Hippocampal substrate induces in vitro neuronal differentiation of neurogenic human OE-MSCs

OE-MSCs are located within the olfactory lamina propria beneath the olfactory basal lamina. Transcript and membrane protein analyses have shown that OE-MSCs are closely related to bone marrow mesenchymal stem cells (BM-MSCs), but exhibit a high-level expression of genes involved in neurogenesis when compared with BM-MSCs (17). In vitro and under appropriate culture conditions, OE-MSCs are prone to give spheres expressing Nestin, glial fibrillary acid protein (GFAP), and polysialylated neural cell adhesion molecule (PSA-NCAM) markers (16, 17), which are also strongly expressed in neural stem cells (Figure 1, A–C). Moreover, OE-MSCs are found to express III–β-tubulin (Figure 1D) and can be differentiated into MAP2 neuronal cells (17).

Figure 1 In vitro assessment of neurogenic characteristics of OE-MSCs. Olfactory stem cells (A) gave rise to spherical clusters (B), expressing the GFAP neural stem cell marker (C), when grown in appropriate medium. Cells from spheres were then allowed to differentiate into neurons expressing the III–β-tubulin neuron marker (D). Freshly prepared mouse hippocampal slices were cultivated on culture insert (E) and loaded with sphere-derived GFP+ OE-MSCs (F). 3 weeks after culture at the air-liquid interface, some GFP+ OE-MSCs (green) adopted a neuron-like shape (G) and gave rise to MAP 2 -expressing neurons (H, white arrows). Scale bars: 100 μm (A, B, C, and D); 500 μm (E); 200 μm (F); 20 μm (G); 50 μm (H).

In a proof-of-principle experiment, we determined whether, as previously described with mesenchymal stem cells (27), the hippocampus could provide a suitable environment for in vitro neural differentiation of human OE-MSCs. Dissociated sphere-derived OE-MSCs, constitutively expressing GFP, were laid on the top of freshly prepared mouse hippocampal slices cultivated on inserts (Figure 1, E and F). Three weeks after grafting, we observed that surviving GFP+ transplanted cells were still on the top of organotypic hippocampal cultures and some of them (nonquantified) displayed an interneuron-like morphology (Figure 1G) and/or expressed MAP2 mature neuronal markers (Figure 1H). Not a single GFAP-expressing cell was ever found within the exogenous cell population. Some grafted human OE-MSCs exhibited electrophysiological properties consistent with immature neurons (data not shown). With these encouraging in vitro results in hand, we chose to graft OE-MSCs into an in vivo mouse model of ibotenic acid–lesioned hippocampus.

Human OE-MSCs restore learning and memory after transplantation in injured hippocampus

For this first series of experiments, 3 major choices were made: (a) memory dysfunction was induced by provoking cell death within the hippocampal cornu ammonis (CA) and dentate gyrus (DG) layers using ibotenic acid, an excitotoxic NMDA agonist (28); (b) all GFP+ OE-MSCs were derived from a single stem cell originating from a sphere and grafted within the lesion site; and (c) no immunosuppressant was delivered to mice transplanted with human stem cells, in order to avoid a putative confounding factor.

Twenty-four hours after hippocampal lesion was induced, brains were screened using MRI (Figure 2, A and B) and animals without signs of bilateral inflammation within the hippocampus were excluded from the study (n = 4). The extent of neurodegeneration, assessed by visualizing edema volume on each scan, was 2.5 mm (± 0.5) when the anteroposterior axis was considered. The pyramidal and granular layers (CA1-3, DG) were significantly damaged (Figure 2, C–F). To better appreciate cellular loss efficiency, we performed region-specific lesion (stratum pyramidale of the CA1 and upper part of the stratum granulosum layer DG) of a hippocampus and observed a reduction of the cell layer density when compared with neighboring intact layers (stratum pyramidale of the CA2/CA3 and lower part of the stratum granulosum layer DG) of the same hippocampus (Figure 2, G and H). Mnesic performances of each mouse were assessed 3 weeks after lesion and 4 weeks after transplantation, either in the olfactory tubing maze or the Morris water maze.

