Journal of Neuroscience Research 69:837 847 (2002)

Flow Cytometric Analysis of Neural Stem Cells in the Developing and Adult Mouse Brain
Ayako Murayama,13 Yumi Matsuzaki,1,3 Ayano Kawaguchi,4 Takuya Shimazaki,1,3* and Hideyuki Okano1,3
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Department of Physiology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan Department of Cell Biology and Neuroscience (A1), Osaka University Graduate School of Medicine, Suita, Osaka, Japan 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Saitama, Osaka, Japan 4 Cell Asymmetry, Center for Developmental Biology, RIKEN, Kobe, Hyogo, Japan

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We rst examined stagedependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) proles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC prole. With development, their number decreased in the FSChigh fractions but increased in the FSClow/SSChigh fraction, whereas NSCs were signicantly concentrated in the fraction containing the largest cells (about 20 m in diameter) at any stage, which were mostly the cells with the highest nestin-enhancer activity. Furthermore, we demonstrated that, at all stages examined, the side population (SP), dened as the Hoechst 33342 low/ negative fraction, which is known to be a stem cellenriched population in bone marrow, was also enriched for Notch1-positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large-cell fraction, however, but were concentrated instead in the FSClow/mid fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24low/ PNAlow fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.
2002 Wiley-Liss, Inc.

Although NSCs have come to be dened experimentally as neurosphere-initiating cells (Reynolds and Weiss, 1992), the lack of available methodologies for their prospective identication or purication has resulted in our having a poorly developed understanding of their biology, compared to the current understanding of hematopoietic stem cells (HSCs). Although selective markers for NSCs have been developed, i.e., Musashi1 (Sakakibara et al., 1996; Sakakibara and Okano, 1997; Kaneko et al., 2000), Nestin (Hockeld and McKay, 1985; Lendahl et al., 1990), and Sox1 (Pevny et al., 1998), antibodies against these intracellular molecules cannot be used for the isolation of living NSCs. McLaren et al. (2001) have recently characterized xed neurosphere-derived cells by uorescence-activated cell sorting (FACS) and found that Nestin-expressing cells contain relatively larger cells. In recent years, several new methods to identify and isolate live NSCs have been developed. We established previously a reporter gene carrying enhanced green uorescent protein (EGFP) under the control of the neuralspecic enhancer for the nestin gene (nestin-EGFP) (Roy et al., 2000a,b; Kawaguchi et al., 2001; Sawamoto, 2001). FACS analysis showed that nestin-EGFP expression correlated with the mitotic index, multipotency, and density of neurosphere-initiating cells, thereby permitting the highyield enrichment of neural stem cells from the cerebral cortex at E14.5 (Kawaguchi et al., 2001).
Contact grant sponsor: Japan Science and Technology Corporation; Contract grant sponsor: Ministry of Education, Science, Sports, Culture and Technology; Contract grant sponsor: Ministry of Health, Labour and Welfare. *Correspondence to: Takuya Shimazaki, Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: shimazak@sc.itc.keio.ac.jp Received 2 April 2002; Revised 8 May 2002; Accepted 8 May 2002

Key words: neural stem cells; neurosphere; uorescent activated cell sorting; forward scatter; side scatter; nestin-EGFP transgenic mice; side population; Notch1; CD24; peanut agglutinin

There is increasing interest in neural stem cells (NSCs) as therapeutic reagents for the damaged brain.
2002 Wiley-Liss, Inc.

Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/jnr.10339

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The rst example of immunoselection using a surface antigen was reported by Johansson et al. (1999), using an antibody to Notch1 to enrich for NSCs from adult mouse brain. Uchida et al. (2000) succeeded subsequently in isolating a population enriched for human fetal NSCs by sorting CD133() CD34() CD45() cells. Rietze et al. (2001) reported the isolation of one type of adult mouse NSC from the periventricular area by gating a diameter larger than 12 m and collecting only cells that did not exhibited peanut agglutinin (PNA)-binding activity, a marker for mouse HSCs (Salner et al., 1982) and that expressed low levels of CD24, a marker for neuronal progenitors and ependymal cells (Calaora et al., 1996; Shewan et al., 1996; Doetsch et al., 1999). They found that 80% of the cells in this population were neurosphereinitiating cells, and thus had properties of stem cells in vitro. Goodell et al. (1996) reported a new method for the isolation of HSCs from adult mouse bone marrow as a fraction that showed low red and blue DNA dye Hoechst 33342 uorescence when analyzed by dual wavelength FACS analysis. These cells have been called the side population (SP). In addition, the SP cells exhibiting the highest dye efux activity were the most primitive (Goodell et al., 1997). These Hoechst 33342 low-staining cells could no longer be detected after treatment with verapamil or reserpine (Goodell et al., 1996; Zhou et al., 2001). SP cells have also been identied in the adult bone marrow of several species (Goodell et al., 1997)., There has been enthusiastic interest recently in discovering whether putative stem cells from solid tissues also share this SP phenotype (Gussoni et al., 1999; Jackson and Goodell. 1999; Seale et al., 2000). SP cells in the CNS, however, have yet to be characterized. To establish a dened strategy for the prospective identication of NSCs, we have characterized the NSCs using the ratio of cell size, measured as forward scatter (FSC) to granularity, measured as side scatter (SSC), i.e., FSC/SSC. Using this prole, we have compared cells from developing mouse brains according to nestin-EGFP expression and SP phenotype.
MATERIALS AND METHODS Animals The nestin-EGFP transgenic mice were described previously (Kawaguchi et al., 2001). Female C57BL/6 J mice were crossed to male nestin-EGFP mice to obtain transgenic mouse embryos. We used heterozygous mice for all of the experiments presented in this paper. For dating pregnancies, the date the vaginal plug was observed was dened as embryonic day 0.5 (E0.5). Cell Preparation for FACS Brain tissues were dissected from transgenic or wild-type mice, placed in serum-free medium composed of a 1:1 mixture of Dulbeccos modied Eagles medium (DMEM) and F-12 (Gibco, Grand Island, NY), and triturated using a Gilson P1000 pipette in Media hormone mix (MHM) medium, which is

