[Stem Cells for Retina Replacement]. / Künstliche Netzhaut aus Stammzellen.

In ophthalmology, regenerative medicine is rapidly becoming a reality. Cell based treatment strategies in end stage retinal degeneration may be of therapeutic value, whatever the mechanism of disease mechanism. However, while corneal transplantation is commonly performed with excellent results, many obstacles must be overcome before retinal transplants can become clinically useful. The major problems are the production of appropriate transplants and functional integration in situ. New technologies allow the production of autologous transplants by inducing pluripotency in adult somatic cells. Driven by this development, exciting new research has been conducted on the development of artificial retinal tissue for basic research and transplantation. This article reviews this progress and discusses its clinical utility.

"Miniguts" from plucked human hair meet Crohn's disease.

Human pluripotent stem cells represent a powerful tool to study human embryonic development and disease but also open up novel strategies for cell replacement therapies. Their capacity to give rise to every cell type of the human body, meanwhile, enables researchers to generate high yields of mesodermal, ectodermal, but also endodermal-derived tissues such as hepatic, pancreatic, or intestinal cells. Another progress in the field came with the advent of 3-dimensional culture conditions, so-called organoids, which facilitate maturation of stem cells and in turn more faithfully recapitulate human tissue architecture. While several studies reported the derivation of organoid cultures from adult intestinal tissue, the derivation of intestinal organoids derived from plucked human hair of Crohn's disease patients has not been reported. The current research project reports such successful generation and characterization of induced pluripotent stem cells (iPSCs) derived from hair sheet keratinocyte cultures of a patient with Crohn's disease. Stepwise differentiation along the intestinal lineage showed no differences in intermediate stages such as definitive endoderm formation. We also directed the patterned primitive gut tube toward intestinal organoids resembling the cellular architecture of human "miniguts". As expected from current pathophysiological knowledge on Crohn's disease, there were no obvious morphological differences in the "miniguts" derived from healthy control and diseased patient-induced pluripotent stem cells. Taken together, our platform will enable for detailed and complementary phenotyping of the pathophysiology of Crohn's disease in a novel disease-in-a-dish format.

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes.

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

[Induced pluripotent stem cells. A new resource in modern medicine]. / Induzierte pluripotente Stammzellen. Eine neue Ressource in der modernen Medizin.

Pluripotent stem cells possess a remarkable unlimited self-renewal capacity and offer unparalleled in vitro differentiation potential. This provides a unique model system not only to study early human development but also gives renewed hope in terms of developing cell therapies and regenerative medicine. S. Yamanaka, a medical doctor and researcher, reported the possibility of reprogramming somatic cells to so-called induced pluripotent stem cells via the ectopic expression of four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc. This Nobel Prize winning work has since revolutionized stem cell research and paved the way for countless new avenues within regenerative medicine. This includes disease modeling in a patient-specific context with the ultimate aim of individually tailored pharmaceutical therapy. Additionally, genetic correction studies have rapidly increased in basic science and thus there is hope that these can be effectively and efficiently translated into clinical applications. Addressing the medical community this review gives a broad general overview about the state of the research field and possible clinical applications of pluripotent stem cells.

The Kvß2 subunit of voltage-gated potassium channels is interacting with ProSAP2/Shank3 in the PSD.

The postsynaptic density is an electron dense meshwork composed of a variety of molecules facilitating neuronal signal transmission. ProSAP2/Shank3 represents a crucial player at postsynaptic sites, assembling large multimeric platforms and anchoring numerous other molecules, thereby linking the functional synapse with the cytoskeleton. ProSAP2/Shank3 is also implicated in the pathogenesis of numerous diseases, including autism spectrum disorders. KvBeta2 (Kvß2) on the other hand serves as a regulatory subunit of voltage-gated potassium channels. Kvß2 is located at various sites in the neuron including the axon (binding to Kv1.2), the dendrites (binding to Kv4.2) and the synapse. Binding of Kvß2 to either Kv1.2 or Kv4 modulates not only the channel conformation but directs targeting of the channel protein complex to distinct loci within the cell. Thus an interaction between ProSAP2 and Kvß2 could have important roles at diverse cellular compartments and moreover during maturation stages. We report here on the direct protein-protein interaction of the postsynaptic density anchoring molecule ProSAP2 and the potassium channel subunit Kvß2, initially identified in a yeast-two-hybrid-screen. Furthermore, we characterize this interaction at synapses using primary hippocampal neurons in vitro.

[Possible applications of new stem cell sources in neurology]. / Anwendungsmöglichkeiten neuer Stammzellquellen in der Neurologie.

