Human neural progenitor cell engraftment increases neurogenesis and microglial recruitment in the brain of rats with stroke

Abstract

Main objectives: Stem cell transplantation is to date one of the most promising therapies for chronic ischemic stroke. The human conditionally immortalised neural stem cell line, CTX0E03, has demonstrable efficacy in a rodent model of stroke and is currently in clinical trials. Nonetheless, the mechanisms by which it promotes brain repair are not fully characterised. This study investigated the cellular events occurring after CTX0E03 transplantation in the brains of rats that underwent ischemic stroke.

Methods: We focused on the endogenous proliferative activity of the host brain in response to cell transplantation and determined the identity of the proliferating cells using markers for young neurons (doublecortin, Dcx) and microglia (CD11b). So as to determine the chronology of events occurring post-transplantation, we analysed the engrafted brains one week and four weeks post-transplantation.

Results: We observed a significantly greater endogenous proliferation in the striatum of ischemic brains receiving a CTX0E03 graft compared to vehicle-treated ischemic brains. A significant proportion of these proliferative cells were found to be Dcx+ striatal neuroblasts. Further, we describe an enhanced immune response after CTX0E03 engraftment, as shown by a significant increase of proliferating CD11b+ microglial cells.

Conclusions: Our study demonstrates that few Dcx+ neuroblasts are proliferative in normal conditions, and that this population of proliferative neuroblasts is increased in response to stroke. We further show that CTX0E03 transplantation after stroke leads to the maintenance of this proliferative activity. Interestingly, the preservation of neuronal proliferative activity upon CTX0E03 transplantation is preceded and accompanied by a high rate of proliferating microglia. Our study suggests that microglia might mediate in part the effect of CTX0E03 transplantation on neuronal proliferation in ischemic stroke conditions.

Figure 1. Effect of CTX0E03 one week post-transplantation.

A) Ki67+ cells were counted in the ipsilateral striatum (area delineated by a dotted line). Graph B represents the total numbers of proliferative cells per mm³ (Ki67-positive) present in the striatum of animals from Protocol 1, calculated with a stereology microscope. C) and D) Left: The percentage of Ki67+/Dcx+ (C) or Ki67+/CD11b+ (D) double-positive cells over the total number of Ki67+ cells is shown; Right: Total numbers of Ki67+/Dcx+ (C) or of Ki67+/CD11b+ (D) cells present in the striatum are shown. NS: not significant; ***: p<0.001.

Figure 2. Stroke affects the organisation of the dorsal tail of the SVZ.

A) Pictures of the four groups of animals from Protocol 2 are represented (a–d). Pictures in a′, b′, c′ and d′ represent a magnification of the dorsal SVZ from a, b, c and d respectively. B) The volume of the dSVZ was evaluated for each group by stereology; C) The density of Ki67+ cells is shown for each group. NS: Not significant; **: p<0.01; ***: p<0.001.

Figure 3. CTX0E03 increase the number of proliferative cells in the striatum four weeks post-transplantation.

A) Representative pictures from the four groups of Protocol 2 are shown. A strong increase of Ki67 immunostaining was observed in the striatum of animals from the MCAO/cells group (arrows). B) The total numbers of Ki67+ cells per mm³ in the striatum are presented. A statistically significant increase of Ki67 immunostaining was observed in the MCAO/cells group. ***: p<0.001.

Figure 4. CTX0E03 engraftment increases the number of proliferating neuroblasts in the striatum of stroke animals.

A) Immunohistochemistry for Ki67 (green) and Dcx (red) was performed on brain sections from each group (Protocol 2). Some Dcx+ neuroblasts were found to be proliferating, in particular in the MCAO/cells group (arrows). B) Top: The total numbers of Ki67+/Dcx+ cells per mm³ are represented. A strong increase in the number of proliferative neuroblasts in the MCAO/cells group is observed; Bottom: The percentage of Dcx+ cells within the proliferative (Ki67+) population is represented for each group. The MCAO condition leads to an increased proportion of neuroblasts within the proliferative pool of cells present in the striatum. NS: not significant; **: p<0.01.

Figure 5. CTX0E03 lead to microglial proliferation, regardless of stroke.

A) Immunohistochemistry for Ki67 (green) and CD11b (red) were performed. A visible increase of microglial staining (CD11b) can be observed in the Sham/cells and MCAO/cells, as compared to the Sham/NAC and MCAO/NAC groups (low magnification pictures). B) Quantification of the total number (top graph) and relative proportion (bottom graph) of proliferative microglia (Ki67/CD11b double-positive cells) are represented. More proliferating microglial cells were found in the MCAO/cells groups as compared to the other conditions. NS: not significant; *: p<0.05.

Figure 6. Representation of the total number of proliferative cells at 1 week and 4 weeks post-transplantation.

Total numbers of Ki67+ (A), Ki67+/Dcx+ (B) and Ki67+/CD11b+ (C) cells per mm³ in the striatum are shown for MCAO/NAC and MCAO/cells groups, one week and four weeks post-transplantation. A) In the absence of cells, the number of proliferative cells in the striatum decreased dramatically between the two time-points (grey volume). In the presence of CTX0E03, the number of Ki67 cells in the striatum decreased more slowly (white volume). B) As for the total number of Ki67+ cells, the numbers of proliferating neuroblasts was maintained by the presence of CTX0E03 (white) as compared to the loss observed in NAC-injected animals (grey). C) Only few microglial cells proliferate at the two time points observed (grey), whereas CTX0E03 cells lead to increased microglial proliferation (white) that decreases with time.