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TISSUE-SPECIFIC STEM CELLS |
a Department of Neurology, University of Ulm, Ulm, Germany;
b Molecular Neurobiology Laboratories; McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA;
c Department of Neurology, Technical University of Dresden, Dresden, Germany;
d Department of Neurology, University of Leipzig, Leipzig, Germany;
e Department of Anatomy and Cell Biology, University of Ulm, Ulm, Germany;
f Clinical Neurochemistry, Department of Child and Youth Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany;
g Division of Biology, California Institute of Technology, Pasadena, California, USA
Key Words. Adult neurogenesis • Neural stem cells • Dopaminergic differentiation • Parkinson’s disease • Electrophysiology • Neuroregeneration
Correspondence: Alexander Storch, M.D., Technical University of Dresden, Department of Neurology, Fetscherstrasse 74, 01307 Dresden, Germany. Telephone: +49-351-458-2532; Fax: +49-351-458-4352; e-mail: alexander.storch{at}neuro.med.tu-dresden.de
Received on April 26, 2005;
accepted for publication on December 9, 2005.
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ABSTRACT |
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-aminobutyric acid), glutamate, and NMDA (N-methyl-d-aspartate). Clonal analysis demonstrated that individual NSCs retain the capacity to generate both glia and neurons. After a multistep differentiation protocol using co-culture conditions with PA6 stromal cells, a small number of cells acquired morphological and functional properties of dopaminergic neurons in culture. Here, we demonstrate the existence of adult tNSCs with functional neurogenic and dopaminergic potential, a prerequisite for future endogenous cell replacement strategies in Parkinson’s disease.
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INTRODUCTION |
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Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types and may also replace or repair diseased brain tissue. Multipotent NSCs with the potential to generate mature cells of all neural lineages, including astroglia, oligodendroglia, and neurons, have been consistently demonstrated within the hippocampus and the SVZ of lateral ventricles [20–26]. These cells grow in vitro as spherical floating clusters (neurospheres) in the presence of epidermal growth factor (EGF) [21, 27]. Previous work has reported that SVZ NSCs correspond to a rare population of relatively quiescent cells. More recently, it has been suggested that not the ciliated ependymal cells correspond to the NSCs [24] but that SVZ astrocytes act as NSCs in both the normal and regenerating adult brain [28]. Recent studies demonstrated the presence of multipotent central nervous system (CNS) stem cells in non-neurogenic regions of the adult brain, including the spinal cord and the ventricular neuraxis [17, 18]. In contrast to SVZ NSCs, the proliferation of these NSCs depends on both EGF and fibroblast growth factor-2 (FGF-2) [18, 26]. Lie and coworkers described the isolation of multipotent NSCs from the adult substantia nigra with neurogenic differentiation capacity in vitro and after transplantation into the adult hippocampus [16]. However, the authors neither reported on detailed characterization of these progenitor cells in vitro nor provided data on neuronal subtype specification or functional properties after differentiation. Moreover, similar to all other adult CNS stem cells, these midbrain-derived NSCs did not differentiate into dopaminergic nerve cells [16].
Here, we show the presence of multipotent clonogenic NSCs in the adult mouse tegmentum (tegmental NSCs [tNSCs]) with all major properties of CNS stem cells such as neurosphere formation, expression of Nestin and other NSC markers, as well as significant telomerase activity. They show the capacity to differentiate in vitro into all neural cell lineages, including astroglia, oligodendroglia, and neurons. Moreover, the resulting nerve cells displayed morphological and functional properties of mature neuronal subpopulations, such as GABAergic and dopaminergic neurons.