Figure 2 Lesion assessment. 24 hours after IH ibotenic acid injections, lesion extent was assessed in vivo by MRI (A and B). Examples of 4 axial contiguous T2-weighted images (slice thickness = 500 μm, TE eff = 60 ms, TR = 3000 ms, rare factor = 8; 8 averages) from control (A) and lesioned (B) mice are shown. In (B), the hypersignal (bright intensity) revealed the extent of the injury. Extent of ibotenic acid–induced neuronal death was visualized using cresyl violet staining (C–F). When compared with controls (C and E), lesioned mice exhibited a dramatic cell loss in the whole hippocampus (D). High magnification images of CA1 pyramidal cell body layers in control and lesioned mouse are shown in E and F, respectively. In G (crystal violet staining) and H (Hoechst blue staining), region-specific lesions in stratum pyramidale of the CA1 (red arrow) and upper part of the stratum granulosum layer DG (yellow arrow) demonstrate their efficiency and specificity when compared with neighboring intact layers. Scale bars: 500 μm (C and D); 100 μm (E and F); 250 μm (G and H).

Hippocampal-dependent associative memory assessment in the olfactory tubing maze. We first used the olfactory tubing maze and an associative memory paradigm that we previously devised (29). Three weeks after the lesion was induced, water-deprived mice (n = 24) were trained to associate a banana synthetic odor with a drop of water and a lemon synthetic odor with a nonaversive sound in a dedicated maze (Supplemental Video 1; supplemental material available online with this article; doi: 10.1172/JCI44489DS1). As shown in Figure 3A, lesioned mice (n = 16) were unable to learn the cue-reward associations across the 5 training sessions and responded randomly in comparison with control mice (n = 8) (multiple ANOVA [MANOVA], F [1,22] = 8.68, P = 0.007). Animals exhibiting correct lesions were selected for grafting of human GFP+ OE-MSCs at the initial sites of ibotenic acid delivery (225,000 cells per hemisphere; grafted intrahippocampal [IH] group, n = 8) or culture medium (sham-grafted IH group, n = 8). Four weeks after transplantation, associative memory was retested according to the same procedure. Statistical analyses of the percentage of correct responses revealed a significant group effect (MANOVA, F [2,21] = 16.426; P < 0.001). The group effect was due to significantly impaired performance in lesioned mice, whether sham-grafted or grafted (IH), overall percentage of correct responses being significantly decreased in these 2 groups when compared with control mice (P < 0.001 and P = 0.037, respectively). However, as shown in Figure 3B, transplanted mice significantly improved their ability to perform correct associations when compared with sham-grafted animals (P = 0.021). Interestingly, grafted animals dramatically improved their learning capabilities across the 5 training sessions and exhibited scores close to those of control animals, while sham-grafted animals continued to respond randomly. Additionally, intertrial interval analysis revealed no significant difference between sham-grafted (28.63 s SEM ± 1.44) and grafted (IH) (28.3 s SEM ± 1.75) groups, excluding any bias related to variations in motor function. To confirm the validity of our findings, the olfactory cue–based test was backed up by a visual cue–based memory assessment using the Morris water maze.

Figure 3 IH and ICV transplantation of human OE-MSCs improved hippocampus-dependent learning and memory. Cognitive capacities of mice were assessed in the olfactory tubing maze (A–D) and the Morris water maze (E and F). Associative or spatial memory in mice was assessed 3 weeks after lesion (A, C, and E) and 4 weeks after cell or culture medium transplantation (B, D, and F). (A–D) Mean percentage of correct responses was obtained during 5 training sessions of 20 trials per day. (A and C) Lesioned mice (n = 2 × 16) exhibited significant impairment in an associative memory task when compared with control mice (n = 2 × 8). 4 weeks after cell implantation in the lesioned sites (B) or in the lateral ventricles (D), grafted mice (grafted, n = 2 × 8) demonstrated a significant improvement in associative memory when compared with vehicle-grafted mice (sham-grafted, n = 2 × 8). (E and F) Graphs showing the mean latencies to reach the platform during 5 training sessions of 4 trials per day. (E) Lesioned mice (n = 32) exhibited significant impairment in a visuospatial learning task when compared with control mice (n = 8). 4 weeks after cell implantation in the lesioned sites (grafted IH, n = 8) or in the lateral ventricles (grafted ICV, n = 8), grafted mice demonstrated a significant improvement in spatial learning and memory when compared with vehicle-grafted mice (sham-grafted and dead cells, n = 8, respectively). See also Supplemental Table 1 and Supplemental Videos 1 and 2. *P < 0.05; **P < 0.01; #P < 0.001.

