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. 2015 Oct 7;6:212.
doi: 10.3389/fneur.2015.00212. eCollection 2015.

Transplanted Neural Progenitor Cells From Distinct Sources Migrate Differentially in an Organotypic Model of Brain Injury

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Free PMC article

Transplanted Neural Progenitor Cells From Distinct Sources Migrate Differentially in an Organotypic Model of Brain Injury

Kapinga P Ngalula et al. Front Neurol. .
Free PMC article

Abstract

Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs) obtained from rats to repair damaged neocortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0-3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ) for excitatory cells, ganglionic eminence (GE) for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ) or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically derived NPCs from the VZ/SVZ migrated less extensively. In the injured neocortex, transplanted cells moved predominantly into the wounded area. NPCs derived from the GE tended to be immunoreactive for GABAergic markers while those derived from the neocortex were more strongly immunoreactive for other neuronal markers such as MAP2, TUJ1, or Milli-Mark. Cells transplanted in vitro acquired the electrophysiological characteristics of neurons, including action potential generation and reception of spontaneous synaptic activity. This suggests that transplanted cells differentiate into neurons capable of functionally integrating with the host tissue. Together, our data suggest that transplantation of neural progenitor cells holds great potential as an emerging therapeutic intervention for restoring function lost to brain damage.

Keywords: cerebral cortex; development; interneuron; neuronal migration; rat.