DMEM/F-12 containing insulin (25 g/ml), transferrin (100 g/ml), progesterone (20 nM), sodium selenate (30 ng), putrescine (60 nM), and HEPES (5 mM) (all from Sigma, St. Louis, MO) (Shimazaki et al., 2001). In the case of the adult brains, before trituration, the periventricular zone was dissected and incubated with dispase (250 U; Becton Dickinson Labware, Two Oak Park, Bedford, MA) and collagenase D (1 mg/ml; Roche, Mannheim, Germany) for 30 min in a CO2 incubator, triturating every 10 min. The protease reactions were stopped by chelation with EDTA (10 mM) and dilution with 0.9 M sucrose in 0.5 HBSS. The mixture was spun at 200 g for 7 min to remove the myelin, and the supernatant was discarded (Johansson et al., 1999). The cell pellet was washed once with DMEM/F-12, then resuspended in MHM medium containing recombinant human epidermal growth factor (EGF; 20 ng/ml) (R&G, Minneapolis, MN) and recombinant human basic broblast growth factor (bFGF; 20 ng/ml) (Genzyme TECHNE, Minneapolis, MN). For Hoechst staining, the neural cells were resuspended at 106 cells/ml in MHM medium containing EGF and bFGF, then incubated with 4 g/ml (for embryos) or 5 g/ml (for postnatal and adult mice) Hoechst 33342 (Sigma) for 90 min at 37C. When reserpine was used, the cells were stained as described in the presence of 2.8 M reserpine (Sigma). After Hoechst staining, the cells were pelleted and resuspended in MHM medium containing EGF and bFGF. For immunostaining, the suspensions were incubated for 20 min on ice with PE-conjugated anti-CD24, -CD45, -c-Kit, or -Sca-1 (1:100; eBioscience) and FITC-conjugated peanut agglutinin (PNA) (1:100; Vector Laboratories, Burlingame, CA), and then washed once in excess MHM medium. Cell Sorting Dissociated cells were spun, resuspended in MHM medium containing 1 g/ml propidium iodide (PI), and ltered through 40 m nylon mesh. Sorting and analyses were carried out on a FACSVantage SE ow cytometer (Becton-Dickinson, San Jose, CA). Dead cells were excluded by gating on forward and side scatter and by eliminating PI-positive events. The cells harvested from wild-type mice were used to set the background uorescence. Viable cells were sorted into MHM medium containing EGF and bFGF. Cell Culture For neurosphere cultures, sorted cells were treated as described previously (Reynolds and Weiss, 1992; Kawaguchi et al., 2001; Shimazaki et al., 2001). In brief, the cells were sorted into MHM medium, and counted. A 1:1 cocktail of this cell suspension and neurosphere-conditioned medium was plated onto each well at 10 cells/l, which is below the cell density at which virtually all spheres are clonal (Hulpas et al., 1997). The number of spheres was counted approximately 14 days later. After mechanical dissociation of each sphere into single cells, each pool of the cells derived from single sphere were cultured again for secondary sphere formation. For differentiation assays, spheres at 10 14 days in vitro were plated onto poly-L-ornithine (PO)-coated coverslips and cultured for another 57 days in DMEM/F-12 containing 1% fetal bovine serum (FBS). Cells were observed under an inverted uorescent microscope (IX70,

