Andreas Kurtz

The angiogenic factor midkine is aberrantly expressed in NF1 -deficient Schwann cells and is a mitogen for neurofibroma-derived cells

Loss of the tumor suppressor gene NF1 in neurofibromatosis type 1 (NF1) contributes to the development of a variety of tumors, including malignant peripheral nerve sheath tumors (MPNST) and benign neurofibromas. Of the different cell types found in neurofibromas, Schwann cells usually provide between 40 and 80%, and are thought to be critical for tumor growth. Here we describe the identification of growth factors that are upregulated in NF1−/− mouse Schwann cells and are potential regulators of angiogenesis and cell growth. Basic fibroblast growth factor (FGF-2), platelet-derived growth factor (PDGF) and midkine (MK) were found to be induced by loss of neurofibromin and MK was further characterized. MK was induced in human neurofibromas, schwannomas, and various nervous system tumors associated with NF1 or NF2; midkine showed an expression pattern overlapping but distinct from its homolog pleiotrophin (PTN). Immunohistochemistry revealed expression of MK in S-100 positive Schwann cells of dermal and plexiform neurofibromas, and in endothelial cells of tumor blood vessels, but not in normal blood vessels. Furthermore, MK demonstrated potent mitogenic activity for human systemic and brain endothelial cells in vitro and stimulated proliferation and soft agar colony formation of human MPNST derived S100 positive cells and fibroblastoid cells derived from an NF1 neurofibroma. The data support a possible central role for MK as a mediator of angiogenesis and neurofibroma growth in NF1.

Histone deacetylase controls adult stem cell aging by balancing the expression of polycomb genes and jumonji domain containing 3

Aging is linked to loss of the self-renewal capacity of adult stem cells. Here, we observed that human multipotent stem cells (MSCs) underwent cellular senescence in vitro. Decreased expression of histone deacetylases (HDACs), followed by downregulation of polycomb group genes (PcGs), such as BMI1, EZH2 and SUZ12, and by upregulation of jumonji domain containing 3 (JMJD3), was observed in senescent MSCs. Similarly, HDAC inhibitors induced cellular senescence through downregulation of PcGs and upregulation of JMJD3. Regulation of PcGs was associated with HDAC inhibitor-induced hypophosphorylation of RB, which causes RB to bind to and decrease the transcriptional activity of E2F. JMJD3 expression regulation was dependant on histone acetylation status at its promoter regions. A histone acetyltransferase (HAT) inhibitor prevented replicative senescence of MSCs. These results suggest that HDAC activity might be important for MSC self-renewal by balancing PcGs and JMJD3 expression, which govern cellular senescence by p16INK4A regulation.

Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: Temporally distinct expression of midkine and pleiotrophen

Heparin‐binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin‐binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF‐2) and one of its receptors (FGFR1/ flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF‐2 during EAE and determined the cellular origin of FGF‐2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF‐2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF‐2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed co‐localization of FGF‐2 to activated microglia/ macrophages with strongest expression in the macrophage‐rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/ microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/ macrophage in an autocrine fashion, to induce the expression of other factors like insulin‐like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/ or regeneration of oligodendrocytes within the lesion area. GLIA 24:390–397, 1998.

Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis

Fibroblast growth factors (FGF)-1 and -2 are found in most embryonic and adult normal and tumor tissues, where they are immobilized in the extracellular matrix (EM). Mobilization of these FGFs is part of a tightly controlled process resulting in the activation of high-affinity receptors. Recently, we have shown that a novel human FGF-binding protein (FGF-BP) mediates the release of immobilized FGF-2 from the EM. Here we isolated genomic and cDNA clones of the mouse FGF-BP homologue and studied its expression during embryonic development and skin carcinogenesis. The murine gene contains two exons that generate a 1.2 kb mRNA and predicts an 18 kDa secreted protein that is 63% identical to its human homologue. FGF-BP mRNA expression during embryogenesis is restricted to skin, intestine and lung. In the developing skin, FGF-BP expression starts at embryonic day 9, reaches peak levels perinatally and is downregulated during postnatal development. Develepment regulation in the intestine is similar, but in lungs and ovaries high expression was also observed in the adult. FGF-BP mRNA expression in the adult skin is dramatically increased during early stages of carcinogen-induced transformation in vivo and by ras-activation in vitro. Finally, mouse FGF-BP binds to FGF-2 and can function as a modulator of FGF in FGF-responsive cells. Our results suggest a potential function of FGF-BP during development and tumorigenesis.