Figures

Figure 1
Figure 1
Model of injury and transplantation. (A) is an organotypic culture obtained on postnatal day 1 (P1), that also sustained an injury. The arrow is the approximate site of a transplant into the SVZ/VZ. (A′) shows the boxed area in (A) and contains a higher power view of the injury outlined by arrowheads; the arrow points to the deep end of the lesion. (B) is an organotypic culture obtained at P1 without injury. For analysis, the neocortex was divided into regions designated as medial, middle, and lateral. (B′) is a higher magnification of the boxed in region in (B) and delineates laminar distinctions in the cortical plate used to define the positions of the transplanted migrating cells. The arrow in (A′) represents the approximate site of cells injected into a lesion and the arrow head in (B′) represents the approximate site of cells injected into the VZ/SVZ of a slice with no lesion. (C) is a coronal section of E16 brain, (C′) is higher magnification of the boxed in region in (C) and represents the developing cortical wall. This is the region used for preparing the cell suspension made from the embryonic neocortex. The circle in C encloses the site used for preparing the GE cell suspension. CP: cortical plate, U: upper layer, M: middle layer, L: lower layer, IZ: intermediate zone, SVZ, subventricular zone; VZ, ventricular zone; LV, lateral ventricle. Scale bar = 500 μm (A–C), 100 μm (A′–C′).
Figure 2
Figure 2
Examples of cells migrating after transplantation into normal cultures. (A–C) are examples of different slices that received injections of acute cell suspensions obtained from the GE (A,B) or the neocortex (C). All populations of transplanted cells (ganglionic eminence, mixed, or neocortical) showed similar distributions throughout the cortex. (D) shows morphologies acquired by the transplanted cells. Scale bar = 500 μm (A), 100 μm (B–C), 10 μm (D).
Figure 3
Figure 3
The distribution of transplanted cells in normal cultures. (A) shows the mediolateral distribution of cells transplanted into normal cortex after 7 days in culture. Significantly fewer cells reached lateral portions of the cortical slice compared with transplanted cells migrating into the middle and medial regions of the slice. (B) illustrates the distribution of cells that migrated away from the injection site. The bars to the right of the vertical line show that of the cells migrating away from the injection site, a greater percentage reached the cortical plate (CP) than those remaining in the intermediate zone (IZ). Significantly fewer cells obtained from the neocortex alone, however, reached the cortical plate compared with cells obtained from the GE or the mixed population (*p < 0.01). The bars to the left of the vertical line show the distribution of cells that reached the cortical plate and moved into the upper (U), middle (M), or lower (L) cortical layers. Of the cells that reach the cortical plate, fewer cells resided in the lower layers of the cortical plate (#p < 0.05). Compared with the GE and mixed populations, a significantly smaller percentage of neocortically derived cells reached the upper layers (*p < 0.001) and a greater percentage of cells remained in the lower layers (*p < 0.001). See Table 1 for the numbers of cells in each group. (Two-way ANOVA followed by the Holm–Sidak pairwise comparison, error bars = SEM).
Figure 4
Figure 4
Transplanted cell distribution in injured cultures. (A) shows examples of transfected GFP cell entering or in the injured area. An arrowhead points to a cell seeming to extend processes within the injured area. (B) is a graph of the distribution of CMDiI labeled cells transplanted into the SVZ of an injured culture that migrated into both injured and the non-injured regions of host slice. Significantly more cells migrated into the injured region (#p < 0.001) compared to the areas of no injury. Fewer neocortical cells reached the injured area compared to GE and mixed (*p < 0.05) cell populations. More of the neocortically derived cells remained in the IZ compared with the other two populations (*p < 0.05). (Two-way ANOVA followed by a post hoc pairwise comparison, Holm–Sidak, error bars = SEM). (C,D) Cells transplanted in the injury remained in place and did not show any migration. (D) is a higher powered image of the boxed in region in B. [Scale bar (A) = 100 μm; (C) = 500 μm; (D) = 20 μm]. IZ, intermediate zone; GE, cells derived from the ganglionic eminence; Ctx, cells derived from the embryonic neocortex; Mix, cells derived from a mixed population of GE and neocortically derived cells.
Figure 5
Figure 5
Phenotype of transplanted cells. The transplanted cells acquired different phenotypes as shown by immunoreactivity against different neuronal and glial markers. The cells shown in (A–C) derive from the GE and are GABA+. Cells shown in (D–I) derive from the neocortex, and the cells shown in (J–L) are a mixed population of transplanted cells; the cells in (D–L) and (P–R) are immunoreactive for neuronal markers, MAP2 in (D–I), Milli-Mark in (J–L), and TUJ in (P–R). The cells in (M–O) show GFAP+ cells (green) in the host slice and a transplanted cell from the GE labeled with CMDiI (red), which is not GFAP immunoreactive. (D–F) is a transfected GFP+ (green) cell and (G–I) are CMDiI+ (red). (J–K) are CMDiI+ (red) and Milli-Mark+ (green). Scale bar = [(A–C,G–I,P–R) 10 μm] and [(D–F,J–O) 50 μm].
Figure 6
Figure 6
Transplanted cells immunoreactive for different markers. This graph represents the percentage of the total number of transplanted cells labeled with CMDiI that were counted for each marker, which were also double labeled for a specific antibody. The total number of counted cells for each marker can be seen in Table 2. In general, similar numbers of transplanted cells from derived from each source (GE, Ctx, Mixed) were immunoreactive for the neuronal markers, Tuj1 (TUJ) and Milli-Mark. GE-derived cells were more likely to differentiate into cells immunoreactive for GABA, while neocortically derived cells are more immunoreactive for MAP2 (*p < 0.01). Very few transplanted cells from any source are immunoreactive for GFAP (#p > 0.05). We used a two-way ANOVA followed by pairwise comparisons with the Holm–Sidak test, error bars = SEM.
Figure 7
Figure 7
Electrophysiological recordings. (A) is the Current–frequency relationship of a GFP+ transplanted neuron. The recorded cell showed a linear increase in firing frequency as a function of depolarizing current. The inset in the graph shows the response of the cell to a 14pA depolarization. Note the lack of spike frequency adaptation, a characteristic of fast spiking interneurons. (B) Intracortical stimulation evoked postsynaptic currents in the same neuron. The black trace is the average of 10 individual sweeps (each sweep overlaid in gray). The prolonged barrage of PSPs impinging upon the cell suggests that the transplanted neuron had become integrated into the host circuitry. The image of the brain drawing above shows a schematic representation of the position of a stimulating electrode and site of recording of a labeled cell. (C) Percentage of GFP cells that demonstrated spontaneous synaptic inputs (n = 15), and (D) percentage of GFP cells that showed synaptic activity after electrical stimulation (n = 11). (E–G) An Avidin–Rhodamine reaction cell recording, (E) shows GFP, (F) shows the Neurobiotin reaction and (G) merge of the two. Scale bar: 20 μm.

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