INTRODUCTION

It was usually regarded that the neurogenesis has terminated in the central nervous system of the adult mammals. However, the recent researches found that some neuronal stem cells maintained activity in adult, especially in the dentate gyrus of hippocampus and the subventricular zone (SVZ)1,2, and the neuronal precursors derived from these stem cells could migrate into the olfactory bulb through the rostral migration stream (RMS) and differentiate into local interneurons3,4. With their potential to produce neurons, now SVZ cells have been widely used to study the mechanism of neuronal migration and brain repair in adult mammalian brain 5,6. It has been known that the portion of SVZ is the largest pool of neuronal precursors in many adult vertebrates7,8. Usually, the neuronal precursor cells derived from the neuronal stem cells lay in or adjacent to the ventricular zone (VZ), and migrate outside SVZ9. Therefore, it was considered that the wall of the brain ventricles played an important role in the diffusion and activation of the transplanted cells10. Obviously, the transplant study of the adult SVZ cells will, not only understand the mechanism of migration and survival of the neuronal precursor cells in adult, but also provide a base for improving strategies of neuronal replacement.

It has been reported that the SVZ precursors from the adult mice could differentiate into neurons or glia in vitro11. Also, the neuronal precursors from neonatal mouse SVZ have been showed to be able to migrate and differentiate into neurons in the multiple regions of the embryonic brain, mainly in the olfactory bulb(OB), septum, thalamus, hypothalamus and cerebellum12. More recently, Snyder et al13 reported that the transplanted stem cells from the human embryo could differentiate into neurons in the forebrain of the embryonic primate. These findings raised a question whether transplanted precursors from adult brain were able to survive and differentiate into mature neurons in the developing recipient brain. Transplants of adult SVZ cells have not been revealed to migrate into and differentiate within non-OB central nervous system, even though the local migration and incorporation occur with neonatal SVZ cells grafted into neonatal striatum12. Furthermore, it is of interest whether the adult SVZ precursors have the same plasticity or potential as the embryonic precursors. To test this hypothesis, we injected the dissociated SVZ cells from the adult into the lateral ventricles of the mouse brain at the different development time. Here was showed that the grafts from the adult SVZ cells were able to incorporate into the developing brain and differentiate into neurons. It is hoped that the present results will contribute to the research for the potential of adult SVZ precursors in the treatment strategies of the brain trauma and neural dysfunction.

MATERIALS AND METHODS

Preparation of SVZ donor cells

Fifty-seven pregnant Kun Ming(KM)mice were used for these experiments (supplied by the Experimental Animal Center of the Medical Department of Peking University).

The 3-5 months old male mice (n=38) were anesthetized with 0.3% sodium pentobarbital (40mg/Kg body weight) and perfused through the ascending aorta with the sterilized D-Hanks. The SVZ explants were dissected from a coronal slice (1 ∼ 2mm thick) extending between the anterior commissure and the third ventricle. A strip of tissue (about 0.1 mm wide; 2 ∼ 4 mm long) was extracted along the lining of the lateral ventricle under the corpus callosum and cut with scalpels into pieces. After digested with 0.06 mg/ml papain at 37°C for 30 min, the donor tissues were rinsed with 1640 medium and then dispersed into single cell suspension. The donor cells were labeled with PKH26 (Sigma) in the Diluent C store solution (1:100) for 10min at room temperature and treated with the equal volume of serum about 1min to block the PKH26 reaction. After washed three times in 1640 medium, the donor cells were centrifuged and resuspended in 1640 medium at 50, 000cells/μ. Before transplant, Trypan blue (0.1%) was sometimes added into the donor cell suspension to check the injection position in the recipient embryos.

The grafted SVZ cell types were determined with NeuN (neuron-specific nuclear protein, Sigma) and GFAP (glial fibrillary acidic protein, Jackson Corporation) immunostaining by using smear method, and double stained with 0.1% Evans blue. Three microscopic fields on the same smear slide were selected randomly for cell type count and statistic analysis.

Recipient mice and group

The donor cells were injected into the lateral ventricle of embryos in the pregnant KM mice (n=57). The date of transplant operation was planned on E15, E17 and P0 (E0: vaginal date, P0: date of birth), separately. The recipient subjects were sacrificed on P3, P20, P30 and P90, separately. Ten experimental groups were divided by the date of transplant (left by slant) and the date of sacrifice (right by slant), indicated as E15/P3, E17/P3, P0/P3 and etc.

Transplant operation

The timed-pregnant KM mice were anesthetized with sodium pentobarbital and injected 120-250 μ 2.5% MgSO4 to relax the smooth muscle. An incision was made along the abdominal midline to expose the uterine horns on the warm pad (37°C. Every embryo was kept upward to show the forebrain ventricles and injected about 1 μ donor cells suspension into the lateral ventricles, one by one. In P0 group, the cell suspension was injected directly into the lateral ventricle.

