@article{HetzAcikgoezVossetal.2014,
  author    = {Hetz, Susan and Acikgoez, Ali and Voss, Ulrike and Nieber, Karen and Holland, Heidrun and Hegewald, Cindy and Till, Holger and Metzger, Roman and Metzger, Marco},
  title     = {In Vivo Transplantation of Neurosphere-Like Bodies Derived from the Human Postnatal and Adult Enteric Nervous System: A Pilot Study},
  series = {PLOS ONE},
  volume    = {9},
  journal   = {PLOS ONE},
  number    = {4},
  doi       = {10.1371/journal.pone.0093605},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116793},
  pages     = {e93605},
  year      = {2014},
  abstract  = {Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via 'bystander' mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.},
  language  = {en}
}
@article{HassounaOttWuestefeldetal.2016,
  author    = {Hassouna, I. and Ott, C. and W{\"u}stefeld, L. and Offen, N. and Neher, R. A. and Mitkovski, M. and Winkler, D. and Sperling, S. and Fries, L. and Goebbels, S. and Vreja, I. C. and Hagemeyer, N. and Dittrich, M. and Rossetti, M. F. and Kr{\"o}hnert, K. and Hannke, K. and Boretius, S. and Zeug, A. and H{\"o}schen, C. and Dandekar, T. and Dere, E. and Neher, E. and Rizzoli, S. O. and Nave, K.-A. and Sir{\´e}n, A.-L. and Ehrenreich, H.},
  title     = {Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus},
  series = {Molecular Psychiatry},
  volume    = {21},
  journal   = {Molecular Psychiatry},
  number    = {12},
  doi       = {10.1038/mp.2015.212},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186669},
  pages     = {1752-1767},
  year      = {2016},
  abstract  = {Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of similar to 20\%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a \(^{15}\)N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated \(^{15}\)N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.},
  language  = {en}
}
@phdthesis{Guenther2018,
  author    = {G{\"u}nther, Katharina},
  title     = {Generation of early human neuroepithelial progenitors from primary cells for biomedical applications},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150348},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Patient-specific induced pluripotent stem cells (iPSCs) emerged as a promising cell source for disease modeling and drug screening as well as a virtually unlimited source for restorative therapy. The thesis deals with three major topics to help realizing biomedical applications with neural stem cells. To enable the generation of transgene-free iPSCs, alternatives to retroviral reprogramming were developed. Hence, the adaptation and evaluation of reprogramming using excisable lentiviral constructs, Sendai virus (SeV) and synthetic mRNA-based methods was assessed in the first part of this thesis. hiPSCs exhibit the pluripotency markers OCT4, SSEA-4, TRA1-60 which were confirmed by immunofluorescence and flow cytometry. Besides, the potential to differentiate in cell types of all three germ layers was detected, confirming pluripotent identity of proliferating colonies resulting from various reprogramming strategies. However, major differences such as high efficiency with SeV in contrast to a relatively low efficiency with mRNA in regard to passage number and the phenotype of starting fibroblasts were observed. Furthermore, a prolonged clone- and passage-dependent residual presence of viral RNA genes was identified in SeV-iPSCs for up to 23 passages using RT-PCR underlining the importance of careful monitoring of clone selection. In contrast, viral-free reprogramming by synthetic mRNA represents a fully non-integrative approach but requires further refinement to be efficiently applicable to all fibroblasts. The second part of this thesis deals with the establishment of a rapid monolayer approach to differentiate neural progenitor cells from iPSCs. To achieve this, a two-step protocol was developed allowing first the formation of a stable, primitive NPC line within 7 days which was expanded for 2-3 passages. In a second step, a subsequent adaptation to conditions yielding neural rosette-like NPCs followed. Both neural lines were demonstrated to be expandable, cryopreservable and negative for the pluripotency marker OCT4. Furthermore, a neural precursor identity including SOX1, SOX2, PAX6, Nestin was confirmed by immunofluorescence and quantitative RT-PCR. Moreover, the differentiation resulted in TUJ1-positive neurons and GFAP-positive astrocytes. Nonetheless, the outcome of glial differentiation from primitive NSCs remained low, whereas FGF/EGF-NPCs were efficiently differentiated into GFAP-positive astrocytes which were implicated in a cellular model of the blood brain barrier. The third and major objective of this study was to generate human early neural progenitor cells from fetal brain tissue with a wide neural differentiation capacity. Therefore, a defined medium composition including small molecules and growth factors capable of modulation of crucial signaling pathways orchestrating early human development such as SHH and FGF was assessed. Indeed, specific culture conditions containing TGFβ inhibitor SB431542, SHH agonist Purmorphamine, GSK3β inhibitor CHIR99021 and basic FGF, but no EGF enabled robust formation of early neuroepithelial progenitor (eNEP) colonies displaying a homogeneous morphology and a high proliferation rate. Moreover, primary eNEPs exhibit a relatively high clonogenicity of more than 23 \% and can be monoclonally expanded for more than 45 passages carrying a normal karyotype. Characterization by immunofluorescence, flow cytometry and quantitative RT-PCR revealed a distinct NPC profile including SOX1, PAX6, Nestin and SOX2 and Prominin. Furthermore, primary eNEPs show NOTCH and HES5 activation in combination with non-polarized morphology, indicative of an early neuroepithelial identity. Microarray analysis unraveled SOX11, BRN2 and other HES-genes as characteristic upregulated genes. Interestingly, eNEPs were detected to display ventral midbrain/hindbrain regional identity. The validation of yielded cell types upon differentiation indicates a strong neurogenic potential with more than 90 \% of TUJ1-positive neurons. Moreover, astrocytes marked by GFAP and putative myelin structures indicating oligodendrocytes were identified. Electrophysiological recordings revealed functionally active neurons and immunofluorescence indicate GABAergic, glutamatergic, dopaminergic and serotonergic subtypes. Additionally, putative physiological synapse formation was observed by the presence of Synapsin and PSD-95 as well as by ultrastructural examination. Notably, rare neurons stained positive for the peripheral neuronal marker Peripherin suggesting the potential of eNEPS to give rise to cells of neural tube and neural crest origin. By the application of specific differentiation protocols an increase of TH-positive neurons or neural crest-derivatives such as putative A- and C-sensory neurons and mesenchymal cells was identified. Taken together, primary eNEPs might help to elucidate mechanisms of early human neurodevelopment and will serve as a novel source for cell replacement and further biomedical applications.},
  subject      = {progenitors},
  language  = {en}
}