Multipotent Neural Stem Cells from the Adult Tegmentum with Dopaminergic Potenti - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:08 |只看该作者 |倒序浏览 |打印

作者：Andreas Hermanna,b,c, Martina Maisela,c, Florian Wegnerd, Stefan Liebaua,e, Dong-Wook Kimb, Manfred Gerlachf, Johannes Schwarzd,g, Kwang-Soo Kimb, Alexander Storchc作者单位：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

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      【摘要】
   Neurogenesis in the adult brain occurs within the two principal neurogenic regions: the hippocampus and the subventricular zone of the lateral ventricles. The occurrence of adult neurogenesis in non-neurogenic regions, including the midbrain, remains controversial, but isolation of neural stem cells (NSCs) from several parts of the adult brain, including the substantia nigra, has been reported. Nevertheless, it is unclear whether adult NSCs do have the capacity to produce functional dopaminergic neurons, the cell type lost in Parkinson¡¯s disease. Here, we describe the isolation, expansion, and in vitro characterization of adult mouse tegmental NSCs (tNSCs) and their differentiation into functional nerve cells, including dopaminergic neurons. These tNSCs showed neurosphere formation and expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, and Olig2, the NSC markers Nestin and Musashi1, and the proliferation markers Ki67 and BrdU (5-bromo-2-deoxyuridine). The cells showed typical propidium iodide¨Cfluorescence-activated cell sorting analysis of slowly dividing cells. In the presence of selected growth factors, tNSCs differentiated into astroglia, oligodendroglia, and neurons expressing markers for cholinergic, GABAergic, and glutamatergic cells. Electrophysiological analyses revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels, action potentials, as well as currents induced by GABA (-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. 0 i* h' S* }/ l- F) u. {+ c5 E' f
      【关键词】 Adult neurogenesis Neural stem cells Dopaminergic differentiation Parkinsons disease Electrophysiology Neuroregeneration0 @5 [" x* _8 v7 {5 u- ]
   INTRODUCTION- c2 X" G, j5 U1 d7 i. o. Q! e

Neurogenesis in the adult mammalian brain is a generally accepted phenomenon in two discrete neurogenic regions: the hippocampus and the subventricular zone (SVZ) of the lateral ventricles .
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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 .
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Here, we show the presence of multipotent clonogenic NSCs in the adult mouse tegmentum (tegmental NSCs ) 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.

MATERIALS AND METHODS
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Sphere Culture

Adult mice (6¨C10-week-old male C57BL/6 mice) were killed by cervical dislocation, and their brains were removed and placed into ice-cold Hanks¡¯ balanced saline solution supplemented with 1% penicillin/streptomycin and 1% glucose (all from Gibco, Tulsa, OK, http://www.invitrogen.com). The tegmental tissue (midbrain and hindbrain), including the subependymal zone/SVZ of the aqueduct and fourth ventricle, was then aseptically prepared using a dissection microscope. The meninges were carefully removed from the tissue after preparation of the tegmental area. For comparative studies with NSCs from adult¡¯s principal neurogenic regions, we isolated the hippocampus from the same mice as described previously . The cells were added to 25-cm2 flasks (2¨C3 x 106 viable cells per flask) in serum-free Neurobasal (NBa) medium containing 1% glutamate, 2% B27 supplement, and 1% penicillin/streptomycin (all from Gibco) supplemented with 20 ng ml¨C1 of both mitogens FGF-2 and EGF (both from Sigma). After 10¨C20 days, sphere formation was observed. These neurospheres were expanded for an additional 3¨C12 weeks (in total, five to 10 passages) before terminal differentiation was initiated. The medium was changed once a week, and growth factors were added twice a week. For BrdU labeling, cells were incubated for 30 hours with 10 µM BrdU.
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Differentiation Conditions3 f: y2 V# s' k! R) [+ n
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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¨C2.0 x 105 cells cm¨C2 in NBa medium containing 1% glutamate, 2% B27 supplement, and 1% penicillin/streptomycin supplemented with 0.5 µmol l¨C1 all-trans-retinoic acid (Sigma), 10 ng ml¨C1 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¨Cderived 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 ) 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).2 n1 R! X( b0 }  f: c
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Table 1. Differentiation capacity of adult tegmental neural stem cells( m  }3 q7 W: c% R
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Clonal Analysis. h1 s8 l- U  s9 S! n' K5 _, G) R7 r
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Clonal analysis was performed according to Uchida and coworkers . In brief, tNSCs were serially diluted into expansion medium in 96-multiwell plates. On the next day, single cell¨Ccontaining wells (assessed microscopically) were expanded by supplementing the medium to 50% with conditioned medium containing growth factors. Cells were expanded for 3¨C6 weeks and then differentiated using feeder cell¨Cfree differentiation protocol 1.. x( u; ^: {2 P1 b  x7 l