Immunohistochemistry

The anesthetized recipient mice were perfused with 0.9% saline followed by 4% polyformaldehyde in PBS. The brains were removed and stored in 4% polyformaldehyde at 4°C for additional 4 h, and then transferred to 20% sucrose for 24 h. The sagittal brain sections were cut at 16mm with microtome and every eighth sections were collected. For the GFAP immunostaining, the sections were treated with 3% normal goat serum for 1-3 h at 4°C to block the non-specific staining and then incubated in the rabbit anti-GFAP antibody 1:500 in PBS overnight at 4°C. After rinsing three times in PBS, sections were incubated in the goat anti-rabbit IgG-FITC (Jackson Corporation) 1: 100 for 2 h at room temperature. For NeuN immunostaining, the sections were treated with normal goat serum same as for GFAP staining and incubated in the mouse anti-NeuN monoclonal antibody 1:500 in PBS at 4°C for 48 h, and rinsed and incubated in the goat anti-mouse IgG-FITC (Jackson Corporation) 1:85 for 2 h at room temperature. The negative control sections were incubated in the same diluted solution without the primary antibody but the other procedures were same as above.

Observation and statistics

PKH26 presents the red fluorescence (Max=567nm) excited by green rays (Max=551nm). DNA fluorescent dye, Hoechst 33342, was used to label background cells to confirm the PKH26 labeled cells. Hoechst 33342 presents blue fluorescence excited by ultraviolet rays. The double-labeled cells with PKH26 and NeuN or GFAP were also observed and counted, separately. Images were acquired by SPOT-II and statistics analysis were performed with SPSS8.0 (ANOVA and LSD).

RESULTS

The phenotypes of the donor cells from adult SVZ

The donor cells on smear slides were immunostained by NeuN and GFAP. The results showed that NeuN-positive cells accounted for 3.1% (X=9.46/305.3, n=6), GFAP-positive cells accounted for 2.5% (X=10.3/411.67, n=6), and the rest represent NeuN and GFAP-negative cells.

The grafted SVZ cells incorporate into multiple regions in the developing mouse brain

To reveal the morphology of the living cells, the grafted SVZ cells were labeled with fluorescent lipophilic dye PKH26 before transplantation. PKH26-labeled cells were detected in multiple regions of the recipient brain (Tab 1). With different surviving times, the PKH26-labeled cells were remarkably detected in the SVZ of embryonic or neonatal brains in all groups. The main regions where graft-derived cells existed were the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum, pons and the retro-cortex etc.. Only a few PKH26 labeled cells were detected in the internal capsule and the area behind hippocampus. Only in E17/P30, there were a large quantity of labeled cells in olfactory bulb (Fig 1, Fig 2).

Table 1 Distribution of PKH26-positive cells in the recipient brain
Full size table
Figure 1
figure 1

Observation of the PKH26-labeled cells transplanted in E17 The recipient was sacrificed in P30 and the brain was sectioned sagittally. PKH26-labeled cells show red fluorescence (Max=567nm) excited by green rays (Max=551nm). (a) is a photograph at low scale (100× to show the distribution of PKH26-labeled cells in the olfactory bulb; (b) is high-power photograph (400×)of the selected part in (a). (c) and (d) are further high-power photograph(Bar=50 μm) of selected part in (b), showing the morphology of PKH26-labeled cells.

Full size image
Figure 2
figure 2

Observation of the PKH26-labeled cells coupled with Hoechst 33342 staining in E17/P30 The PKH26-labeled cells (red) are showed in (a); Hoechst 33342-positive cells (blue) are showed in (b). Arrows in (a) and (b) show the same cells that are double-labeled for both PKH26 and Hoechst 33342. Left and right panels show the same sections. Bar=100 μm

Full size image

The differentiation of the grafted precursors in the developing mouse brain

The PKH26 stained sections were double-labeled with mouse anti-NeuN monoclonal antibody or rabbit anti-GFAP antibodies in the recipient brain sections. Most of the PKH26-labeld cells also showed another fluorescence. align=right This indicated that some grafted cells may had neuronal or astrocytic phenotypes in the recipient brains (Fig 3). In many cases, cell morphology was detected with 0.1% Evans blue, the lipophilic dye. The statistics analysis of the double-labeled cells in P30 and P90 showed that the NeuN-positve cells in SVZ, cerebellum and olfactory bulb represent 12.6%, 23.1% and 28.2%, respectively. The GFAP-positive cells in SVZ, cerebellum and thalamus represent 21.66%, 20.12% and 18.74%, respectively.