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MATERIALS AND METHODS |
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Differentiation Conditions
Induction of neural differentiation (protocol 1) was initiated by plating the cells on poly-L-lysine (PLL)-coated glass coverslips at a concentration of 1.5–2.0 x 105 cells cm–2 in NBa medium containing 1% glutamate, 2% B27 supplement, and 1% penicillin/streptomycin supplemented with 0.5 µmol l–1 all-trans-retinoic acid (Sigma), 10 ng ml–1 brain-derived neurotrophic factor (BDNF; Promega, Madison, WI, http://www.promega.com), and 100 µM dibuteryl(db)-cAMP (Sigma). Cells were differentiated for 14 days. For analyzing the differentiation capacity of tNSCs, several other combinations of growth factors/cytokines were used (Table 1
). The following substances were used: 10% fetal bovine serum (Biochrom AG, Berlin, http://www.biochrom.de), 100 pg/ml interleukin (IL)-1b, 1 ng/ml IL-11, 10 ng/ml glial cell line–derived neurotrophic factor, 1 ng/ml leukemia inhibitory factor (all from Sigma), 10 ng/ml FGF-4, 10 ng/ml FGF-8, and 10 ng/ml sonic hedgehog (Shh; all from R&D Systems, Minneapolis, http://www.rndsystems.com). For co-culture analyses, various feeder cells (mouse embryonic fibroblasts [MEFs]), PA6 stromal cells, as well as cortical and striatal astrocytes from embryonic day 14.5 [E14.5] rat embryos) were cultured on gelatine-coated cover slides as described earlier [29, 30]. Before plating the tNSCs on the feeder layers, the cells were incubated with expansion media supplemented with 500 ng/ml Shh and 100 ng/ml FGF-8 for 48 hours ("pre-stimulation"). When co-culture was started, confluent feeder cells where washed twice with phosphate-buffered saline (PBS) and tNSCs were seeded at a concentration of 1.5–2.0 x 105 cells cm–2 in Glasgow’s modified Eagle’s medium (Gibco) containing 1% glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol ("differentiation medium" [DM]) supplemented with 10% knockout-serum replacement (Gibco). Medium change was performed on day 4 and every other day after that. After 7 days, the medium was changed to "differentiation medium" supplemented with 1% N2 (DM-N2 medium; Gibco).
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Flow Cytometry
tNSCs were treated with Accumax (Gibco) and washed with PBS. Dead cells were excluded from analysis by forward-scatter gating. Samples were processed using a FACSCalibur flow cytometer, and analyses were performed with the Cellquest software (both from Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Antibodies were used as follows: CD34 –fluorescein isothiocyanate (FITC) 1:10, CD45-phycoerythrin (PE) 1:10, CD24-FITC 1:200 (all from Chemicon, Temecula, CA, http://www.chemicon.com), rat anti-prominin1 monoclonal antibody 13A4 1:300 (BD Biosciences, San Diego, http://www.bdbiosciences.com), and mSSEA1 1:5 (kindly provided by Dr. Beltinger, University of Ulm) followed by fluorescence-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Propidium iodide (PI)–fluorescence-activated cell sorting (FACS) was performed as previously described [32]. In brief, the cells were dissociated by trypsin (Gibco) and washed with PBS. The cells were fixed with 80% ice-cold ethanol and incubated at –20°C for 30 minutes. The cells were washed with PBS and PI-stained (0.5 mg/ml PI in 0.038 M sodium citrate buffer [pH 7.0]) for 30 minutes in the dark (37°C). Dead cells were excluded from analysis by forward-scatter gating. A minimum of 10,000–12,000 events were acquired for each sample.
Immunocytochemistry
Cell cultures were fixed in 4% paraformaldehyde in PBS or with 4% paraformaldehyde/PBS followed by ice-cold acidic ethanol and HCl for BrdU staining. Immunocytochemistry was carried out using standard protocols. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: mouse anti-Tuj1 1:500 or rabbit anti-Tuj1 1:2,000 (Covance, Richmond, CA, http://www.covance.com), sheep or rabbit anti–tyrosine hydroxylase (TH) 1:200 (Pel-Freez, Rogers, AR, http://www.pel-freez.com), rabbit anti-serotonin (5-HT) 1:2,500 (DiaSorin, Stillwater, MN, http://www.diasorin.com), rabbit anti-cholineacetyltransferase 1:500, mouse anti-galactocerebrosidase C (GalC) 1:750, rabbit anti–glial fibrillary acidic protein (GFAP) polyclonal 1:1,000, mouse anti-Nestin monoclonal 1:500, anti-CD-24-FITC 1:200, rabbit anti-neurogenin2 1:500, rabbit anti-neuroD1 1:500, rabbit anti-Musashi1 1:500 (all from Chemicon), rabbit anti-Ki-67 1:500 (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), rabbit anti–-aminobutyric acid (GABA) 1:1,000, rabbit anti-glutamate 1:10,000 (both from Sigma), mouse anti-GFAP monoclonal 1:1,000, mouse anti–monoclonal antimicrotubule-associated protein 2ab (MAP2ab) 1:300 (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), rat anti–prominin1-FITC monoclonal antibody 13A4 1:800 (BD Biosciences), rat anti–PSA-NCAM (polysialylated neural cell adhesion molecule) 1:250 (BD Pharmingen), rabbit anti-olig2 1:1,000 (kindly provided by Dr. Takebayashi; [33]) and fluorescence-labeled secondary antibodies (Jackson ImmunoResearch Laboratories), and rat anti-BrdU 1:40 with fluorescence-labeled secondary antibody (both from Abcam, Cambridge, U.K., http://www.abcam.com). Images were captured using a fluorescence microscope (Axiovert 135; Zeiss, Oberkochen, Germany, http://www.zeiss.com) or a Leica TCS/NT confocal microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com) equipped with krypton, krypton/argon, and helium lasers.