Hippocampal-dependent reference memory assessment in the Morris water maze. To evaluate visuospatial learning and reference memory, a second series of mice (n = 32) were trained to find an immersed platform in the Morris water maze (ref. 30 and Supplemental Video 2). Lesions were found to induce dramatic deficits in spatial learning as shown by the significant difference in the mean escape latencies between control and lesioned groups (MANOVA, F [1,38] = 4.66; P = 0.037) (Figure 3E). As shown in Figure 3E, at day 5, control mice reached the submerged platform significantly faster (31.9 s SEM ± 3.9) when compared with lesioned mice (46.2 s ± 2.1) (ANOVA, F [1,38] = 9.307; P = 0.004), while no difference was observed in the swimming speed between control (11.10 cm/s ± 0.87) and lesioned mice (10.98 cm/s ± 0.36). Twenty-four hours later, probe test showed a significantly longer time spent in the platform quadrant (Q1) by control mice, whereas no difference was observed in the lesioned group (Supplemental Table 1). The latter data indicate a reference memory deficit, consecutive to the lesion. After confirming the lesion efficiency, a strictly similar protocol was applied for both grafted IH and sham-grafted IH groups. Moreover, we used an additional control group, which included mice grafted with dead OE-MSCs at the initial lesion sites (dead cells IH group, n = 8). Four weeks after transplantation, MANOVA of the latencies to reach the platform revealed a significant group effect (F [3,28] = 4.41; P = 0.012). The group effect was due to impaired performance in sham-grafted IH (P = 0.023) and dead cells IH groups (P = 0.024): latencies were significantly increased in these 2 groups when compared with control group, whereas no significant difference was observed for the grafted IH group (P = 0.406) (Figure 3F). Again, we observed a positive effect of OE-MSCs grafts, as indicated by the significant decrease in the latencies to reach the platform across the 5 training sessions, when compared with both sham-grafted IH and dead cell groups. Noticeably, swimming speed analysis revealed no intergroup difference (ANOVA, F [3,34] = 2.465; P = 0.081). Probe test analyses revealed a significantly longer time spent in the platform quadrant (Q4) by both control and grafted IH groups, whereas no significant difference was observed in lesioned and dead cell groups (Supplemental Table 1).

Transplantation of human OE-MSCs restores synaptic transmission and long-term synaptic plasticity

Electrophysiological recordings were performed in order to substantiate behavioral data. At the end of the cognitive tests, 20 mice were sacrificed, acute hippocampal slices were prepared, and integrity of the trisynaptic hippocampal circuitry (loop DG-CA3-CA1) was tested using a multielectrode array including 60 extracellular electrodes. Electrical stimulation was delivered in the DG cell body layers and evoked responses (field excitatory postsynaptic potentials [fEPSPs]) were measured in CA3 and CA1 subfields (Figure 4A). Compared with control mice, almost no EPSP was elicited in CA3 and CA1 in slices from sham-grafted animals. In contrast, although of smaller amplitude when compared with control group, fEPSPs were recorded in CA1 following DG stimulation in slices from grafted animals (Figure 4B).

Figure 4 Recovery of excitatory synaptic transmission and LTP after human OE-MSC transplantation in lesioned hippocampi. 5 weeks after cell grafting, acute hippocampal slices were prepared and synaptic transmission was evaluated with a multi-electrode array. (A) Schematic diagram illustrating the positioning of hippocampal slices on a 60-electrode array. fEPSPs were evoked along the hippocampal circuitry by delivering stimulations (red electrode) in the DG. Recording electrodes (1 to 6) were located in CA1 and CA3 subfields. (B) Representative fEPSPs from control, sham-grafted, and grafted mice. A partial recovery of evoked fEPSPs was observed in slices from grafted but not in vehicle-treated mice. (C–F) The same setting was used to record CA1 LTP. Illustrative examples of LTP triggered by high-frequency stimulation are shown in control (C), sham-grafted (D), and grafted (E) mice. fEPSP amplitude was normalized against control values and plotted. (F) Group data for LTP in control (n = 5), sham-grafted (n = 7), and grafted (n = 8) mice; mean values of LTP levels (measured 40 minutes after HFS) are indicated with an horizontal bar (each animal is represented by a circle). (G) Basal synaptic transmission in control (n = 4), sham-grafted (n = 4), and grafted (n = 4) mice. The input/output curve was generated by applying increasing stimulation intensities and by plotting the fEPSP amplitude as a function of the corresponding fiber volley amplitude. (H) Group data for paired-pulse facilitation in control (n = 4), sham-grafted (n = 4), and grafted (n = 4) mice. *P < 0.05; **P < 0.01.