Figure 3
figure 3

Double-labeled cells in the olfactory bulb (a, b) and SVZ (c, d) Arrows in (a) and (b) show the same cells that are double-labeled with PKH26 (red) (a) and NeuN-positive cells (b) in E17/P30 groups. Arrows in (c) and (d) show the same cells that are double- labeled with PKH26(c) and GFAP (d) in E15/P90 groups. Bar=100 μm

Full size image

DISCUSSION

The previous studies demonstrated that the neuronal precursors derived from embryos could migrate into almost all regions of central nervous system in the embryonic brain in mice12,14,15. The grafted SVZ cells from neonatal mice also were able to survive in the embryonic brain but migrate more restrictedly than that from embryos, i.e. without migrating to the cortex and hippocampus16. Our transplanted studies revealed that the SVZ cells from the adult mice could survive in the developing brain (E15, E17 and P0) and migrate into multiple regions, but with the survival ability lower than the precursors from neonate12. The present result suggests that the donor neuronal precursors from adult have a similar activity in the developing recipient brain in the embryonic and neonatal mice; and that the potential of the neuronal precursors correlates with the recipient age, decreasing with aging.

In adult rodent, the SVZ precursors are proved to migrate into the OB along rostral migratory stream (RMS) in chain and then move to the different layers of OB in radial direction, but without migrating outside of OB16. Our studies showed that the SVZ cells from the adult migrated not only into OB but also into multiple regions out of OB in the developing brain in mice. It is possible that the migration of the donor cells was affected by some specific site factors in the recipient brain. In the present studies, the graft-derived SVZ cells migrating to OB were found only in E17/P30 (Fig 1, Fig 2), not in the other experimental groups. It is unlikely that this OB incorporation was caused by a misplaced injection into the parenchyma of RMS, as cells were injected bilaterally in lateral ventricular and were labeled in bilateral OB of the recipient brains. It is possible that the development of OB neurons originates in E17 and finishes in P30. In this critical period, either the expression of some factors may induce the migration to OB, or the SVZ provides the optimum condition for the formation of OB precursor cells. However, This hypothesis requires further investigations.

In all experimental groups, PKH26-labeled cells were always found in SVZ of the recipient brains. This indicated that the SVZ is a starting point of the grafted cells to migrate outward, that this region is important for survival of the grafted cells. Our results show that there was different number of PKH26- labeled cells in the different regions of the recipient brain. It is possible that the plasticity differs in the different regions of the developing brain. From two reasonable explanations, one is that the grafted cells alter their direction or quantity of migration after undergoing a specific selection, which is controlled by some specific factors before or after a critical development time. This specific selection has been reported in the songbird canary, which the migration of their neuronal precursor cells significantly depended on the spatial and temporal selection with the season variation17. The other is that some grafted cells, which have neither the definite direction to migrate nor the correct neuronal network to incorporate in, maybe occur the programmed cell death (apoptosis) at the different rate during migration and after entering different brain regions. It is maybe either absent of a proper local cue or presence of inhibitory signal18. Even though there has been not any direct evidence on apoptosis in the developing brain. Our results revealed that the quantity of the PKH26-labeled cells decreases in E15/P90 compared to the other experimental groups.

Double-labeling by immunohistochemistry showed that some grafted cells differentiated into neurons or astrocytes after migrating into the target regions (Fig 3). Of the determination of the donor cell phenotypes (3.1% neurons and 2.5% glia), it was not established whether the double-labeled neurons or gila in the recipient brain came directly from the grafted neurons but not the grafted neuronal precursors. According to the statistics analysis of the quantity of the double-labeled cells in P30 and P90 groups, the percentage of neuron or glia in the recipient sections was higher (21.3% and 20.2%) than in the donor cell smeared slides. This result confirmed that some grafted neuronal precursors expressed neuronal phenotype after migrating into the special target. However, it is uncertain whether these grafted cells with neuronal phenotype establish synaptic connection with surrounding neurons, and whether they develope functions of a mature neuron.

The previous evidence showed that the SVZ cells migrated only into OB in the normal adult mice. Our transplant studies demonstrated that the SVZ cells from adult could migrate into multiple regions and differentiate into neurons in the developing brain in mice, and that the survival quantity of the donor SVZ cells correlated with the development time of the recipient brain. In the present researches, the grafted cells, including the nestin-positive cells from hippocampus transplanted into the cerebellum18, the original cells from striatum transplanted into the developing cortex19, the original cells expressing lacZ gene from mice cerebellum transplanted into hippocampus of neonatal rats or mice20, were found to have the same phenotypes as the surrounding cells. All together these indicate that the local cues play an important role in migration and differentiation of the precursor cells in central nervous system. It would be interesting to investigate how the plasticity of the precursor cells is induced, and what factors control the plasticity of the precursor cells in adult. The answers to these questions will also help to develop the treatment of the degeneration and damage of central nervous system in adult.