 the protein composition of embryonic CSF is more complex than that of adult CSF [22], our results indicate that adult CSF has the capacity to influence the behaviour of adult NSCs in the adult brain, too.
CSF as a beneficial environment for cell survival and growth has also been postulated by in vivo studies, investigating for example the behaviour of fetal NSCs after injection in the fourth ventricle of spinal cord lesioned rats with a good survival of grafted cells within the CSF [38, 39], as well as in a recent in vitro study of our own group with fetal rat NSCs [31]. In the present study, CSF had a general stimulating effect demonstrated by the faster loss of self-renewing capacity and stronger cell extension outgrowth. Adult hNSCs differentiated predominantly into astrocytes (38% in CSF, 25% in standard media) when treated with CSF and to a lower extend into oligodendrocytes and neurons. After differentiation for 7 days, we found 9% of the cells to be β-tubulin+ in CSF. In standard culture media, 31% of the cells were β-tubulin III+. Therefore, our data strongly suggest inhibitory effects of CSF on neurogenesis of ahNSCs, but promoting effects on astroliogenesis. Possible factors in CSF influencing differentiation behaviour of NSCs are bone morphogenetic proteins (BMPs): Monoclonal antibodies against BMP7 were for example shown to inhibit CSF induced dendritic outgrowth of neurons. BMP4 was shown to induce neuronal differentiation of NSCs by activating the ERK and inhibiting the GSK3β pathway. Both described effects could be blocked by Noggin, a BMP inhibitor [40, 41]. These described effects of BMPs, do have an influence on neuronal differentiation only in fetal NSCs from rat. Until recently, it remained elusive whether these BMPs may also play a role in the observed effects of adult human CSF on astroglial differentiation, but we could show that parts of the CSF mediated effects on fetal rat NSCs could be blocked by Noggin[31]. BMPs thus seem to be at least a part of the soluble factors in the CSF influencing stem cell behaviour in fetal rat NSCs. However, in the present work, we could not find any inhibiting effects of Noggin on CSF survival effects on adult human NSCs. This raises the question whether BMPs influence NSC behaviour only during ontogenesis and therefore have no influence on NSCs derived from the adult brain or whether BMPs might differential effects in human and rat cell systems.
It is well accepted that lumbar CSF is different from ventricular CSF because of both passive diffusion processes across the blood-CSF-barrier and suggested active secretion processes during the cranio-caudal circulation [42]. Consistently, all herein described CSF effects can only be attributed to leptomeningeal CSF. Whether ventricular CSF has similar same effects on NSC behaviour remains elusive. The use of CSF out of ventricular drainages is however problematic, as ventricular drainages are used in patients with obstructed CSF circulation (for example cerebral aqueduct stenosis) or other reasons of elevated brain pressure with defect blood-brain- and blood-CSF-barrier (for example after major stroke, after intracerebral bleedings or inoperable brain tumours). Furthermore, CSF out of ventricular drainages is often contaminated by blood and altered by inflammatory processes, which is why it cannot be used for the examination of healthy CSF effects.
Conclusions
Together, our results demonstrate that adult leptomeningeal CSF has a trophic influence on adult human NSCs: survival and differentiation into astroglial cells are promoted. Thus, our data point to a pivotal role of CSF in regulating adult neurogenesis under physiological and presumably also under pathological conditions as suggested previously [28–30]. Future experiments are warranted to determine which compounds within the CSF, besides BMPs, might be responsible for the effects on NPC behaviour. It also needs to be investigated whether there are different CSF compounds influencing the behaviour of NSCs originating from fetal or adult brain. This might give us the opportunity to influence resident NSCs of the SVZ to induce their proliferation as well as to promote their migration to pathologically altered brain regions.