RNA Extraction and Quantitative Real-Time RT-PCR Analysis
Total cellular RNA was extracted from tNSCs and 14-day differentiated tNSCs (differentiation protocol 1) using RNAeasy total RNA purification kit followed by treatment with RNase-free DNase (Qiagen, Hilden, Germany, http://www.qiagen.com). Quantitative real-time one-step reverse transcription–polymerase chain reaction (RT-PCR) was carried out using the LightCycler System (Roche, Mannheim, Germany, http://www.roche.com), and amplification was monitored and analyzed by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. One microliter (50 ng) of total RNA was reverse-transcribed and subsequently amplified using Quanti-Tect SYBR Green RT-PCR Master mix (Qiagen) and 0.5 µmol l–1 of both sense and antisense primers. Tenfold dilutions of total RNA were used as external standards. Standards and samples were simultaneously amplified. After amplification, melting curves of the RT-PCR products were acquired to demonstrate product specificity. The results are expressed relative to the housekeeping gene HMBS (hydroxymethylbilane synthase). Primer sequences, lengths of the amplified products, and melting point analyses are summarized in Table 2.
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Electrophysiology
Cells were investigated 7–20 days after differentiation using standard whole-cell patch-clamp technique. Data were recorded using an EPC-7 or EPC-9 amplifier (HEKA Electronics Inc., Lambrecht/Pfalz, Germany, http://www.heka.com) and pClamp data acquisition software (Axon Instruments, Union City, CA, http://www.moleculardevices.com) essentially as described previously [38, 39]. Extracellular solution contained (in mmol l–1) 142 NaCl, 8.1 KCl, 1 CaCl2, 6 MgCl2, 10 HEPES, and 10 D-glucose (pH 7.4; 320 mOsm). Pipette solution contained (in mmol l–1) 153 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.3; 305 mOsm). Using these solutions, borosilicate pipettes had resistances of 6–10 M
. Seal resistances in the whole-cell mode were between 0.1 and 1 G. For recordings of GABA-induced inward currents, the solutions were balanced giving an equilibrium potential of 0 mV for chloride ions. For recordings of glutamate-induced inward currents, the external bath solution contained (in mmol–1) 162 NaCl, 1.2 CaCl2, 2.4 KCl, 0.01 glycine, 11 glucose, 10 HEPES (pH 7.3; 320 mOsm). Picrotoxin (100 µM; Sigma) and strychnine (2 µM; Sigma) were added to inhibit ligand-gated chloride channels. The internal solution contained (in mmol–1) 95 CsCl, 6 MgCl2, 1 CaCl2, 10 HEPES, and 11 EGTA (pH 7.2; 300 mOsm). Drugs were applied rapidly via gravity with an SF-77B perfusion fast-step system (Warner Instruments LLC, Hamden, CT, http://www.warnerinstruments.com). Data were analyzed using pClamp 8.0, Microsoft Excel 97 (Microsoft, Redmond, WA, http://www.microsoft.com), and Origin 5.0 software. Resting membrane potentials (RMP) were determined immediately after gaining whole-cell access. Action potentials (APs) were elicited by applying increasing depolarizing current pulses (10-pA current steps). The afterhyperpolarization (AHP) amplitude was measured from peak to beginning of plateau reached during the current injection, AP duration was measured at half amplitude, and time to peak AHP from spike onset.
