How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

Aims: To investigate neural stem cell (NSC) interactions with striatal tissue following engraftment and the effects of growth factors. Materials & methods: Organotypic striatal slice cultures established from neonatal rats were used as an ex vivo model system. Survival, integration and differentiation of grafted NSCs from the previously generated C17.2 clone and host tissue response were investigated weekly for 28 days in vitro. To direct grafted cells towards a neuronal lineage, the role of growth factor supplementation and serum-free culturing conditions was studied using neural stem cells overexpressing neurotrophin-3 and Neurobasal™/B27 culture medium. Results: Following engraftment, NSCs gradually integrated morphologically and formed a part of the host 3D cytoarchitecture. Compared with nongrafted cultures, NSC engraftment increased the overall survival of the organotypic cultures by 39%, and reduced the host cell necrosis by more than 80% (from 2.1 ± 0.5% to 0.3 ± 0.1%), the host cell apoptosis by more than 60% (from 1.4 ± 0.4% to 0.5 ± 0.1%) and the reactions to mechanical trauma by 30% (estimated by nestin and glial fibrillary acidic protein immunohistochemistry) 7 days after engraftment. Elevated neurotrophin-3 production in NSCs and serum-free culturing conditions directed grafted NSCs towards a neuronal lineage as indicated by increased Tuj1 and Map2ab expression. However, this did not alter the survival of organotypic cultures. Conclusions: NSC engraftment was associated with rescue of imperiled host cells and reduction of host cell gliosis. These NSC effects were not related to the addition of growth factors, suggesting that other factors are involved in the supportive effects of the host following NSC engraftment.

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Gap-junctional intercellular communication between grafted neural stem cells (NSCs) and host cells seems to be essential for many of the functional and beneficial interactions after NSC engraftment. Gap-junctional communication is also known to increase in the central nervous system after hypoxia and ischemia. We therefore hypothesized that controlled hypoxic preconditioning of murine NSCs (C17.2) before the engraftment is a reliable method to increase connexin 43 expression and improve subsequent graft and host communication. Data indicated that 3-h exposure to hypoxia increased the number of connexin 43 aggregates in treated NSCs by 31%. This was paralleled by enhanced hemichannel function showed by faster calcein dye efflux and an augmentation of the early functional graft and host communication.

Re‐formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell‐mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neuronal network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell‐cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. Curr. Protoc. Stem Cell Biol. 23:2D.13.1‐2D.13.26. © 2012 by John Wiley & Sons, Inc.

Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc.

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