Background The existence of neurogenesis in the hippocampus of adult nonhuman primates has been confirmed in recent years, however, the biological properties of adult neural stem cells or neural progenitor cells (NPCs) from this region remain to be extensively explored. The present work was to investigate on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro.
Methods NPCs isolated from the hippocampus of adult cynomolgus monkeys were expanded in vitro in serum-free media containing growth factors, and were then allowed to differentiate by removing mitotic factors. The expansion capacity of NPCs and their differentiation potential were assayed by immunohistochemical and immunocytochemical analysis.
Results During primary culture, NPCs underwent cell division, proliferation and aggregation to form neurospheres that were growing in suspension. Without mitotic stimulation, most neurospheres adhered to the culture dish and started to differentiate. Eventually, nearly 12% of the differentiated cells expressed neuron specific marker- β III-tubulin (Tuj1) and 84% expressed astrocyte specific marker-fibrillary acidic protein (GFAP). In addition, the expression of a neural stem cell marker, nestin, was found both in NPCs and in the subgranular zone of adult monkey hippocampus, where NPCs were originally derived.
Conclusions NPCs from the hippocampus of adult cynomolgus monkeys can be expanded to some extent in vitro and are capable of differentiating into neurons and astrocytes. Further experiments to promote the in vitro proliferation capacity of NPCs will be required before adult NPCs can be used as a useful cell model for studying adult neurogenesis and cell replacement therapy using adult stem cells.

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Most neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, are progressive in nature and few effective therapies exist to delay disease progression or to promote recovery. To date, there have been limited options for repairing an impaired central nervous system (CNS). Nonetheless, rapid advances in stem cell biology have raised the possibility of replacing degenerative or lost neurons by transplantation therapy.¹

So far, cells used in experimental transplantations are obtained from various sources including dissociated fetal mesencephalic cells, neurons derived from embryonic stem cells and fetal neural stem cells (NSCs). However, the limitation of access and ethical concerns of using embryo derived cells have prompted the search for alternative cell sources, especially the cells originated from adult brains. The existence of neurogenesis has been demonstrated in specific neurogenic regions of adult mammalian CNS: the subgranular zone (SGZ) in the dentate gyrus of hippocampus and the subventricular zone (SVZ) of lateral ventricles.^2-5 Cells isolated from these regions bear NSC characteristics, such as able to self-renew and capable of differentiating into specialized cell types of the CNS (neurons, astrocytes, oligodendrocytes).^4,6 Furthermore, within the adult brain, newly generated neurons have been found to be capable of migrating, extending long axonal projections, functionally integrating into pre-existing host neural networks, and contributing to reconstruction of brain functions in an adult CNS environment.^1,7,8

In the brain of nonhuman primates, active neurogenesis has been confirmed in the hippocam- pus,^3,9 yet the biological properties of NSCs from the adult primate hippocampus still remain to be extensively explored. In contrast, neural progenitor cells (NPCs) have been isolated and expanded from other adult mammalian CNS even including non-neurogenic region (cortex) of adult macaque monkey. 10-14 The present work focuses on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro.

METHODS

Animals
Three male pathogen-free cynomolgus monkeys (Macaca Fascicularis), 3.5 to 4.5 years of age and 2.9 to 3.7 kg of body weight were used in this study. Animal care and use were conducted at the Primate Research Center of Wincon TheraCells Co. (Nanning, China), under a protocol approved by the Institutional Animal Care and Use Committee (IACUC). All animals had continuous water supply and were fed with a “primate diet” twice daily supplemented with fresh fruits and vegetables. The animals were conditioned for at least one month prior to the start of the experiment.

Isolation of the hippocampus from adult cynomolgus monkeys
The monkeys were euthanized by intravenous administration of sodium pentobarbital and the brain was removed and immersed in ice-cold Hank's balanced salt solutions (Invitrogen, USA) immediately. Overlying meninges and blood vessels were stripped away, and the hippocampus was aseptically isolated by cutting open along the dentate fissure in brain bottom using an eye scissor and rolling outwardly by a sterile bamboo stick.