Determination of Dopamine, GABA, and Serotonin Production and Release
For determination of dopamine production and release, media were supplemented with 100 µmol l–1 tetrahydrobiopterin and 200 µmol l–1 ascorbate 2 days prior to medium harvest. Dopamine levels were determined in medium, and extracellular buffer was stabilized with EGTA/glutathione solution as reported previously [29] and stored at –80°C until analysis. Dopamine was assayed by reverse-phase high-performance liquid chromatography (HPLC) with an electrochemical detector as previously described [40]. GABA and serotonin were assayed by HPLC with fluorescence detection [40], employing pre-column derivatisation with ortho-phthaldialdehyde and an automatic HPLC system (Kontron Instruments, Neufahrn, Germany). The excitation and emission wave lengths of the fluorescence detector were set at 330 and 450 nm, respectively.
Cell Counting and Statistics
For quantification of the percentage of cells producing a given marker, in any given experiment the number of positive cells of the whole well surface was determined relative to the total number of DAPI-labeled nuclei. In a typical experiment, a total of 500–1,000 cells were counted per marker. Statistical comparisons were made by Dunnett’s t test. If data were not normally distributed, a non-parametric test (Mann-Whitney U test) was used for comparisons of results. All data are expressed as mean ± SEM.
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RESULTS |
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). FACS analysis revealed that the phenotype of the tNSCs was CD24low/–, CD34–, CD45–, Prominin1–, and SSEA1– (Fig. 1B). Furthermore, we used quantitative real-time RT-PCR and immunocytochemistry on unfixed or fixed neurospheres to investigate the expression pattern of several early and late neuroectodermal genes (Fig. 2A, 2C; for complete names of genes and encoded proteins, refer to Table 2
). tNSCs expressed high levels of early neuroectodermal markers such as Nestin, an intermediate filament protein present in CNS stem cells (Fig. 1A, 1C) [21, 41], and the CNS stem cell marker Musashi1 [42], as well as the proneural genes Olig2, Neurogenin2, and NeuroD1 on both the mRNA and protein levels (Fig. 1A, 1C). Proneural genes, which encode transcription factors of the basic helix-loop-helix class, are important factors for NSC self-renewal, multipotency, and commitment to neural lineages of NSCs [43–48]. Confocal microscopy of the spheres revealed that nearly all (>95%) living cells without nuclear condensation (DAPI stain) expressed Nestin (Fig. 1A) with some cells (<5%) being double-positive for Nestin and GFAP (data not shown). Co-labeling with anti-olig2 antibody further demonstrated co-expression of both early neuroectodermal markers in nearly all living cells (Fig. 1A). Furthermore, the proneural genes Neurogenin2 and NeuroD1, as well as the NSC marker Musashi1, were expressed in nearly all cells within the neurospheres as shown by confocal microscopy (Fig. 1A). The neurogenic fate-determining transcription factor Pax6 mRNA was also expressed in these neurospheres (Fig. 1C). Only very few cells were weakly positive for CD24 or PSA-NCAM protein (data not shown). Markers for mature neural cell types, such as MBP, PLP, NTRK1, MAP2, and TH, were undetectable or very low at the mRNA level; at the protein level, MAP2ab, GalC, and TH were absent (data not shown). This phenotype is similar to that of NSCs derived from adult mouse SVZ or hippocampus [20, 22, 23]. tNSCs could be passaged and cultured as secondary and tertiary neurospheres without changing morphology and phenotype for 3–12 weeks (~5–10 passages).
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for details of cell cycle distribution). In addition, we used the proliferation marker Ki67 as well as BrdU incorporation to identify DNA-synthesizing cells (Figs. 1A and 2B). Ki67 was detected in the nucleus of proliferating cells in all active phases of the cell cycle from the late G1-phase through the M-phase but is absent in non-proliferating and early G1-phase cells and in cells undergoing DNA repair [49–51]. As expected, total Ki67 staining (representing late G1 through M phase) was higher than total BrdU staining at each time point. When the percentage of G1-phase cells was subtracted from total Ki67 staining, the resulting percentage was very similar to that of BrdU-stained cells (Fig. 2B).
To evaluate cell death by apoptosis during cultivation of tNSCs, we used PI-FACS analysis and DAPI staining. After 4–12 weeks, an average number of 4% ± 1% of cells were found by PI-FACS analyses to be apoptotic. Histograms representing FACS analysis and DAPI staining of apoptotic events in tNSCs are shown in Figure 2B. Spontaneously apoptotic cells exhibited chromatin condensation and margination, sometimes followed by appearance of apoptotic bodies, as revealed by DAPI staining, and were negative for Ki67 (data not shown) as well as for most other markers tested such as Nestin and Olig2 (Fig. 1A).