Then we investigated whether this improvement was associated with changes in LTP in the CA1 subfield, by applying a train of high-frequency stimulation (100 Hz, 1 s) to the Schaffer collateral pathway. Forty minutes after high-frequency stimulation, LTP magnitude was 90% ± 6% (n = 5) in control animals (Figure 4C), while it was almost completely abolished in sham-grafted animals (4% ± 9%, n = 7) (t test; P < 0.001) when signals could be recorded in the CA1 area (Figure 4D). In contrast, a significant recovery of LTP (35% ± 10%, n = 8) was observed in grafted animals when compared with sham-grafted (t test; P = 0.0119) (Figure 4, E and F). However, LTP in grafted animals remained significantly lower when compared with LTP recorded in control animals (Figure 4F) (t test; P = 0.0019). Additionally, EPSPs zeroed after perfusion of NBQX, an AMPA receptor antagonist, confirming the synaptic nature of recorded EPSPs (data not shown).

In parallel, we investigated whether lesioning and cell grafting altered basal synaptic transmission and short-term synaptic plasticity. Basal synaptic transmission was studied by generating the input/output curve. The fEPSP amplitude obtained at various stimulus intensities was plotted against the corresponding fiber volley amplitude (Figure 4G). In control animals, we observed a linear relationship with a slope value of 1.92 (n = 4; r2 = 0.91). Lesioning the animals led to a change in the synaptic signals, as the slope of the curve was flattened to 1.20 (n = 4; r2 = 0.94). In grafted animals, the slope was 1.13 (n = 4; r2 = 0.71), not significantly different from values obtained in sham-grafted animals. Short-term synaptic plasticity was evaluated by pairing stimuli at intervals ranging from 25 to 400 ms. No significant change was detected in sham-grafted and grafted animals when compared with control group, although in lesioned animals, there was a lower paired-pulse ratio for the interstimulus intervals of 200, 300, and 400 ms (Figure 4H).

Human OE-MSCs survive, migrate, differentiate into neurons, and stimulate endogenous neurogenesis in mouse lesioned hippocampus

The functional recovery following transplantation was associated with an exogenous neurogenesis. Five weeks after transplantation, we found that 60,000 to 90,000 exogenous cells (survival rate ranging from 13% to 20%) had settled within the hippocampus. A large proportion of GFP+ cells were distributed along the different damaged hippocampal fields, including CA1, CA3, and DG (Figure 5, A–C). While 69% ± 11% of the OE-MSCs were expressing III–β-tubulin, an immature neuron marker, and 0.9% ± 0.4% were positive for MAP2, a mature neuron marker (Figure 5, D–F), none of the exogenous cells were immune-positive for the astrocytic marker GFAP (Figure 5G). Moreover, GFP+ cells were also observed in other cerebral areas, especially in cortices above the hippocampus (Supplemental Video 3). Not a single exogenous cell was found in peripheral structures such as kidneys, liver, or lung (data not shown). An additional experiment, based on transplantation of human OE-MSCs in unilaterally unlesioned hippocampus, demonstrated that stem cells migrated toward the contralateral lesioned hippocampus (Figure 5, H–K). Interestingly, transplantation of OE-MSCs in bilaterally unlesioned hippocampi showed that cells do not migrate and stay within the tract generated by the injecting needle.