Primary cell culture of NPCs
The isolated hippocampus tissue was washed in three consecutive culture dishes containing fresh 4oC Hank's solutions plus 1% Penicillin/Streptomycin (Invitrogen), and then transferred to a 60-mm tissue dish containing 2 ml Hank's solutions. After the tissue was mechanically dissociated into small pieces, 2 ml of 0.25% trypsin-EDTA (Invitrogen) solutions were added and the dish was incubated at 37 ?C for 45 minutes. During trypsin incubation, the tissue pieces were lightly triturated by a fire-polished Pasteur pipette every 10 minutes. After trypsin digestion, dissociated cells in suspension were centrifuged down at 1000 r/min for 5 minutes, washed in Hank's solutions three times, and were then filtered through a 300-mesh stainless steel sieve to remove large pieces of debris. After filtration, cells were resuspended in DMEM/F12 (Combination of Dulbecco's Modified Eagle Medium with Ham's F-12, 1:1, Invitrogen) medium supplemented with 1% N2 (Invitrogen), 0.075% Bovine Serum Albumin Fraction (BSA, Invitrogen), 1% Penicillin/ Streptomycin, 1.25 μ g/ml insulin (Sigma, USA), 1.25 μ g/ml transferrin (Sigma, USA), 20 ng/ml epidermal growth factor (EGF, R&D Systems) and 20 ng/ml basic fibroblast growth factor (bFGF-2, R&D Systems). Finally, individually suspended cells were seeded in 45 mm culture dishes and maintained at 37 ?C (O₂/CO₂, 95:5) in an incubator (Forma, USA). Half of the media was replaced with fresh media at two-day intervals. The proliferation and differentiation of cells were observed using an inverted microscope (Nikon ECLIPSE TS-100, Nikon Corp., Japan) and photo images were obtained using an attached digital camera (Nikon E4500, Nikon Corp., Japan).

Neurospheres differentiation
To initiate differentiation, individual neurospheres formed from primary hippocampal cell culture were seeded one per well in 24-well-plates (Corning) coated with poly-ornithine and laminin (Sigma, USA), and maintained in serum-free medium DMEM/F12 supplemented with 2% B27 (Invitrogen) and 1% penicillin/streptomycin. Under this condition, neurospheres were inclined to grow as a monolayer and started to differentiate. The differentiation was allowed to proceed for 14 days with 50% fresh media changes every 2 days. On the 14th day, an immunocytochemical analysis of the remaining cells was performed.

Immunohistochemistry and immunocytochemistry
Immunohistochemistry
Hippocampus tissue was first fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) for 48 hours at 4 ?C. It was then transferred to 20% sucrose in 0.1 mol/L phosphate-buffered saline (PBS) for 24 hours at 4 ?C, followed by 30% sucrose in 0.1 mol/L PBS for 3 days at 4 ?C. Afterwards, it was quickly frozen in liquid-nitrogen cooled isopentane. The frozen tissue was sectioned 20 μ m by a cryostat (Shando cat No. 06206, England). For immunohistochemical labeling, the sections were blocked with 1% normal goat serum in 0.1 mol/L PBS for 1 hour at room temperature, and then incubated with primary antibody (rabbit anti-human nestin, Chemicon, 1:100) overnight at 4 ?C, followed by secondary antibody incubation (Texas red-conjugated goat anti-rabbit IgG, Jackson ImmunoResearch, 1:200) for 2 hours at room temperature. The sections were washed three times for 5 minutes each in 0.01 mol/L PBS after each step of antibody staining. SYTO 11 (Molecular Probes) was used for nuclei counterstaining. Finally, the sections were mounted with fluorescent mounting medium and viewed with a fluorescent microscope (Nikon 2000 U, Nikon Corp., Japan) and a digital camera (Spot RT, USA).