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Olympus, Japan) equipped with a cooled CCD digital camera (SPOT2, Seki Technotron, Japan). Fluorescence Analysis for Cryosections Transgenic mouse brains were xed in 4% paraformaldehyde in PBS at 4C overnight, immersed in 20% sucrose in PBS at 4C overnight, and then embedded in O.C.T. compound (Sakura Finetechinical Co. Ltd., Tokyo, Japan). Frozen sections (14-m thick) were washed with PBS and examined with a uorescence microscope. The images for EGFP were obtained in the green channel. Immunocytochemistry Cells attached to PO-coated coverslips were xed in 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were rinsed with PBS three times and incubated in PBS containing 10% goat serum and 0.01% Triton X-100 for blocking, then incubated at 4C overnight with the following antibodies: anti-nestin (mouse IgG, 1:500; Developmental Studies Hybridoma Bank), anti-GFP (rabbit IgG, 1:400; MBL, Nagano, Japan), anti--III-tubulin (mouse IgG, 1:100; Sigma), anti-GFAP (rabbit, 1:2; Dako, Carpinteria, CA), and O4 (mouse IgM, 1:2000; Chemicon, Temecula, CA). After three washes with PBS, the cells were incubated with secondary antibodies conjugated with uorescein isothiocyanate (FITC), Texas red, or AMCA (all from Jackson ImmunoResearch, West Grove, PA) for 1 hr at room temperature. For anti-Notch1 (M-20) (goat IgG, 1:100; Santa Cruz, CA) staining, we used Biotinylated anti-goat IgG (1:400; Jackson) as the second antibody, and then enhanced the signal using the VECTASTAIN elite ABC kit (Vector) followed by TSA Fluorescence Systems (NEN Life Science Products, Inc., Boston, MA). After three washes with PBS, the samples were mounted on slides and examined with a universal uorescence microscope (Axiophot 2; Carl Zeiss, Oberkochen, Germany).

RESULTS Characterization of Neural Stem Cells by FACS Based on the FSC/SSC Prole To characterize neural stem cells (NSCs) prospectively without using transgene-derived uorescence (Roy et al., 2000a,b; Kawaguchi et al., 2001), we used physical parameters, which are forward scatter (FSC), representing cell size, and side scatter (SSC), representing cellular granularity. McLaren et al. (2001) previously characterized xed neurosphere-derived cells by FACS analysis relying on the FSC/SSC prole and using several intracellular markers of neural cells. Here, we investigated various characteristics of NSCs in vivo during CNS development to identify stem cells prospectively at each stage. We rst examined the stage-dependent changes in the FSC/SSC proles in the developing striatum (Fig. 1), where NSCs appear to be active throughout the life of mammals (Morshed et al., 1994). To gain more insight into the characteristics of NSCs during neurogenesis, we focused on the three stages, at embryonic days (E) 11.5, 14.5, and early postnatal days (P) 13, which represent the beginning, ourishing, and ending of embryonic neurogenesis. To measure the FSC/SSC prole, we set up seven individual gates along the prole, named GATEs 2 8, as

shown in Figure 1A. We then measured the frequency of cells at the different developmental stages that were sorted into the different gates (Fig. 1B). The frequency of cells in GATE 4 markedly diminished between E14.5 and the early postnatal days (36.9 2.8 to 9.5 4.0% of the total cells). In contrast, the percentage of cells in GATE 3 increased from E14.5 to P13 (2.8 0.5 to 23.2 4.4% of total cells). Because GATE 4 occupied a dominant population in the embryonic stage, this fraction seemed to contain more immature cells, compared to GATE 3. To determine which population of cells actually contained the NSCs, we carried out neurosphere formation assays. This assay involves a culture system in which NSCs selectively proliferate to form multicellular aggregates called neurospheres (Reynolds and Weiss, 1992). For this assay, the sorted cells were counted and plated at a density of 10 cells/l, then the number of neurospheres whose diameter was 50 m 10 14 days after plating was counted. Observation under a microscope showed that GATE 7 contained much more telophase cells undergoing cytokinesis (44.6%) than the other GATEs (containing less than 16.5% in GATE8), so we eliminated this fraction from the assay to maintain the accuracy of this study. As shown in Figure 2A, neurosphere-initiating cells were distributed mainly to GATEs 4, 5, and 8 during the embryonic stages, and to GATEs 5, 6, and 8 at P13. In contrast, virtually no neurospheres were formed from the cells in GATEs 2 and 3 at any developmental stage (n 6 10). Interestingly, the distribution of neurosphereinitiating cells to GATE 4 decreased with development (1.5 0.2, 1.0 0.0, and 0.2 0.1-fold of that in the unfractionated cells, at E11.5, 14.5, and P13, respectively). In contrast, cells in GATE 6 showed very low neurosphere-forming activity during the embryonic stages (0.4 0.1 and 0.2 0.0-fold, at E11.5 and 14.5, respectively) whereas the percentage of neurosphere-forming cells increased at P1-3 (0.9 0.2-fold). GATE 8 appeared to have the highest ratio of neurosphere-initiating cells at any developmental stage. In particular, it showed a significant enrichment of neurosphere-initiating cells (3.3 0.6-fold) at E14.5. The ratio, however, decreased at P1-3 (1.7 0.2-fold). To conrm these neurosphere-initiating cells were actually NSCs, their multipotency and self-renewal capacity were examined in vitro. Individual neurospheres were allowed to differentiate after plating and were cultured for 57 days in the absence of mitogen, then processed for indirect- immunocytochemistry with antibodies against neuronal (III-tubulin), astrocytic (GFAP), and oligodendrocytic (O4) markers. All of the colonies examined (n 520 in each fraction) contained these three cell types (Fig. 2B), indicating that the sorted neurosphere-initiating cells were multipotent. In addition, to examine the self-renewal capacity of the sorted cells, primary neurospheres were individually transferred into separate wells and dissociated into single cells, then cultured for 14 days and assessed for