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 ¨Cfluorescein 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)¨Cfluorescence-activated cell sorting (FACS) was performed as previously described ) for 30 minutes in the dark (37¡ãC). Dead cells were excluded from analysis by forward-scatter gating. A minimum of 10,000¨C12,000 events were acquired for each sample.; A8 i4 Z0 R+ P% q+ }. Z

Immunocytochemistry) _& G( V  o7 h  K& I4 j+ d

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¨Ctyrosine 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¨Cglial 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¨C-aminobutyric acid (GABA) 1:1,000, rabbit anti-glutamate 1:10,000 (both from Sigma), mouse anti-GFAP monoclonal 1:1,000, mouse anti¨Cmonoclonal antimicrotubule-associated protein 2ab (MAP2ab) 1:300 (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), rat anti¨Cprominin1-FITC monoclonal antibody 13A4 1:800 (BD Biosciences), rat anti¨CPSA-NCAM (polysialylated neural cell adhesion molecule) 1:250 (BD Pharmingen), rabbit anti-olig2 1:1,000 (kindly provided by Dr. Takebayashi; ) 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.
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RNA Extraction and Quantitative Real-Time RT-PCR Analysis/ i9 e  H: q- x6 C% D' T
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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¨Cpolymerase 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¨C1 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.

Table 2. Primers for quantitative real-time reverse transcription¨Cpolymerase chain reaction
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Telomerase Activity

A highly sensitive in vitro assay known as the quantitative real-time telomeric repeat amplification protocol .

Electrophysiology

Cells were investigated 7¨C20 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 . Extracellular solution contained (in mmol l¨C1) 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¨C1) 153 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.3; 305 mOsm). Using these solutions, borosilicate pipettes had resistances of 6¨C10 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¨C1) 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¨C1) 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
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For determination of dopamine production and release, media were supplemented with 100 µmol l¨C1 tetrahydrobiopterin and 200 µmol l¨C1 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 , 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.1 f$ ^) R) A2 g

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¨C1,000 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 ¡À SEM.
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RESULTS
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Isolation and In Vitro Characterization of Neurosphere-Forming Cells from the Adult Tegmentum

To isolate tNSCs, the tegmentum (midbrain and hindbrain) was selectively dissected from adult C57BL/6 mice using a dissection microscope. Cells were isolated and cultured in uncoated flasks in serum-free NBa medium supplemented with EGF and FGF-2. After 10¨C20 days in vitro, the cells formed small spheres with all typical morphological properties of neurospheres (Fig. 1A). FACS analysis revealed that the phenotype of the tNSCs was CD24low/¨C, CD34¨C, CD45¨C, Prominin1¨C, and SSEA1¨C (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) . tNSCs could be passaged and cultured as secondary and tertiary neurospheres without changing morphology and phenotype for 3¨C12 weeks (~5¨C10 passages).6 Y" P- w* ^# O& Y/ ]/ A+ \