Immunocytochemistry
Prior to fixation, neurospheres were cultured onto coverslips coated with 0.1% gelatin (Sigma, USA) for 24 hours and rinsed in PB. Cell cultures and neurospheres were fixed in 0.1 mol/L phosphate-buffered 4% paraformaldehyde for 20 minutes. After blocked with 3% normal goat serum for 40 minutes at room temperature, the specimens were incubated overnight at 4?C with primary antibodies followed by 1 hour at room temperature with secondary antibodies, washed with 0.01 mol/L PBS and coverslipped with fluorescent mounting medium. Double immuno-fluorescent staining was conducted by using primary antibodies at following dilutions: mouse anti-human β III-tubulin (Tuj1) 1:1000 (Promega), rabbit anti-human glial fibrillary acidic protein (GFAP) 1:1000 (Promega), rabbit anti-human nestin 1:100, mouse anti-human neuronal nuclear antigen (NeuN) 1:1000 (Chemicon). The secondary antibodies used for immunofluorescent labeling were diluted at 1:200 (Texas red-conjugated goat anti-rabbit IgG, Cy2-conjugated goat anti-mouse IgG, Texas red-conjugated goat anti-mouse IgG, Cy2-conjugated goat anti-rabbit IgG, Jackson ImmunoResearch). Nuclei counterstaining was performed using 4', 6'-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma, USA). Fluorescent images of monolayer cells were captured using a fluorescent microscope system (see above), and images of neurosphere were obtained using a confocal microscope (BioRad MRC1024, USA). The final images were rendered by Adobe Photoshop.

RESULTS

Expansion of NPCs from the hippocampus of adult cynomolgus monkeys
In order to examine the growth potential of cells isolated from the hippocampus of adult cynomolgus monkeys, cells were cultured and monitored over an extended period of time. During the 1st week, the round viable cells stuck to the dish surface 3 － 4 days after plating and gradually increased in size (Fig. 1a). In the 2nd week, some cells began to divide, giving rise to new cells (Fig. 1b), and starting to extend neurites of various shape and length. By the end of 2nd week, newly generated cells tended to aggregate (Fig. 1c). In the 3rd week, cells started to form spherical clusters (Fig. 1d), which continued to grow as aggregates in mass and number, eventually gave rise to neurospheres (Fig. 1e). In the 4th week, neurospheres became larger, some reaching up to 250 μ m in diameter and starting to become detached from dish (Fig. 1f). Most floating neurospheres formed between the 15th and 22nd day after plating. In the 5th week, some neurospheres tended to adhere to dish again, but could be resuspended by a slight triturating. In the 6th and 7th week, neurospheres with coarse surface became much easier to adhere to the dish and difficult to be resuspended by triturating (Fig. 1g). Once adherence happened, spontaneous differentiation initiated with radial processes growing out from the neurospheres and extending scatteredly (Fig. 1h). During the 8th and 9th week, more neurospheres differentiated spontaneously, the processes emanating from the neurospheres formed prominent fiber bundles and a surrounding glia-like monolayer (Fig. 1i). By the end of the 9th week, most cells stopped proliferation and became completely differentiated (Fig. 1j).

Double-labeled immunofluorescent examination of GFAP-Tuj1, nestin-Tuj1, GFAP-NeuN and nestin-NeuN in the differentiated cultures revealed that most of the cells expressed nestin, some express Tuj1 and others express GFAP. However, there were no cells labeled with NeuN. These results indicated that the neurospheres were multipotent, able to differentiate to neurons and astrocytes (Fig. 2). Quantitative analysis revealed that nearly 12% cells were immunoreactive to Tuj1 and 84% GFAP-positive cells were detected.

Identification of NPCs in the hippocampus of adult cynomolgus monkeys
To examine where NPCs exist in adult hippocampus of cynomolgus monkeys, hippocampal tissue sections were directly labeled with an anti-nestin antibody (Fig. 3a-c). Nestin-positive cells were detected in a region between the granule cell layer and the hilus (or CA4), which is consistent with previous studies showing that the progenitor cells in adult hippocampus resided in the SGZ, where proliferative activity was most robust. 3,20 Correspondingly, when neurospheres were labeled with anti-nestin antibody, most of the NPCs appeared to be nestin-positive (Fig. 3d). So, it seems that isolated NPCs were from the same region of the hippocampus.

