Functional Stem Cell Integration Assessed by Organotypic Slice Cultures

Abstract

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.

Astrocytes release prostaglandin E2 to modify respiratory network activity

“…This ratio is slightly higher than what is observed in acutely frozen and sectionend brainstems, where 55 ± 6% of the pFRG/RTN cells (n = 3) and 53 ± 4% of the preBötC cells (n = 6) were astrocytes. The larger proportion of astrocytes compared to neurons could in part be due to reactive gliosis induced by the organotypic brainstem slice culture preparation ( Stoppini et al, 1991 ; Herlenius et al, 2012 ; Forsberg et al, 2016 ). However, in the living brainstem slice cultures, 40 ± 12% of the active cells in pFRG/RTN were astrocytes (42 ± 27 astrocytes out of 106 ± 62 active cells per network), whereas only 20 ± 9% of active cells in the preBötC were astrocytes (29 ± 17 out of 146 ± 39 active cells per network; Figure 2c and e , p<0.05 when comparing the pFRG/RTN and the preBötC, n = 41; see Supplementary file 2 for a list of all statistical tests used for all comparisons in the present study).…”

Astrocytes release prostaglandin E2 to modify respiratory network activity

“…This ratio is slightly higher than what is observed in acutely frozen and sectionend brainstems, where 55 ± 6% of the pFRG/RTN cells (n = 3) and 53 ± 4% of the preBö tC cells (n = 6) were astrocytes. The larger proportion of astrocytes compared to neurons could in part be due to reactive gliosis induced by the organotypic brainstem slice culture preparation (Stoppini et al, 1991;Herlenius et al, 2012;Forsberg et al, 2016). However, in the living brainstem slice cultures, 40 ± 12% of the active cells in pFRG/RTN were astrocytes (42 ± 27 astrocytes out of 106 ± 62 active cells per network), whereas only 20 ± 9% of active cells in the preBö tC were astrocytes (29 ± 17 out of 146 ± 39 active cells per network; Figure 2c and e, p<0.05 when comparing the pFRG/RTN and the preBö tC, n = 41; see Supplementary file 2 for a list of all statistical tests used for all comparisons in the present study).…”

CO2-evoked release of PGE2 modulates sighs and inspiration as demonstrated in brainstem organotypic culture

“…The brainstem slices were kept in culture for 7–21 days in vitro (DIV) before fixation or live imaging experiments. For a detailed protocol, see Herlenius and colleagues ( Herlenius et al, 2012 ).…”