Figure 1. Characteristics of adult tNSCs during in vitro expansion. (A): Morphology and marker expression of adult tNSCs during expansion in the presence of EGF and FGF-2. Spheres were cultured for 2¨C3 hours to allow attachment and then stained for Nestin (b, i), Ki67 (d), Olig2 (e, i), Neurogenin2 (f, j), NeuroD1 (g, k), and/or Musashi1 (h, l). For BrdU staining (c), the spheres were cultured in the presence of 10 µM BrdU for 30 hours before plating. Nuclei were counterstained with DAPI. Confocal images (i¨Cl) confirm the presence of the respective marker protein in nearly all cells within the neurosphere. Arrows mark Ki67¨C/DAPI  cells (d) and apoptotic cells not expressing Nestin/Olig2 (e), respectively. Scale bars = 15 µm (d), 30 µm (e, i, j¨Cl), 50 µm (b, c, f¨Ch), and 200 µm (a). (B): Flow cytometry of tNSCs cultured for 4¨C12 weeks (five to 10 passages). Cells were labeled with fluorescence-coupled antibodies against CD24, CD34, CD45, prominin1, and SSEA1 or immunoglobulin isotype control antibodies. Cells were analyzed using a FACSCalibur flow cytometer. Black line, control immunoglobulin; red line, specific antibody. (C): Quantitative transcription profile of tNSCs. Top panel: Representative real-time RT-PCR analysis using the LightCycler technique. Plot of the fluorescence versus the cycle number obtained from SYBR Green detection of serially diluted NES mRNA (encoding for nestin). The crossing line represents the position of the threshold. Diagram on right shows the standard curve obtained by plotting cycle number of crossing points versus dilution factor. Bottom panel: Quantitative real-time RT-PCR analyses of NSC markers (NES, MSl1), proneural genes (NeuroD1, Neurog2, Olig2), and the neuronal transcription factor Pax6 in tNSCs during expansion. Expression levels are expressed relative to the housekeeping gene HMBS. Data shown are mean values ¡À SEM from at least three independent experiments. For primers, complete names of genes, and melting curve analyses demonstrating the specificity of amplified PCR products, see Table 2. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor; FGF, fibroblast growth factor; HMBS, hydroxymethylbilane synthase; PCR, polymerase chain reaction; RT-PCR, reverse transcription¨Cpolymerase chain reaction; tNSC, tegmental neural stem cell.

Figure 2. Cell cycle distribution, proliferation potential, and apoptosis of adult tNSCs. (A): PI-FACS analysis of tNSCs showing the cell cycle distribution typical for slowly dividing cells. (B): Quantitative data of PI-FACS analyses, Ki67 immunostaining, BrdU staining, and nuclei with chromatin clumping and fragmentations (apoptotic nuclei) in DAPI stainings. (See Fig. 2A for Ki67, BrdU, and DAPI stainings of neurospheres.) Results are the mean ¡À SEM from three to six independent experiments. (C): Telomerase activity in tNSCs after 3 and 12 weeks, respectively, as well as adult cortical tissue and mouse ESCs during expansion measured by the quantitative real-time telomeric repeat amplification protocol. Telomerase activity was normalized to protein content. Results are mean values ¡À SEM from three independent experiments. #p 9 X$ [- _5 W/ H8 t7 T
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Cell Cycle Distribution, Proliferation Potential, and Apoptosis of tNSCs6 X" H! u* N/ A  B% N

Cell cycle analysis using PI-FACS demonstrated that, besides apoptosis (see below), there was a large fraction of tNSCs distributed in G1, S, and G2/M phases (see Fig. 2A, 2B 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 . 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).3 J, d/ }9 z4 w

To evaluate cell death by apoptosis during cultivation of tNSCs, we used PI-FACS analysis and DAPI staining. After 4¨C12 weeks, an average number of 4% ¡À 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).
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Telomerase is inactive in most somatic cells but present in various stem cell populations .( X3 W4 ?1 |6 Z9 T