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Fig. 1. FCS/SSC prole of mouse striatal cells at various developmental stages. Striatal cells were dissociated mechanically. The dissociated single cells were then suspended in medium supplemented with 1 g/ml propidium iodide (PI). By multiparameter ow cytometry, all the cells in the PI-negative live cell gate were plotted according to the FSC and SSC two-dimensional prole. A: Representative FSC/SSC

prole of striatal cells from E11.5, E14.5 amd P1 mice. GATEs 2 8 were dened based on the FSC/SSC signal intensity. B: The frequency of the cells in the GATE 2 8 subfractions at E11.5 (white), E14.5 (light gray), and P13 (deep gray). The data give the mean SEM of three independent experiments.

secondary neurosphere formation. Virtually all the wells had secondary neurospheres whose appearance resembled that of the primary neurospheres (Fig. 2C). Thus, the original neurosphere-initiating cells had both self-renewal capacity and multipotency, indicating that they were neural stem cells. These results indicate that NSCs have diverse characteristics in their physical parameters as assessed using the FCS/SSC prole and can be concentrated to some extent. Characterization of Neural Progenitors According to the FSC/SSC Prole Using nestin-EGFP Transgenic Mice We found previously that NSCs as well as neurosphere-initiating progenitor cells are most concen-

trated in the pool of the cells that show the brightest uorescence derived from the nestin-EGFP transgene in the developing cerebral cortex at E14.5 (Kawaguchi et al., 2001). To learn the correlation between the FSC/SSC prole and nestin-EGFP expression in neural progenitors, we used the same transgenic mice for more detailed analysis. We rst examined the stage-dependent changes in the expression pattern of EGFP in the striatum (Fig. 3A). Under the uorescence microscope, EGFP-positive cells were located within the ventricular zone (VZ) of the striatum during neurogenesis (E11.5, E14.5, and P2) in a manner resembling the distribution of EGFP-positive cells in the developing cerebral cortex, as described previously (Kawaguchi et al., 2001). In the adult, the brightest EGFP-

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Fig. 2. Distribution of neural stem cells to cellular subfractions isolated from the striatum based on the FSC/SSC prole. A: Neurosphere formation was assessed for the cellular subfractions, which are indicated in Figure 1A. The numbers of neurospheres (50 m in diameter) per 2 103 cells were counted after 10 14 days. Values are the mean and the SEM of determinations from 6 9 wells of two or three independent experiments. The data presented were calculated by dividing the number of neurospheres from each fraction by the number from the unfractionated population. B: III-tubulin neurons (red), GFAP

astrocytes (blue) and O4 oligodendrocytes (green) were generated by sorted neurosphere-initiating cells, indicating their multipotency. Individual primary neurospheres were allowed to differentiate for 57 days in the absence of mitogen and processed for the triple-labeling immunocytochemistry. Scale bar 50 m. C: Formation of secondary neurospheres by single cells from each primary neurosphere, indicating self-renewal expansion of sorted neurosphere-initiating cells. Scale bar 50 m.

expressing cells were restricted to the ependymal cell layer, with a few moderately bright cells scattered within the whole periventricular area. Interestingly, in the corpus callosum, there were weakly positive EGFP cells along the putatively myelinated bers and bright nestin-EGFPpositive cells among these bers.

To examine the stage that nestin-EGFP uorescence faithfully corresponded to the expression of the Nestin protein (Hockeld and McKay, 1985; Dahlstrand et al., 1995), we carried out double-labeling immunocytochemistry of EGFP and Nestin on the striatal cells from the transgenic mice. We conrmed the coincidental expres-

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Fig. 3. nestin-EGFP expression in the developing and adult striatum. A: Photomicrographs of EGFP uorescence in coronal sections from forebrains of E11.5, E14.5, P2, and adult nestin-EGFP transgenic mice. ST, striatum; LV, lateral ventricle; cc, corpus callosum; pia, pia side. Scale bar 100 m. B: Double-labeling immunocytochemistry of EGFP and Nestin on the striatal cells from the transgenic mice at P2. After birth, the amount of colocalization decreased gradually. An arrow

indicates a cell expressing neither EGFP nor Nestin. Arrowheads indicate cells expressing only EGFP but not nestin. Scale bar 50 m. C: Comparison of nestin-EGFP expression in E11.5, E14.5, and P1 striatum. Gates were dened by the intensity of EGFP uorescence as EGFP (milk-white), EGFP (light green), and EGFP (deep green). The mean frequency in each gate was calculated as percentage of total live cells (mean SEM of 212 independent experiments).

