Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures.

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.

Functional stem cell integration assessed by organotypic slice cultures.

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.

Dynamic changes in connexin expression following engraftment of neural stem cells to striatal tissue.

Gap-junctional intercellular communication between grafted neural stem cells (NSCs) and host cells seem to be essential for many of the beneficial effects associated with NSC engraftment. Utilizing murine NSCs (mNSCs) grafted into an organotypic ex vivo model system for striatal tissue we examined the prerequisites for formation of gap-junctional couplings between graft and host cells at different time points following implantation. We utilized flow cytometry (to quantify the proportion of connexin (Cx) 26 and 43 expressing cells), immunohistochemistry (for localization of the gap-junctional proteins in graft and host cells), dye-transfer studies with and without pharmacological gap-junctional blockers (assaying the functionality of the formed gap-junctional couplings), and proliferation assays (to estimate the role of gap junctions for NSC well-being) to this end. Immunohistochemical staining and dye-transfer studies revealed that the NSCs already form functional gap junctions prior to engraftment, thereby creating a substrate for subsequent graft and host communication. The expression of Cx43 by grafted NSCs was decreased by neurotrophin-3 overexpression in NSCs and culturing of grafted tissue in serum-free Neurobasal B27 medium. Cx43 expression in NSC-derived cells also changed significantly following engraftment. In host cells the expression of Cx43 peaked following traumatic stimulation and then declined within two weeks, suggesting a window of opportunity for successful host cell rescue by NSC engraftment. Further investigation of the dynamic changes in gap junction expression in graft and host cells and the associated variations in intercellular communication between implanted and endogenous cells might help to understand and control the early positive and negative effects evident following neural stem cell transplantation and thereby optimize the outcome of future clinical NSC transplantation therapies.

Graft and host interactions following transplantation of neural stem cells to organotypic striatal cultures.

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.