Telomerase is inactive in most somatic cells but present in various stem cell populations [52–54]. Quantification of telomerase activity in tNSCs using the telomeric repeat amplification protocol [34, 35] showed stable telomerase activity in tNSCs over the 3-month expansion period with significantly higher activity compared to that measured in mouse cortical tissue (negative control) but lower activity compared to that in mouse ESCs (Fig. 2C). Together, investigations of the proliferation characteristics revealed the typical pattern of slowly dividing cells with a small amount of spontaneous apoptosis within the tNSC neurospheres, similar to fetal mouse mesencephalic NSCs [55].
Differentiation Capacity of Adult tNSCs
Differentiation of tNSCs was initiated after 3–12 weeks by removal of mitogens, plating the cells onto poly-L-lysine, and addition of BDNF, db-cAMP, and retinoic acid (protocol 1, Fig. 1A). After 14 days, the majority of cells (71% ± 27%) acquired morphologic and phenotypic characteristics of astrocytes (GFAP+), 6% ± 5% acquired those of oligodendrocytes (GalC+), and 5% ± 2% those of mature neurons (MAP2ab+; n = 8; Fig. 3B). GFAP, GalC, and Tuj1/MAP2ab were never found in the same cell (Figs. 1D and 4A). Consistently, quantitative RT-PCR of tNSCs after differentiation using protocol 1 for 14 days confirmed the differentiation of tNSCs into mature neuroectodermal cells (Fig. 3E). The NSC marker gene NES (encoding Nestin) was downregulated sixfold, whereas mRNA levels of NTRK1, GFAP, MBP, PLP, and NF increased six- to 430-fold during the differentiation process (Fig. 3E). Most mRNA levels of markers for mature neural cell types in differentiated tNSCs were lower compared with those in adult mouse cortex (Fig. 3E). No dopaminergic or serotoninergic cells could be detected using protocol 1. Replacement of BDNF/db-cAMP/retinoic acid by various combinations of growth factors/cytokines known to induce the dopaminergic phenotype in several murine stem cell types [38] led to changes in the differentiation capacity into astroglial, oligodendroglial, and neuronal cells, but no TH+ neurons were observed (Table 1). For analyzing the differentiation capacity of acutely isolated tNSCs before neurosphere formation (7 days after preparation), we used the BrdU incorporation assay. The tNSCs were incubated during the expansion phase with BrdU (10 µM) for 30 hours. Then, differentiation was initiated using protocol 1. We were able to detect BrdU+/Tuj1+ neurons and BrdU+/GFAP+ glial cells (Fig. 3C), demonstrating that these neurons and glial cells were de novo–generated from proliferating progenitor cells.
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). In fact, protocol 4 is similar to the protocol for dopaminergic differentiation of mouse ESCs reported by Kawasaki and coworkers [30]. There were no relevant differences in the differentiation capacity of tNSCs into astroglia and neurons with respect to the various differentiation conditions (Table 1). In contrast, only protocol 4 was able to induce dopaminergic neurogenesis in a small percentage of cells (2.4% ± 1.4% of Tuj1+ cells; n = 3), showing typical morphological features of Tuj1+/TH+ dopaminergic neurons with small irregularly shaped soma and bipolar or multipolar branching processes with varicosities (Fig. 4A, 4B). Using quantitative RT-PCR, we consistently demonstrated expression of various dopaminergic marker genes, including NR4A2 (encoding the nuclear receptor Nurr1), TH (TH), DDC (dopa-decarboxylase), and DAT (dopamine transporter), in tNSCs differentiated using protocol 4 (Fig. 4C). No dopaminergic neurogenesis could be observed in cultures without PA6 cells (including protocols using PA6 conditioned media), and there were no differences in the amounts of glutamatergic, GABAergic, cholinergic, and serotoninergic neurons in cultures with PA6 cells compared with PA6 cell–free cultures (Fig. 4B; Table 1). Remarkably, the morphology of the different nerve cell subtypes was significantly different with large soma and multiple neurites in cholinergic cells and relatively small soma with two to three neurites in GABAergic and glutamatergic neurons as described for primary cultures (Fig. 4A). In contrast, protocol 4 also induced glutamatergic, GABAergic, and cholinergic neurons out of corresponding adult mouse hippocampal NSCs but was unable to produce TH+ dopaminergic cells out of hippocampal NSCs (data not shown).
