Ladewig, Julia: Lineage Selection and Enhanced Tissue Integration of Functional and Cryopreservable Human Embryonic Stem Cell-Derived Neurons. - Bonn, 2009. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Julia Ladewig}},
title = {Lineage Selection and Enhanced Tissue Integration of Functional and Cryopreservable Human Embryonic Stem Cell-Derived Neurons},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2009,
month = may,

note = {In this study, a human Doublecortin (DCX) promoter-based lineage selection strategy is presented for the generation of purified human embryonic stem (hES) cell-derived immature neurons. Stable transfection of long-term self-renewing hES cell derived neural stem cells (lt-hESNSC) with a neuronal specific DCX-EGFP construct and subsequent selection allowed the generation of clonal hES cell-derived long-term self-renewing neural stem cell lines, which show specific and abundant expression of EGFP exclusively in immature neurons. Fluorescence activated cell sorting (FACS) enabled the enrichment of DCX-EGFP-positive immature human neurons at purities of up to over 95%. Selected neurons were amenable to replating and functionally mature in vitro.
Considering that the applicability of purified hES cell-derived neurons would largely benefit from an efficient cryopreservation technique, defined freezing conditions were devised involving caspase inhibition, which enabled the storage of hES cell-derived neurons, and yielded post thawing survival rates up to 83%. The cryopreserved neurons were amenable to replating, and developed physiological properties comparable to their non-cryopreserved counterparts. Combined with the established lineage selection protocol, this cryopreservation technique enabled the generation of human neurons in a ready-to-use format for a large variety of biomedical applications.
Migration studies in transwell chambers and transplantation into rodent CNS tissue revealed an enhanced migratory and integration potential of the DCX-EGFP purified immature neurons. In contrast, transplants comprising neural stem cells (NSCs) or a mixture of neurons and neural stem/progenitor cells showed restricted migration into the host tissue, accompanied by core formation. Many components are involved in the cellular and molecular mechanisms of neuronal migration, such as the extracellular matrix, integrins, cell adhesion molecules, cell junctions as well as soluble factors and their receptors. Experiments performed in the context of this study provided first evidence that one possible underlying mechanism for the effect of cluster formation and restricted emigration of donor neurons from neural stem/progenitor-containing grafts might be chemoattractive interactions between the transplanted neural stem/progenitor cells and the immature neurons. In detail, human neurons showed a pronounced chemoattractive migration towards undifferentiated NSCs in vitro. In addition, the two populations expressed complementary sets of chemoattractants and their respective receptors. Moreover, first results indicated that interfering with the suggested chemoattractive mechanisms might reduce this proposed auto-attraction between neural stem/progenitor cells and immature neurons. These preliminary data open the possibility to pharmacologically interfere with this auto-attraction mechanism. In particular, the antiangiogenic drug endostatin, which interferes with the chemoattractant-mediated migration of endothelial cells, appears to be sufficient to induce enhanced migration out of mixed neural cell populations in vitro. These data should offer a starting point for the development of pharmacological strategies to enhance tissue integration in neural transplantation.},

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