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sion of these proteins in the striatum at E14.5. After birth, however, the amount of colocalization decreased gradually (Fig. 3B). We next examined the nestin-EGFP expression using FACS analysis. Fig. 3C shows FACS proles of the nestinEGFP expression in striatal cells from developing mouse brains. We classied nestin-EGFP-expressing cells into three categories according to the uorescence intensity of the nestin-EGFP: negative (EGFP), weakly positive (low) (EGFP), and positive (EGFP). The EGFP population diminished from 62.3 6.7% at E11.5 to 12.3 1.4% in the adult. In contrast, the percentage of EGFP cells increased with development. When we sorted the nestin-EGFP striatal cells according to the FSC/SSC prole (Fig 1), the GATE 4, 5, 7, and 8 fractions predominantly consisted of EGFP cells (62.793.7%) at all stages tested (Fig. 4). In GATE 6, EGFP cells became the majority at P13 (74.4 3.1%). In contrast, EGFP and EGFP made up the major population of the FSClow/SSClow (GATEs 2 and 3) fraction, which contained few NSCs. This result indicates that the fractionation of neural cells according to their FSC/SSC prole correlates well with sorting them by their nestin-EGFP intensity. Collectively, the NSCs and the nestin-EGFP-positive progenitor cells can be selectively extracted as FSChigh/SSClow gated cells (in GATEs 4 and 8) from the embryonic brain. Characterization of the SP in the CNS The SP is known to be composed mainly of stem cells in several organs, especially in the hematopoietic system (Goodell et al., 1996; Gussoni et al., 1999). To further characterize the fractions isolated using the FSC/ SSC prole, we analyzed the distribution of SP cells to the different GATEs, although SP cells in the central nervous system (CNS) have not been well characterized yet. Cells from the striatum of embryonic/postnatal animals were stained with 4 g/ml (for embryos) or 5 g/ml (for neonates and adults) of Hoechst 33342 and analyzed as described previously (Goodell et al., 1996). As with the staining of bone marrow cells, FACS analysis of the striatal cells exhibited an SP fraction that displayed low staining with Hoechst 33342 and a main population (MP) of cells that was more brightly stained with the dye (Fig. 5A). These striatal SP cells disappeared upon addition of reserpine (Fig. 5A), as is the case for bone-marrow SP cells (Zhou et al., 2001). The characterization of striatal SP cells exhibited several features that distinguished them from bone-marrow SP cells. They included c-Kit positive cells, but were mostly negative for Sca-1 and CD45 (data not shown), which are surface markers that are expressed on bone-marrow SP cells (Goodell et al., 1997). We next examined the correlation between the nestin-EGFP expression level and the Hoechst staining prole in striatal cells from the transgenic mice. We found that the SP cells consisted of both EGFP and EGFP cells, and a few SP cells were in the EGFP fraction in the embryonic striatum (Fig. 5B). We then examined whether the SP cells expressed neural progenitor markers. Sorted

Fig. 4. Nestin-EGFP expression in FSC/SSC-based subfractions (GATE 2-8). EGFP uorescence intensity for each FSC/SSC-based subfraction of embryonic/postnatal striatal cells is presented as described in the legend for Figure 3 (mean SEM of 312 independent experiments).

SP cells were attached to PO-coated coverslips by centrifugation followed by xation. The attached cells were then used for Notch1 immunocytochemistry, as Notch1 is known to be preferentially expressed in NSCs (Johansson et al., 1999). As shown in Figure 5C, the SP cell fraction was signicantly rich in Notch1-positive cells for all of the developmental stages (more than 60% of the SP cells); in contrast, the percentage of Notch1-positive cells in the total cells decreased from 55 9% at E11.5 to only 7 4% at P13. It has been shown recently that neural stem cells in the periventricular area of the adult mouse brain are highly

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Fig. 5. Characterization of brain SP cells. Dissociated cells were stained with Hoechst 33342 before FACS analysis, as described in Materials and Methods. Representative FACS dot plot showing the presence and phenotypes of SP cells. A: Small gated cell population identies the SP cells (1% of the E14.5 striatal population) that signicantly decreased in the presence of reserpine B: Mean nestin-EGFP uorescence intensity of wild-type (control), total striatal, and striatal SP cells. C: Fluorescent microscopic images of Notch1 expression in sorted SP cells

from the P2 striatum. The indicated values are the percentage of Notch1-positive cells at each developmental stage (n 2 or 3). DF: The distribution of adult striatum SP cells according to their expression of CD24 and PNA in the periventricular area. The SP FACS plot of adult striatal cells (D) and of the 12m CD24low/PNAlow fraction (E), and PNA/FSC proles and CD24/PNA expression of the SP population (F) are shown. Scale bar 50 m.