Human Embryonic Stem Cell Lines and Their Use in International Research

Research in human pluripotent stem cells, including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), is one of the most dynamic research fields. Despite the high public attention, especially for hESC research, there is only scattered information on the number of hESC lines and the degree, dynamics, and diversification of their use on a global level. In this study we present data on the current number of publicly disclosed hESC lines, on the extent and impact of experimental work involving hESCs, and on the use of specific hESC lines in international research. The results are based on the evaluation of nearly 1,000 research papers published by the end of 2008, which describe experimental work on hESCs, and of a comprehensive database of published hESC lines. The average impact of hESC research papers is high at 7.422, with a predominance of research output by the United States. Of at least 1,071 original hESC lines derived up to November 2009 at 87 institutions in 24 countries, only a fraction is thoroughly characterized. Our data show the global predominance of a few hESC lines in research, but also reveal remarkable country‐specific differences. Comparison of hESC and hiPSC application did not show a diminished role for hESC research, but rather revealed that, up to this time, both fields continue to expand, exist independently, and partially overlap. STEM CELLS 2010;28:240–246

Adoptive T‐Cell Therapy of a Lung Transplanted Patient with Severe CMV Disease and Resistance to Antiviral Therapy

Infections with cytomegalovirus (CMV) can induce severe complications after transplantation, particularly in patients resistant to virostatic therapy. Adoptive transfer of CMV‐specific T‐cell lines has demonstrated promising results in patients after hematopoietic stem cell transplantation. However, the generation of specific T‐cell lines ex vivo and their function in vivo is complicated in solid organ transplant (SOT) recipients. Here, we present the successful adoptive transfer of autologous CMV‐specific T cells to a lung transplant recipient with ganciclovir‐resistant CMV‐pneumonia requiring mechanical ventilation. Infused T cells rapidly expanded in vivo and efficiently inhibited viral replication as confirmed by extensive longitudinal immunological monitoring. After full recovery, the patient was released from the clinic. After 4 weeks, the infection reappeared and persisted at a low level even after a second T‐cell infusion. Our experimental data indicate that this could be the consequence of the late differentiated phenotype of the infused T cells and therefore their insufficient longevity in vivo. In summary, our report signifies the high therapeutic potential of adoptive immunotherapy in the treatment of SOT recipients when all other measures show no effect. Further studies have to elucidate the most potent strategies to generate antigen‐specific T cells with high functional capacity and robust long‐term persistence.

Dominant-Negative Fibroblast Growth Factor Receptor Expression Enhances Antitumoral Potency of Oncolytic Herpes Simplex Virus in Neural Tumors