Stem cells provide broad possibilities in modern science and medicine. This counts not only for investigations of developmental aspects but also for cell-based therapies, pharmacotoxicological testing and improvements in personalized medicine. The recent described techniques of induced pluripotent stem cells, directly induced neural stem cells and directly induced neurons are a major step forward by providing new possibilities for research on neurological diseases. Nevertheless, a variety of questions remain open regarding stem cell-based therapeutic strategies including tumorigenicity and phenotypical stability in the receptor brain. The major hope is that the new stem cell-based neural cell systems will help to understand the pathophysiology of neurodegenerative diseases. The future will show whether and how stem cells will lead to successful restorative therapies and/or to suitable cell models for neurological diseases.

The postsynaptic density protein Abelson interactor protein 1 interacts with the motor protein Kinesin family member 26B in hippocampal neurons.

Abelson interactor protein 1 (Abi-1) localizes to postsynaptic densities (PSDs) of excitatory synapses and was shown to be transported from the PSD to the nucleus and back depending upon synaptic activation. We employed a yeast-two-hybrid screen to search for putative transport molecules. We found Kif26B a member of the Kif family of transport proteins that has not been characterized in the central nervous system as a direct interaction partner of Abi-1. We delineated a proline-rich motif within the cargo-binding domain of Kif26B to be responsible for this protein-protein interaction. Kif26B was able to recruit Abi-1 to the microtubule network and we found that the expression of Kif26B is responsible for the localization of Abi-1 to PSDs in maturing neurons. Taken together we report that Abi-1 is a cargo of Kif26B in primary hippocampal neurons, pointing to a role of this transport molecule in the movement of Abi-1 between different cell compartments. Additionally, we provide the first detailed investigation of Kif26B and its cargo molecules in neuronal cells.

Methylphenidate exerts no neurotoxic, but neuroprotective effects in vitro.

Methylphenidate (MPH) is the most common used drug in child and adolescent psychiatry. Despite of this fact, however, little is known about its exact pharmacological mechanisms. Here we investigated the toxic effects of MPH in vitro in human embryonic kidney (HEK-293) cells stably expressing the human dopamine transporter (HEK-hDAT cells) and in cultured rat embryonic (E14.5) mesencephalic cultures. MPH alone (up to 1 mM) affected neither the growth of HEK-hDAT cells nor the survival of dopaminergic (DA) neurons in primary cultures after treatment up to 72 h. No differences in neuronal arborisation or in the density of synapses were detected. 1-methyl-4-phenylpyridinium (MPP(+)) showed no toxic effect in HEK-293 cells, but had significant toxic effects in HEK-hDAT cells and DA neurons. MPH (1 microM - 1 mM) dose-dependently reduced this cytotoxicity in HEK-hDAT cells and primary mesencephalic DA neurons. The presented results show that application of MPH alone does not have any toxic effect on DA cells in vitro. The neurotoxic effects of MPP(+) could be significantly reduced by co-application of MPH, an effect that is most likely explained by MPH blocking the DAT.

5'-heterogeneity of the human excitatory amino acid transporter cDNA EAAT2 (GLT-1).

TWO novel transcripts of the human excitatory amino acid transporter EAAT2 (GLT-1) were cloned using PCR cloning techniques. Comparative sequence analysis of the 5'-untranslated region (UTR) of five EAAT2 transcripts led to the definition of five putative 5'-untranslated exons that are combined in a variable manner. Alternative splicing is a likely mechanism for the 5' complexity of the EAAT2 cDNA. The potential functional meaning of this finding such as differential expression of the EAAT2 transcripts in the central nervous system remains to be demonstrated.

Alternative splicing of the glutamate transporter EAAT2 (GLT-1).

The human glutamate transporter EAAT2 (GLT-1) is of major importance for synaptic glutamate reuptake, and reportedly, a candidate gene for neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease and epilepsy. Here we report the polymerase chain reaction (PCR) cloning of two novel EAAT2 transcripts, named EAAT2-C1 and EAAT2-C2, which originate from alternative splicing of the human EAAT2 gene. EAAT2-C1 results from skipping of the protein coding exon eight. In contrast, EAAT2-C2 is characterized by usage of internal splice sites in the exons five and six. The splicing events lead to a deletion of 45 and 107 amino acids, respectively, located in the C-terminal and central part of the putative protein.

Pertussis toxin inhibits contractions but not endothelium-dependent relaxations of rabbit pulmonary artery in response to acetylcholine and other agonists.