Clonal Analysis of Adult tNSCs
To determine whether individual tNSCs could generate both neurons and glia, neurospheres were dissociated and individual cells were isolated by the single-cell culture technique in ultra low–attachment multiwell-plates. Single cells were cultured in expansion medium for 3–6 weeks. One third of the single cells proliferated to generate secondary neurospheres (Fig. 3D). Clonal cells were then differentiated for 14 days using differentiation protocol 1. Double immunostaining revealed that each of these clonally derived neurospheres differentiated into neurons and astrocytes (Fig. 3D). In addition to the BrdU incorporation experiments (Fig. 3C), these results demonstrate that neurons and glia are de novo–generated from proliferating tNSCs.
Functional Properties of Differentiated Adult tNSCs
To analyze the functional properties of differentiated tNSCs, we performed whole-cell voltage-clamp recordings to measure voltage-gated sodium and potassium currents and current-clamp recordings to measure the capacity to generate action potentials after differentiation for 10–20 days using protocol 1. The RMP of differentiated tNSCs was –46 ± 4 mV (n = 34), the input resistance was 536 ± 45 M (n = 48), and the cell membrane capacity was 14.7 ± 1.3 pF (n = 48). Of differentiated tNSCs, 83 out of 95 recorded cells (87.4%) expressed a sustained outward current of a few hundred pA up to 5 nA (Fig. 5A). These currents showed a voltage dependence and kinetics characteristic for A-type currents as well as tetraethylammonium-sensitive delayed rectifier potassium channels (Fig. 5A, 5B). In 67 out of 95 cells (70.5%), we could identify inward currents of up to 2 nA with voltage dependence and kinetics typical for voltage-activated sodium channels (Fig. 5C, 5D). These currents were tetrodotoxin (TTX)-sensitive (Fig. 5C). Current-clamp recordings revealed that differentiated tNSCs generated TTX-sensitive APs with a duration of 9.6 ± 0.2 ms (n = 8) and a short AHP (Fig. 5E). The length of AHPs was 22.7 ± 0.8 ms, and the maximal amplitude 9.8 ± 0.4 mV (n = 8).
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). These currents could be antagonized by co-application of bicuculline and enhanced by co-application of diazepam (Fig. 6C). The characteristics of GABA-induced currents in our cells are typical for GABAA receptors. Furthermore, glutamate induced fast inward currents in a dose-dependent manner with an EC50 value of 8 µM (Fig. 6D, 6E). Nine out of 10 cells expressing glutamate-induced currents also showed functional NMDA receptors with amplitudes of approximately 27% of the mean glutamate amplitude (Fig. 6F). Because there is no reliable evidence for functional glutamate receptors of the NMDA subtype in glial cells [56], these data further demonstrate functional properties of mature nerve cells.
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), but only small amounts of dopamine were found in extracellular buffer or after stimulation of the cells with 56 mM KCl for 45 minutes. (Fig. 7B), consistent with the amounts of TH+ cells and the expression of various dopaminergic marker genes, including the key enzymes of dopamine synthesis (TH and dopa-decarboxylase; Fig. 4). Under the same conditions, we detected GABA production and potassium-dependent release of GABA by differentiated tNSCs (Fig. 7B). There was no dopamine production by undifferentiated tNSCs or tNSCs differentiated using protocol 1. Consistent with the immunocytochemical data, we did not find the neurotransmitter serotonin in any sample.
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DISCUSSION |
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–7). Their potential to proliferate was slower compared with embryonic/fetal NSCs, whereas their differentiation capacity was similar to that of NSCs derived from fetal rodent or human mesencephalon [29, 38, 55, 57, 58, 60].
