expression patterns for cellular phenotypes.