Guanine nucleotide binding proteins (G proteins) sensitive to pertussis toxin (PTX) mediate the muscarinic receptor responses in several tissues. Therefore, the present study sought to investigate whether smooth muscle contractions and/or endothelium-dependent relaxations in response to acetylcholine (ACh) and other agonists were sensitive to PTX. In endothelium-denuded rabbit pulmonary artery rings, ACh, clonidine and serotonin produced concentration-dependent contractions which were markedly inhibited in nominally Ca+(+)-free medium and abolished in the presence of ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.2 mM). In endothelium-denuded arterial rings obtained from rabbits treated in vivo with PTX (5 micrograms/kg i.v., 5 days before sacrifice) maximum contractions to ACh, clonidine and serotonin were inhibited by 77, 67 and 35%, respectively. Contractions induced with KCl (10-40 mM) were also abolished in Ca+(+)-free medium, but they were not affected by PTX. Endothelium-dependent relaxations of phenylephrine-contracted pulmonary arteries in response to ACh adenosine triphosphate and substance P were also reduced or abolished upon removal of extracellular Ca++. However, the endothelium-dependent relaxations were not affected by PTX. These data demonstrate that contractions of pulmonary arterial smooth muscle cells after stimulation through muscarinic receptors, alpha adrenoceptors and serotonin receptors require the influx of extracellular Ca++. This receptor-stimulated Ca++ influx is likely to be regulated by a PTX-sensitive G protein. Also, the induction of release of relaxing factor from endothelial cells of the pulmonary artery via muscarinic, purinergic or substance P receptors requires extracellular Ca++. However, in these cells, a different mode of signal transduction, insensitive to PTX, seems to be involved.

The inhibition of alpha 1-adrenoceptor-mediated contractions of rabbit pulmonary artery by Ca2+-withdrawal, pertussis toxin and N-ethylmaleimide is dependent on agonist intrinsic efficacy.

Contractions were induced in rings of rabbit pulmonary artery with the preferential alpha 1-adrenoceptor agonists, phenylephrine, methoxamine and St 587 [2-(2-chloro-trifluoromethyl-phenylimino)imidazolidine and the preferential alpha 2-adrenoceptor agonists, clonidine and B-HT 920 [6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d) azepine] [corrected]. Phenylephrine and methoxamine acted as full agonists whereas St 587, clonidine and B-HT 920 were partial agonists (intrinsic activities 0.62, 0.38 and 0.42, respectively). Experiments with alpha 1- and alpha 2-adrenoceptor antagonists indicated that the receptors involved are of the alpha 1 type only. Removal of extracellular Ca2+ inhibited maximal contractions to phenylephrine and methoxamine by 30% and 49%, respectively. The remaining contraction components of the full agonists were abolished by the "intracellular Ca2+ antagonist" TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate]. Contractions to St 587, clonidine and B-HT 920 were virtually abolished in Ca2+-free medium. Pretreatment of the donor rabbits with pertussis toxin (2.5 micrograms/kg i.v., 5-6 days before sacrifice) attenuated the efficacies of the full agonists, phenylephrine and methoxamine by only 24% and 17%, respectively, whereas maximal contractions to the partial agonists, St 587, clonidine and B-HT 920, were inhibited by 46%, 61% and 75%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

LY 83583 interferes with the release of endothelium-derived relaxing factor and inhibits soluble guanylate cyclase.

LY 83583 (6-anilino-5,8-quinolinedione) has been reported to lower intracellular cyclic GMP by an unknown mechanism. The objective of the present study was to investigate the effect of LY 83583 on different types of vasorelaxation and to study its mechanism of action. Low concentrations of LY 83583 (less than or equal to 0.1 microM) inhibited endothelium-dependent relaxations of rabbit aortic strips induced by acetylcholine or by the calcium ionophore A23187. Higher concentrations (greater than or equal to 0.3 microM) were required to produce partial inhibition of relaxation to sodium nitroprusside and glyceryl trinitrate. Cyclic AMP-mediated relaxations, induced by isoprenaline or forskolin, were not affected by LY 83583 (10 microM). The site of interference of LY 83583 with endothelium-dependent relaxation was examined with endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells that were grown on microcarrier beads and stimulated by superfusion with ATP or thimerosal. EDRF in the superfusate was detected by endothelium-denuded segments of rabbit femoral artery, which responded with dilation and, simultaneously, by purified soluble guanylate cyclase (GC) in test tubes, which was activated by EDRF. When LY 83583 was added to the glutathione-containing GC-assay or to the superfusate from cultured endothelial cells, it did not affect stimulation of soluble GC by EDRF but it slowly reversed the dilator response of the arterial detector segment. Superfusion of cultured endothelial cells with LY 83583 (1 microM), rapidly and reversibly inhibited EDRF release.(ABSTRACT TRUNCATED AT 250 WORDS)