NSCs are often defined in vitro by the presence of the CNS stem cells marker Nestin [41, 61, 62], a class IV intermediate filament protein [61, 63, 64] and a marker for precursor cells in the adult brain [24, 61, 65–67]. Nestin is highly expressed in tNSCs during in vitro expansion. Furthermore, the RNA-binding protein Musashi1 is involved in post-transcriptional gene regulation in neuroprogenitors and/or NSCs and is used as a marker of those cell types [42]. Consistently, Musashi1 is expressed in tNSCs both at the mRNA and protein levels (Fig. 1). A large body of knowledge exists on lineage determination in NSCs, because unlike many types of stem cells, NSCs undergo self-renewal and multilineage differentiation in culture. Proneural genes, encoding transcription factors of the basic helix-loop-helix class, such as NeuroD1, Neurogenin2, and Olig2, are both necessary and sufficient to initiate the development of neural lineages and to promote the generation of progenitors that are committed to neural differentiation [43–45]. Proneural genes are therefore expressed mostly in neuroprogenitor cells, including NSCs in early neuroectodermal tissues [23, 46–48]. Consistently, tNSCs expressed mRNA and protein of NeuroD1, Neurogenin2, as well as Olig2 (Fig. 1). For example, NeuroD1 and Neurogenin2 are considered neuronal differentiation genes and promote exit from the cell cycle and inhibit gliogenesis [68–70]. In addition, Olig2 is a pivotal factor for neurogenesis and oligodendroglial specification of adult NSCs in vitro [23, 71]. Although Olig2 showed no co-localization with adult CNS stem cell markers in vivo, it becomes highly upregulated under expansion conditions with high levels of EGF and FGF-2 (Fig. 1 and [23]). Olig2 is required for the proliferation and self-renewal of neurosphere cells, as shown by interference analysis [23]. The transcription factor Pax6 is the major neurogenic determinant for radial glial cells and adult NSCs [23, 72–74]. Indeed, neurogenic differentiation in neurospheres fully depends on the expression of the transcription factor Pax6, independent of the region of origin [23]. Analysis of Pax6 expression in tNSCs demonstrated Pax6 expression during the expansion phase, further confirming their neurogenic potential (Fig. 2C). Together, tNSCs expressed a typical pattern of early neuroectodermal and/or NSC marker genes during the expansion phase in vitro, including major factors for proliferation and self-renewal as well as neurogenic differentiation, such as proneural genes and Pax6.
Further evidence for the stem cell–like nature of tNSCs within the neurospheres is the demonstration of clonogenicity of individual cells in the single-cell culture assay showing the potential of clonally derived neurospheres to differentiate into both neurons and astrocytes. In addition, these results demonstrate that neurons and glia are de novo–generated from proliferating cells (Fig. 3C, 3D). Finally, showing proliferation by PI-FACS analysis, BrdU labeling and Ki67 immunostaining as well as the expression of telomerase [54], these tNSCs fulfill the major characteristics of multipotent NSCs. After removal of mitogens, plating the cells onto PLL, and incubation with BDNF, db-cAMP, and retinoic acid for 14 days, tNSCs differentiate into all major cell types of the CNS, namely mature (MAP2ab+) neurons, oligodendrocytes, and astrocytes with an approximate ratio of 1:1.5:14, respectively. Replacement of the mentioned growth factors with various combinations of growth factors and/or cytokines did not lead to major changes of this differentiation capacity (Table 1). In the presence of selected growth factors (see Materials and Methods for details), tNSCs differentiated into cells expressing the markers for cholinergic, GABAergic, or glutamatergic neurons with similar amounts of the distinct neuronal subpopulations with respect to the various differentiation protocols. Thus, differentiation of tNSCs into these neuronal subtypes did not depend on pre-stimulation with FGF-8 and Shh or co-culture with feeder cells such as MEFs, astrocytes, or PA6 cells (Fig. 4B). Interestingly, no serotoninergic cells could be observed with any differentiation protocol tested.
We further demonstrated that a high percentage of differentiated adult tNSCs acquired functional properties of nerve cells, such as generation of TTX-sensitive sodium channels and action potentials similar to differentiated fetal mouse mesencephalic NSCs [38] or adult NSCs derived from mouse SVZ [75]. A subset of these cells expressed currents with the characteristics of functional GABAA receptors as well as ionotropic glutamate receptors, including the NMDA-subtype known to be expressed exclusively by neuronal cells [56]. Together, this in vitro differentiation capacity of tNSCs is similar to that of NSCs derived from fetal brain as well as the principal neurogenic regions (hippocampus and SVZ of the lateral ventricles) of the adult brain of various species [21, 31, 41, 76–78]. Zhao and colleagues demonstrated neurogenesis in the adult substantia nigra of C57BL/6 mice in vivo [7]. Two other studies did not find newly born neurons in the adult substantia nigra of normal or lesioned rodents [13, 16]. Moreover, Lie and colleagues found the production of glial cells, but not of neuronal cells, from adult substantia nigra progenitor cells in vivo [16]. In contrast, the in vitro differentiation capacity of these substantia nigra NSCs seems to include neuronal cell lineages [16]. However, this study reported only differentiation into immature neurons (NeuN+ or Tuj1+ cells) and did not show any functionality or neuron subtype specification [16].
