Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases

Abteilung für Neurologie

Comparative analyses of the neurogenic capacity of human neuroprogenitor populations derived from neural and mesodermal tissue Dissertation zur Erlangung des Doktorgrades Dr. hum. biol. de

Lack of vascular endothelial growth factor receptor-2/Flk1 signaling does not affect substantia nigra development

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Lack of hypoxia-inducible factor-1α (HIF1α) impairs midbrain dopaminergic neurogenesis which could be rescued by vascular endothelial growth factor (VEGF) via VEGFR-2 signaling. Here, we conditionally inactivated the VEGFR-2, encoded by the fetal liver kinase 1 (Flk1) gene, in murine NPCs to determine its role in proliferation and survival in vitro as well as survival of dopaminergic neurons in vivo. Flk1 conditional knock-out (Flk1 CKO) mice showed no general brain phenotype. There was no midbrain-specific impairment of NPC proliferation as seen in HIF1α CKO mice. In the substantia nigra (SN) of adult Flk1 CKO mice, nonbiased stereological cell counts revealed no reduction of TH-positive neurons of Flk1 CKO mice compared with control Cre/wt mice (in which the wild-type Flk1 allele is expressed in parallel with the Cre recombinase allele). In conclusion, VEGF receptor signaling seems not to be relevant to the development and survival of substantia nigra dopaminergic neurons within the hypoxia-HIF1α signaling pathway

Multipotent Neural Stem Cells from the Adult Tegmentum with Dopaminergic Potential Develop Essential Properties of Functional Neurons

Neurogenesis in the adult brain occurs within the two principal neurogenic regions: the hippocampus and the subventricular zone of the lateral ventricles. The occurrence of adult neurogenesis in non-neurogenic regions, including the midbrain, remains controversial, but isolation of neural stem cells (NSCs) from several parts of the adult brain, including the substantia nigra, has been reported. Nevertheless, it is unclear whether adult NSCs do have the capacity to produce functional dopaminergic neurons, the cell type lost in Parkinson's disease. Here, we describe the isolation, expansion, and in vitro characterization of adult mouse tegmental NSCs (tNSCs) and their differentiation into functional nerve cells, including dopaminergic neurons. These tNSCs showed neurosphere formation and expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, and Olig2, the NSC markers Nestin and Musashi1, and the proliferation markers Ki67 and BrdU (5-bromo-2-deoxyuridine). The cells showed typical propidium iodide–fluorescence-activated cell sorting analysis of slowly dividing cells. In the presence of selected growth factors, tNSCs differentiated into astroglia, oligodendroglia, and neurons expressing markers for cholinergic, GABAergic, and glutamatergic cells. Electrophysiological analyses revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels, action potentials, as well as currents induced by GABA (γ-aminobutyric acid), glutamate, and NMDA (N-methyl-d-aspartate). Clonal analysis demonstrated that individual NSCs retain the capacity to generate both glia and neurons. After a multistep differentiation protocol using co-culture conditions with PA6 stromal cells, a small number of cells acquired morphological and functional properties of dopaminergic neurons in culture. Here, we demonstrate the existence of adult tNSCs with functional neurogenic and dopaminergic potential, a prerequisite for future endogenous cell replacement strategies in Parkinson's disease

Initiation of Dopaminergic Differentiation of Nurr1^- Mesencephalic Precursor Cells Depends on Activation of Multiple Mitogen-Activated Protein Kinase Pathways

Interleukin-1 (IL-1) plays a pivotal role in terminal dopaminergic differentiation of midbrain-derived neural precursor cells already committed to the mesencephalic dopaminergic phenotype (named mdNPCs for mesencephalic dopaminergic neural precursor cells). Here we characterized the molecular events in long-term expanded rat nuclear receptor related-1^- (Nurr1^-) mdNPCs in response to IL-1β during their terminal dopaminergic specification. We showed that IL-1β induced a rapid induction of mRNA of dopaminergic key fate-determining transcription factors, such as Nurr1 and Pitx3, and a subsequent increase of tyrosine hydroxylase protein as an early marker for dopaminergic neurons in vitro. These effects of IL-1β were specific for mdNPCs and were not observed in striatal neural precursor cells (NPCs). Surprisingly, IL-1β did not activate the NF-κB pathway or the transcription factor activating protein 1 (AP-1), but inhibition of nuclear translocation of NF-κB by SN50 facilitated IL-1β-induced Nurr1 expression and dopaminergic differentiation of mdNPCs. Incubation of mdNPCs with IL-1β led to a rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein (MAP) kinases within 1 to 3 hours, whereas Jun kinase was not phosphorylated in response to IL-1β. Consistently, inhibition of the ERK1/2 pathway or p38 MAP kinase blocked Nurr1 upregulation and further dopaminergic specification of mdNPCs, but not differentiation into MAP2ab^+ neurons. IL-1 receptor antagonist did not block early dopaminergic differentiation events, suggesting that the effects of IL-1β are not mediated through activation of IL-1 receptor type I. Our results indicate that induction of terminal dopaminergic specification of Nurr1^- mdNPCs by IL-1β depends on activation of the ERK1/2 and p38 MAP kinase pathway

No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

<div><p>Mutations within the <i>LRRK2</i> gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.</p></div

High incidence of implantable cardioverter defibrillator malfunctions during radiation therapy: neutrons as a probable cause of soft errors

Item does not contain fulltextAIMS: To investigate the behaviour of the implantable cardioverter defibrillator (ICD) function during actual radiotherapy sessions. METHODS AND RESULTS: Fifteen patients with an ICD underwent 17 radiation treatments for cancer [cumulative dose to the tumour was between 16 Gray (Gy) and 70 Gy; photon beams with maximum energies between 6 megaelectronvolt (MeV) and 18 MeV were employed]. During every session, the ICD was programmed to a monitoring mode to prevent inappropriate therapy delivery. Afterwards, the ICDs were interrogated to ensure proper function. Calculated radiation dose at the ICD site was /=10 MeV, and boron-10 which is present in the integrated circuit

LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines.

<p><b>A:</b> RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. <b>B:</b> In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. <b>C:</b> Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.</p

Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons.

<p><b>A-B:</b> Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; <b>C-H:</b> Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. <b>C1-H2:</b> Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.</p