Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting

Abstract

Introduction: Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions.

Methods: Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation.

Results: An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells.

Conclusions: The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses.

Figure 1

Longitudinal in vivo bioluminescence imaging of neural stem cell-Luciferase/enhanced green fluorescent protein (NSC-Luc/eGFP) grafts. (A) In vitro characterisation of NSC-Luc/eGFP. Left, representative fluorescence microscopy image of Luciferase/eGFP-expressing NSC (NSC-Luc/eGFP) used in this study. Green colour, direct eGFP fluorescence. Middle histogram overlay, representative flowcytometric analysis of parental NSC and NSC-Luc/eGFP. Open black histogram, control background fluorescence in FL-1 green channel from parental NSC. Filled green histogram, direct eGFP fluorescence in FL-1 green channel from NSC-Luc/eGFP. Right, in vitro bioluminescence analysis of 1 × 10⁵ parental NSC and NSC-Luc/eGFP. Data are expressed as photons/s/sr/cm² from a 5-minute time period (± standard error of the mean (SEM), n = 4). (B) In vivo bioluminescence imaging (BLI) - image aquisition. Representative time course image showing in vivo BLI of mice grafted with 1.5 × 10⁵ NSC-Luc/eGFP in the central nervous system (CNS). Images were acquired at day 1 (n = 25), day 3 (n = 15), day 5 (n = 15), day 7 (n = 10), day 10 (n = 5) and day 14 (n = 5) post-implantation. Regions of interest are drawn on the mouse head, where NSC-Luc/eGFP were injected, and on the mouse shoulder, considered as background signal. A representative time course image was chosen out of five mice imaged for the whole time course. (C) In vivo BLI- image analysis. Quantitative analysis of in vivo BLI analysis at day 1, 3, 5, 7, 10 and 14 post-implantation. BLI signals (in photons/s/sr/cm²) are provided as the mean (± SEM) for all mice analysed, both from the specific region of interest on the mouse head (red bars) and from the control region of interest on the mouse shoulder (blue bars). Significant differences are described in the results section.

Figure 2

Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses. First row, NSC-Luciferase/enhanced fluorescent green protein (Luc/eGFP) graft survival. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 6 to 9 for eGFP/TOPRO3 combination) per mouse analysed at each time point. The provided scale bars indicate 200 μm. Second row, cellular hypoxia. Direct eGFP fluorescence (green) combined with Hypoxyprobe-1staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from two to five mice analysed at each time point. The provided scale bars indicate 50 μm. Third, fourth and fifth row, endogenous glial cell behaviour. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba1) (red, fourth row), S100 calcium binding protein B (S100B) (red, fifth row) or glial fibrillary acidic protein (GFAP) (red, sixth row) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 3 for eGFP/TOPRO3/Iba1 combination, n = 3 for eGFP/TOPRO3/S100B combination and n = 1 for eGFP/TOPRO3/GFAP) per mouse analysed at each time point (n = 4/5). The provided scale bars indicate 50 μm for Iba1 and S100B images and 200 μm for GFAP images. Sixth row, graft site remodelling. Direct eGFP fluorescence (green) combined with myelin base protein (MBP) staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 200 μm.

Figure 3

In vivo cell graft and glial cell behaviour - quantitative analysis. (a) Survival of grafted neural stem cell-Luciferase/enhanced fluorescent green protein (NSC-Luc/eGFP). Presented data are the estimated total number of eGFP-expressing NSC-Luc/eGFP detected within the implant site for each mouse analysed (n = 5 for day 0, n = 5 for day 1, n = 4 for day 3, n = 4 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total number of eGFP-expressing NSC-Luc/eGFP at each time point analysed. The inset graph represents the mean % (± standard error of the mean (SEM)) of NSC-Luc/eGFP graft survival at each time point analysed from day 1 post-implantation. Significant differences are described in the results section. (b-e) Cellular density of ionized calcium binding adaptor molecule 1 (Iba1)^pos microglia and S100 calcium binding protein B (S100B)^pos astrocytes within the implant site and within the implant border. Presented data are the ln(x)-transformed values of estimated total density of microglia and astrocytes within the implant border for each mouse analysed (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total cellular density at each time point analysed. Significant differences are described in the results section. (f) Astrogliosis within the implant site and implant border (both areas are combined here). Presented data indicate the average degree of glial fibrillary acidic protein (GFAP)^pos astrogliosis (based on image coverage after GFAP staining) for each mouse analyses (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average degree of astrogliosis at each time point analysed. Significant differences are described in the results section.

Figure 4

Histological analysis of in vivo neural stem cell (NSC) graft apoptosis. Direct enhanced fluorescent green protein (eGFP) fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with the control immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red, CONTROL STAINING) and the specific immunofluorescence staining for TUNEL+ apoptotic cells (red, TUNEL STAINING) at day 0 and 1 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 100 μm.