DISCUSSION

We have successfully isolated NPCs from the hippocampus of adult cynomolgus monkeys. These cells can be expanded in vitro to grow to neurospheres, which are capable of differentiating into neurons and astrocytes. This result is in accordance with the features of adult NSCs as reported from previous studies on other mammalian brains: multipotent progenitors of the adult brain are proliferative cells with only limited self-renewal that can differentiate into at least two different cell lineages. 10,21-24 In addition, the detection of nestin-positive cells in the hippocampal sections indicates the existence of uncommitted NPCs in the SGZ region of the hippocampus, which is consistent with previously demonstrated evidences.^3,14,25,26

Comparing to NSCs isolated from the hippocampus of human fetus, NPCs from adult monkey hippocampus show several distinct features. First, under similar culture conditions containing growth factors EGF and bFGF, human fetal NSCs can be expanded indefinitely whereas monkey adult NPCs maintain proliferation for 9 weeks in culture. The neurospheres of adult NPCs are prone to adherence and spontaneous differentiation even under growth promoting conditions, which is not observed in human fetal hippocampal NSCs.¹³ In addition, the proliferation of adult NPCs is apparently slower than that of fetal NSCs, as it takes much longer for adult NPCs to form neurospheres from dispersed primary cells (15－22 days) than for fetal NSCs (5－9 days). This is not so surprising since it is believed that despite recent estimates suggest that the number of new neurons produced in adulthood is greater than originally thought, the rate of neurogenesis in adult CNS is lower than that during development.²⁷ Palmer et al 28 had reported that brain tissues from young human after death yielded significantly more cells per gram and these cells had a higher proliferative capacity than that of adult brain tissues. Overall, the characteristics of adult NPCs are consistent with the fact that other adult derived progenitors have limited self-renewal, proliferation and are more prone to differentiation.²²

In this study, the adult NPCs have demonstrated the ability to differentiate into both the neuronal and glial lineages. A somewhat surprising result is that while robust Tuj1 staining is observed in some of the differentiation progeny of NPCs, there was no NeuN labeled cells at all. Tuj1 has been used extensively to label newly generated and immature neurons,^28,29 whereas NeuN is thought to be a pan-neuronal marker that stains mature and postmitotic neurons. ²⁹ So it appears that under the current differentiation condition, even after two weeks of culture, differentiated cells remain at an immature stage. In a previous study on cynomolgus monkeys using BrdU incorporation to label NPCs and their progenies in vivo , NeuN and BrdU were not co-expressed in the same cells until 2 weeks after BrdU injection.³⁰ This indicated that in the adult hippocampus, BrdU labeled NPCs would take at least two weeks to differentiate into NeuN-positive mature neurons. It is not yet clear whether under current in vitro conditions, the differentiation of NPCs to mature neurons is simply delayed or being inhibited. To fully understand the question will need thorough analysis of NPCs differentiation in culture.

In summary, this study provides an initial study of NPCs isolated from the brain of adult nonhuman primates. It is demonstrated that the NPCs can be expanded in culture and are capable of differentiating into neurons and astrocytes. Nonetheless, before adult NPCs can be used as a useful cell model, much more work has to be carried out, not only to promote proliferation so as to generate unlimited cells numbers, but also to insure complete differentiation of NPC into neurons. We believe that further analysis of adult NPCs will lead to the better understanding of neurogenesis in the adult brain and providing a potential source for cell therapy.

Acknowledgements: We thank Dr. ZOU Chun-lin and Dr. GUAN Yun-qian for technical support and figure preparation. We are also grateful to YI De-qiao and YAO Dun-yun for valuable assistance in primate experiments.

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