Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain - PubMed (original) (raw)

Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain

Vladimer Darsalia et al. J Cereb Blood Flow Metab. 2011 Jan.

Abstract

Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours after the insult in the injured striatum, but reached maximum levels 1 to 6 weeks after stroke. Our findings show that the best survival of grafted human NSCs in stroke-damaged brain requires optimum numbers of cells to be transplanted in the early poststroke phase, before the inflammatory response is established. These findings, therefore, have direct clinical implications.

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Figures

Figure 1

Figure 1

Influence of poststroke delay and cell numbers on the survival and migration of grafted neural stem cells (NSCs). (A and B) Number of surviving cells in NSC grafts at 5 weeks after implantation in stroke-damaged (grafted at 48 hours (A and B) and 6 weeks (A) after the insult) or intact brain. (C) Percentage surviving cells of total number of grafted NSCs at 5 weeks after transplantation performed 48 hours after stroke. (D and E) Migration of grafted NSCs as evaluated outside the transplant core in stroke-damaged or intact brain. Means±s.e.m. *P<0.05, one-way analysis of variance followed by Bonferroni post hoc test. (F) Typical appearance of NSC graft. Solid white line depicts core of the graft; dashed white line marks border of migration. Upper inset—high magnification of the core of the graft; lower inset—high magnification of the area close to the border of the graft. Scale bar=1 mm. CC, corpus callosum; LV, lateral ventricle.

Figure 2

Figure 2

Influence of poststroke delay and cell numbers on the proliferation and differentiation of grafted neural stem cells (NSCs). (A and B) Percentage Ki67+ cells of total number of cells in the graft at 5 weeks after transplantation in the stroke-damaged and intact brain. (C and D) Confocal photomicrographs with orthogonal reconstruction of grafted HuNu+ NSCs coexpressing doublecortin (DCX) and HuD, respectively. Number of DCX+ cells (E and F). Percentage DCX+ cells (G and H) or HuD+ cells (I–L) of total number of migrated, HuNu+ cells in the graft at 5 weeks after implantation in the stroke-damaged or intact brain. Scale bar=15 _μ_m. Means±s.e.m. *P<0.05, one-way analysis of variance followed by Bonferroni post hoc test.

Figure 3

Figure 3

Expression of inflammatory markers at different time points after stroke and transplantation of neural stem cells (NSCs). (A, E, and I) Typical appearance of striatal damage after 30 minutes of middle cerebral artery occlusion in the NeuN-stained sections. Photomicrographs showing immunoreactivity for Iba1 (B, F, and J), ED1 (C, G, and K), and major histocompatibility complex (MHC) class II (D, H, and L) in stroke-damaged brain. (M and N) Time course of number of cells expressing Iba1, ED1, and MHC class II in the stroke-damaged (M) and intact (N) striatum. Animals transplanted at 48 hours (A–D) and 6 weeks (E–L) after stroke were killed 4 hours (A–H) or 5 weeks (I–L) thereafter. All animals in stroke groups had received grafts, and all groups (‘stroke-damaged brain' (M) and ‘intact brain' (N)) were given immunosuppressive treatment. Scale bar=1 mm (F) and 15 _μ_m (insets). Means±s.e.m. *P<0.05, one-way analysis of variance followed by Bonferroni post hoc test.

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