Differentiation Capacity of Adult tNSCs" Q9 p0 Z2 Y# R! B
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Differentiation of tNSCs was initiated after 3¨C12 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% ¡À 27%) acquired morphologic and phenotypic characteristics of astrocytes (GFAP ), 6% ¡À 5% acquired those of oligodendrocytes (GalC ), and 5% ¡À 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  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¨Cgenerated from proliferating progenitor cells.
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Figure 3. In vitro differentiation capacity of tNSCs. (A): Schematic diagrams of protocols for differentiation of tNSCs (see Materials and Methods for details). (B): tNSCs differentiated using protocol 1 were stained against markers for astrocytes (GFAP), oligodendrocytes (GalC), or neurons (MAP2ab). Nuclei are counterstained with DAPI (blue). Scale bar = 30 µm. Quantification of differentiation capacity of 14-day cultures of tNSCs differentiated using protocol 1. Data shown are mean values ¡À SEM from eight independent experiments. (C): Immunocytochemical analysis of differentiated tNSCs expanded for only 7 days. Prior to differentiation, tNSCs were incubated with 10 µM BrdU for 30 hours and then differentiated using protocol 1 and double-stained against GFAP or Tuj1 (green) and BrdU (red). Arrows indicate double-labeled cells. Scale bar = 15 µm. (D): Clonal analysis of single tNSCs. Left panel: Secondary neurospheres can be derived from a single tNSC from expanded neurospheres. Scale bar = 200 µm. Right panel: Differentiation capacity of clonally derived neurosphere cells. Progeny of single cell¨Cderived neurospheres can be differentiated into neurons (MAP2ab) and astrocytes (GFAP). Nuclei were counterstained with DAPI (blue). Scale bar = 30 µm. (E): Quantitative transcription profile of tNSCs, differentiated tNSCs using protocol 1, and primary mouse cortical tissue. Results of quantitative real-time RT-PCR analyses of the NSC marker gene Nestin (NES), the neural gene NTRK1, glial genes (GFAP, MBP, PLP), and the neuronal gene NF are displayed. Expression levels are expressed relative to the housekeeping gene HMBS. For primers, complete names of genes, and melting curve analyses demonstrating the specificity of amplified PCR products as listed in Table 2. Results are mean values ¡À SEM from at least three independent experiments. #p
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Figure 4. Neuronal subtype differentiation capacity of tNSCs in vitro established by using differentiation protocols 2¨C4 after an expansion phase of 3¨C12 weeks. (A): Triple immunostaining of tNSCs differentiated using protocol 4 for markers of astroglia (GFAP), neurons (Tuj1), and oligodendroglia (GalC), as well as cholinergic (ChAT), GABAergic (GABA), glutamatergic (glutamate), and dopaminergic (TH) neurons. Nuclei were counterstained with DAPI (blue). Arrows mark double-stained neurons. The inset shows high-magnification confocal image of a Tuj1 /TH  neuron. Scale bars = 30 µm (left panel) or 15 µm. (B): Quantitative data of triple immunostainings of tNSCs differentiated with protocols 2¨C4, respectively. Results are mean values ¡À SEM from at least three independent experiments. (C): Quantitative real-time RT-PCR analyses of the dopaminergic marker genes nuclear receptor Nurr1 (NR4A2), TH (TH), dopa-decarboxylase (DDC), and dopamine transporter (DAT) on tNSCs differentiated using protocol 4 (closed bars) and negative control (open bars). Expression levels are expressed relative to the housekeeping gene HMBS. For primers, complete names of genes, and melting curve analyses, see Table 2. Results are mean values ¡À SEM from at least three independent experiments. ##p
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Next, we tested various multistep protocols with pre-stimulation using FGF-8 and/or Shh for 48 hours in expansion medium before plating the cells on PLL or feeder layers of several cell types, including astroglia, MEFs, and PA6 stromal cells (protocols 2¨C4; Fig. 3A). In fact, protocol 4 is similar to the protocol for dopaminergic differentiation of mouse ESCs reported by Kawasaki and coworkers . 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% ¡À 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¨Cfree 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).
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Clonal Analysis of Adult tNSCs
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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¨Cattachment multiwell-plates. Single cells were cultured in expansion medium for 3¨C6 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¨Cgenerated from proliferating tNSCs.3 U% V- v, h/ p" b- E, U8 N" N
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Functional Properties of Differentiated Adult tNSCs