Figure 5 Human OE-MSCs transplanted into lesioned mouse hippocampi survived, migrated, and differentiated into neurons. (A) 5 weeks after transplantation, exogenous GFP+ human OE-MSCs were present in the different fields (CA1, CA3, DG) of the lesioned hippocampus. (B and C) GFP+ human OE-MSCs were mostly found in pyramidal (CA3, B) and granule cell layers (DG, C). (D) Within these layers, a high proportion (69%) of GFP+ human OE-MSCs (green) expressed III–β-tubulin (red) (white arrows in D). (E) High magnification of the merged picture of human GFP+ OE-MSCs (green) expressing III–β-tubulin (red). (F) A small proportion of human GFP+ OE-MSCs (green) expressed MAP2 (white arrow), a marker for mature neurons (red). (G) No GFP+ human OE-MSC (green) was ever found to express the astrocytic marker GFAP (red). (H) 4 weeks after lesioning the right hippocampus, GFP+ OE-MSCs were transplanted into the intact hippocampus (i.e., left hemisphere). At day 0 (D 0 ) after transplantation, cells formed clusters and were only observed within the injection site as a cell cluster (I). At D 4 after transplantation, numerous GFP+ cells were observed migrating outside the injection site toward the contralateral lesioned hippocampus (J). At D 7 after transplantation, few GFP+ OE-MSCs were observed inside the contralateral hippocampus (K). Scale bars: 250 μm (A); 200 μm (J); 100 μm (B, C, D, F, G, I, and K); 20 μm (E). See also Supplemental Video 3.

To quantify the impact of human OE-MSCs on endogenous hippocampal neurogenesis, newborn cells were labeled with the thymidine analog BrdU. In each group (n = 5 per group), the percentage of newly formed neurons expressing BrdU and NeuN was determined 5 weeks after the last BrdU injection (Figure 6, A–E). No GFP+ cell was found to be BrdU positive and no tumor formation was ever observed. In confirmation, we found that not a single exogenous cell was positive for Ki67 (data not shown). Moreover, we performed a comparative CFSE proliferation assay showing that, in our culture conditions and prior transplantation, sphere-derived cells are not proliferative or are poorly proliferative (Supplemental Figure 1). Additionally, we quantified a significant increased neurogenesis within the subgranular zone and granule cell layers of the DG in both sham-grafted (P = 0.032) and grafted groups (P = 0.008) when compared with control group. The number of BrdU+/NeuN+ cells was approximately 1.5-fold and 2.5-fold higher in the sham-grafted group and in the grafted group, respectively. Moreover, newly generated neurons were significantly more numerous in the grafted group when compared with the sham-grafted group (P = 0.008). We also labeled immature neuronal cells with doublecortin (DCX) antibody, in order to assess the state of the endogenous neurogenesis just before sacrifice, 5 weeks after transplantation (Figure 6, F–H). Surprisingly, no GFP+ exogenous cell was found to be DCX positive (Figure 6H). We observed a 1.8-fold increase in the number of endogenous DCX-positive cells in the grafted group when compared with either sham-grafted or control groups (P < 0.01) (Figure 6I).

Figure 6 Human OE-MSCs stimulated endogenous neurogenesis after transplantation in lesioned hippocampi. 5 weeks after grafting, brain sections of mice injected with BrdU twice a day during 3 days following cell implantation (n = 5 for each group) were immunostained with anti-NeuN (green) and anti-BrdU antibodies (red) (A–D). Quantification of BrdU+/NeuN+ cells in DG indicated an increased number of mitotic cells in grafted (IH) mice when compared with sham-grafted (IH) (P < 0.05) and control (P < 0.01) mice (E). DCX immunohistochemistry revealed the presence of immature neurons in the DG of control (G) and grafted (IH) (F, H) mice. As shown in (H), no GFP+ human OE-MSC was found to express DCX. Lesioned mice with IH transplant of human OE-MSCs (grafted [IH], n = 5) exhibited a higher percentage of DCX-positive cells when compared with vehicle-treated (sham-grafted [IH], n = 5) and control (n = 5) mice (I). (E and I). Scale bars: 10 μm (A–C); 100 μm (D, F, G, and H). *P < 0.05; **P < 0.01.