concentrated in the cells with a diameter larger than 12 m (12 m) and in the CD24low/PNAlow fraction (Rietze et al., 2001). FACS analysis for the adult striatal cells with CD24, PNA, and Hoechst33342 staining showed that the 12 m CD24low/PNAlow fraction included some SP cells, although not all, indicating that SP cells are at least present in the pool of NSCs in the periventricular area of the adult brain (Fig. 5DF).

Finally, we examined how the SP cells were distributed using the FSC/SSC prole (Fig. 6). SP cells were concentrated in the GATE 4 or 5 fraction during the embryonic stages (30.9% and 65.2% in GATE 4, and 46.9% and 15.2% in GATE 5, at E11.5 and E14.5 respectively). Postnatally, the percentage of the GATE 2 cells in SP increased markedly (29.7%), but GATE 5 cells decreased (5.8%) (Fig. 6A). In contrast, there were no de-

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Fig. 6. Comparison of the FSC/SSC prole and the Hoechst Red/ Blue FACS dot plot of striatal cells. A: Representative FSC/SSC prole of total striatal cells from E14.5 embryos and striatal SP cells from E11.5, E14.5, and P13 mice. B: Representative FACS dot plot of Hoechst Red/Blue for each GATE 2 8 subfraction of E14.5 striatal cells.

tectable SP cells in GATE 8 predominantly consisted of cells that were in S-G2M phase (Fig. 6A,B). Thus, the SP cells are distributed to a relatively small cell fraction during the CNS development. These results suggest that the NSC population may contain SP cells but is not identical. DISCUSSION In mammalian CNS development, NSCs may be responsible for the generation of multiple types of neurons and glia regulated spatially and temporally by cell intrinsic and extrinsic factors. To elucidate exactly how this cytogenesis is regulated, prospective identication and characterization of NSCs in each developmental stage is crucial. We investigated developmental proles of NSCs by FACS using four different parameters: physical parameters (FSC and SSC), nestin enhancer driven-EGFP (nestin-EGFP) expression, and live Hoechst staining, and demonstrated that the characteristics of NSCs are not uniform and change developmentally. In the view of the physical parameters, FSC/SSC, the characteristics of NSCs seem diverse. The fractionation of striatal cells combined with the neurosphere formation assay clearly showed that the stem cell population could be divided into various types according to size and cellular granularity (GATEs 4 8 in Fig. 1). In addition, the distribution of NSCs to each gate changed with development. In particular, the percentage of NSCs in GATEs 4 and 8 decreased signicantly between E14.5 and P13, when the amount of gliogenesis is rising, whereas the percentage of NSCs in GATEs 5 and 6 increased, indicating NSCs gain more cellular granularity in this period (Fig. 2A). It may be possible that GATE 6 may contain adult-specic NSCs, because neurosphereinitiating cells and nestin positive cells in GATE 6 are specically increase at postnatal stage. There are some evidences that adult NSCs and fetal NSCs have different function and competence to several cytokines (see review by Temple, 2001). Note that the FSC/SSC prole of cells from the adult striatum was quite different from that of embryos, so we were not able to compare adult NSCs with embryonic and early postnatal ones by this parameter. We have shown previously that the brightest 10% of the EGFP uorescent cells (nestin-EGFP10%) from the cortex of nestin-EGFP transgenic mouse embryos contains the highest concentration of NSCs (Kawaguchi et al., 2001). In this study, we further characterized the nestin-EGFP10% cells using the FSC/SSC prole. In the FSC/SSC prole, nestin-EGFP10% cells were concentrated more in GATEs 4, 7, and 8, where the NSCs were concentrated (Fig. 2A), compared to nestin-EGFP cells at E14.5 (Fig. 4). By microscopic analysis, many cells in GATE 7 were in telophase undergoing cytokinesis (44.6%), whereas Hoechst 33342 staining showed that the majority of GATE 7 and 8 cells were in S-G2M at all stages examined (Fig. 6C). When cells from the E14.5 striatum in GATE 7 were sorted and cultured, the percentage of neurosphere-initiating cells was high as expected (2.67fold of non-gated live cells). GATE 8 also contained some telophase cells (16.5%), but they were less granular than