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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¨C20 days using protocol 1. The RMP of differentiated tNSCs was ¨C46 ¡À 4 mV (n = 34), the input resistance was 536 ¡À 45 M (n = 48), and the cell membrane capacity was 14.7 ¡À 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 ¡À 0.2 ms (n = 8) and a short AHP (Fig. 5E). The length of AHPs was 22.7 ¡À 0.8 ms, and the maximal amplitude 9.8 ¡À 0.4 mV (n = 8).9 `/ ~) V' |/ f- X& V8 ?
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Figure 5. Electrophysiological properties of adult tegmental NSCs differentiated using protocol 1 for 10 days. For voltage-clamp measurements (A¨CD), cells were held at ¨C70 mV and depolarized to  50 mV with increasing amplitudes in steps of 10 mV. (A): Representative traces from a differentiated tNSC displaying inward and sustained outward currents. Potassium outward currents were markedly reduced by application of TEA (30 mM). (B): Current-voltage (I¨CV) relationships of the outward currents in steady state at the end of the voltage steps in the absence () and presence of 30 mM TEA (). Data are mean values ¡À SEM from three to 11 independent experiments. (C¨CD): Fast inward currents could be completely blocked by application of TTX (1 µM) and displayed the characteristic I¨CV 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 ¨C65 mV, and TTX-sensitive action potentials peaked at approximately  40 mV. Abbreviations: TEA, tetraethylammonium; tNSC, tegmental neural stem cell; TTX, tetrodotoxin.
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To further characterize the functional properties of differentiated tNSCs, we measured the response of tNSCs differentiated for 7 days using protocol 1 to various concentrations of GABA (10¨C1,000 µM), glutamate (1¨C300 µM), or N-methyl-D-aspartate (NMDA; 100 µM). GABA elicited inward currents in a dose-dependent manner with a half-maximal effective concentration (EC50 value) of 42 µM (Fig. 6A, 6B). 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 , these data further demonstrate functional properties of mature nerve cells.' N$ z5 R8 b1 I+ z3 Q0 u4 v0 D# l
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Figure 6. Electrophysiological properties of ligand-gated ion channels in adult tegmental neural stem cell (tNSCs) after in vitro differentiation for 7 days using differentiation protocol 1. The holding potential was ¨C70 mV in whole-cell voltage-clamp mode, and substances were applied with a rapid perfusion system every 30 seconds. (A): Representative recordings of GABA-induced inward currents after applications of increasing concentrations of GABA (10¨C1,000 µM). (B): GABA dose-response curve with an half-maximal effective concentration (EC50) value = 41.5 ¡À 1.1 µM. Data are mean values ¡À SEM from five independent experiments. (C): GABA-induced currents could be antagonized by co-application of bicuculline and enhanced by co-application of diazepam. (D): Representative recordings of glutamate-induced inward currents after applications of increasing concentrations of glutamate (1¨C300 µM). (E): Glutamate dose-response curve with an EC50 value = 8.2 ¡À 1.8 µM. Data are mean values ¡À SEM from eight independent experiments. (F): NMDA-induced currents could be elicited in nine out of 10 cells expressing glutamate-induced currents (mean NMDA amplitude was 27% of mean glutamate amplitude). Abbreviations: GABA, -aminobutyric acid; NMDA, N-methyl-D-aspartate.
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Neurotransmitter (dopamine and GABA) production and release were studied on tNSCs differentiated using protocol 4. HPLC electrochemical detector analysis showed significant amounts of dopamine in media conditioned by differentiated tNSCs for 2 days (38.3 ¡À 10.7 pg/ml; n = 4; Fig. 7A, 7B), 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.* B7 X  k% T: I$ A- i( O
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Figure 7. Neurotransmitter (dopamine, GABA) production and release of tNSCs differentiated on PA6 cells using protocol 4. (A): Representative chromatograms of HPLC-ECD determination of dopamine in medium conditioned for 2 days by tNSCs on PA6 cells (protocol 4, red) and PA6 cells alone (blue). Standard is presented in green. (B): Quantification of dopamine and GABA production in medium conditioned for 2 days (blue bars), in extracellular buffer conditioned for 45 minutes (red bars), and in extracellular buffer 56 mM KCl conditioned for 45 minutes (green bars). Abbreviations: DOPAC, dihydroxyphenyl acetic acid; GABA, -aminobutyric acid; HPLC-ECD, high-performance liquid chromatography¨Celectrochemical detector; KCl, potassium chloride; tNSC, tegmental neural stem cell.1 `8 x! L+ k3 r9 O; l' r
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DISCUSSION