Methods
Collection of adult human leptomeningeal CSF
The CSF samples were taken for diagnostic purpose from adult patients in the Neurological Clinic of the Ernst Moritz Arndt University of Greifswald by a lumbar puncture. Scientific use of CSF samples was approved by the local Ethics Committee. All patients gave a written informed consent for the diagnostic procedure. Lumbar puncture was performed by standard protocols. The final diagnosis of all patients was Idiopathic Normal Pressure Hydrocephalus (NPH). CSF was only used if all standard parameters were normal (cell count, glucose-, lactate. and albumin-content, immunoglobulin-quotient). Contamination with blood was excluded. In all patients, tumors and infectious diseases were excluded. Surplus CSF from diagnostic samples of all patients was spun down to remove remaining cells, pooled and analyzed to exclude non-sterility and presence of inflammatory markers. The pooled CSF was normal in all standard parameters; contamination with blood was excluded once again. The exact parameters were as follows: Cell count <1/μl, no blood contamination, normal protein of 369 mg/l, normal glucose of 3.6 mmol/l, normal lactate of 2.0 mmol/l, normal ferritine of 7.2 μg/l, normal albumin quotient of 5.0 × 10-3, normal immunoglobulin quotients: IgG 2.1 × 10-3, IgA 1.1 × 10-3, IgM <0.2 × 10-3. Pooled CSF samples were aliquoted and frozen at -80° until used.
Isolation and propagation of adult human neural stem cells (ahNSCs)
Adult human hippocampal tissue was obtained from routine epilepsy surgery procedures (selective amygdalo-hippocampectomia) following informed consent of the patients. All procedures were in accordance with the Helsinki convention and approved by the ethics committees at the EMA University of Greifswald (III UV 77/06) and at Dresden University of Technology (EK47032006). The tissue was stored in ice-cold Hank's balanced salt solution (HBSS) supplemented with 11 mM glucose and 1% penicillin/streptomycin. In all patients, tumours and infectious diseases were excluded by means of high-resolution magnetic resonance imaging and screening for inflammatory markers. Additionally, neuropathological tissue examination did not reveal evidence for tumour formation. For expansion of neurospheres, tissue samples were dissociated using trypsin, DNase and mechanical trituration similar as described previously [19]. Several media and supplements were tested like DMEM, DMEM/F12, Neurobasal (all from Gibco), P4-8F (Athena) with and without N2 or B27 supplements (Gibco) and growth factors. Best results concerning especially the amount of primary neurospheres per dissected hippocampal tissue and the early passage propagation was achieved by Knock-Out DMEM medium, supplemented with 10% KO supplement; 0.5 mM glutamine; 1% penicillin/streptomycin (all from Gibco) and 20 ng/ml both EGF and FGF-2 (from Sigma-Aldrich)(KO/DMEM). Therefore this medium was chosen as control expansion medium. Cultures were incubated at 37°C in a humidified atmosphere and lowered O2 conditions of 5% CO2, 92% N2 and 3 ± 2% O2. Fresh medium was added once a week, growth factors twice a week. For BrdU labelling, cells were incubated with 10 μm BrdU for 72 h. For immunocytochemistry studies of neurospheres, these were allowed to attach for 2-4 h before been fixed as described below.
Differentiation conditions
Cells were differentiated by plating them onto poly-L-lysine-coated chamber slides or 6-well-plates in P4-8F (standard differentiation media; from AthenaES, Baltimore, MD, USA, the albumin-content of P4-8F of 250 μg/ml matches the normal albumin content of healthy adult lumbar CSF, the glucose content of P4-8F is in a physiological concentration of 7 mmol/l) or in CSF without adding any growth factors. Some of the differentiation experiments were conducted in the presence of 150 ng/ml recombinant Noggin (R&D System, Minneapolis, MN). For studying cell survival, proliferation and neurite outgrowth cells were allowed to differentiate for up to 72 hours, for investigating cell fate decisions for up to 7 days respectively. Half of the media was changed every third day. For investigating cell morphology, survival rate and marker expression by immunocytochemistry, the cultures were fixed from 0 h up to 72 h and 14 days after starting the differentiation process.
Immunostainings
For immunocytochemistry, cell cultures were fixed in 4% paraformaldehyde in PBS or with 4% paraformaldehyde/PBS followed by ice-cold acidic ethanol and 2N HCL for BrdU staining. Immuncytochemistry was carried out using standard protocols. Cell nuclei were counter stained with 4,6-diamidino-2-phenylindole (DAPI). To determine the self renewing potential of NPCs, Ki67 expression and BrdU incorporation were used. Ki67 is detected in the nucleus of proliferating cells in all active phases of the cell cycle from the late G1 phase though the M-phase [43, 44]. BrdU marks cells within the S-phase of the cell cycle [45]. Antibodies and dilutions were as follows: rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal 1:1000 (Chemicon International, Temecula, CA, USA), mouse anti-microtubuli associated protein (MAP2ab) monoclonal 1:100 (Pharmingen, San Diego, CA, USA), mouse anti-galactocerebroside (GalC) monoclonal 1:500 (