Human OE-MSCs grafted within the lateral ventricles, survive, migrate, differentiate, and restore mnesic capacities

The positive results obtained during this first series of experiments led us to modify 1 of our 3 initial options. Using the same animal model, we decided to graft GFP+ OE-MSCs, derived from a single stem cell, within the cerebrospinal fluid of lateral ventricles. As previously described, we first confirmed the lesion efficiency in both tests (Figure 3, C and E). Four weeks after transplantation, behavioral assessment using the olfactory tubing maze revealed a significant group effect (MANOVA, F [2,21] = 34.839; P < 0.001). The group effect was due to significantly impaired performance in lesioned mice (sham-grafted intracerebroventricular [ICV] group), with an overall percentage of correct responses significantly decreased in this group when compared with control (P < 0.001) and grafted ICV groups (P < 0.001). Across the 5 training sessions, grafted animals improved their mnesic capacities significantly, while sham-grafted animals remained unable to perform the task (Figure 3D). Intertrial interval analysis revealed no significant difference between sham-grafted (31.12 s SEM ± 2.16) and grafted (ICV) (28.42 s SEM ± 1.7) groups. In parallel, analysis of latencies in the Morris water maze test revealed a significant group effect (F [3,28] = 10.473; P < 0.001). The group effect was due to significantly impaired performance in sham-grafted (P = 0.009) and dead cell groups (P = 0.009), with latencies significantly increased in these 2 groups, when compared with control and grafted ICV groups. No significant difference was observed between grafted ICV and control groups (P = 1) (Figure 3F). Whereas no significant difference was observed in the swimming speed between grafted ICV and both sham-grafted (P = 0.1) and control (P = 1) groups, similar analysis revealed that grafted mice with dead cells were significantly faster when compared with those in the grafted ICV group (P = 0.01). Probe test analyses revealed a significantly longer time spent in the platform quadrant (Q4) by both control and grafted ICV groups, while no significant difference was observed in lesioned and dead cell groups (Supplemental Table 1).

Improved mnesic performances were also associated with the presence of GFP+ cells in the different layers of the lesioned hippocampi (Figure 7, A and B, and Supplemental Video 4). An extensive exogenous neurogenesis was observed, with stem cells differentiating into cells expressing neuron-specific markers (Figure 7, C and D). No GFP+/GFAP+ cell was ever encountered. Exogenous cells were found in cortical areas but remained always immunonegative for the mature neuronal marker MAP2 (Figure 7E), and no GFP+ cell was found in peripheral structures. Conversely, GFP+ cells exhibiting an undifferentiated morphology and negative for neural markers (III–β-tubulin, MAP2, GFAP) were found at the margin of ventricular areas (Figure 7F). Regarding these data, we expected a migratory potential of OE-MSCs in response to specific signals generated by a lesion.

Figure 7 Human OE-MSCs transplanted into the cerebrospinal fluid (CSF) of hippocampus-lesioned mice survived at least 5 weeks, migrated, and differentiated into neurons. (A) Increased density of GFP+ cells within the pyramidal cell body layers demonstrated the ability of human OE-MSCs to migrate from the CSF toward the injury zone. (B) As demonstrated by confocal image reconstitution using projection transparency (see also Supplemental Video 4), exogenous GFP+ cells were also remarkably distributed within the granule cell body layers of the DG. The insert indicates that not a single human OE-MSC expressed the astrocytic marker GFAP (red). (C and D) Within the CA3 field, some pyramidal cell–like and interneuron-like GFP+ human OE-MSCs (green) expressed III–β-tubulin (red) (white arrows in C, yellow arrow in D), but others were immunonegative for this immature neuronal marker (green arrows in D). (E) Exogenous GFP+ cells migrated as well in other cerebral areas (cortical area in E), but remained immunonegative for the mature neuronal marker MAP2 (red). (F) GFP+ cells exhibiting an undifferentiated morphology were found at the margin of the ventricular areas. Scale bars: 100 μm (A, C, D, E, and F); 30 μm (B, insert). See also Supplemental Video 4.

Lesioned hippocampi overexpress genes involved in cell chemoattraction

In order to understand OE-MSC homing, a comparative gene expression profile of lesioned and control hippocampi (3 vs. 3) was performed using mouse pangenomic DNA microarrays 4 weeks after surgery. In total, 114 transcripts with a fold change above 2.5 were found upregulated in lesioned hippocampi. The list of overexpressed genes indicates that 53% were involved in immune and inflammatory processes, 22% in cell metabolism, and 27% in various other processes (Supplemental Figure 2). Interestingly, among overexpressed genes, 8% code for chemokines and 2 of them, Ccl2 and Cxcl10, have a fold change above 10 (Table 1). Moreover, cytokines such as Spp1 and C3, known for their chemotactic properties, were also upregulated with a fold change of 11 and 162.5, respectively.