Here, we provide evidence for the existence of multipotent NSCs in the adult mouse tegmentum (midbrain and hindbrain region) with the capacity to differentiate in vitro into functional nerve cell types, including dopaminergic neurons. We isolated and cultured NSCs from the adult tegmentum (tNSCs) of C57BL/6 mice using the neurosphere culture technique similar to those used for isolation and propagation of fetal mesencephalic NSCs .

NSCs are often defined in vitro by the presence of the CNS stem cells marker Nestin . 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.% G  x; v* B! ?) _4 f: E5 X( ^% U+ U
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Further evidence for the stem cell¨Clike 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¨Cgenerated 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 , 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 .% K; d* |9 |: I& K; [. _* l( L

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 ) 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.
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Zhao and colleagues demonstrated dopaminergic neurogenesis in the adult substantia nigra of C57BL/6 mice in vivo . 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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SUMMARY6 ]4 i  U- K2 Z: @# k( C
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We demonstrated the presence of multipotent clonogenic NSCs in the tegmentum of adult mice with the differentiation capacity similar to embryonic/fetal mesencephalic NSCs, including specification into functional nerve cells such as dopaminergic and GABAergic neurons. Previous reports suggest that these cells were derived from the SVZ of the aqueduct and/or fourth ventricle, a structure that is nearby the substantia nigra pars compacta (A9 region) where the degenerating dopaminergic neurons in Parkinson¡¯s disease (PD) are located. Recent studies failed to conclusively demonstrate dopaminergic neurogenesis in the adult mammalian midbrain in vivo , but our results suggest that NSCs residing in the adult midbrain/hind-brain area can give rise to new functional (dopaminergic) neurons when exposed to appropriate environmental signals. The presented developmental potential of adult tNSCs is one prerequisite for potential future endogenous cell replacement strategies in PD. However, future studies are warranted to clarify the mechanisms for controlling proliferation and dopaminergic differentiation of these adult tNSCs. The presented novel cell culture system provides a powerful tool for investigating these molecular mechanisms of adult neurogenesis and dopaminergic differentiation.

ACKNOWLEDGMENTS

We thank Oana M. Popa, Snezana Maljevic, and Holger Lerche for fruitful discussions; and Thomas Lenk for technical assistance. This work was supported in part by grants from the Interdisciplinary Center for Clinical Research Ulm (Interdiszlplinäres Zentrum f¨¹r Klinische Forschung ; Polish-German Cooperation in Neuroscience Program) to A.S. A.H. was supported by an IZKF fellowship as a member of the graduate college GRK460, Ulm.8 I/ s8 \: P9 H- A' p; }! S; @

DISCLOSURES: c3 D% c4 [3 t
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The authors indicate no potential conflicts of interest., R( I/ V/ U* z  X* s  b# G
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