The analysis of neuronal subtype differentiation of tNSCs revealed that without pre-stimulating the cells with FGF-8 and Shh and subsequent co-culture with PA6 stromal cells, no TH+ dopaminergic neurons could be achieved. However, after a multistep differentiation protocol using pre-stimulation and subsequent co-culture of tNSCs with PA6 cells, a small subset of cells not only acquired typical morphological features of dopaminergic cells but also produced the neurotransmitter dopamine, demonstrating the differentiation of tNSCs into functional dopaminergic neurons (Figs. 4, 7). No dopaminergic neurogenesis was detected using other cell types for feeder layer cultures, such as cortical or striatal astrocytes known to induce neurogenesis in adult NSCs [78] or MEFs, normally used for proliferation of ESCs. These data suggest a crucial role of PA6 cells (or the so-called stromal cell–derived inducing activity [30]) on dopaminergic neurogenesis not only for ESCs but also for adult tNSCs. However, in contrast to ESCs, tNSCs seem to need an additional priming step to become dopaminergic, such as the pre-incubation procedure with FGF-8 and Shh used in the present study.
Zhao and colleagues demonstrated dopaminergic neurogenesis in the adult substantia nigra of C57BL/6 mice in vivo [7]. A subsequent study was not able to reproduce these results or demonstrate newly generated dopaminergic neurons by the BrdU incorporation assay in normal or 6-hydroxydopamine–lesioned animals [13]. In contrast to the latter results established in vivo, we were able to show dopaminergic specification of isolated adult tNSCs in vitro. This fact is in contrast to previous results on adult NSCs not reporting dopaminergic specification of adult NSCs in vitro independent of the region of origin of the NSCs [7, 13, 16, 78]. The reasons for these discrepancies are likely the following: (a) The in vivo differentiation capacity may not reflect the entire lineage potential of resident progenitor cells, because regional environmental factors appear to restrict in situ differentiation to distinct neural lineages [16–18]. We established a multistep differentiation protocol including co-culturing the tNSCs with PA6 stromal cells known to have potent effects on dopaminergic differentiation of ESCs in vitro [30] to provide an environment sufficient for dopaminergic specification in vitro of isolated and long-term expanded adult tNSCs. However, the amounts of dopaminergic neurons generated from adult tNSCs are very low (
2.5% of all neurons or 0.25% of all cells, which is five- to 10-fold less than differentiated fetal mesencephalic NSCs derived from rodents or humans) [29, 38, 79]. The knowledge of the specific factors produced by PA6 stromal cells, which induce dopaminergic specification, will help to further optimize the efficiency of dopaminergic specification of adult NSCs. High yields of dopaminergic cells would be essential for characterization of the intracellular mechanisms of adult dopaminergic neurogenesis as well as the NSC-derived dopaminergic neurons. (b) We used, for the first time, NSCs from the whole adult tegmentum and not just from the substantia nigra or the principal neurogenic regions of the adult brain [7, 16, 78]. Future studies are warranted to further define the exact region within the adult tegmentum from which "pre-dopaminergic" NSCs can be generated.
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) and presence of 30 mM TEA (
). Data are mean values ± SEM from three to 11 independent experiments. (C–D): Fast inward currents could be completely blocked by application of TTX (1 µM) and displayed the characteristic I–V relationship of voltage-gated sodium channels (n = 7). (E): Current-clamp recordings of the same cell recorded in (C) in response to depolarizing current pulses of increasing amplitude. The resting membrane potential was approximately –65 mV, and TTX-sensitive action potentials peaked at approximately +40 mV. Abbreviations: TEA, tetraethylammonium; tNSC, tegmental neural stem cell; TTX, tetrodotoxin.