Purpose: Oncolytic herpes simplex viruses (HSV) appear to be a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Fibroblast growth factor (FGF) signaling is important for the growth and migration of endothelial and tumor cells. Here, we examine the strategy of arming oncolytic HSV with a dominant-negative FGF receptor (dnFGFR) that targets the FGF signaling pathway. Experimental Design: A mouse Nf1:p53 malignant peripheral nerve sheath tumor (MPNST) cell line expressing dnFGFR was generated by transfection. The effects of dnFGFR expression on cell growth and migration in vitro and tumor formation in vivo were determined. The dnFGFR transgene was then inserted into oncolytic HSV G47Δ using a bacterial artificial chromosome construction system. Antitumoral and antiangiogenic activities of bG47Δ-dnFGFR were examined. Results: MPNST 61E4 cells expressing dnFGFR grew less well than parental control cells. bG47Δ-dnFGFR showed enhanced killing of both tumor (human U87 glioma and F5 malignant meningioma cells and murine MPNST 61E4 and 37-3-18-4 cells) and proliferating endothelial cells (human umbilical vascular endothelial cell and Py-4-1) in vitro compared with the control vector bG47Δ-empty without inhibiting viral replication. In vivo, bG47Δ-dnFGFR was more efficacious than its nonexpressing parent bG47Δ-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 MPNST tumors in nude mice. Conclusions: By using multiple therapeutic mechanisms, including destruction of both tumor cells and tumor endothelial cells, an oncolytic HSV encoding dnFGFR enhances antitumor efficacy. This strategy can be applied to other oncolytic viruses and for clinical translation.

Age related changes of the extracellular matrix and stem cell maintenance

Aging is characterized by reduced tissue and organ function, regenerative capacity, and accompanied by a decrease in tissue resident stem cell numbers and a loss of potency. The impact of aging on stem cell populations differs between tissues and depends on a number of non cell-intrinsic factors, including systemic changes associated with immune system alterations, as well as senescence related changes of the local cytoarchitecture. The latter has been studied in the context of environmental niche properties required for stem cell maintenance. Here, we will discuss the impact of the extracellular matrix (ECM) on stem cell maintenance, its changes during aging and its significance for stem cell therapy. We provide an overview on ECM components and examples of age associated remodeling of the cytoarchitecture. The interaction of stem cells with the ECM will be described and the importance of an intact and hospitable ECM for stem cell maintenance, differentiation and stem cell initiated tissue repair outlined. It is concluded that a remodeled ECM due to age related inflammation, fibrosis or oxidative stress provides an inadequate environment for endogenous regeneration or stem cell therapies. Means to provide adequate ECM for stem cell therapies and endogenous regeneration and the potential of antioxidants to prevent ECM damage and promote its repair and subsequently support regeneration are discussed.

Concerted Regulation of CD34 and CD105 Accompanies Mesenchymal Stromal Cell Derivation from Human Adventitial Stromal Cell

Mesenchymal stromal cells (MSC) have been intensively studied for innovative therapeutic applications. MSC in vitro are characterized by plastic-adherent proliferation, their specific immunophenotype and multipotency, whereas MSC progenitors in vivo are described as perivascular cells. Whether MSC progenitors acquire in vitro MSC characteristics upon in vitro culture is still unclear. This question can be experimentally accessed by analyzing changes in cellular properties that occur during the early in vitro culture phase, the MSC derivation phase. Here, we examined dynamics in morphology, proliferation, and expression of surface markers used for MSC characterization (such as CD34, CD105, CD146, and CD271) in tight kinetics during the MSC derivation phase of adipose tissue-derived MSC (AT-MSC). Using multiparametric flow cytometry, we identified 3 major ex vivo stromal vascular cell subsets: CD34+ CD146-CD271(+/-) adventitial stromal cell-like cells (AdSC), CD34- CD146+ CD271(+/-) pericyte-like cells (PC), and CD34+ CD31+ CD146+ endothelial cells. Of these subsets, only AdSC, but not PC gave rise to MSC under MSC culture conditions. At day 4 of culture, AdSC became fibroblastoid and upregulated CD105, CD146, and CD271. Following this phenotypic transition, AdSC commenced proliferation and downregulated CD34. In our study, we demonstrate that AdSC are more clonogenic AT-MSC progenitors than PC. Moreover, we, for the first time have dissected the phenotypic transitions from MSC progenitors to in vitro MSC during the MSC derivation phase using multiparametric flow cytometry. Hence, we propose a model describing how de novo acquisition of the typical MSC morphology by AdSC is accompanied by concerted regulation of surface marker expression upon in vitro culture.

Ral overactivation in malignant peripheral nerve sheath tumors.

ABSTRACT Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.