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the GATE7 cells (Fig. 1). These results suggest that the NSCs in GATE 8 may be in the S phase and early mitotic phases, i.e., prophase and metaphase. The mitotic index of the nestin-EGFP10% cells as assessed by BrdU labeling showed that the nestin-EGFP10% fraction is the most likely to be in the S phase in the E14.5 cortex (Kawaguchi et al., 2001); this observation supports the above possibility. If this is true, the reduction in the frequency of NSCs in GATE 8 during the early postnatal period may reect an extension of the cell-cycle time of the NSCs. In addition, the difference in the cell-cycle phase may be one reason why the size of NSCs is diverse. Moreover, because the neurosphere-formation assay picks up only primitive cells that have proliferative capacity in response to mitogens such as EGF or FGF2, it is possible that the phase of the cell-cycle the NSCs are in affects their viability and responsiveness to the mitogens. In this case, cells in GATE 8 and in the nestin-EGFP10% fraction should show the highest frequency of neurosphere formation at all developmental stages. We may be able to obtain a cell population enriched in NSCs by collecting cells in early mitosis. It is known that quiescent hematopoietic stem cells are concentrated in the SP; these bone-marrow cells have a high level of dye efux activity (Goodell et al., 1996, 1997). We sorted SP cells from the developing striatum and analyzed their FSC/SSC prole and nestin-EGFP expression (Figs. 5 and 6). The major population of SP cells occupied GATEs 2 6. Interestingly, the SP population did not contain nestin-EGFP10% cells. Consistent with this, GATE 8 that were occupied mostly by nestinEGFP10% cells (56.7 8.5%) from the embryonic striatum did not contain SP cells. Almost 60% of the striatal SP cells expressed Notch 1 protein (Fig. 5C), which is known to be preferentially expressed in NSCs (Johansson et al., 1999). Unlike HSCs, NSCs retain almost no neurosphere-initiating ability after Hoechst staining in the presence of 2% serum, an almost 30-fold drop from unstained cells. Because of this technical difculty, we cannot conclude whether the SP population contains NSCs, although it is possible that some SP cells in striatum are dormant NSCs; HSCs from normal bone marrow are largely quiescent (Spangrude et al., 1991) and they have been shown to exist among hematopoietic SP cells (Goodell et al., 1996). Interestingly, GATE 2 cells that did not contain neurosphere-initiating cells also included SP cells. It would be possible to speculate that those SP cells include also other type of stem cells such as pluripotent stem cells identied in other adult tissues (Sanchez-Ramos et al., 2000; Woodbury et al., 2000; Toma et al., 2001). Given these observations, the characteristics of NSCs are unlikely to be uniform and appear to alter with development, making it difcult to isolate a cell fraction that contains only NSCs, at least using only a few parameters and markers. In recent studies, unexpected types of cells have been shown to have features as NSCs, e.g., ependymal cells (Johansson et al., 1999), GFAP-positive astrocytes (type B cells) in the adult brain (Doetsch et al., 1999), and radial ber-possessing cells in the embryonic brain

(Malatesta et al., 2000; Ourednik et al., 2001; Miyata et al., 2001). Moreover, recent reports indicate a possible heterogeneity of adult NSCs: oligodendrocyte progenitor cells (O2A progenitor cells) in the optic nerve can be dedifferentiated back to stem-like cells in vitro (Kondo and Raff, 2000), and type B cells progeny (type C cells) are stimulated by EGF to convert into NSCs in the adult SVZ (Doetsch et al., 2001). These reports suggest that there are many types of NSCs in the developing and adult CNS; therefore, we will have to analyze several pools of NSCs with different features to understand exactly what characteristics dene NSCs, what types of NSC exist, and how they contribute to CNS histogenesis and maintenance. The present study should provide some insight into the considerations required for the prospective identication and characterization of distinct types of NSCs in various developmental stages, and also for the analysis of their cell-cycle transition during development. Clarication of these characteristics will be required for further understanding of the regulatory mechanisms underlying the self-renewal and differentiation of NSCs. ACKNOWLEDGMENTS We thank Drs. T. Miyata, K. Sawamoto, and M. Sakaguchi for helpful discussion and Dr. M. Osawa and H. Yamasaki for technical instructions for the cell sorting. We also thank Dr. K. Yoshiya and T. Kojima for technical assistance. REFERENCES
Calaora V, Chazal G, Nielsen PJ, Rougon G, Moreau H. 1996. MCD24 expression in the developing mouse brain and zones of secondary neurogenesis in the adult. Neuroscience 73:581594. Dahlstrand J, Lardelli M, Lendahl U. 1995. Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system. Brain Res Dev Brain Res 84:109 129. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703716. Doetsch FK, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A. 2001. EGF induces conversion of transit amplifying neurogenic precursors into multipotential invasive cells in the adult brain. Soc Neurosci Abstr 894. 4: http://sfn.scholarone.com/itin2001/ Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:17971806. Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria MA, Paradis G, Grupp SA, Mulligan RC. 1997. Dye efux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:13371345. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC. 1999. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401:390 394. Hockeld S, McKay RDG. 1985. Identication of major cell classes in the developing mammalian nervous system. J Neurosci 5:3310 3328. Hulpas R, Tiarks C, Reilly J, Hsidh CC, Recht L, Quesenberry PJ. 1997. In vitro cell-density dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free condition. Exp Neurol 148:147 156.

Various Characteristics of Neural Stem Cells

Jackson KA, Mi T, Goodell MA, 1999. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 96:1448214486. Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J. 1999. Identication of a neural stem cell in the adult mammalian central nervous system. Cell 96:2534. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H. 2000. Musashi1: an evolutionarily conserved marker for CNS stem cells and neural progenitor cells. Dev Neurosci 22:138 150. Kawaguchi A, Miyata T, Sawamoto K, Takashita N, Murayama A, Akamatsu W, Ogawa M, Okabe M, Tano Y, Goldman SA, Okano H. 2001. nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol Cell Neurosci 17:259 273. Kondo T, Raff M. 2000. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289:1754 1757. Lendahl U, Zimmerman LB, McKay RDG. 1990. CNS stem cells express a new class of intermediate lament protein. Cell 60:585595. Malatesta P, Hartfuss E, Gotz M. Isolation of radial glial cells by uorescentactivated cell sorting reveals a neuronal lineage. Development 127:5253 5263. McLaren FH, Svendsen CN, van der Meide P, Joly E. 2001. Analysis of neural stem cells by ow cytometry: cellular differentiation modies pattern of MHC expression. J Neuroimmunol 112:35 46. Miyata T, Kawaguchi A, Okano H, Ogawa M. 2001. Asymmetric inheritance of radial glial bers by cortical neurons. Neuron 31:727741. Morshead CM, Reynolds BA, Craig CG, McBurney MW, Staines WA, Morassutti D, Weiss S, van der Kooy D. 1994. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13:10711082. Ourednik V, Ourednik J, Flax JD, Zawada WM, Hutt C, Yang C, Park KI, Kim SU, Sidman RL, Freed CR, Snyder EY. 2001. Segregation of human neural stem cells in the developing primate forebrain. Science 293:1820 1824. Pevny LH, Sockanathan S, Placzek M, Lovell-Badge R. 1998. A role for SOX1 in neural determination. Development 125:19671978. Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett. 2001. Purication of a pluripotent neural stem cell from the adult mouse brain. Nature 412:736 739. Reynolds BA, Weiss S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:17071710. Roy NS, Benraiss A, Wang S, Fraser RAR, Goodman R, Couldwell WT, Nedergaard M, Kawaguchi A, Okano H, Goldman SA. 2000a. Promotertargeted selection and isolation of neural progenitor cells from the adult human ventricular zone. J Neurosci Res 59:321331. Roy NS, Wang S, Jiang L, Kang J, Restelli C, Fraser RAR, Couldwell WT, Kawaguchi A, Okano H, Nedergaard M, Goldman SA. 2000b. In vitro neurogenesis by neural progenitor cells prospectively identied and viably sorted from the adult human hippocampus. Nat Med 6:271277.

847

Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nataga T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H. 1996. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 176:230 242. Sakakibara S, Okano H. 1997. Expression of neural RNA-binding proteins in the postnatal CNS: implications of their roles in neuronal and glial cell development. J Neurosci 17:8300 8312. Salner AL, Obbagy JE, Hellman S. 1982. Differing stem cell self-renewal of lectin-separated murine bone marrow fractions. J Natl Cancer Inst 68: 639 641. Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W. 2000. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 164:247 256. Sawamoto K, Nakao N, Kakishita K, Ogawa Y, Toyama Y, Yamamoto A, Yamaguchi M, Mori K, Goldman SA, Itakura T, Okano H. 2001. Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene. J Neurosci 21:38953903. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. 2000. Pax7 is required for the specication of myogenic satellite cells. Cell 102:777786. Shewan D, Calaora V, Nielsen P, Cohen J, Rougon G, Moreau H. 1996. mCD24, a glycoprotein transiently expressed by neurons, is an inhibitor of neurite outgrowth. J Neurosci 16:2624 2634. Shimazaki T, Shingo T, Weiss S. 2001. The ciliary neurotrophic factor/ leukemia inhibitory factor/gp130 receptor complex operates in the maintenance of mammalian forebrain neural stem cells. J Neurosci 21:7642 7653. Spangrude GJ. 1991. Hematopoietic stem-cell differentiation. Curr Opin Immunol 3:171178. Temple S. 2001. The development of neural stem cells. Nature 414:112 117. Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD. 2001. Isolation of multipotent adult stem cells from dermis of mammalian skin. Nat Cell Biol 3:778 784. Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL. 2000. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 97:14720 14725. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61:364 370. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld CG, Osawa M, Nakauchi H, Porrentino BP. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and a molecular determinant of the side-population phenotype. Nat Med 7:1028 1034.